United States Patent Application: 0200040047

( 106 of 1848 )

United States Patent Application	20200040047
Kind Code	A1
SOLLNER; Christian ; et al.	February 6, 2020

INTERACTION OF DRAXIN AND Y-NETRINS

Abstract

This invention relates to extracellular protein-protein interactions and their possible therapeutic uses. More particularly, this invention describes the interaction between Draxin, particularly fragments binding to .gamma.-Netrins comprising SEQ ID NO.:1, 2 or 3, and variants thereof, with .gamma.-Netrins, and the use of this interaction to disrupt .gamma.-Netrin/Netrin receptor interactions. The invention also relates to diagnostic and/or therapeutic uses of Draxin or fragments or variants thereof, as well as to an antibody against Draxin inhibiting binding of Draxin to .gamma.-Netrins. Further, the invention relates to fragments of .gamma.-Netrins, in particular Draxin-binding Netrin1-fragments comprising SEQ ID NO.: 51 and variants thereof, as well as to an antibody against .gamma.-Netrins inhibiting binding of .gamma.-Netrins to Netrin receptors.

Inventors:

SOLLNER; Christian; (Nurtingen, DE) ; GAO; Xuefan; (Tubingen, DE) ; NUSSLEIN-VOLHARD; Christiane; (Tubingen-Bebenhausen, DE)

Applicant:

Name	City	State	Country	Type
Max-Planck-Gesellschaft zur Foerderung der Wissenschaften e.V.	Muenchen		DE

Assignee:

Max-Planck-Gesellschaft zur Foerderung der Wissenschaften e.V.
Muenchen
DE

Family ID:

49999766

Appl. No.:

16/553536

Filed:

August 28, 2019

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number
15113878	Jul 25, 2016	10435442
PCT/EP2015/051088	Jan 21, 2015
16553536
62049643	Sep 12, 2014

Current U.S. Class: 1/1

Current CPC Class: C07K 14/475 20130101; C07K 14/4703 20130101; C07K 14/47 20130101; C07K 16/18 20130101; C07K 2319/30 20130101; C07K 14/461 20130101; A61K 38/00 20130101; C07K 2317/76 20130101

International Class: C07K 14/47 20060101 C07K014/47; C07K 14/475 20060101 C07K014/475; C07K 16/18 20060101 C07K016/18; C07K 14/46 20060101 C07K014/46

Foreign Application Data

Date	Code	Application Number
Jan 23, 2014	EP	14152341.5

Claims

1-15. (canceled)

16. Draxin-binding peptide comprising (i) at least 20 consecutive amino acids from the sequence KACDCHPVGAAGKTCNQTTGQCPCKDGVTGITCNRCANGYQQSRSP IAPCIKIPIAPP (SEQ ID NO.: 51) or (ii) a variant thereof having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO.: 51; wherein said peptide has a length of up to 580 amino acids and is optionally fused to a heterologous peptide or polypeptide.

17. The peptide according to claim 16, wherein said peptide comprises a sequence selected from the group consisting of SEQ ID NO.: 45, SEQ ID NO.: 48, a peptide corresponding to SEQ ID NO.: 45 or SEQ ID NO.: 48 from another species, or a variant thereof having a sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% thereto.

18. The peptide according to claim 16, which is fused to a heterologous peptide or polypeptide.

19. The peptide according to claim 16, in combination with a carrier suitable for use in medicine.

20. The peptide according to claim 16, wherein said variant contains at least one non-naturally occurring substitution modification relative to SEQ ID NO.:51.

21. The peptide according to claim 18, wherein said peptide is fused to a functional fragment of an immunoglobulin (Ig).

22. The peptide according to claim 21, wherein said functional fragment of an immunoglobulin (Ig) is an Ig Fc fragment.

23. The peptide according to claim 22, wherein said Ig Fc fragment is a human Ig Fc fragment.

24. The peptide according to claim 23, wherein said human Ig Fc fragment is a human IgG Fc fragment.

25. A method for treating a condition associated with, accompanied by or mediated by pathologic, decreased, .gamma.-Netrin expression or activity, or for treating a condition associated with, accompanied by or mediated by pathologic, increased, Draxin expression or activity, comprising administering a draxin-binding peptide comprising (i) at least 20 consecutive amino acids from the sequence KACDCHPVGAAGKTCNQTTGQCPCKD GVTGITCNRCANGYQQSRSP IAPCIKIPIAPP (SEQ ID NO.: 51) or (ii) a variant thereof having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO.: 51, which is optionally fused to a heterologous peptide or polypeptide,

26. The method according to claim 25, wherein said peptide has a length of up to 500, up to 400, up to 300, up to 250, up to 200, up to 150, up to 100, up to 75 or up to 60 amino acids.

27. The method according to claim 25, wherein said peptide comprises at least 20, at least 30, at least 40 or at least 50 consecutive amino acids from SEQ ID NO.:51 or the complete SEQ ID NO.:51, or a variant thereof having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO.: 51.

28. The method according to claim 25, wherein said condition is a cardiovascular disease, which is selected from the group consisting of ischemia/reperfusion (I/R) injury; myocardial infarction; mitochondrial damage; neointimal formation and restenosis; vascular injury or vascular dysfunction; vascular smooth muscle cell migration and proliferation; apoptosis of endothelial progenitor cells, procure induced restenosis; and hypertension.

29. The method according to claim 25, wherein said peptide is administered as an active agent in a pharmaceutical composition together with at least one pharmaceutically acceptable carrier.

30. The method according to claim 25, further comprising administering at least one additional therapeutically active agent, wherein the additional therapeutically active agent is selected from the group consisting of decoy Netrin receptors, cytostatic agents, cytotoxic agents, statins, antihyperlipidemic agents, anti-coagulant agents, kinase inhibitors and angiogenesis modulators.

31. The method according to claim 28, wherein said ischemia/reperfusion (I/R) injury is selected from the group consisting of renal I/R injury and I/R injury of cardiac tissue; and said myocardial infarction is an infarct resulting from I/R injury.

32. An antibody, functional fragment or derivative thereof directed against Draxin, wherein said antibody, functional fragment or derivative thereof inhibits binding of Draxin to at least one .gamma.-Netrin, wherein said antibody or functional fragment or derivative thereof is directed against an epitope between amino acids 209-284, amino acids 226-257, or amino acids 232-252, of human Draxin.

33. An antibody, functional fragment or derivative thereof directed against Netrin1, which inhibits binding of at least one .gamma.-Netrin to at least one Netrin receptor, and which is directed against an epitope between amino acids 285-451 of human Netrin1 or a corresponding epitope in another species.

34. The antibody according to claim 32, wherein said corresponding epitope in another species is an epitope located in the EGF1-3 domains of a zebrafish .gamma.-Netrin.

35. The antibody or fragment or derivative thereof according to claim 32, in combination with a pharmaceutically acceptable carrier.

Description

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional of U.S. Ser. No. 15/113,878 filed Jul. 25, 2016, which is a 35 U.S.C. 371 National Phase Entry Application from PCT/EP2015/051088, filed Jan. 21, 2015, which claims the benefit of U.S. Patent Application No. 62/049,643 filed on Sep. 12, 2014 and European Patent Application No. 14152341.5 filed Jan. 23, 2014, the disclosure of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates to extracellular protein-protein interactions and their possible therapeutic uses. More particularly, this invention describes the interaction between Draxin, particularly fragments binding to .gamma.-Netrins comprising SEQ ID NO.:1, 2 or 3, and variants thereof, with .gamma.-Netrins, and the use of this interaction to disrupt .gamma.-Netrin/Netrin receptor interactions. The invention also relates to diagnostic and/or therapeutic uses of Draxin or fragments or variants thereof, as well as to an antibody against Draxin inhibiting binding of Draxin to .gamma.-Netrins. Further, the invention relates to fragments of .gamma.-Netrins, in particular Draxin-binding Netrin1-fragments comprising SEQ ID NO.: 51 and variants thereof, as well as to an antibody against .gamma.-Netrins inhibiting binding of .gamma.-Netrins to Netrin receptors.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 16, 2018, filed in the parent application Ser. No. 15/113,878 is named 57261 PUSWO_ST25.txt and is 87,752 bytes in size.

BACKGROUND OF THE INVENTION

[0004] The laminin-related netrin protein family in humans comprises 5 members. Three of them, Netrin1, Netrin3, and Netrin4 are secreted proteins. NetrinG1 and NetrinG2 instead are linked to the cell surface by a GPI anchor. The LamNT domain and the EGF domains of Netrin1 and Netrin3 are derived from the .gamma. chain of Laminin1. In the context of the present invention, these Netrins are thus referred to as ".gamma.-Netrins". In contrast, the corresponding domains of Netrin4, NetrinG1 and NetrinG2 are homologous to the domains present in the .beta. chain of Laminin1 (Moore et al., 2007).

[0005] Netrin1 is a diffusible, laminin-related protein identified as neuronal guidance cue during development of the nervous system. Netrin1 mediates its biological effects through binding to receptors, which belong to the so-called dependence receptors, e.g. deleted in colorectal cancer (DCC) and uncoordinated-5-homolog (UNCSH). Recently, it has been found that Netrin1 is expressed outside the nervous system and contributes to the patterning of developing epithelial tissues such as mammary gland, pancreas, and lung by regulating diverse processes including adhesion, motility, proliferation, and differentiation of cells.

[0006] Numerous tumors have been described to express cell surface receptors belonging to the DCC- and UNC5-family. These receptors are binding to the secreted ligand Netrin1 in the extracellular space and serve, in addition to their well-established neurodevelopmental function, as dependence receptors in cancers (Castets et al., 2012; Mehlen et al., 2011). In tumors they can regulate tumor cell survival in a Netrin1 dependent manner. Netrin1 itself is known to be upregulated by many tumor types and has been suggested to act as an oncogene (Arakawa, 2004; Fitamant et al., 2008). If Netrin1 is not bound to dependence receptors of the DCC- and UNC5-family, the receptors cannot form dimers or multimers, which in turn triggers the activation of a pro-apoptotic pathway.

[0007] In several studies, decoy Netrin receptor fragments have been used to disrupt Netrin/Netrin receptor interactions in order to induce pro-apoptotic signaling. For example, such receptor fragments have been used in cancer cell lines (Delloye-Bourgeois et al., 2009; Fitamant et al., 2008) and in animal models (Fitamant et al., 2008; Paradisi et al., 2013; Paradisi et al., 2009) to induce cancer cell death. However, using a fragment of a Netrin receptor causes interference at a relatively late stage of the signaling cascade, namely just before dimerization of the receptor. Moreover, even when using high concentrations of these decoy receptors, a residual binding of Netrin to the full length receptor cannot be prevented.

[0008] It was thus an object of the invention to provide compounds that can be used for interfering with the binding of .gamma.-Netrins, in particular Netrin1 to at least one of its receptors, which at least partially overcome the disadvantages of the prior art.

[0009] Draxin is a secreted protein described to be involved in axon guidance decisions (Islam et al., 2009). In contrast to Netrins 1-3, which are present in vertebrates and invertebrates, Draxin can only be found in vertebrate genomes. The amino acid sequence of human Draxin is shown as SEQ ID NO.: 4. In zebrafish, there exist two Draxin isoforms (DraxinA and DraxinB); their amino acid sequences are represented by SEQ ID NO.: 5 and SEQ ID NO.: 6.

[0010] By using an extracellular protein-protein interaction screen assay (AVEXIS), the present inventors identified Draxin as a novel direct binding partner for Netrin1. Furthermore, by using an AVEXIS based competition assay, the inventors were able to show that Draxin or Draxin protein fragments can compete with Netrin receptors for binding to Netrin1.

[0011] The present invention therefore provides specific peptides binding to .gamma.-Netrins (".gamma.-Netrin-binding peptides"), particularly to Netrin1, as well as antibodies directed against .gamma.-Netrins (".gamma.-Netrin-binding antibodies"), particularly against Netrin1, which may be used for interfering with .gamma.-Netrin/Netrin receptor binding, in particular Netrin1/Netrin receptor binding. The invention further provides Draxin-binding peptides as well as Draxin-binding antibodies inhibiting binding of Draxin to .gamma.-Netrins, in particular to Netrin1.

DETAILED DESCRIPTION OF THE INVENTION

[0012] In one aspect, the present invention relates to peptides which bind to at least one .gamma.-Netrin, with a high specificity and a high affinity. Importantly, the affinity of Draxin to the .gamma.-Netrins, in particular to Netrin1, is significantly higher than the affinity of .gamma.-Netrins, in particular of Netrin1 to Netrin receptors.

[0013] Accordingly, the invention provides a .gamma.-Netrin-binding peptide, comprising (i) the sequence EVMPTLDMALFDWTDYEDLKP (SEQ ID NO.: 1), or (ii) the sequence DVAPTFNMALFDWTDYEDMRP (SEQ ID NO.: 2), or (iii) the sequence EVMPTLDMTLFDWTDYEDMKP (SEQ ID NO.: 3), or (iv) a variant thereof having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO.: 1, 2 and/or 3, wherein said peptide has a length of up to 328 amino acids and is optionally fused to a heterologous peptide or polypeptide. Preferably, the .gamma.-Netrin is Netrin1.

[0014] The .gamma.-Netrin-binding peptide is characterized in that it binds with high affinity to Netrins derived from the .gamma. chain of Laminin1, comprising in particular human Netrin1 and human Netrin3 as well as Netrins 1a, 1b and 2 from zebrafish (Danio rerio). In particular, the .gamma.-Netrin-binding peptide of the invention binds to human Netrin1 and to zebrafish Netrin1a (also referred to as Ntn1a) and Netrin1b (also referred to as Ntn1b). In preferred embodiments, it is therefore referred to as "Netrin1-binding peptide".

[0015] In some embodiments, the peptide with a length of up to 328 amino acids comprises any one of SEQ ID NO.: 1, 2, 3 or a variant thereof having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO.: 1, 2 and/or 3 and is free of any further heterologous peptides.

[0016] In other embodiments, the peptide with a length of up to 328 amino acids comprises any one of SEQ ID NO.: 1, 2, 3 or a variant thereof having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO.: 1, 2 and/or 3 and is fused to a heterologous peptide or polypeptide as defined below.

[0017] The minimal sequence of the .gamma.-Netrin-binding peptide, in particular Netrin1-binding peptide is represented by

TABLE-US-00001 SEQ ID NO.: 1 EVMPTLDMALFDWTDYEDLKP, SEQ ID NO.: 2 DVAPTFNMALFDWTDYEDMRP, and SEQ ID NO.: 3 EVMPTLDMTLFDWTDYEDMKP, respectively.

[0018] In some embodiments, the peptide has a length of up to 328 amino acids, up to 324 amino acids, up to 320 amino acids, up to 250 amino acids, up to 200 amino acids, up to 100 amino acids, up to 50 amino acids, or up to 30 amino acids. According to other embodiments, the peptide has a length of up to 150 amino acids or up to 125 amino acids. Fragments comprising up to 50 amino acids are preferred according to some embodiments.

[0019] In certain preferred embodiments, a variant of SEQ ID NO.: 1, SEQ ID NO.: 2 or SEQ ID NO.: 3 have a sequence identity of at least 70% to SEQ ID NO.: 1, 2 and/or 3. In further embodiments the level of sequence identity may be at least 90%, or even at least 95% to SEQ ID NO.: 1, 2 and/or 3.

[0020] A "variant" in the context of the present invention is any peptide whose amino acid sequence varies in at least one position from the respective reference peptide, but retains the biological activity of the reference peptide; for example, a variant of SEQ ID NO.: 1 differs in at least one amino acid therefrom, and retains the .gamma.-Netrin-, particularly Netrin1-binding activity. In particular, variants of SEQ ID NO.: 1, 2 and 3 differ in 1, 2, 3, 4, 5, 6 or 7 amino acids from SEQ ID NO.: 1, SEQ ID NO.: 2 and/or SEQ ID NO.: 3, provided they retain the .gamma.-Netrin-, particularly Netrin1-binding activity. Variations will usually be generated by amino acid substitutions. Particularly, a variant according to the invention will be characterized in that it has been changed to contain at least one non-naturally occurring substitution modification relative to the respective reference peptide.

[0021] The peptidic compounds of the present invention comprise a linear backbone of amino carboxylic acids linked by peptide, i.e. carboxamide bonds. Preferably, the amino carboxylic acids are .alpha.-amino carboxylic acids and more preferably L-.alpha.-amino carboxylic acids, unless indicated otherwise. Any amino acid of the sequences disclosed herein may be replaced either by an unmodified canonical proteinogenic L-amino acid, or by an unmodified canonical proteinogenic D-amino acid. Also envisaged are substitutions with non-canonical proteinogenic amino acids, in particular with ornithine, 2,4-diamino butyric acid, 2,3-diamino propionic acid, selenocysteine, pyrrolysine, hydroxyproline, O-phosphoserine, O-phosphotyrosin, .gamma.-carboxyglutamic acid, .gamma.-aminobutyric acid, norleucine, .epsilon.-aminohexanoic acid, and with other posttranslationally modified amino acids, e.g. amino acids with amidated carboxyl groups (at C-termini of peptides), amino acids with alkylated (e.g. methylated) side chains, amino acids with an amino side chain group (such as lysine and ornithine) with modifications at one or both of the hydrogen atoms of the amino side chain group, for example with a lipophilic moiety attached via a carboxamide bond, etc.

[0022] The percent sequence identity may be determined according to the following formula:

I=n:L

wherein I is the identity in percent, n is the number of identical amino acids between a given sequence and a comparative sequence as shown e.g. in SEQ ID NOs.: 1, 2 and 3, and L is the length of the comparative sequence. Importantly, when calculating the percent sequence identity according to this formula, an alignment of the two sequences shall be carried out without gaps between complementary portions and over the whole length of the comparative sequence.

[0023] In specific embodiments, the invention provides a .gamma.-Netrin-binding peptide, comprising (i) the sequence EVMPTLDMALFDWTDYEDLKP (SEQ ID NO.: 1), or (ii) the sequence DVAPTFNMALFDWTDYEDMRP (SEQ ID NO.: 2), or (iii) the sequence EVMPTLDMTLFDWTDYEDMKP (SEQ ID NO.: 3), or (iv) a variant thereof having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO.: 1, 2 and/or 3, wherein said peptide has a length of up to 100 amino acids and is optionally fused to a heterologous peptide or polypeptide.

[0024] According to some embodiments, the .gamma.-Netrin-binding peptide, in particular the Netrin1-binding peptide, comprises any one of SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14 or SEQ ID NO.: 15 or a corresponding fragment of another species. Preferably, the peptide comprises any one of SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15 or a corresponding fragment of another species, more preferably SEQ ID NO.: 15 or a corresponding fragment of another species.

[0025] In some preferred embodiments, the .gamma.-Netrin-binding peptide, in particular the Netrin1-binding peptide, comprises a sequence which has a length of 22 amino acids. This 22-amino-acid (22aa) sequence may for example be SEQ ID NO.:16, or it may be SEQ ID NO.: 14, or it may be SEQ ID NO.: 17, or it may be a variant of any of these sequences, e.g. a corresponding fragment from another species with an additional amino acid residue, preferably glycine (Gly) at the N-terminus. Variants of SEQ ID NO.: 16, 14, 17 have a sequence identity of at least 70% to SEQ ID NO.: 16, 14 and/or 17. In further embodiments, the level of sequence identity may be at least 90%, or even at least 95% to SEQ ID NO.: 16, 14 and/or 17. In particular, a variant of SEQ ID NO.: 16, 14, 17 may differ in 1, 2, 3, 4, 5, 6 or 7 amino acids from SEQ ID NO.: 16, SEQ ID NO.: 14 and/or SEQ ID NO.: 17, provided they retain the .gamma.-Netrin-binding activity.

[0026] Also encompassed by the invention is any variant of any one of SEQ ID NOs.: 7-15 or a corresponding fragment of another species having a sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% thereto, as long as the binding activity to .gamma.-Netrins, preferably to Netrin1, is maintained. In some preferred embodiments, the level of sequence identity is at least 70%. In further preferred embodiments, the level of sequence identity is at least 90%.

[0027] In some embodiments, the .gamma.-Netrin-binding peptide, in particular the Netrin1-binding peptide, according to the invention is fused to a heterologous peptide or polypeptide. A "heterologous peptide or polypeptide" in the context of the invention is any peptide with a length of at least 4 amino acids, which originates from another species as compared to the .gamma.-Netrin-binding peptide or which is an artificial, i.e. non-naturally occurring peptide or polypeptide. Examples of such heterologous (poly)peptides are protein tags, in particular epitope tags such as the Myc-tag and the HA-tag, affinity tags such as FLAG, poly(His), chitin binding protein (CBP), maltose binding protein (MBP) and glutathione-S-transferase (GST), enzymes such as alkaline phosphatase, luciferase, horseradish peroxidase and .beta.-galactosidase, or fluorescent proteins such as GFP, RFP and the like. Other examples are sequences that direct peptides attached thereto to specific locations inside cells or to the extracellular space (signal sequences); specific examples of such sequences are represented by SEQ ID NOs.: 70 and 71.

[0028] Further preferred examples of heterologous (poly)peptides are immunoglobulins (Ig) or functional fragments of immunoglobulins, such as Fv, scFv, Fab, Fab', F(ab')2, Fc, diabodies, minibodies, domain antibodies (dAb), camelid antibodies, nanobodies (VHH), disulfide stabilized Fv fragments (dsFv) and CDR-comprising peptides. The immunoglobulins or fragments thereof may be of any isotype, e.g. of the IgA-, IgD-, IgE, IgG or IgM-type. A functional structure analogous to the isotype G of immunoglobulins (IgG) is preferred. Among these, immunoglobulins or fragments thereof of the IgG1-, IgG2-, IgG3-, or IgG4-type are preferred.

[0029] In particularly preferred embodiments, the heterologous peptide or polypeptide is an Ig Fc fragment, for example an Fc fragment from mouse, rat, chicken, rabbit or human, with human Ig Fc fragments being preferred. Still more preferably, the human Ig Fc fragment is a human IgG Fc fragment, e.g. a human IgG1, IgG2, IgG3, or IgG4 Fc fragment.

[0030] The heterologous (poly)peptide may be conjugated to the .gamma.-Netrin-binding peptide directly or via a spacer of suitable length. Suitable spacers are e.g. heterologous peptide linkers having a length of from 10 to 50, preferably from 10 to 30 amino acid residues. It is further preferred for the peptide linkers to be flexible linkers without a secondary structure. For example, suitable peptide linkers consist of at least 80% or at least 90%, preferably at least 95% or completely of glycine and/or serine residues. Particularly suitable are peptide linkers which contain a plurality of sequences SGGGG (SEQ ID NO 82).

[0031] In some preferred embodiments of the invention, the inventive .gamma.-Netrin-binding peptides, particularly the Netrin1-binding peptides, are competitive with Netrin receptors and can even release a .gamma.-Netrin, e.g. human Netrin1 or zebrafish Netrin1a or 1 b, bound to a Netrin receptor.

[0032] In a further aspect, the invention relates to a .gamma.-Netrin-binding peptide as defined herein for use in medicine. A "use in medicine" in the context of the invention may be a use in therapy and/or a use in diagnostics. Preferably, the .gamma.-Netrin-binding peptide, in particular the Netrin1-binding peptide, according to the invention is for use in human medicine, but it may also be used for veterinary purposes.

[0033] In particular, a .gamma.-Netrin-binding peptide comprising (i) the sequence EVMPTLDMALFDWTDYEDLKP (SEQ ID NO.:1), or (ii) the sequence DVAPTFNMALFDWTDYEDMRP (SEQ ID NO.:2), or (iii) the sequence EVMPTLDMTLFDWTDYEDMKP (SEQ ID NO.:3), or (iv) a variant thereof having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO.:1, 2 and/or 3, which is optionally fused to a heterologous peptide or polypeptide, is for use in the prevention or treatment of a condition associated with, accompanied by or mediated by pathologic, particularly increased, .gamma.-Netrin expression or activity. Preferably, the .gamma.-Netrin is Netrin1.

[0034] A "pathologic expression or activity" of .gamma.-Netrin, in particular Netrin1, as referred to herein is generally meant to encompass all situations, in which the ratio of expression and/or activity between Draxin and .gamma.-Netrin and/or between .gamma.-Netrin and at least one Netrin receptor is abnormal.

[0035] In particular, a "pathologic expression" means any level of expression, either on the DNA or the protein level, that differs from that of a normal healthy subject. Preferably, a pathologic .gamma.-Netrin expression may be or may lead to a decreased .gamma.-Netrin protein level or, more preferably, an increased .gamma.-Netrin protein level, particularly an increased Netrin1 protein level, in a given (extracellular) environment of an organism. In accordance with preferred embodiments of the invention, the presence of an increased .gamma.-Netrin level, particularly protein level, more particularly Netrin1 protein level, is defined as lying between 1 and 2 standard deviations above the average of healthy adults. Similarly, the presence of a decreased .gamma.-Netrin protein level is defined as lying between 1 and 2 standard deviations below the average of healthy adults. For example, the .gamma.-Netrin level in patient samples may be increased or decreased by a factor of at least 1,3 as compared to control samples from healthy adults.

[0036] A "pathologic activity" as referred to herein means in particular any activity of a protein that is abnormal. Preferably, a pathologic .gamma.-Netrin activity may be a decreased or increased activity, e.g. binding to Netrin receptors with a lower or higher affinity. In accordance with preferred embodiments of the invention, the presence of an increased .gamma.-Netrin activity, particularly Netrin1 activity, is defined as lying between 1 and 2 standard deviations above the average of healthy adults. Similarly, the presence of a decreased .gamma.-Netrin activity is defined as lying between 1 and 2 standard deviations below the average of healthy adults. For example, the .gamma.-Netrin activity in patient samples may be increased or decreased by a factor of at least 1,3 as compared to control samples from healthy adults.

[0037] A control group of "healthy adults" according to the invention can be determined by the skilled person with routine experimentation. For example, "healthy adults" for use as a possible control group herein may be healthy persons between 25 and 65 years of age, having a body mass index of 20-25, which may be sex-matched, if applicable.

[0038] The .gamma.-Netrin-binding peptide for use as defined above may e.g. be a full-length Draxin from any vertebrate species, in particular human Draxin, rat Draxin, mouse Draxin, zebrafish Draxin A or zebrafish Draxin B. It may also be a variant of a full-length Draxin protein, characterized in that the variant differs in at least one, at least five, at least ten, at least 20 or even more amino acids from the complete sequence of the respective full-length Draxin protein, provided it retains the .gamma.-Netrin-, in particular the Netrin1-binding activity. In preferred embodiments, the peptide is however significantly shorter as compared to full-length Draxin. For example, the peptide may have a length of up to 328, up to 324, up to 320, up to 300, up to 250, up to 200, up to 150, up to 100, up to 75 or up to 50 amino acids. In some preferred embodiments, the peptide has a length of up to 50 amino acids, e.g. 21 amino acids or 22 amino acids and is derived from zebrafish DraxinA. The peptide may or may not be fused to a heterologous peptide or polypeptide. Suitable peptides or polypeptides for fusion with the .gamma.-Netrin-binding peptide are those described herein above, preferably protein tags, immunoglobulins (Ig) or functional fragments of immunoglobulins, wherein Ig Fc fragments, particularly human Ig Fc fragments are preferred. Still more preferably, the human Ig Fc fragment is a human IgG Fc fragment, e.g. a human IgG1, IgG2, IgG3, or IgG4 Fc fragment.

[0039] In some embodiments of the invention, the condition associated with, accompanied by or mediated by pathologic, particularly increased, .gamma.-Netrin, preferably Netrin1 expression or activity is a hyperproliferative disease. A hyperproliferative disease according to the invention may be a tumor disease, a premalignant, non-neoplastic or non-malignant hyperproliferative disorder. In particular, the condition is a tumor disease.

[0040] An exemplary hyperproliferative disease which may be prevented or treated using the .gamma.-Netrin-binding peptides, particularly Netrin1-binding peptides according to the invention is neoplastic lesions found in human inflammatory bowel disease (IBD).

[0041] Exemplary tumor diseases, which may be prevented or treated using the .gamma.-Netrin-binding peptides, particularly Netrin1-binding peptides according to the invention, are selected from the group consisting of breast cancer, renal cancer, liver cancer, prostate cancer, colorectal cancer, lung cancer, neuroblastoma, meningioma of the brain, pituitary adenoma, glioma, glioblastoma, acute myeloid leukemia, sarcoma, melanoma, ovarian adenocarcinoma, renal adenocarcinoma, uterus adenocarcinoma, stomac adenocarcinoma, kidney adenocarcinoma and rectal adenocarcinoma, pancreatic cancer, inflammation driven cancers, particularly colorectal cancer, colorectal cancer associated with IBD, colorectal cancer associated with ulcerative colitis, colorectal cancer associated with Crohn's disease, and tumors derived from inflammatory bowel disease, including metastatic and particularly aggressive forms of these tumor diseases.

[0042] According to one embodiment, the tumor disease is neuroblastoma.

[0043] Preferably, the tumor disease is selected from the group consisting of pancreatic cancer, colorectal cancer, breast cancer, particularly metastatic breast cancer, and lung cancer, particularly non-small cell lung cancer.

[0044] In other embodiments of the invention, the condition associated with, accompanied by or mediated by pathologic .gamma.-Netrin, particularly Netrin1 expression or activity is a cardiovascular disease, in particular atherosclerosis.

[0045] In still other embodiments of the invention, the condition associated with, accompanied by or mediated by pathologic .gamma.-Netrin, particularly Netrin1 expression or activity is a neurological disorder, e.g. spinal cord injury.

[0046] The .gamma.-Netrin-binding peptide, particularly Netrin1-binding peptide for use according to the invention may, according to a further aspect of the invention, also be present as an active agent in a pharmaceutical composition together with at least one pharmaceutically acceptable carrier. The present invention thus relates to a pharmaceutical composition comprising a .gamma.-Netrin, particularly Netrin1-binding peptide or a salt or solvate thereof and at least one pharmaceutically acceptable carrier. Optionally, the pharmaceutical composition further comprises pharmaceutically acceptable excipients and/or adjuvants. Concentrations of these carriers, excipients and/or adjuvants, if used, are in a range that is physiologically acceptable.

[0047] A "carrier" as used herein must be physiologically acceptable and retain the therapeutic properties of the substance with which it is administered. Standard acceptable pharmaceutical carriers and their formulations are known to the skilled person. The carriers used will differ according to the administration route. Examples are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatine, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting-point wax, cocoa butter, water, alcohols, polyols, glycerol, vegetable oils, buffers etc.

[0048] As exemplary excipients, disintegrators, binders, fillers, and lubricants may be mentioned. Examples of disintegrators include agar-agar, algins, calcium carbonate, cellulose, colloid silicon dioxide, gums, magnesium aluminium silicate, methylcellulose, and starch. Examples of binders include microcrystalline cellulose, hydroxymethyl cellulose, hydroxypropylcellulose, and polyvinylpyrrolidone. Examples of fillers include calcium carbonate, calcium phosphate, tribasic calcium sulfate, calcium carboxymethylcellulose, cellulose, dextrin, dextrose, fructose, lactitol, lactose, magnesium carbonate, magnesium oxide, maltitol, maltodextrins, maltose, sorbitol, starch, sucrose, sugar, and xylitol. Examples of lubricants include agar, ethyl oleate, ethyl laureate, glycerin, glyceryl palmitostearate, hydrogenated vegetable oil, magnesium oxide, stearates, mannitol, poloxamer, glycols, sodium benzoate, sodium lauryl sulfate, sodium stearyl, sorbitol, and talc.

[0049] Usual stabilizers, preservatives, wetting and emulsifying agents, consistency-improving agents, flavour-improving agents, salts for varying the osmotic pressure, buffer substances, solubilizers, diluents, emollients, colorants and masking agents and antioxidants come into consideration as pharmaceutical adjuvants.

[0050] In yet a further aspect of the invention, the .gamma.-Netrin-binding peptide, particularly Netrin1-binding peptide for use according to the invention may be widely combined with other therapeutically active agents, especially to achieve a synergistic effect in therapy and/or to be able to reduce the dosage of one or more active ingredients. Thus, the peptide is, in some embodiments, for use together with at least one additional therapeutically active agent, wherein the additional therapeutically active agent is particularly selected from the group consisting of decoy Netrin receptors, cytostatic agents, cytotoxic agents, statins, antihyperlipidemic agents, anti-coagulant agents, kinase inhibitors and angiogenesis modulators. Especially preferred therapeutic agents for combination with the .gamma.-Netrin-binding peptide are cytostatic agents and cytotoxic agents.

[0051] A combination of a .gamma.-Netrin-binding peptide, particularly a Netrin1-binding peptide, with at least one additional therapeutically active agent can be applied either by separate administration of the active ingredients to the patient or in the form of combination products in which a plurality of active ingredients are present in one pharmaceutical preparation. When the active ingredients are administered by separate administration of the active ingredients, this can be done simultaneously or consecutively.

[0052] The peptides according to the invention, either in an isolated form or as a pharmaceutical composition will typically be administered to a subject in need thereof, in particular a human subject. They will typically also be administered in a therapeutically effective amount, i.e. in an amount sufficient to achieve the desired effect. For example, one desired effect to be achieved by administration of .gamma.-Netrin-binding peptides or anti-.gamma.-Netrin antibodies or fragments or derivatives described herein may be to block, inhibit and/or neutralize one or more biological function of .gamma.-Netrins, in particular of Netrin1, such as the binding to one or more Netrin receptor(s).

[0053] Administration of suitable compositions may be effected in different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, oral, intradermal, intranasal or intrabronchial administration. Administration may also be conducted directly at the target site.

[0054] The necessary amount to be effective depends on a number of factors, such as the choice of the specific compound, the intended use, the administration route and the clinical condition of the patient. An appropriate "effective" amount in any individual case may be determined by a skilled person in the art, e.g. a skilled physician, using routine experimentation. An exemplary "therapeutically effective amount" of a peptide according to the invention may be about 0.01 mg to 50 mg/dose, preferably 0.1 mg to 10 mg/dose.

[0055] In yet a further aspect, the invention relates to an antibody or fragment or derivative thereof directed against Draxin, which inhibits binding of Draxin to .gamma.-Netrins, in particular to Netrin1.

[0056] In yet a further aspect, the invention relates to an antibody or fragment or derivative thereof directed against Netrin1, which inhibits binding of at least one .gamma.-Netrin, in particular of Netrin1, to at least one Netrin receptor, and which is directed against an epitope between amino acids 285-451 of human Netrin1. In particular, the antibody inhibits binding of at least one .gamma.-Netrin, preferably Netrin1 to at least one of DCC (Deleted in colorectal cancer), DSCAM, DSCAM-L1, PTPRF (protein tyrosine phosphatase, receptor-type F), NEO (Neogenin), ADORA2B (adenosine A2B), Nope (Neighbor Of Punc E1 1), and members of the UNCSH family (Uncoordinated-5 homologues; UNC5H1, UNC5H2, UNC5H3, UNC5H4).

[0057] The term "antibody" as used herein particularly refers to molecules comprising at least one immunoglobulin heavy chain and at least one immunoglobulin light chain. Each heavy and light chain may comprise a variable and a constant domain. The antigen-binding site may be formed from the variable domains of a heavy and a light chain. A variable region (also referred to as variable domain) comprises complementarity determining regions (CDRs), e.g. a CDR1, a CDR2 and a CDR3 region, and framework regions (FRs) flanking the CDRs. The term "complementarity determining region" is readily understood by the skilled person (see, for example, Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSHL Press, Cold Spring Harbor, N.Y., 1988; incorporated herein by reference in its entirety) and refers to the stretches of amino acids within the variable domain of an antibody that primarily make contact with the antigen and determine antibody specificity. This region is also known as the hypervariable region.

[0058] The term "(functional) antibody fragment or derivative thereof" as used herein encompasses fragments of antibodies, especially of human or humanized antibodies, such as portions of the above-mentioned antibodies which comprise at least one antigen-binding site. Examples of antibody fragments according to the invention include Fab fragments, Fab' fragments, F(ab').sub.2 fragments, Fv fragments, diabodies or single chain antibody molecules and other fragments as long as they exhibit the desired capability of binding to their target antigen, e.g. Draxin or at least one .gamma.-Netrin. As exemplary single chain antibody molecules, scFv and nanobodies (VHH) are mentioned.

[0059] In case of antibodies or fragments or derivatives thereof directed against Draxin, only those antibodies, fragments and derivatives which inhibit the binding of Draxin to at least one .gamma.-Netrin, preferably to Netrin1 are encompassed by the invention.

[0060] Likewise, in case of antibodies or fragments or derivatives thereof directed against .gamma.-Netrin(s), only those antibodies, fragments and derivatives which inhibit the binding of at least one .gamma.-Netrin, preferably Netrin1, to at least one Netrin receptor are encompassed by the invention.

[0061] Such inhibition can be determined by the skilled person via routine experimentation, e.g. via binding assays.

[0062] The term "bind" or "binding" of e.g. an antibody as used herein means an at least temporary interaction or association with or to e.g. a target antigen, e.g. Draxin or a .gamma.-Netrin, comprising fragments thereof containing an epitope.

[0063] In preferred embodiments, the antibody directed against Draxin (i.e. the anti-Draxin antibody) according to the invention binds to an epitope on Draxin, which is located between amino acid residues 209-284, preferably between amino acid residues 226-257, of a zebrafish Draxin, preferably zebrafish DraxinA. More preferably, the antibody binds to an epitope between amino acids 232-252 of zebrafish DraxinA.

[0064] In further preferred embodiments, the anti-Draxin antibody according to the invention binds to an epitope on Draxin, which is located in a region of human Draxin corresponding to amino acid residues 209-284, preferably between amino acid residues 226-257, of zebrafish Draxin, preferably zebrafish DraxinA. More preferably, the antibody binds to an epitope between amino acids 222-243 of human Draxin.

[0065] In preferred embodiments, the antibody directed against .gamma.-Netrin (i.e. the anti-.gamma.-Netrin antibody) according to the invention binds to an epitope on Netrin1, which is located between amino acid residues 285-451 (i.e. EGF1-3), preferably between amino acid residues 341-451 (i.e. EGF2-3), of human Netrin1 or a corresponding epitope in another .gamma.-Netrin and/or another species, preferably to an epitope located in the EGF1-3 domains, preferably the EGF2-3 domains of zebrafish Netrin1a and/or Netrin1b. More preferably, the antibody binds to an epitope between amino acids 404-451 (i.e. EGF3) of a human .gamma.-Netrin, preferably human Netrin1 or to an epitope located in the EGF3 domain of a zebrafish .gamma.-Netrin, preferably zebrafish Netrin1a or Netrin1b.

[0066] The antibody or fragment or derivative thereof according to the invention may be derived from any antibody-producing animal species. Preferably, it is a mouse, rat or human antibody or functional antibody fragment or antibody derivative. The antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, and/or a recombinant antibody. Monoclonal antibodies, in particular human or humanized monoclonal antibodies are preferred.

[0067] A monoclonal antibody (also referred to as mAB) is a single molecular species of antibody and is usually produced by creating hybrid antibody-forming cells from a fusion of nonsecreting myeloma cells with immune spleen cells. Polyclonal antibodies, by contrast, are produced by injecting an animal (such as a rodent, rabbit or goat) with an antigen, and extracting serum from the animal. A chimeric antibody is an antibody in which the variable domain of e.g. a murine antibody is combined with the constant region of a human antibody. Recombinant antibodies are obtained via genetic engineering without having to inject animals. Human antibodies according to the invention may be prepared using transgenic mice or by phage display; these methods are well known in the art.

[0068] In yet a further aspect, the antibodies or fragments or derivatives thereof according to the invention are for use in medicine (as defined above), particularly human medicine. For example, they may be used in diagnostics to determine qualitatively or quantitatively their respective antigens; they may also be used as a diagnostic agent for diseases with pathologic, in particular increased target expression.

[0069] In certain embodiments of yet a further aspect, the anti-.gamma.-Netrin antibody (including its functional fragments and derivatives as defined herein) is for use in the prevention or treatment of a condition associated with, accompanied by or mediated by pathologic, particularly increased, .gamma.-Netrin, particularly Netrin1 expression or activity, as defined herein above. This condition is preferably a hyperproliferative disease, in particular a tumor disease.

[0070] Exemplary tumor diseases, which may be prevented or treated using the anti-.gamma.-Netrin antibody, preferably anti-Netrin1-antibody, or fragments or derivatives thereof according to the invention, are selected from the group consisting of breast cancer, renal cancer, liver cancer, prostate cancer, colorectal cancer, lung cancer, neuroblastoma, meningioma of the brain, pituitary adenoma, glioma, glioblastoma, acute myeloid leukemia, sarcoma, melanoma, ovarian adenocarcinoma, renal adenocarcinoma, uterus adenocarcinoma, stomac adenocarcinoma, kidney adenocarcinoma and rectal adenocarcinoma, pancreatic cancer, inflammation driven cancers, particularly colorectal cancer, colorectal cancer associated with IBD, colorectal cancer associated with ulcerative colitis, colorectal cancer associated with Crohn's disease, and tumors derived from inflammatory bowel disease, including metastatic and particularly aggressive forms of these tumor diseases.

[0071] Preferably, the tumor disease is selected from the group consisting of pancreatic cancer, colorectal cancer, breast cancer, particularly metastatic breast cancer, and lung cancer, particularly non-small cell lung cancer. In another embodiment, the tumor disease is neuroblastoma.

[0072] The anti-.gamma.-Netrin antibody, preferably anti-Netrin1-antibody or fragment or derivative thereof may also be present in the form of a pharmaceutical composition as defined above, and may also be used in combination with further pharmaceutically active agents, particularly one or more agent selected from the group consisting of decoy Netrin receptors, cytostatic agents, cytotoxic agents, statins, antihyperlipidemic agents, anti-coagulant agents, kinase inhibitors and angiogenesis modulators. Especially preferred therapeutic agents for combination with the anti-.gamma.-Netrin-antibody are cytostatic agents and cytotoxic agents.

[0073] In certain embodiments of yet a further aspect, the anti-Draxin antibody (including its functional fragments and derivatives as defined herein) is for use in the prevention or treatment of a condition associated with, accompanied by or mediated by pathologic, particularly decreased, .gamma.-Netrin, particularly Netrin1 expression or activity, as defined herein above. Likewise, the anti-Draxin antibody (including its functional fragments and derivatives as defined herein) may be used in the prevention or treatment of a condition associated with, accompanied by or mediated by pathologic, particularly increased, Draxin expression or activity.

[0074] This condition may be a cardiovascular disorder, in particular a cardiovascular disorder which can be prevented or treated by increasing .gamma.-Netrin, in particular Netrin1 expression and/or activity. Exemplary cardiovascular disorders are selected from ischemia/reperfusion (I/R) injury, e.g. renal I/R injury, I/R injury of cardiac tissue; myocardial infarction, particularly infarcts resulting from I/R injury; mitochondrial damage; neointimal formation and restenosis; vascular injury or vascular dysfunction; vascular smooth muscle cell migration and proliferation; apoptosis of endothelial progenitor cells, procure induced restenosis; and hypertension.

[0075] A "pathologic expression or activity" of Draxin is generally meant to encompass all situations, in which the ratio of expression and/or activity between Draxin and any .gamma.-Netrin is abnormal. Particularly, a pathologic Draxin expression may be or may lead to a decreased Draxin protein level or, preferably, an increased Draxin protein level in a given (extracellular) environment of an organism. A pathologic Draxin activity may be an decreased or increased activity, e.g. binding to a .gamma.-Netrin, particularly Netrin1 with a lower or higher affinity.

[0076] In yet a further aspect, the present invention relates to peptides which bind to Draxin, in particular to human Draxin and/or zebrafish DraxinA and/or zebrafish DraxinB, with a high specificity and a high affinity.

[0077] Accordingly, the invention provides a Draxin-binding peptide comprising (i) at least consecutive amino acids from the sequence KACDCHPVGAAGKTCNQTTGQCPCKDGVTGITCNRCANGYQQSRSPIAPCIKIPIA PP (SEQ ID NO.: 51) or (ii) a variant thereof having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO.:51, wherein said peptide has a length of up to 580 amino acids and is optionally fused to a heterologous peptide or polypeptide.

[0078] In particular, the Draxin-binding peptide comprises at least 20, at least 30, at least 40, at least 50 consecutive amino acids from SEQ ID NO.:51 or the complete SEQ ID NO.: 51 or a variant thereof having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO.:51.

[0079] In some embodiments, the peptide has a length of up to 580 amino acids, up to 500 amino acids, up to 400 amino acids, up to 300 amino acids, up to 250 amino acids, up to 200 amino acids, up to 100 amino acids, up to 75 amino acids, or up to 60 amino acids. Fragments comprising up to 60 amino acids are preferred according to some embodiments.

[0080] In some embodiments, the peptide with a length of up to 580 amino acids comprises SEQ ID NO.: 51 or a variant thereof having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO.: 51 and is free of any further heterologous peptides.

[0081] In other embodiments, the peptide with a length of up to 580 amino acids comprises SEQ ID NO.: 51 or a variant thereof having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO.: 51 and is fused to a heterologous peptide or polypeptide as defined above, preferably protein tags, immunoglobulins (Ig) or functional fragments of immunoglobulins, wherein Ig Fc fragments, particularly human Ig Fc fragments are preferred. Still more preferably, the human Ig Fc fragment is a human IgG Fc fragment, e.g. a human IgG1, IgG2, IgG3, or IgG4 Fc fragment.

[0082] In some embodiments, the peptide may also be fused to a heterologous signal sequence, e.g. a human Netrin1-fragment comprising SEQ ID NO.: 51 may be fused to a zebrafish signal sequence (comprising e.g. SEQ ID NO.: 70 or 71), as shown in SEQ ID NOs.: 68 and 69.

[0083] In a further aspect, the invention relates to a Draxin-binding peptide as defined herein for use in medicine, particularly human medicine.

[0084] In certain embodiments of yet a further aspect, the Draxin-binding peptide described herein is for use in the prevention or treatment of a condition associated with, accompanied by or mediated by pathologic, particularly decreased, .gamma.-Netrin, in particular Netrin1 expression or activity, as defined herein above. Likewise the Draxin-binding peptide may be used in the prevention or treatment of a condition associated with, accompanied by or mediated by pathologic, particularly increased, Draxin expression or activity.

[0085] This condition may be a cardiovascular disorder, in particular a cardiovascular disorder which can be prevented or treated by increasing .gamma.-Netrin, in particular Netrin1 expression and/or activity. Exemplary cardiovascular disorders are selected from ischemia/reperfusion (I/R) injury, e.g. renal I/R injury, I/R injury of cardiac tissue; myocardial infarction, particularly infarcts resulting from I/R injury; mitochondrial damage; neointimal formation and restenosis; vascular injury or vascular dysfunction; vascular smooth muscle cell migration and proliferation; apoptosis of endothelial progenitor cells, procure induced restenosis; and hypertension.

[0086] The Draxin-binding peptide for use according to the invention may, according to a further aspect of the invention, also be present as an active agent in a pharmaceutical composition together with at least one pharmaceutically acceptable carrier. The present invention thus relates to a pharmaceutical composition comprising an Draxin-binding peptide thereof and at least one pharmaceutically acceptable carrier and, optionally, pharmaceutically acceptable excipients and/or adjuvants.

[0087] Still a further aspect of the present invention is a method for the treatment of a condition associated with, accompanied by or mediated by pathologic, particularly increased or decreased .gamma.-Netrin, in particular Netrin1 expression or activity, comprising administering a .gamma.-Netrin-binding peptide, a Draxin-binding peptide, an anti-.gamma.-Netrin antibody and/or an anti-Draxin antibody as described herein above to a subject, particularly a human subject in need thereof. In particular, this subject suffers from a hyperproliferative disorder as defined above.

[0088] The invention also refers to a nucleic acid molecule, preferably an isolated nucleic acid molecule, encoding a .gamma.-Netrin-binding peptide, particularly the Netrin1-binding peptide, the Draxin-binding peptide, and the antibodies or fragments or derivatives thereof directed against .gamma.-Netrin, in particular Netrin1, and/or Draxin as described above. The term "nucleic acid molecule" encompasses a natural DNA or RNA or a recombinantly or synthetically produced DNA, RNA or LNA or a recombinantly produced chimeric nucleic acid molecule comprising any of these nucleic acids either alone or in combination. For example, the nucleic acid may be cDNA or genomic DNA corresponding to an entire gene or a substantial portion thereof or to fragments and derivatives thereof. The nucleotide sequence may correspond to the naturally occurring nucleotide sequence or may contain single or multiple nucleotide substitutions, deletions or additions. The nucleic acid may also be fused to another nucleic acid. The nucleic acid molecule of the invention may be in operative linkage to an expression control sequence, i.e. to a sequence which is necessary to effect the expression of coding nucleic acid sequences. Such expression control sequences may include promoters, enhancers, ribosomal binding sites and/or transcription termination sequences. Specific examples of suitable expression control sequences are known in the art.

[0089] The nucleic acid molecule of the invention may be located on a vector which may additionally contain a replication origin and/or a selection marker gene. Examples of vectors are plasmids, cosmids, phages, viruses etc.

[0090] Further, the invention refers to a recombinant cell, which comprises the nucleic acid molecule as described above. The nucleic acid molecule may be introduced into the recombinant cell by transformation, transfection or transduction according to any method known in the art. The recombinant cell may e.g. be a prokaryotic or eukaryotic cell. Preferably, the cell is a mammalian cell, e.g. a hamster, rabbit, or human cell. Preferably, the cell is a human cell.

[0091] The .gamma.-Netrin-binding peptide, the Draxin-binding peptide, and the antibodies or fragments or derivatives thereof directed against .gamma.-Netrin and/or Draxin of the invention may be prepared by a method, wherein the cell as described above is cultured under conditions which allow expression of the antibody encoding nucleic acid molecule. The antibody may be collected from the cultured cell or the culture supernatant. Preferably, the antibody is prepared from a mammalian, particularly from a human cell.

[0092] Also encompassed by the invention are salts and solvates, preferably pharmaceutically acceptable salts and solvates of the disclosed peptides, in particular of the .gamma.-Netrin-, preferably Netrin1-binding peptides and the Draxin-binding peptides.

[0093] Pharmaceutically acceptable salts may include, but are not limited to, acid addition salts and basic salts. Examples of acid addition salts include chloride, sulfate, hydrogen sulfate, (hydrogen) phosphate, acetate, citrate, tosylate or mesylate salts. Examples of basic salts include salts with inorganic cations, e.g. alkaline or alkaline earth metal salts such as sodium, potassium, magnesium or calcium salts and salts with organic cations such as amine salts.

[0094] A "solvate" is a complex of a peptide of the invention or a salt thereof with solvent molecules, e.g. organic solvent molecules and/or water.

BRIEF DESCRIPTION OF THE FIGURES

[0095] FIG. 1. Draxin directly binds Netrin1.

[0096] A) AVEXIS screen results showing that the DraxinA prey protein, in addition to the positive control bait Matn4 (+), only binds to Netrin1a in a protein library consisting of 171 bait proteins. B) Interaction network of the zebrafish Netrin1 and Draxin paralogs. Consecutive screen results show that the interaction is also conserved for the paralogs.

[0097] FIG. 2. The binding of Draxin to Netrin1 is conserved for the human homologs and detectable across human and zebrafish proteins.

[0098] Heatmap showing results from the AVEXIS assay. The screen has been performed in both bait-prey orientations using pentameric prey proteins. Absorbance at 486 nm (A486 nm) has been measured after 1-hour incubation (black: A486 nm>0.1=binding, grey: A486 nm 0.08-0.1=weak binding, 2 repeats). B) Network view of the results.

[0099] FIG. 3. Draxin protein alignment using the Clustal W method.

[0100] The alignment of human, mouse, chick, and zebrafish DraxinA and DraxinB protein shows that the N-terminal half of the protein is poorly conserved. In the most C-terminal part of the protein a conserved 10-cysteine containing region can be found, which resembles the cysteine-rich region present in Dickkopf proteins.

[0101] FIG. 4. A conserved 21aa DraxinA derived peptide is sufficient for the binding to Netrin1a.

[0102] A) By using a set of truncated and deletion containing monomeric DraxinA preys the binding interface of DraxinA to Netrin1a has been mapped down to a 21 amino acid region (aa232-aa252). The protein fragments binding to Netrin1a are indicated in black, weak binding ones in grey, and non binding fragments are depicted in white. B) and C): Using a similar approach the DraxinA binding interface in Netrin1a has been narrowed down to the third EGF-domain containing region (aa401-aa458).

[0103] FIG. 5. Multiple species alignment of the Draxin derived 21 aa peptide sequence.

[0104] The figure shows that the Netrin1 binding peptide is highly conserved in vertebrates.

[0105] FIG. 6. DraxinA inhibits the binding of Netrin1a to Netrin receptors.

[0106] A) Schematic representation of the AVEXIS based competition assay. B) Purified full-length Draxin inhibits the binding of of the Netrin1a bait to the DCC-prey proteins. C) This effect can also be seen for the Unc5b and Neo1 (D) netrin receptors. Equal amounts of BSA (D) are not able to inhibit the binding between Netrin and Neo1. E) Draxin is not able to interfere with the binding of RGMc to Neo1. RGMc is another known Neo1 ligand. (% binding: binding with inhibitor/binding without inhibitor .times.100%, error bars indicate mean.+-.s.d.; n=4)

[0107] FIG. 7. A 21 amino acid fragment of Draxin is sufficient to outcompete Netrin/Netrin receptor interactions.

[0108] A) Full-length DraxinA-hFc and the 21aa peptide fused to the hFc region are able to outcompete DCC for binding to Netrin1a. In contrast the hFc fusion of full-length DraxinA with a deletion of aa231-252 is not able to compete for binding. None of the 3 DraxinA-hFc fusion protein versions is able to block the binding between other known tested receptor-ligand pairs: (B) Cntn1a/Ptprz1b, (C) Vasn/Islr2, and (D) EphB4a/EphrinB2a. (% binding: binding with inhibitor/binding without inhibitor .times.100%, error bars show mean.+-.s.d.; n=3)

[0109] FIG. 8. The binding of the 21aa DraxinA derived peptide is highly specific.

[0110] A) A pentameric DraxinA aa232-252-prey protein has been screened against a library consisting of 141 bait proteins. We only observed binding to the Netrin1a-bait. The positive control (+) corresponds to Matn4. B) The DraxinA aa232-252-bait has been screened against 191 pentameric prey proteins and only showed binding to the Netrin1a prey.

[0111] FIG. 9. Draxin outcompetes receptor bound Netrin1a.

[0112] A) Scheme of the experimental design. B) The inhibitory effect of DraxinA is not decreased by already preformed Netrin1a/DCC complexes.

[0113] FIG. 10: Protein alignment of the third EGF domain of Netrins.

[0114] Protein alignment of the third EGF domain of human Netrin1, Netrin3, Netrin4 and zebrafish Netrin1a and Netrin1b shows that this domain is highly conserved for the .gamma.-chain netrins but not for Netrin4 which belongs to the laminin1 .beta.-chain derived netrins. In agreement with the protein alignment data our AVEXIS binding data indicate that only .gamma.-chain derived Netrins can bind to Draxin.

[0115] FIG. 11: In vivo detection of the Draxin-Netrin1a interaction in zebrafish embryos.

[0116] A) Schematic illustration of the assay design. mRNAs encoding the indicated fluorophore tagged genes were injected into one-cell stage zebrafish embryos and imaged at sphere stage (4 hours post fertilization (hpf)). The imaging plane corresponded to a region approximately 15 .mu.m beneath the enveloping layer of the embryos.

[0117] B) Single section confocal images of the embryos. (Ba, Ba', Ba'') embryos injected with 100 pg Draxin-super folder GFP (sfGFP) mRNA displayed uniform distribution of Draxin-sfGFP protein in the extracellular space. In contrast injection of 100 pg of Netrin1a-sfGFP mRNA (Bb, Bb', Bb'') resulted in dense membrane associated speckles positive for Netrin1a-sfGFP protein. In (Ba) and (Bb) memRFP has been used to label the cell surface. Upon co-injections of 200 pg Draxin-sfGFP mRNA and 200 pg of Netrin1a-mCherry mRNA (Bc, Bc', Bc'') Draxin-sfGFP and Netrin1a-mCherry proteins co-localize into membrane associated spots. (n=7; arrowheads point to examples of co-localization; scale bars correspond to 10 .mu.m).

[0118] FIG. 12: In situ detection of the Draxin-Netrin1a interaction in zebrafish using an affinity probe.

[0119] A) Schematic illustration of the experiment. A Draxin.sub.aa209-284-hFc-fusion protein was generated in HEK293-6E cells as a probe to detect endogenous netrins. Mildly fixed 48 hpf embryos were incubated with the affinity probe (Draxin.sub.aa209-284-hFc) and the signal detection has been carried out by using a fluorophore tagged anti human IgG antibody.

[0120] B) Results of the in situ detection experiments. The signal from Draxin.sub.aa209-284-hFc-fusion probe was detectable in the floor plate region in wild type (also abbreviated as wt) fish (Ba), and was not detectable in netrinla and netrinlb double-knockdown embryos (Bb). The shh:GFP transgenic line has been used for the experiments to visualize floor plate cells. Arrowheads point to the Draxin.sub.aa209-284-hFc derived signal. Scale bar in (Ba''), 20 .mu.m; applies to all panels. (n>10)

[0121] FIG. 13: Heatmap depicting binding results between human DRAXIN and Netrin signaling system members.

[0122] AVEXIS was used to test pairwise binding events in both bait/prey orientations between human DRAXIN, the derived 21 amino acid peptide, human Netrin family members, and two representative Netrin receptors (DCC, UNC5B). The Matn4-bait served as an internal prey-protein control and was used for normalization of the A486 nm values; conditioned medium (CM) serves as negative control; n=3.

[0123] FIG. 14: Surface Plasmon Resonance Analysis of Draxin Binding.

[0124] Binding of Draxin to immobilised recombinant human Netrin1, UNC5B, and DCC was monitored using Surface Plasmon Resonance experiments on a Siacore 3000 instrument. See text for details.

[0125] The invention will be further illustrated by the following examples.

EXAMPLES

[0126] Methods

[0127] AVEXIS Based Library Screen

[0128] To detect extracellular protein-protein interactions in a high-throughput manner we used the AVEXIS assay as described in Bushell et al. (Bushell et al., 2008) with minor modifications.

[0129] In brief: A zebrafish protein library enriched for mainly in neuronal tissues expressed secreted proteins and extracellular domains of cell surface proteins has been assembled. The library consists of prey and bait proteins. Preys are composed of the extracellular domain (ECD) of interest followed by a CD4 tag (rat Cd4d3+4) and a pentamerization domain derived from the rat Comp protein followed by .beta.-lactamase. For the bait proteins the ECDs are fused to a CD4 tag and a biotinylation peptide. All proteins for the screen have been expressed by transient transfection of Human Embryonic Kidney (HEK293-6E) cells (Durocher et al., 2002) grown in Freestyle medium (Invitrogen) containing 1% FCS. Supernatants have been harvested 6 days post transfection. The bait proteins have been dialyzed against HBS (140 mM NaCl, 5 mM KCl, 2 mM CaCl.sub.2), 1 mM MgCl2, 10 mM HEPES, pH 7.4) to remove free biotin. The proteins in the supernatants have been quantified and normalized.

[0130] For the AVEXIS screen the supernatant dilution factors have been adjusted to values allowing faithful detection of the interaction between Vasn (Slit-like2) and Islr2 (Sollner and Wright, 2009) with determined KD of 12 .mu.M and a very short half-life (t1/2 0.16 s) (in preparation). The bait proteins have been immobilized on streptavidin coated 96 well microtiter plates (one bait/well) and incubated for 1 hour at room temperature. After 3 wash steps using HBS as wash buffer the baits have been probed by 50.mu.l of normalized prey proteins. After one-hour incubation the non-bound preys have been washed away by 2 washes with HBST (0.05% Tween) followed by two HBS washes. Then 50.mu.l of nitrocefin/well (0.1 mg/ml) has been added and incubated for 1 h at room temperature. Then the absorbance at 486 nm of each well has been measured using a .mu.Quant spectrophotometer (BIO-TEK Instruments, INC). As a positive control for the prey proteins we used the Matn4 ECD as mono-biotinylated bait protein. Matn4 has been shown to bind to the coiled-coil pentamerization domain of Comp (Mann et al., 2004), which is present in all recombinant pentameric prey proteins of our AVEXIS library.

[0131] In the primary screen interactions were `called` if the absorbance (at 486 nm) of a well after 1 h of incubation was .gtoreq.0.1 and 3 Sigma above the standard deviations of the mean of all wells. All interactions detected in the primary screen have been retested in a validation screen by using independently produced batches of proteins.

[0132] Domain Mapping Experiments

[0133] For the binding interface mapping experiments we used monomeric prey proteins. The concentration of the monomeric preys has been determined as described for the AVEXIS assay (.beta.lactamase enzymatic activity). After normalization, the monomeric preys have been screened against a set of proteins from the library to identify and remove promiscuous binders caused for example through improper domain boundary design. Both prey and bait orientations were tested in the domain mapping screen and two repeats for each orientation had been carried out.

[0134] Protein Purification

[0135] His-tagged full-length zebrafish Draxin protein (DraxinCD4d3+46.times.His) has been expressed in HEK293-6E cells and affinitiy purified from tissue culture supernatants using HisTrap HP columns (GE healthcare). The correct size of the purified protein has been checked on a protein gel.

[0136] AVEXIS Based Competition Assay

[0137] The procedure of the competition assay is based on the AVEXIS assay. Netrin1 receptors were used as prey proteins together with 6.times.Histagged purified Draxin (potential antagonist), and probed against Netrin1a bait proteins. The indicated concentrations of the potential inhibitors have been added together with prey proteins. For the competition tests with purified Draxin the concentration of the Netrin receptor prey proteins has been adjusted to an identical threshold binding concentration.

[0138] hFc Fusion Protein Normalization for the Competition Assay

[0139] We determined the concentration of ECD-hFc fusion proteins in tissue culture supernatants by ELISA, using the human IgG Fc fragment (Calbiochem) as a reference. Dilution series of the ECD-hFc containing supernatants have been incubated over night at 4.degree. C. on 96 well Maxisorp plates (Nunc). After 3 PBS washes the plates were blocked with 0.5% BSA containing PBS (1 h). After additional 2 PBS washes and 1-hour incubation with an anti-human IgG (Fc specific) antibody fused to alkaline phosphatase (SIGMA) the plates have been washed 3 times with PBS. The detection has been carried out by addition of 50 .mu.l/well of the AP substrate p-nitrophenylphosphate (SouthernBiotech). The substrate turnover has been determined by measuring the absorbance at 405 nm.

[0140] In Vivo Binding Assay

[0141] To reveal whether the interaction between Draxin and Netrin1a is detectable in vivo, an mRNA overexpression assay was designed to visualize the localization of the two proteins in zebrafish embryos. Constructs of full-length zebrafish Draxin and Netrin1a fused with fluorescent proteins were generated by using the Gateway.RTM. cloning system (Life Technologies).

[0142] Before used in the in vivo detection assay, the coding sequences of the generated fusion proteins were cloned into AVEXIS plasmids, expressed as preys and tested for activity against bait proteins of the corresponding binding partner. Following constructs were selected for the in vivo binding test: Draxin and Netrin1a C-terminally fused to superfolder-GFP (Draxin-sfGFP, Ntn1a-sfGFP) and Netrin1a C-terminally fused with mCherry (Ntn1a-mCherry). The corresponding capped mRNAs were synthesized using the mMESSAGE mMACHINE SP6 or T7 Transcription Kit (Ambion) according to manufacturer instructions. For the injections, zebrafish embryos were dechorionated using 1 mg/ml Pronase (Roche, 11459643001) and then injected with 1 nl mRNA into the cell center at one cell stage. Draxin-sfGFP or Netrin1a-sfGFP mRNAs were injected at 100 pg/embryo in combination with 10-15 pg/embryo of membrane-tagged RFP (mRFP) to label the cell membranes. The same amount of mRNA (100-200 pg/embryo) was injected in the Draxin-sgGFP and Netrin1a-mCherry coexpression experiments. The injected embryos have been cultured at 28.5.degree. C. in agarose-coated dishes. At sphere stage (4 hpf), embryos were immobilized in 1% low-melting-point agarose in glass-bottom Petri dishes with the animal pole facing the coverslip. The imaging plane corresponded to a region approximately 15 .mu.m beneath the enveloping layer of the embryos. Single plane confocal images of the embryos were taken using a Zeiss LSM 780 NLO microscope.

[0143] In Situ Detection of Draxin Binding Partners

[0144] A netrin binding fragment of Draxin (aa209-284) fused to the Fc region of human IgG (Draxin.sub.aa209-284-hFc) has been expressed in HEK293-6E cells and used as an affinity probe to detect binding partners in zebrafish embryos. Draxin.sub.aa209-284-hFc in situ staining has been done in whole mount wild type and netrin-1 knockdown zebrafish embryos. At 46 hpf wild type zebrafish embryos were dechorionated by for 2 hours at RT incubation in 0.1 mg/ml Pronase. At 48 hpf embryos were prefixed for 10 min at RT in 4% (w/v) paraformaldehyde (PFA) containing 1% Triton X-100 (v/v). After 3 washes (each 20 min) with PBS+1% Triton X-100 the embryos were blocked for 4 h at RT in PBS containing 0.2% BSA and 0.5% Triton X-100. Overnight incubation at 4.degree. C. with HEK293-6E supernatant containing the Draxin-hFc fusion protein was followed by 3 short washes (10 min each) with PBST. Subsequently the embryos were post-fixed in 4% PFA (4 h at RT or overnight at 4.degree. C.), rinsed shortly 3 times with PBST and incubated for 4 h at RT with an Alexa Fluor 568 goat anti human IgG antibody (Invitrogen, 1:250 dilution). After 3 washes (30 min each) in PBST the embryos were mounted in glycerol for visualization using a Zeiss LSM 510 microscope. A shorter version of Draxin containing the Netrin1a binding site had to be used because the full-length version of Draxin-hFc caused uniform background staining in zebrafish embryos probably by unspecific binding to glycosaminoglycans (GAGs) present on cell surfaces. A series of additional control ECD-hFc proteins have been tested, only Draxin.sub.aa209-284-hFc displayed binding to the extracellular space of the floor plate.

[0145] Knockdown of netrin1a and netrin1b in Zebrafish Embryos

[0146] Morpholino antisense oligonucleotides (Gene-Tools) have been used to generate zebrafish with reduced netrin protein expression levels. The following morpholino sequences have been used to knockdown ntn1a ATGATGGACTTACCGACACATTCGT-3', SEQ ID NO.: 80) and ntn1b (5'-CGCACGTTACCAAAATCCTTATCAT-3', SEQ ID NO.: 81). In previous studies both morpholinos had been shown to efficiently knockdown the corresponding genes (Kastenhuber et aL, 2009; Suli et al.; 2006).

[0147] Surface Plasmon Resonance (SPR)

[0148] Surface Plasmon Resonance (SPR) experiments were performed on a Biacore 3000 (GE Healthcare) at 25.degree. C. using a SA sensor chip in 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.005% Surfactant P20 (HBS-P) running buffer at a flow rate of 30 .mu.l/min. The instrument was used according to manufacturer's instructions.

Example 1: DraxinA Physically Interacts with Netrin1a

[0149] Using a protein-protein interaction screen assay, designed to identify direct interactions within a protein library consisting of secreted proteins and extracellular domains of cell surface proteins (Bushell et al., 2008), we carried out a large-scale screen involving more than 40,000 binding experiments. The library we used for the screen was strongly enriched for zebrafish proteins known to be expressed in the developing nervous system. During this screen we identified a novel interaction between two secreted proteins with known function in axon guidance, Netrin1a and DraxinA. In the primary screen a DraxinA prey protein has been tested for binding against a library consisting of 171 bait proteins, including a positive control. The DraxinA prey protein specifically bound to the Netrin1a bait (FIG. 1A) and did not bind to any additional proteins of the library. The interaction has been confirmed in both bait-prey orientations in a validation screen using new protein samples. Interestingly, both netrin1 and draxin are duplicated in zebrafish. In subsequent binding assays we were able to show that the Netrin1/Draxin interaction is also conserved for the paralogs Netrin1b and DraxinB (FIG. 1B).

[0150] The AVEXIS assay is able to detect very transient and weak interactions due to the avidity effect caused by the use of pentameric prey proteins. Hence, in order to test whether the interaction between Netrin1a and Draxin is transient or rather stable we used monomeric prey proteins and probed them against the corresponding binding partners. Using this approach, we confirmed the interaction between Netrin1a and Draxin suggesting that this interaction is based on strong binding between the two proteins.

Example 2: The Interaction Between Draxin and Netrin1 is Conserved for the Human Homologs

[0151] Next we asked the question whether the interaction between Netrin1 and Draxin is conserved. By using the corresponding human homologs NTN1 and DRAXIN we were able to show that the interaction is indeed conserved. In addition, we observed that zebrafish Netrin1a was able to bind to human Draxin and vice versa (FIG. 2). This strongly indicates that this newly identified interaction is conserved within vertebrate species underscoring the biological relevance of this interaction.

Example 3: The Netrin1 Binding Region of Draxin has been Mapped Down to a 21Aa Motif

[0152] Next, we narrowed down the region in DraxinA required for binding to Netrin1a. Zebrafish DraxinA consists of 360 amino acids (aa). The first 23 aa are part of the signal peptide. This sequence is followed by a poorly conserved N-terminal half of the protein (FIG. 3). In contrast, the C-terminal half of the protein is highly conserved and ends with a 10 cysteine-containing domain (aa285-aa360). In terms of cyteine spacing this domain is similar to domains present in the Wnt antagonist Dkk1 (Glinka et al., 1998).

[0153] To map down the Netrin1a binding region in DraxinA we generated a series of DraxinA truncations and deletions and tested them for binding against Netrin1a (FIG. 4). Using this approach we were able to narrow down the binding region to a 21aa DraxinA protein fragment. In addition, a full-length version of DraxinA lacking these 21aa completely lost the ability to bind to Netrin1a. Additional removal of 5aa from the N-terminal or C-terminal end of the 21aa stretch caused a dramatic reduction of the binding ability to Netrin1a. Interestingly, the Netrin-binding 21aa stretch (aa232-252) of DraxinA is highly conserved cross vertebrate species (FIG. 5). It is noteworthy that, this conserved 21aa region is also highly specific for Draxin and cannot be found in other proteins.

Example 4: Netrin1a Domain Mapping

[0154] Netrin1a is a multi-domain containing protein composed of 603 amino acids. It consists of a laminin N-terminal domain (LamNT) encoded by amino acid 44-282 followed by 3 laminin-type epidermal growth factor-like domains (aa284-450), and a C-terminal domain (C345C) encoded by amino acid 486 to 594. In order to map the DraxinA binding region in Netrin1a we generated a set of truncated Netrin1a fragments and probed them in the AVEXIS assay for binding against DraxinA. Using this approach we were able to narrow down the binding region to a fragment consisting of amino acid 401-458 (FIG. 4B). This fragment encodes the third laminin-type EGF domain. The third EGF-domain of Netrin1a is highly conserved in vertebrate Netrin1 homologs. For example, only a single amino acid exchange is present in this domain between zebrafish Netrin1a (CDCHPVGAAGKTCNQTTGQCPCKDGV TGITCNRCANGYQQSRSPIAPC; SEQ ID NO: 64) and human Netrin1 (CDCHPVGAAGKTCNQTTGQCPCKDGVTGITCNRCAKGYQQSRSPIAPC; SEQ ID NO: 65) proteins,

[0155] Interestingly, the third EGF domain of Netrin1 has recently been shown to be required for Netrin receptor binding (Finci et al., 2014; Xu et al., 2014). These findings offer a mechanistic explanation for our observed competition assay results.

Example 5: DraxinA is Able to Inhibit the Binding of Netrin1a to Netrin Receptors

[0156] By using an AVEXIS-based competition assay (FIG. 6 A) we tested whether the binding of DraxinA to Netrin1a has an influence on Netrins ability to bind to Netrin receptors. First we confirmed that we reliably detected the binding of Netrin1a to Netrin receptors of the DCC/Neo1 and Unc5 families with the AVEXIS method. We also tested whether Draxin is able to bind to the corresponding Netrin receptors. Contrary to previous findings (Ahmed et al., 2011), we were not able to detect direct binding between Draxin and any of the tested Netrin receptors using the AVEXIS platform. This has also been confirmed by a recent publication (Haddick et al. 2014). In the competition assay the extracellular domains (ECDs) of Netrin receptors have been used as prey proteins together with purified full length DraxinA and probed for binding against Netrin1a bait proteins. Using this strategy, we observed a DraxinA concentration dependent inhibition of the binding between Netrin1a and Netrin receptors (FIG. 6B,C,D). The inhibition is specific for DraxinA. Furthermore, DraxinA (FIG. 6 E) is not able to block the binding of RGMc to Neo1, another reported ligand of the corresponding receptor (Bell et al., 2013).

Example 6: The 21Aa DraxinA Fragment Fused to the Human Fc Region of IgG is Sufficient to Block the Binding of Netrin1a to Netrin Receptors

[0157] Next we assayed whether the 21aa DraxinA fragment fused to the human Fc tag (Draxin.sub.aa232-252-hFc) is sufficient to outcompete Netrin/Netrin receptor interactions.

[0158] We compared the effect of Draxin.sub.aa232-252-hFc with full-length Draxin-hFc and a version of DraxinA where the 21aa Netrin1 binding motif has been deleted (DraxinA.DELTA..sub.aa231-252-hFc) in the competition assay for their ability to interfere with the binding of Netrin1a to Dcc. The results show that Draxin.sub.aa232-252-hFc has a similar efficiency in inhibiting the binding of Netrin1a to Dcc as the DraxinA full-length version (Draxin-hFc). The DraxinA version with the 21 aa deletion (DraxinA.DELTA..sub.aa231-252-hFc) is not able to compete for binding to Netrin1a (FIG. 7). In addition we used our set of different Draxin-hFc proteins to test whether they have an effect on other known interactions (Cntn1a/Ptprz1b, Vasn/Islr2, EphB4a/EphrinB2a). None of the 3 Draxin-hFc versions was able to inhibit any of the tested interactions (FIG. 7 B,C,D).

[0159] Taken together, our results show that the 21aa region is necessary for the competition and that the Draxin.sub.aa232-252-hFc fusion protein is also sufficient to outcompete Netrin receptors for Netrin1a binding.

Example 7: The Binding of the 21Aa DraxinA Fragment to Netrin1a is Highly Specific

[0160] In order to determine the binding specificity of the 21aa DraxinA peptide we used the AVEXIS assay and screened the 21aa fragment as bait and prey against proteins from our library. In this screen against a set of 141 bait proteins the DraxinA.sub.aa232-252-prey only bound to the Ntn1a-bait and to the positive control bait Matn4 (FIG. 8A). Similar results we obtained for the DraxinA.sub.aa232-252-bait protein, which only interacted with the Ntn1a-prey in a screen against a set of 191 different prey proteins (FIG. 8B). These findings indicate that the binding of this short 21 aa peptide to Netrin1a is highly specific.

Example 8: DraxinA Outcompetes Receptor Bound Netrin 1a

[0161] Netrin1 binds with high affinity (K.sub.d's in the low nM range) to its receptors of the DCC- and Unc5-family (Leonardo et al., 1997). Hence, we asked the question whether already bound Netrin1a could be displaced from the receptors by DraxinA. In order to do so we carried out an AVEXIS based competition assay and tested three different settings (FIG. 9A). In one experimental setting Netrin1a-baits were preincubated with purified DraxinA before addition of the DCC prey. In the second set of experiments the Netrin1a-baits were incubated with a mixture of DraxinA and DCC prey proteins, and in the third set of experiments Netrin1a-baits have been preincubated with DCC preys followed by the addition of purified DraxinA as inhibitor. We did not observe a difference in the %-binding response between these 3 sets of experiments. These findings show that already formed DCC/Netrin1a complexes can be disrupted by the addition of DraxinA. These findings indicate that DraxinA has a higher affinity for Netrin1a than the Netrin receptor DCC.

Example 9: In Vivo Detection of the Draxin-Netrin Interaction in Zebrafish Embryos

[0162] To independently confirm the Draxin/Netrin1a interaction and to test whether both proteins are able to interact in vivo, we made use of transient protein overexpression experiments in zebrafish embryos. mRNAs encoding Draxin fused to superfolder GFP (Draxin-sfGFP) and Netrin1a tagged with mCherry (Netrin1a-mCherry) or superfolder GFP (Netrin1a-sfGFP) have been injected into one-cell stage zebrafish embryos. The distribution of the corresponding fluorophore tagged proteins has been analyzed in sphere stage zebrafish embryos (4 hours post fertilization) (FIG. 11A). At this developmental stage the extracellular space width between the cells is very large, ideally suited to visualize the localization of secreted proteins. Upon injection of mRNA encoding Draxin-sfGFP we observed an evenly distributed signal outside the cells in the extracellular milieu of 4 hpf zebrafish embryos (FIG. 11 Ba). In contrast thereto, the distribution of Netrin1a-sfGFP was restricted to cell surface sub-domains (FIG. 11 Bb).

[0163] When Draxin-sfGFP was coexpressed with Netrin1a-mCherry, Draxin-sfGFP re-located to Netrin1a-mCherry positive membrane associated densities (FIG. 11 Bc). This indicated that localized Netrin1a-mCherry was able to capture diffusible Draxin-sfGFP.

[0164] The mRNA overexpression experiments showed that Draxin and Netrin1a are able to interact with each other in vivo. To further support this we used another strategy aiming to detect the distribution of endogenous Draxin interaction partners at developmental stages relevant for axon guidance decisions. From our binding assay with monomeric prey proteins we already had hints that the interaction between Draxin and Netrin is of high-affinity. Thus, we fused a netrin-binding fragment of the Draxin-ECD (aa209-284) to the human Fc region to generate an affinity probe. First, we tested this probe on zebrafish embryos from different developmental stages. After very gentle fixation the embryos were incubated with HEK293-6E cell supernatants containing the recombinant soluble Draxin.sub.aa209-284-hFc protein.

[0165] Using an Alexa-Fluor 568 anti human IgG antibody to detect in situ bound Draxin.sub.aa209-284-hFc we only detect a signal in close proximity to the floor plate (FIG. 12A, 12Ba). Because floor plate cells express Netrin1a and Netrin1b, we had indications that the signal detected by using the Draxin affinity probe indeed corresponds to in the extracellular space localized netrin. To prove this observation, we compared 48 hpf wt embryos with netrinla and netrinlb double-knockdown embryos (FIG. 12Ba, 12Bb). In double-knockdown embryos the signal from the bound affinity probe was barely detectable compared to non-injected siblings, indicating that the Draxin.sub.aa209-284-hFc probe indeed detected netrin. Taken together, the results from our mRNA overexpression and Draxin affinity probe experiments provide strong evidence that Draxin and Ntn1a are able to interact in vivo in zebrafish embryos.

Example 10: Human DRAXIN/Netrin-Signaling Network

[0166] To determine the binding specificity of DRAXIN/Netrin interactions, we carried out a pairwise binding screen between human DRAXIN and human Netrin family members. Except Netrin-5, we included all human Netrin family members consisting of two secreted .gamma.-Netrins (Netrin-1 and Netrin-3) and one secreted (Netrin-4) and two GPI-linked .beta.-Netrins (Netrin-G1 and Netrin-G2) in our binding study. Human DRAXIN and a 21 amino acid Netrin binding fragment derived thereof (SEQ ID NO.: 1) bound to Netrin-1 and Netrin-3 but not to human .beta.-Netrin family members (FIG. 13).

[0167] These experiments confirm for human proteins the Draxin/.gamma.-Netrin binding specificity within the Netrin family and showed that the human 21 amino acid DRAXIN fragment (SEQ ID NO.: 1), like its zebrafish counterpart (SEQ ID NOs.: 3), is sufficient for binding.

Example 11: Validation of the Draxin/Netrin1 Interaction by Surface Plasmon Resonance

[0168] Recombinant human Draxin was purchased from R&D systems. Biotinylated recombinant human UNC5B, Netrin1 and DCC were produced recombinantly using the described mammalian expression system (HEK293-6E). Biotinylated proteins were immobilised on the SA coated sensor chip and Draxin was injected sequentially in increasing concentrations (0 nM, 1.2 nM. 2.3 nM, 4.7 nM, 9.4 nM, 18.8 nM) for 3 min. Dissociation was allowed for 5 min in HBS-P. Binding was monitored and an interaction of Draxin to immobilised Netrin-1 was observed with a binding constant KO of approximately 20 to 100 nM. No binding of Draxin to UNC5B and DCC was detected (FIG. 14).

REFERENCES

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Sequence CWU 1

1

82121PRTHomo sapiens 1Glu Val Met Pro Thr Leu Asp Met Ala Leu Phe Asp Trp Thr Asp Tyr1 5 10 15Glu Asp Leu Lys Pro 20221PRTDanio rerio 2Asp Val Ala Pro Thr Phe Asn Met Ala Leu Phe Asp Trp Thr Asp Tyr1 5 10 15Glu Asp Met Arg Pro 20321PRTDanio rerio 3Glu Val Met Pro Thr Leu Asp Met Thr Leu Phe Asp Trp Thr Asp Tyr1 5 10 15Glu Asp Met Lys Pro 204349PRTHomo sapiens 4Met Ala Gly Pro Ala Ile His Thr Ala Pro Met Leu Phe Leu Val Leu1 5 10 15Leu Leu Pro Leu Glu Leu Ser Leu Ala Gly Ala Leu Ala Pro Gly Thr 20 25 30Pro Ala Arg Asn Leu Pro Glu Asn His Ile Asp Leu Pro Gly Pro Ala 35 40 45Leu Trp Thr Pro Gln Ala Ser His His Arg Arg Arg Gly Pro Gly Lys 50 55 60Lys Glu Trp Gly Pro Gly Leu Pro Ser Gln Ala Gln Asp Gly Ala Val65 70 75 80Val Thr Ala Thr Arg Gln Ala Ser Arg Leu Pro Glu Ala Glu Gly Leu 85 90 95Leu Pro Glu Gln Ser Pro Ala Gly Leu Leu Gln Asp Lys Asp Leu Leu 100 105 110Leu Gly Leu Ala Leu Pro Tyr Pro Glu Lys Glu Asn Arg Pro Pro Gly 115 120 125Trp Glu Arg Thr Arg Lys Arg Ser Arg Glu His Lys Arg Arg Arg Asp 130 135 140Arg Leu Arg Leu His Gln Gly Arg Ala Leu Val Arg Gly Pro Ser Ser145 150 155 160Leu Met Lys Lys Ala Glu Leu Ser Glu Ala Gln Val Leu Asp Ala Ala 165 170 175Met Glu Glu Ser Ser Thr Ser Leu Ala Pro Thr Met Phe Phe Leu Thr 180 185 190Thr Phe Glu Ala Ala Pro Ala Thr Glu Glu Ser Leu Ile Leu Pro Val 195 200 205Thr Ser Leu Arg Pro Gln Gln Ala Gln Pro Arg Ser Asp Gly Glu Val 210 215 220Met Pro Thr Leu Asp Met Ala Leu Phe Asp Trp Thr Asp Tyr Glu Asp225 230 235 240Leu Lys Pro Asp Gly Trp Pro Ser Ala Lys Lys Lys Glu Lys His Arg 245 250 255Gly Lys Leu Ser Ser Asp Gly Asn Glu Thr Ser Pro Ala Glu Gly Glu 260 265 270Pro Cys Asp His His Gln Asp Cys Leu Pro Gly Thr Cys Cys Asp Leu 275 280 285Arg Glu His Leu Cys Thr Pro His Asn Arg Gly Leu Asn Asn Lys Cys 290 295 300Phe Asp Asp Cys Met Cys Val Glu Gly Leu Arg Cys Tyr Ala Lys Phe305 310 315 320His Arg Asn Arg Arg Val Thr Arg Arg Lys Gly Arg Cys Val Glu Pro 325 330 335Glu Thr Ala Asn Gly Asp Gln Gly Ser Phe Ile Asn Val 340 3455360PRTDanio rerio 5Met Val Ala Pro Gly Leu Cys Gln Leu Phe Ile Leu Leu Leu Ile Thr1 5 10 15Leu Ser His Thr Leu His Ser Ser Glu Ile Ser Ser Asp Asn Phe Lys 20 25 30Gln Ser Leu Thr Pro Ser Thr Thr Thr Ser Lys Glu His Pro Glu Thr 35 40 45Gly Leu Thr Gly Gly Arg Gln Gln Lys Arg His Trp Ser Gly Lys Glu 50 55 60Arg Asp Ser Ala Gly Leu Phe Ser Gln Arg His Met Asp Arg Leu Glu65 70 75 80Asp Asp Gly Thr Ser Met Glu Gly Leu Ser Pro Val Arg Leu Glu Met 85 90 95Gly Pro Gly Asp Thr Met Lys Ala Glu Val His Gly Glu Val Arg Ala 100 105 110Ser Ala Gln Met Arg Gln Gly Ser His Pro Ala Glu Gly Glu Leu Asn 115 120 125Arg Lys Gly Arg Arg His Ser His Arg Leu Leu Ala Glu His Arg Lys 130 135 140His Gly Gly Lys Lys Asp Lys Gly Arg Gly Lys Gly Asp Leu Ser Asp145 150 155 160Pro Glu Pro Glu Leu Asp Ser Leu Leu Lys Asp Leu Asn Ala Phe Glu 165 170 175Asp Gly Leu Asn Thr Ser Pro Pro Asn Tyr Asn Ser Val Pro Leu Asn 180 185 190Glu Val Pro Ser Pro Leu Ser Pro Ile Leu Val Thr Thr Ala Ile Lys 195 200 205Gly His Pro Pro Thr Leu Pro Pro Ala Ser Thr Lys Pro Gln Lys Ser 210 215 220Ser Gln Gly Arg Thr Gln Gly Glu Val Met Pro Thr Leu Asp Met Thr225 230 235 240Leu Phe Asp Trp Thr Asp Tyr Glu Asp Met Lys Pro Ala Asp Ser Trp 245 250 255Pro Ser Asn Lys Arg Lys Asp Lys Arg Arg Ser Lys Asn Lys Ser Asn 260 265 270Gly Asn Thr Thr Thr Glu Ala Gly Ile Val Glu Pro Cys Asp His His 275 280 285Leu Asp Cys Leu Ser Gly Ser Cys Cys Asp Leu Arg Glu Phe Glu Cys 290 295 300Lys Pro His Asn Arg Gly Leu Asn Asn Lys Cys Phe Asp Asp Cys Met305 310 315 320Cys Glu Glu Gly Leu Arg Cys Tyr Ala Lys Phe His Arg Lys Arg Arg 325 330 335Val Thr Arg Arg Arg Gly Arg Cys Val Asp Pro Glu Ser Val Asn Ser 340 345 350Asn Gln Gly Ala Phe Ile Thr Val 355 3606351PRTDanio rerio 6Met Ala Val Ser Cys Trp Tyr Phe Ala Leu Phe Leu Ile Phe Asp Leu1 5 10 15Met Thr Met Thr Leu Gly Thr Asn Thr His His Asn Ser Pro Met Glu 20 25 30Val Phe Ser Glu Asn Ile Ile Ile Pro Pro Lys Pro Glu Ala Ser Ile 35 40 45His His His Thr His Gln Arg Thr Asp Arg Gly Arg Lys Glu Arg Met 50 55 60Thr Ala Ser Gln Leu Arg Glu Arg Pro Arg Ile Ala Ile Phe His Thr65 70 75 80Gln Asn Glu Gly Pro Asp Leu Glu Gly Leu Ser Pro Val Arg Leu Glu 85 90 95Met Glu Pro Ala Asp Lys Arg Arg Val Met Thr Pro Arg Lys Lys Thr 100 105 110Phe Met Gly Ser Asp Ser Leu Ile Gln Glu Lys Met Asn Ile Ser Pro 115 120 125Gly Ala Glu Thr Pro Glu Lys Ala Met Arg Arg Pro Thr Val Arg Lys 130 135 140Val Phe Gly Gly His Ile Thr Arg Ala Pro His Glu Glu Glu Ser Leu145 150 155 160Ala Ser Gly Lys Lys Arg Arg Val Ser Phe Asp Gln Arg Leu Asn Lys 165 170 175Ala Ser Phe Gly Ser Pro Thr Glu Pro Val Leu Pro Ala Ala Thr Val 180 185 190Gly Thr Phe Ile Leu Pro Ile Thr Ala Ala Val Asp Gly Asn Pro Asn 195 200 205Pro Ser Ser Glu Pro Gln Val Arg Arg Tyr Leu Gly Gly Asp Val Ala 210 215 220Pro Thr Phe Asn Met Ala Leu Phe Asp Trp Thr Asp Tyr Glu Asp Met225 230 235 240Arg Pro Gly Asp Lys Lys Gln Tyr Ser Lys Lys Gln Gly Ser Glu Lys 245 250 255Gln Ala Thr Gln Ser Pro Ser Thr Gly Leu Val Arg Leu Thr Ser Glu 260 265 270Asn Asn Val Cys Lys His His Leu Asp Cys Leu Pro Gly Ser Cys Cys 275 280 285Asn Leu Arg Lys His Val Cys Glu Leu His Asn Arg Gly Phe Asn Asn 290 295 300Lys Cys Tyr Asp Ser Cys Met Cys Glu Glu Gly Leu Arg Cys Tyr Ala305 310 315 320Lys Ser His Arg His Tyr Arg Ile Thr Arg Lys Lys Gly Gln Cys Val 325 330 335Asp Pro Glu Asp Leu Asn His Ala Val Ser Arg Trp Met Gln Met 340 345 350775PRTDanio rerio 7His Pro Pro Thr Leu Pro Pro Ala Ser Thr Lys Pro Gln Lys Ser Ser1 5 10 15Gln Gly Arg Thr Gln Gly Glu Val Met Pro Thr Leu Asp Met Thr Leu 20 25 30Phe Asp Trp Thr Asp Tyr Glu Asp Met Lys Pro Ala Asp Ser Trp Pro 35 40 45Ser Asn Lys Arg Lys Asp Lys Arg Arg Ser Lys Asn Lys Ser Asn Gly 50 55 60Asn Thr Thr Thr Glu Ala Gly Ile Val Glu Pro65 70 75866PRTDanio rerio 8His Pro Pro Thr Leu Pro Pro Ala Ser Thr Lys Pro Gln Lys Ser Ser1 5 10 15Gln Gly Arg Thr Gln Gly Glu Val Met Pro Thr Leu Asp Met Thr Leu 20 25 30Phe Asp Trp Thr Asp Tyr Glu Asp Met Lys Pro Ala Asp Ser Trp Pro 35 40 45Ser Asn Lys Arg Lys Asp Lys Arg Arg Ser Lys Asn Lys Ser Asn Gly 50 55 60Asn Thr65948PRTDanio rerio 9His Pro Pro Thr Leu Pro Pro Ala Ser Thr Lys Pro Gln Lys Ser Ser1 5 10 15Gln Gly Arg Thr Gln Gly Glu Val Met Pro Thr Leu Asp Met Thr Leu 20 25 30Phe Asp Trp Thr Asp Tyr Glu Asp Met Lys Pro Ala Asp Ser Trp Pro 35 40 451043PRTDanio rerio 10His Pro Pro Thr Leu Pro Pro Ala Ser Thr Lys Pro Gln Lys Ser Ser1 5 10 15Gln Gly Arg Thr Gln Gly Glu Val Met Pro Thr Leu Asp Met Thr Leu 20 25 30Phe Asp Trp Thr Asp Tyr Glu Asp Met Lys Pro 35 401150PRTDanio rerio 11Gln Gly Arg Thr Gln Gly Glu Val Met Pro Thr Leu Asp Met Thr Leu1 5 10 15Phe Asp Trp Thr Asp Tyr Glu Asp Met Lys Pro Ala Asp Ser Trp Pro 20 25 30Ser Asn Lys Arg Lys Asp Lys Arg Arg Ser Lys Asn Lys Ser Asn Gly 35 40 45Asn Thr 501232PRTDanio rerio 12Gln Gly Arg Thr Gln Gly Glu Val Met Pro Thr Leu Asp Met Thr Leu1 5 10 15Phe Asp Trp Thr Asp Tyr Glu Asp Met Lys Pro Ala Asp Ser Trp Pro 20 25 301327PRTDanio rerio 13Gln Gly Arg Thr Gln Gly Glu Val Met Pro Thr Leu Asp Met Thr Leu1 5 10 15Phe Asp Trp Thr Asp Tyr Glu Asp Met Lys Pro 20 251422PRTDanio rerio 14Gly Glu Val Met Pro Thr Leu Asp Met Thr Leu Phe Asp Trp Thr Asp1 5 10 15Tyr Glu Asp Met Lys Pro 201521PRTDanio rerio 15Glu Val Met Pro Thr Leu Asp Met Thr Leu Phe Asp Trp Thr Asp Tyr1 5 10 15Glu Asp Met Lys Pro 201622PRTHomo sapiens 16Gly Glu Val Met Pro Thr Leu Asp Met Ala Leu Phe Asp Trp Thr Asp1 5 10 15Tyr Glu Asp Leu Lys Pro 201722PRTDanio rerio 17Gly Asp Val Ala Pro Thr Phe Asn Met Ala Leu Phe Asp Trp Thr Asp1 5 10 15Tyr Glu Asp Met Arg Pro 20181047DNAHomo sapiens 18atggctgggc ctgccatcca caccgctccc atgctgttcc tcgtcctcct gctgcccctg 60gagctgagcc tggcaggcgc ccttgcacct gggacccctg cccggaacct ccctgagaat 120cacattgacc tcccaggccc agcgctgtgg acgcctcagg ccagccacca ccgccggcgg 180ggcccgggca agaaggagtg gggcccaggc ctgcccagcc aggcccagga tggggctgtg 240gtcaccgcca ccaggcaggc ctccaggctg ccagaggctg aggggctgct gcctgagcag 300agtcctgcag gcctgctgca ggacaaggac ctgctcctgg gactggcatt gccctacccc 360gagaaggaga accgacctcc aggttgggag aggaccagga aacgcagcag ggagcacaag 420agacgcaggg acaggttgag gctgcaccaa ggccgagcct tggtccgagg tcccagctcc 480ctgatgaaga aggcagagct ctccgaagcc caggtgctgg atgcagccat ggaggaatcc 540tccaccagcc tggcgcccac catgttcttt ctcaccacct ttgaggcagc acctgccaca 600gaagagtccc tgatcctgcc cgtcacctcc ctgcggcccc agcaggcaca gcccaggtct 660gacggggagg tgatgcccac gctggacatg gccttgttcg actggaccga ttatgaagac 720ttaaaacctg atggttggcc ctctgcaaag aagaaagaga aacaccgcgg taaactctcc 780agtgatggta acgaaacatc accagccgaa ggggaaccat gcgaccatca ccaagactgc 840ctgccaggga cttgctgcga cctgcgggag catctctgca caccccacaa ccgaggcctc 900aacaacaaat gcttcgatga ctgcatgtgt gtggaagggc tgcgctgcta tgccaaattc 960caccggaacc gcagggttac acggaggaaa gggcgctgtg tggagcccga gacggccaac 1020ggcgaccagg gatccttcat caacgtc 1047191080DNADanio rerio 19atggtggctc ctggcttgtg tcaactcttc attctacttc ttataacact gtctcacact 60ttgcacagct ctgaaatctc atctgacaat ttcaaacaga gtctgacccc ctccaccact 120acctccaagg aacaccctga aacaggactt accggtggca gacaacaaaa gagacactgg 180tcaggcaaag aacgggatag tgctgggctg ttttctcaaa ggcatatgga cagactggag 240gatgatggga caagtatgga gggtctgagc cccgtaaggc tagagatggg acctggggac 300actatgaagg cagaagttca cggtgaggtc agggcttctg ctcaaatgcg tcaaggaagt 360catccagcag aaggagagtt aaatcgtaaa ggcagacgac atagtcacag gcttctggct 420gagcacagaa agcatggagg caaaaaagac aaaggtcgag gtaaagggga tctcagcgac 480cctgaaccag aattagactc cttgctgaag gacttaaatg catttgagga tggtctaaac 540acttctccac ccaattacaa cagtgtccct ctcaatgaag ttccctcccc tctctcccct 600attttggtaa ccacggcaat caaagggcat cccccaacac ttcccccagc ctccaccaaa 660ccccagaagt caagccaagg caggactcaa ggtgaagtga tgcccactct ggacatgacc 720ctctttgact ggactgatta tgaggatatg aagcctgcag acagttggcc atcaaacaaa 780agaaaagata aacgtcgcag caaaaacaag agcaatggaa acacaacaac tgaggctgga 840attgttgaac catgtgacca tcatcttgac tgcctttctg gttcctgttg tgacctcaga 900gaatttgaat gtaaacctca caaccgtggc ctaaataaca agtgttttga tgactgcatg 960tgtgaggagg gtctccgatg ttacgccaag ttccaccgca agcggagagt gacccgaaga 1020cgtggccgct gtgtggaccc tgaatcagtc aacagcaacc aaggagcttt tattaccgtc 1080201053DNADanio rerio 20atggcagttt cctgttggta ttttgcctta tttctcatct ttgacttgat gactatgaca 60ctgggcacca acacccacca caattcacca atggaagtct tcagcgaaaa cataataatc 120ccgcccaagc cagaggcatc catacatcat cacactcatc aaaggacaga cagaggacgc 180aaggagagga tgacagccag tcagctcaga gaaagacccc gcattgccat cttccacacc 240caaaatgaag ggccagacct ggagggcctc agtcccgttc gcttagagat ggaaccggca 300gacaaacgtc gggtaatgac tcctagaaag aagaccttca tgggctctga ttccttaatt 360caagaaaaaa tgaatatttc tcctggtgcc gagactccag agaaggcgat gaggcgtccc 420actgttcgaa aagtgtttgg agggcacatc actagggctc ctcatgagga agagtctttg 480gcatcaggga agaaacggag ggtttctttt gatcaaaggc tcaataaagc ctcctttggg 540agtcccacag agccagtgct tcctgctgcc actgttggca ccttcatatt gcccataact 600gcagcagtcg atgggaatcc aaacccctca agtgaaccgc aggtcagacg ttatttaggt 660ggggatgtgg cgcccacttt taacatggcc ttatttgact ggacggatta tgaagatatg 720aggcctggag ataaaaagca atattcgaaa aagcaaggtt ctgaaaaaca ggctacacaa 780agcccaagca ctggacttgt gagacttaca tcagaaaaca atgtctgtaa acatcatctg 840gattgtctgc caggttcctg ctgcaatctc agaaagcatg tgtgtgagct tcataaccgt 900ggcttcaata acaagtgcta tgacagctgc atgtgtgagg aaggacttcg gtgctatgca 960aaatcacaca gacattaccg catcacccgc aaaaagggac agtgtgttga tcctgaggac 1020ctaaatcatg cagtcagcag atggatgcag atg 105321303DNADanio rerio 21atggtggctc ctggcttgtg tcaactcttc attctacttc ttataacact gtctcacact 60ttgcacagct ctgaagggca tcccccaaca cttcccccag cctccaccaa accccagaag 120tcaagccaag gcaggactca aggtgaagtg atgcccactc tggacatgac cctctttgac 180tggactgatt atgaggatat gaagcctgca gacagttggc catcaaacaa aagaaaagat 240aaacgtcgca gcaaaaacaa gagcaatgga aacacaacaa ctgaggctgg aattgttgaa 300cca 30322101PRTDanio rerio 22Met Val Ala Pro Gly Leu Cys Gln Leu Phe Ile Leu Leu Leu Ile Thr1 5 10 15Leu Ser His Thr Leu His Ser Ser Glu Gly His Pro Pro Thr Leu Pro 20 25 30Pro Ala Ser Thr Lys Pro Gln Lys Ser Ser Gln Gly Arg Thr Gln Gly 35 40 45Glu Val Met Pro Thr Leu Asp Met Thr Leu Phe Asp Trp Thr Asp Tyr 50 55 60Glu Asp Met Lys Pro Ala Asp Ser Trp Pro Ser Asn Lys Arg Lys Asp65 70 75 80Lys Arg Arg Ser Lys Asn Lys Ser Asn Gly Asn Thr Thr Thr Glu Ala 85 90 95Gly Ile Val Glu Pro 10023276DNADanio rerio 23atggtggctc ctggcttgtg tcaactcttc attctacttc ttataacact gtctcacact 60ttgcacagct ctgaagggca tcccccaaca cttcccccag cctccaccaa accccagaag 120tcaagccaag gcaggactca aggtgaagtg atgcccactc tggacatgac cctctttgac 180tggactgatt atgaggatat gaagcctgca gacagttggc catcaaacaa aagaaaagat 240aaacgtcgca gcaaaaacaa gagcaatgga aacaca 2762492PRTDanio rerio 24Met Val Ala Pro Gly Leu Cys Gln Leu Phe Ile Leu Leu Leu Ile Thr1 5 10 15Leu Ser His Thr Leu His Ser Ser Glu Gly His Pro Pro Thr Leu Pro 20 25 30Pro Ala Ser Thr Lys Pro Gln Lys Ser Ser Gln Gly Arg Thr Gln Gly 35 40 45Glu Val Met Pro Thr Leu Asp Met Thr Leu Phe Asp Trp Thr Asp Tyr 50 55 60Glu Asp Met Lys Pro Ala Asp Ser Trp Pro Ser Asn Lys Arg Lys Asp65 70 75 80Lys Arg Arg Ser Lys Asn Lys Ser Asn Gly Asn Thr 85 9025222DNADanio rerio 25atggtggctc ctggcttgtg tcaactcttc attctacttc ttataacact gtctcacact 60ttgcacagct ctgaagggca tcccccaaca cttcccccag cctccaccaa accccagaag 120tcaagccaag gcaggactca aggtgaagtg atgcccactc

tggacatgac cctctttgac 180tggactgatt atgaggatat gaagcctgca gacagttggc ca 2222674PRTDanio rerio 26Met Val Ala Pro Gly Leu Cys Gln Leu Phe Ile Leu Leu Leu Ile Thr1 5 10 15Leu Ser His Thr Leu His Ser Ser Glu Gly His Pro Pro Thr Leu Pro 20 25 30Pro Ala Ser Thr Lys Pro Gln Lys Ser Ser Gln Gly Arg Thr Gln Gly 35 40 45Glu Val Met Pro Thr Leu Asp Met Thr Leu Phe Asp Trp Thr Asp Tyr 50 55 60Glu Asp Met Lys Pro Ala Asp Ser Trp Pro65 7027207DNADanio rerio 27atggtggctc ctggcttgtg tcaactcttc attctacttc ttataacact gtctcacact 60ttgcacagct ctgaagggca tcccccaaca cttcccccag cctccaccaa accccagaag 120tcaagccaag gcaggactca aggtgaagtg atgcccactc tggacatgac cctctttgac 180tggactgatt atgaggatat gaagcct 2072869PRTDanio rerio 28Met Val Ala Pro Gly Leu Cys Gln Leu Phe Ile Leu Leu Leu Ile Thr1 5 10 15Leu Ser His Thr Leu His Ser Ser Glu Gly His Pro Pro Thr Leu Pro 20 25 30Pro Ala Ser Thr Lys Pro Gln Lys Ser Ser Gln Gly Arg Thr Gln Gly 35 40 45Glu Val Met Pro Thr Leu Asp Met Thr Leu Phe Asp Trp Thr Asp Tyr 50 55 60Glu Asp Met Lys Pro6529225DNADanio rerio 29atggtggctc ctggcttgtg tcaactcttc attctacttc ttataacact gtctcacact 60ttgcacagct ctgaacaagg caggactcaa ggtgaagtga tgcccactct ggacatgacc 120ctctttgact ggactgatta tgaggatatg aagcctgcag acagttggcc atcaaacaaa 180agaaaagata aacgtcgcag caaaaacaag agcaatggaa acaca 2253075PRTDanio rerio 30Met Val Ala Pro Gly Leu Cys Gln Leu Phe Ile Leu Leu Leu Ile Thr1 5 10 15Leu Ser His Thr Leu His Ser Ser Glu Gln Gly Arg Thr Gln Gly Glu 20 25 30Val Met Pro Thr Leu Asp Met Thr Leu Phe Asp Trp Thr Asp Tyr Glu 35 40 45Asp Met Lys Pro Ala Asp Ser Trp Pro Ser Asn Lys Arg Lys Asp Lys 50 55 60Arg Arg Ser Lys Asn Lys Ser Asn Gly Asn Thr65 70 7531171DNADanio rerio 31atggtggctc ctggcttgtg tcaactcttc attctacttc ttataacact gtctcacact 60ttgcacagct ctgaacaagg caggactcaa ggtgaagtga tgcccactct ggacatgacc 120ctctttgact ggactgatta tgaggatatg aagcctgcag acagttggcc a 1713257PRTDanio rerio 32Met Val Ala Pro Gly Leu Cys Gln Leu Phe Ile Leu Leu Leu Ile Thr1 5 10 15Leu Ser His Thr Leu His Ser Ser Glu Gln Gly Arg Thr Gln Gly Glu 20 25 30Val Met Pro Thr Leu Asp Met Thr Leu Phe Asp Trp Thr Asp Tyr Glu 35 40 45Asp Met Lys Pro Ala Asp Ser Trp Pro 50 5533156DNADanio rerio 33atggtggctc ctggcttgtg tcaactcttc attctacttc ttataacact gtctcacact 60ttgcacagct ctgaacaagg caggactcaa ggtgaagtga tgcccactct ggacatgacc 120ctctttgact ggactgatta tgaggatatg aagcct 1563452PRTDanio rerio 34Met Val Ala Pro Gly Leu Cys Gln Leu Phe Ile Leu Leu Leu Ile Thr1 5 10 15Leu Ser His Thr Leu His Ser Ser Glu Gln Gly Arg Thr Gln Gly Glu 20 25 30Val Met Pro Thr Leu Asp Met Thr Leu Phe Asp Trp Thr Asp Tyr Glu 35 40 45Asp Met Lys Pro 5035141DNADanio rerio 35atggtggctc ctggcttgtg tcaactcttc attctacttc ttataacact gtctcacact 60ttgcacagct ctgaaggtga agtgatgccc actctggaca tgaccctctt tgactggact 120gattatgagg atatgaagcc t 1413647PRTDanio rerio 36Met Val Ala Pro Gly Leu Cys Gln Leu Phe Ile Leu Leu Leu Ile Thr1 5 10 15Leu Ser His Thr Leu His Ser Ser Glu Gly Glu Val Met Pro Thr Leu 20 25 30Asp Met Thr Leu Phe Asp Trp Thr Asp Tyr Glu Asp Met Lys Pro 35 40 45371809DNADanio rerio 37atggtgagag tctctgatgc tttggtcact ttggtgactc tctgctgtgt gctcaaaggg 60actgtcggcg gatatggaat gagcatgttc gccgctcaga cctccccgcc ggatccgtgt 120tacgacgaga acggacaccc cagaagatgc atccccgact tcgtaaacgc ggcgttcggg 180aaagaagtac gcgcgtccag cacctgcggc aaaacgccga gtcgttactg cgtggtgacc 240gagaaagggg acgaaagaca cagaaactgc cacacgtgcg acgcgtcaga cccaaagaag 300aatcacccac cagcttacct gaccgacctg aacaatcctc acaatctcac ctgctggcag 360tcggacaatt acctccagta tcctcaaaac gtcactttaa ctttatcctt gggcaagaaa 420tttgaggtga cctacgtgag tttgcagttc tgctcacctc gaccggagtc tatggcgatc 480tttaaatcga tggactacgg aaagtcctgg gtgcctttcc agtactactc gacccagtgt 540agaaagatgt acaacaagcc cagcaaagcc acgattacta agcagaacga gcaagaggcc 600atctgcacag attctcacac cgacatgcat cctctctccg gcgggctgat cgcgttcagc 660accctggacg ggcgaccctc cgcgcacgac tttgacaatt cacccgtact tcaggactgg 720gtgaccgcca ctgacattaa ggtgactttc agccgcctgc acactttcgg agacgaaaac 780gaggatgact cggagctggc cagagattcc tatttttacg cagtttccga cctgcaggtt 840ggaggcagat gtaagtgtaa tggacacgca tcacggtgcg tcaaagaccg ggatggaaac 900ctagtgtgcg agtgcaagca caacacagcc ggaccagagt gtgacagatg caaacctttt 960cactatgacc gaccctggca gcgcgcaacc gccagagaag ccaacgaatg tgtcgcctgc 1020aattgtaacc ttcatgcgag gcgctgtcgt ttcaacatgg agctttacaa actctctgga 1080aggaaaagtg gaggagtctg tctgaactgc cgccacaata cagctggtcg ccactgccac 1140tactgcaaag agggctacta tagagacatg tccaagccca tctcccacag aaaggcctgc 1200aaagcctgtg attgccatcc tgtgggggcc gcgggcaaaa cctgtaacca aaccacaggc 1260caatgcccct gtaaagacgg tgtgacgggt atcacatgca accgttgtgc taacggctac 1320cagcagagcc gatcacccat tgccccctgc ataaaaattc ccatcgctcc gccaaccacc 1380actgcaagca gcacagaaga gccatcagac tgtgaatcct actgcaaggc atccaaaggc 1440aagctgaaga tcaatatgaa gaagtactgc aagaaagatt atgccgttca agtccacatc 1500ctgaaagcag ataaagcagg agagtggtgg aagttcaccg tcaacatcat ctctgtttac 1560aaacagggtg aaagccgaat tcgcagagga gaccagttcc tctgggtcag ggcaaaggat 1620gtggcctgca agtgtccgaa gatcaagtcc ggcaagaaat accttctgct ggggaacgac 1680gaggattcgc caggacaaag cggaatggtg gcggacaagg gcagtctggt cattcagtgg 1740agagacactt gggctcggag actccggaag tttcagcaaa gggagaagaa aggaaaatgc 1800aagaaagca 180938603PRTDanio rerio 38Met Val Arg Val Ser Asp Ala Leu Val Thr Leu Val Thr Leu Cys Cys1 5 10 15Val Leu Lys Gly Thr Val Gly Gly Tyr Gly Met Ser Met Phe Ala Ala 20 25 30Gln Thr Ser Pro Pro Asp Pro Cys Tyr Asp Glu Asn Gly His Pro Arg 35 40 45Arg Cys Ile Pro Asp Phe Val Asn Ala Ala Phe Gly Lys Glu Val Arg 50 55 60Ala Ser Ser Thr Cys Gly Lys Thr Pro Ser Arg Tyr Cys Val Val Thr65 70 75 80Glu Lys Gly Asp Glu Arg His Arg Asn Cys His Thr Cys Asp Ala Ser 85 90 95Asp Pro Lys Lys Asn His Pro Pro Ala Tyr Leu Thr Asp Leu Asn Asn 100 105 110Pro His Asn Leu Thr Cys Trp Gln Ser Asp Asn Tyr Leu Gln Tyr Pro 115 120 125Gln Asn Val Thr Leu Thr Leu Ser Leu Gly Lys Lys Phe Glu Val Thr 130 135 140Tyr Val Ser Leu Gln Phe Cys Ser Pro Arg Pro Glu Ser Met Ala Ile145 150 155 160Phe Lys Ser Met Asp Tyr Gly Lys Ser Trp Val Pro Phe Gln Tyr Tyr 165 170 175Ser Thr Gln Cys Arg Lys Met Tyr Asn Lys Pro Ser Lys Ala Thr Ile 180 185 190Thr Lys Gln Asn Glu Gln Glu Ala Ile Cys Thr Asp Ser His Thr Asp 195 200 205Met His Pro Leu Ser Gly Gly Leu Ile Ala Phe Ser Thr Leu Asp Gly 210 215 220Arg Pro Ser Ala His Asp Phe Asp Asn Ser Pro Val Leu Gln Asp Trp225 230 235 240Val Thr Ala Thr Asp Ile Lys Val Thr Phe Ser Arg Leu His Thr Phe 245 250 255Gly Asp Glu Asn Glu Asp Asp Ser Glu Leu Ala Arg Asp Ser Tyr Phe 260 265 270Tyr Ala Val Ser Asp Leu Gln Val Gly Gly Arg Cys Lys Cys Asn Gly 275 280 285His Ala Ser Arg Cys Val Lys Asp Arg Asp Gly Asn Leu Val Cys Glu 290 295 300Cys Lys His Asn Thr Ala Gly Pro Glu Cys Asp Arg Cys Lys Pro Phe305 310 315 320His Tyr Asp Arg Pro Trp Gln Arg Ala Thr Ala Arg Glu Ala Asn Glu 325 330 335Cys Val Ala Cys Asn Cys Asn Leu His Ala Arg Arg Cys Arg Phe Asn 340 345 350Met Glu Leu Tyr Lys Leu Ser Gly Arg Lys Ser Gly Gly Val Cys Leu 355 360 365Asn Cys Arg His Asn Thr Ala Gly Arg His Cys His Tyr Cys Lys Glu 370 375 380Gly Tyr Tyr Arg Asp Met Ser Lys Pro Ile Ser His Arg Lys Ala Cys385 390 395 400Lys Ala Cys Asp Cys His Pro Val Gly Ala Ala Gly Lys Thr Cys Asn 405 410 415Gln Thr Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr Gly Ile Thr 420 425 430Cys Asn Arg Cys Ala Asn Gly Tyr Gln Gln Ser Arg Ser Pro Ile Ala 435 440 445Pro Cys Ile Lys Ile Pro Ile Ala Pro Pro Thr Thr Thr Ala Ser Ser 450 455 460Thr Glu Glu Pro Ser Asp Cys Glu Ser Tyr Cys Lys Ala Ser Lys Gly465 470 475 480Lys Leu Lys Ile Asn Met Lys Lys Tyr Cys Lys Lys Asp Tyr Ala Val 485 490 495Gln Val His Ile Leu Lys Ala Asp Lys Ala Gly Glu Trp Trp Lys Phe 500 505 510Thr Val Asn Ile Ile Ser Val Tyr Lys Gln Gly Glu Ser Arg Ile Arg 515 520 525Arg Gly Asp Gln Phe Leu Trp Val Arg Ala Lys Asp Val Ala Cys Lys 530 535 540Cys Pro Lys Ile Lys Ser Gly Lys Lys Tyr Leu Leu Leu Gly Asn Asp545 550 555 560Glu Asp Ser Pro Gly Gln Ser Gly Met Val Ala Asp Lys Gly Ser Leu 565 570 575Val Ile Gln Trp Arg Asp Thr Trp Ala Arg Arg Leu Arg Lys Phe Gln 580 585 590Gln Arg Glu Lys Lys Gly Lys Cys Lys Lys Ala 595 600391374DNADanio rerio 39atggtaagga ttttggtaac gtgcgtctcc atggtgtcca tcacctcgat ggtgtccggt 60gctcgcggtg gatacgggat gagcatgttc gcggctcagt cctctcctcc ggacccgtgc 120tacgacgaga acgggaaccc cagacgctgc atccccgact tcgtgaactc cgctttcggg 180aaggacgtgc gcgtgtccag cacctgcggc tctcctccgt cgcgctactg cgtggtgacc 240gagaaaggcg aggagagatc gagggactgc aacatctgcg acgccacaga ccccaaaaag 300acccatccgc ccgcatacct gacagacctc aacaaccctc ataacctcac ctgctggcag 360tcggagaact acgtgcaata cccgcagaac gtgactctga ctctgtcttt ggggaagaag 420tttgaagtga cttatgtgag cctccagttc tgctctcctc gccccgaatc catggccatc 480ttcaaatcca tggactatgg gaaaacctgg gtgccttttc agttctactc aacccagtgc 540aagaaaatgt acaacaagcc cagcaaagct gccatcacca agcagaacga gcaggaggcg 600atctgcacgg actctcacac ggacatgcag ccgttaaccg gcggcctcat cgcgttcagc 660acgctggatg gcagaccgtc cgcgcacgac ttcgacaact cgcccgtcct gcaggactgg 720gtcacggcca ccgacatcaa agtgaccttc aaccggctgc acacgttcgg ggatgagaac 780gaggacgatt cggagctcgc cagggactcg tatttttacg cggtgtctga cctgcaggtc 840ggtggacggt gtaagtgtaa cgggcacgcg tcgaagtgcg tgaaggaccg ggaaggaaac 900ctagtgtgcg aatgcaagca caacactgcg ggaccagagt gtgacaggtg taaacccttc 960cactacgacc ggccctggca gcgcgcgact gccagagagg cgaacgagtg tgtcgcttgt 1020cactgtaacc tacatgcccg ccgctgccgc ttcaacatgg agctgtataa gttgtcaggc 1080cgcaggagtg gaggagtctg cctgaactgc agacacaaca ccgccggacg ccactgccac 1140tactgtaaag agggctacta cagagacatg agcaaggcta tatcacaccg acgtgcatgc 1200aaagcctgcg attgtcatcc tgttggtgca gctggtaaga cctgtaacca gacaactgga 1260cagtgtccat gtaaagatgg tgtgactggc atcacctgta atcgctgtgc taaaggatac 1320cagcagagca gatcacccat cgccccctgt atcaaaattc cagttgctgc tccc 137440458PRTDanio rerio 40Met Val Arg Ile Leu Val Thr Cys Val Ser Met Val Ser Ile Thr Ser1 5 10 15Met Val Ser Gly Ala Arg Gly Gly Tyr Gly Met Ser Met Phe Ala Ala 20 25 30Gln Ser Ser Pro Pro Asp Pro Cys Tyr Asp Glu Asn Gly Asn Pro Arg 35 40 45Arg Cys Ile Pro Asp Phe Val Asn Ser Ala Phe Gly Lys Asp Val Arg 50 55 60Val Ser Ser Thr Cys Gly Ser Pro Pro Ser Arg Tyr Cys Val Val Thr65 70 75 80Glu Lys Gly Glu Glu Arg Ser Arg Asp Cys Asn Ile Cys Asp Ala Thr 85 90 95Asp Pro Lys Lys Thr His Pro Pro Ala Tyr Leu Thr Asp Leu Asn Asn 100 105 110Pro His Asn Leu Thr Cys Trp Gln Ser Glu Asn Tyr Val Gln Tyr Pro 115 120 125Gln Asn Val Thr Leu Thr Leu Ser Leu Gly Lys Lys Phe Glu Val Thr 130 135 140Tyr Val Ser Leu Gln Phe Cys Ser Pro Arg Pro Glu Ser Met Ala Ile145 150 155 160Phe Lys Ser Met Asp Tyr Gly Lys Thr Trp Val Pro Phe Gln Phe Tyr 165 170 175Ser Thr Gln Cys Lys Lys Met Tyr Asn Lys Pro Ser Lys Ala Ala Ile 180 185 190Thr Lys Gln Asn Glu Gln Glu Ala Ile Cys Thr Asp Ser His Thr Asp 195 200 205Met Gln Pro Leu Thr Gly Gly Leu Ile Ala Phe Ser Thr Leu Asp Gly 210 215 220Arg Pro Ser Ala His Asp Phe Asp Asn Ser Pro Val Leu Gln Asp Trp225 230 235 240Val Thr Ala Thr Asp Ile Lys Val Thr Phe Asn Arg Leu His Thr Phe 245 250 255Gly Asp Glu Asn Glu Asp Asp Ser Glu Leu Ala Arg Asp Ser Tyr Phe 260 265 270Tyr Ala Val Ser Asp Leu Gln Val Gly Gly Arg Cys Lys Cys Asn Gly 275 280 285His Ala Ser Lys Cys Val Lys Asp Arg Glu Gly Asn Leu Val Cys Glu 290 295 300Cys Lys His Asn Thr Ala Gly Pro Glu Cys Asp Arg Cys Lys Pro Phe305 310 315 320His Tyr Asp Arg Pro Trp Gln Arg Ala Thr Ala Arg Glu Ala Asn Glu 325 330 335Cys Val Ala Cys His Cys Asn Leu His Ala Arg Arg Cys Arg Phe Asn 340 345 350Met Glu Leu Tyr Lys Leu Ser Gly Arg Arg Ser Gly Gly Val Cys Leu 355 360 365Asn Cys Arg His Asn Thr Ala Gly Arg His Cys His Tyr Cys Lys Glu 370 375 380Gly Tyr Tyr Arg Asp Met Ser Lys Ala Ile Ser His Arg Arg Ala Cys385 390 395 400Lys Ala Cys Asp Cys His Pro Val Gly Ala Ala Gly Lys Thr Cys Asn 405 410 415Gln Thr Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr Gly Ile Thr 420 425 430Cys Asn Arg Cys Ala Lys Gly Tyr Gln Gln Ser Arg Ser Pro Ile Ala 435 440 445Pro Cys Ile Lys Ile Pro Val Ala Ala Pro 450 455411374DNADanio rerio 41atggtgagag tctctgatgc tttggtcact ttggtgactc tctgctgtgt gctcaaaggg 60actgtcggcg gatatggaat gagcatgttc gccgctcaga cctccccgcc ggatccgtgt 120tacgacgaga acggacaccc cagaagatgc atccccgact tcgtaaacgc ggcgttcggg 180aaagaagtac gcgcgtccag cacctgcggc aaaacgccga gtcgttactg cgtggtgacc 240gagaaagggg acgaaagaca cagaaactgc cacacgtgcg acgcgtcaga cccaaagaag 300aatcacccac cagcttacct gaccgacctg aacaatcctc acaatctcac ctgctggcag 360tcggacaatt acctccagta tcctcaaaac gtcactttaa ctttatcctt gggcaagaaa 420tttgaggtga cctacgtgag tttgcagttc tgctcacctc gaccggagtc tatggcgatc 480tttaaatcga tggactacgg aaagtcctgg gtgcctttcc agtactactc gacccagtgt 540agaaagatgt acaacaagcc cagcaaagcc acgattacta agcagaacga gcaagaggcc 600atctgcacag attctcacac cgacatgcat cctctctccg gcgggctgat cgcgttcagc 660accctggacg ggcgaccctc cgcgcacgac tttgacaatt cacccgtact tcaggactgg 720gtgaccgcca ctgacattaa ggtgactttc agccgcctgc acactttcgg agacgaaaac 780gaggatgact cggagctggc cagagattcc tatttttacg cagtttccga cctgcaggtt 840ggaggcagat gtaagtgtaa tggacacgca tcacggtgcg tcaaagaccg ggatggaaac 900ctagtgtgcg agtgcaagca caacacagcc ggaccagagt gtgacagatg caaacctttt 960cactatgacc gaccctggca gcgcgcaacc gccagagaag ccaacgaatg tgtcgcctgc 1020aattgtaacc ttcatgcgag gcgctgtcgt ttcaacatgg agctttacaa actctctgga 1080aggaaaagtg gaggagtctg tctgaactgc cgccacaata cagctggtcg ccactgccac 1140tactgcaaag agggctacta tagagacatg tccaagccca tctcccacag aaaggcctgc 1200aaagcctgtg attgccatcc tgtgggggcc gcgggcaaaa cctgtaacca aaccacaggc 1260caatgcccct gtaaagacgg tgtgacgggt atcacatgca accgttgtgc taacggctac 1320cagcagagcc gatcacccat tgccccctgc ataaaaattc ccatcgctcc gcca 137442458PRTDanio rerio 42Met Val Arg Val Ser Asp Ala Leu Val Thr Leu Val Thr Leu Cys Cys1 5 10 15Val Leu Lys Gly Thr Val Gly Gly Tyr Gly Met Ser Met Phe Ala Ala 20 25 30Gln Thr Ser Pro Pro Asp Pro Cys Tyr Asp Glu Asn Gly His Pro Arg 35 40 45Arg Cys Ile Pro Asp Phe Val Asn Ala Ala Phe Gly Lys Glu Val

Arg 50 55 60Ala Ser Ser Thr Cys Gly Lys Thr Pro Ser Arg Tyr Cys Val Val Thr65 70 75 80Glu Lys Gly Asp Glu Arg His Arg Asn Cys His Thr Cys Asp Ala Ser 85 90 95Asp Pro Lys Lys Asn His Pro Pro Ala Tyr Leu Thr Asp Leu Asn Asn 100 105 110Pro His Asn Leu Thr Cys Trp Gln Ser Asp Asn Tyr Leu Gln Tyr Pro 115 120 125Gln Asn Val Thr Leu Thr Leu Ser Leu Gly Lys Lys Phe Glu Val Thr 130 135 140Tyr Val Ser Leu Gln Phe Cys Ser Pro Arg Pro Glu Ser Met Ala Ile145 150 155 160Phe Lys Ser Met Asp Tyr Gly Lys Ser Trp Val Pro Phe Gln Tyr Tyr 165 170 175Ser Thr Gln Cys Arg Lys Met Tyr Asn Lys Pro Ser Lys Ala Thr Ile 180 185 190Thr Lys Gln Asn Glu Gln Glu Ala Ile Cys Thr Asp Ser His Thr Asp 195 200 205Met His Pro Leu Ser Gly Gly Leu Ile Ala Phe Ser Thr Leu Asp Gly 210 215 220Arg Pro Ser Ala His Asp Phe Asp Asn Ser Pro Val Leu Gln Asp Trp225 230 235 240Val Thr Ala Thr Asp Ile Lys Val Thr Phe Ser Arg Leu His Thr Phe 245 250 255Gly Asp Glu Asn Glu Asp Asp Ser Glu Leu Ala Arg Asp Ser Tyr Phe 260 265 270Tyr Ala Val Ser Asp Leu Gln Val Gly Gly Arg Cys Lys Cys Asn Gly 275 280 285His Ala Ser Arg Cys Val Lys Asp Arg Asp Gly Asn Leu Val Cys Glu 290 295 300Cys Lys His Asn Thr Ala Gly Pro Glu Cys Asp Arg Cys Lys Pro Phe305 310 315 320His Tyr Asp Arg Pro Trp Gln Arg Ala Thr Ala Arg Glu Ala Asn Glu 325 330 335Cys Val Ala Cys Asn Cys Asn Leu His Ala Arg Arg Cys Arg Phe Asn 340 345 350Met Glu Leu Tyr Lys Leu Ser Gly Arg Lys Ser Gly Gly Val Cys Leu 355 360 365Asn Cys Arg His Asn Thr Ala Gly Arg His Cys His Tyr Cys Lys Glu 370 375 380Gly Tyr Tyr Arg Asp Met Ser Lys Pro Ile Ser His Arg Lys Ala Cys385 390 395 400Lys Ala Cys Asp Cys His Pro Val Gly Ala Ala Gly Lys Thr Cys Asn 405 410 415Gln Thr Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr Gly Ile Thr 420 425 430Cys Asn Arg Cys Ala Asn Gly Tyr Gln Gln Ser Arg Ser Pro Ile Ala 435 440 445Pro Cys Ile Lys Ile Pro Ile Ala Pro Pro 450 45543684DNADanio rerio 43atggtgagag tctctgatgc tttggtcact ttggtgactc tctgctgtgt gctcaaaggg 60actgtcggcg gatatggagg agacgaaaac gaggatgact cggagctggc cagagattcc 120tatttttacg cagtttccga cctgcaggtt ggaggcagat gtaagtgtaa tggacacgca 180tcacggtgcg tcaaagaccg ggatggaaac ctagtgtgcg agtgcaagca caacacagcc 240ggaccagagt gtgacagatg caaacctttt cactatgacc gaccctggca gcgcgcaacc 300gccagagaag ccaacgaatg tgtcgcctgc aattgtaacc ttcatgcgag gcgctgtcgt 360ttcaacatgg agctttacaa actctctgga aggaaaagtg gaggagtctg tctgaactgc 420cgccacaata cagctggtcg ccactgccac tactgcaaag agggctacta tagagacatg 480tccaagccca tctcccacag aaaggcctgc aaagcctgtg attgccatcc tgtgggggcc 540gcgggcaaaa cctgtaacca aaccacaggc caatgcccct gtaaagacgg tgtgacgggt 600atcacatgca accgttgtgc taacggctac cagcagagcc gatcacccat tgccccctgc 660ataaaaattc ccatcgctcc gcca 68444228PRTDanio rerio 44Met Val Arg Val Ser Asp Ala Leu Val Thr Leu Val Thr Leu Cys Cys1 5 10 15Val Leu Lys Gly Thr Val Gly Gly Tyr Gly Gly Asp Glu Asn Glu Asp 20 25 30Asp Ser Glu Leu Ala Arg Asp Ser Tyr Phe Tyr Ala Val Ser Asp Leu 35 40 45Gln Val Gly Gly Arg Cys Lys Cys Asn Gly His Ala Ser Arg Cys Val 50 55 60Lys Asp Arg Asp Gly Asn Leu Val Cys Glu Cys Lys His Asn Thr Ala65 70 75 80Gly Pro Glu Cys Asp Arg Cys Lys Pro Phe His Tyr Asp Arg Pro Trp 85 90 95Gln Arg Ala Thr Ala Arg Glu Ala Asn Glu Cys Val Ala Cys Asn Cys 100 105 110Asn Leu His Ala Arg Arg Cys Arg Phe Asn Met Glu Leu Tyr Lys Leu 115 120 125Ser Gly Arg Lys Ser Gly Gly Val Cys Leu Asn Cys Arg His Asn Thr 130 135 140Ala Gly Arg His Cys His Tyr Cys Lys Glu Gly Tyr Tyr Arg Asp Met145 150 155 160Ser Lys Pro Ile Ser His Arg Lys Ala Cys Lys Ala Cys Asp Cys His 165 170 175Pro Val Gly Ala Ala Gly Lys Thr Cys Asn Gln Thr Thr Gly Gln Cys 180 185 190Pro Cys Lys Asp Gly Val Thr Gly Ile Thr Cys Asn Arg Cys Ala Asn 195 200 205Gly Tyr Gln Gln Ser Arg Ser Pro Ile Ala Pro Cys Ile Lys Ile Pro 210 215 220Ile Ala Pro Pro22545202PRTDanio rerio 45Gly Asp Glu Asn Glu Asp Asp Ser Glu Leu Ala Arg Asp Ser Tyr Phe1 5 10 15Tyr Ala Val Ser Asp Leu Gln Val Gly Gly Arg Cys Lys Cys Asn Gly 20 25 30His Ala Ser Arg Cys Val Lys Asp Arg Asp Gly Asn Leu Val Cys Glu 35 40 45Cys Lys His Asn Thr Ala Gly Pro Glu Cys Asp Arg Cys Lys Pro Phe 50 55 60His Tyr Asp Arg Pro Trp Gln Arg Ala Thr Ala Arg Glu Ala Asn Glu65 70 75 80Cys Val Ala Cys Asn Cys Asn Leu His Ala Arg Arg Cys Arg Phe Asn 85 90 95Met Glu Leu Tyr Lys Leu Ser Gly Arg Lys Ser Gly Gly Val Cys Leu 100 105 110Asn Cys Arg His Asn Thr Ala Gly Arg His Cys His Tyr Cys Lys Glu 115 120 125Gly Tyr Tyr Arg Asp Met Ser Lys Pro Ile Ser His Arg Lys Ala Cys 130 135 140Lys Ala Cys Asp Cys His Pro Val Gly Ala Ala Gly Lys Thr Cys Asn145 150 155 160Gln Thr Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr Gly Ile Thr 165 170 175Cys Asn Arg Cys Ala Asn Gly Tyr Gln Gln Ser Arg Ser Pro Ile Ala 180 185 190Pro Cys Ile Lys Ile Pro Ile Ala Pro Pro 195 20046468DNADanio rerio 46atggtgagag tctctgatgc tttggtcact ttggtgactc tctgctgtgt gctcaaaggg 60actgtcggcg gatatggacc tgcatcgcct gcctcacccg cttctgtcgc ctgcaattgt 120aaccttcatg cgaggcgctg tcgtttcaac atggagcttt acaaactctc tggaaggaaa 180agtggaggag tctgtctgaa ctgccgccac aatacagctg gtcgccactg ccactactgc 240aaagagggct actatagaga catgtccaag cccatctccc acagaaaggc ctgcaaagcc 300tgtgattgcc atcctgtggg ggccgcgggc aaaacctgta accaaaccac aggccaatgc 360ccctgtaaag acggtgtgac gggtatcaca tgcaaccgtt gtgctaacgg ctaccagcag 420agccgatcac ccattgcccc ctgcataaaa attcccatcg ctccgcca 46847156PRTDanio rerio 47Met Val Arg Val Ser Asp Ala Leu Val Thr Leu Val Thr Leu Cys Cys1 5 10 15Val Leu Lys Gly Thr Val Gly Gly Tyr Gly Pro Ala Ser Pro Ala Ser 20 25 30Pro Ala Ser Val Ala Cys Asn Cys Asn Leu His Ala Arg Arg Cys Arg 35 40 45Phe Asn Met Glu Leu Tyr Lys Leu Ser Gly Arg Lys Ser Gly Gly Val 50 55 60Cys Leu Asn Cys Arg His Asn Thr Ala Gly Arg His Cys His Tyr Cys65 70 75 80Lys Glu Gly Tyr Tyr Arg Asp Met Ser Lys Pro Ile Ser His Arg Lys 85 90 95Ala Cys Lys Ala Cys Asp Cys His Pro Val Gly Ala Ala Gly Lys Thr 100 105 110Cys Asn Gln Thr Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr Gly 115 120 125Ile Thr Cys Asn Arg Cys Ala Asn Gly Tyr Gln Gln Ser Arg Ser Pro 130 135 140Ile Ala Pro Cys Ile Lys Ile Pro Ile Ala Pro Pro145 150 15548121PRTDanio rerio 48Val Ala Cys Asn Cys Asn Leu His Ala Arg Arg Cys Arg Phe Asn Met1 5 10 15Glu Leu Tyr Lys Leu Ser Gly Arg Lys Ser Gly Gly Val Cys Leu Asn 20 25 30Cys Arg His Asn Thr Ala Gly Arg His Cys His Tyr Cys Lys Glu Gly 35 40 45Tyr Tyr Arg Asp Met Ser Lys Pro Ile Ser His Arg Lys Ala Cys Lys 50 55 60Ala Cys Asp Cys His Pro Val Gly Ala Ala Gly Lys Thr Cys Asn Gln65 70 75 80Thr Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr Gly Ile Thr Cys 85 90 95Asn Arg Cys Ala Asn Gly Tyr Gln Gln Ser Arg Ser Pro Ile Ala Pro 100 105 110Cys Ile Lys Ile Pro Ile Ala Pro Pro 115 12049279DNADanio rerio 49atggtgagag tctctgatgc tttggtcact ttggtgactc tctgctgtgt gctcaaaggg 60actgtcggcg gatatggacc tgcatcgcct gcctcacccg cttctaaagc ctgtgattgc 120catcctgtgg gggccgcggg caaaacctgt aaccaaacca caggccaatg cccctgtaaa 180gacggtgtga cgggtatcac atgcaaccgt tgtgctaacg gctaccagca gagccgatca 240cccattgccc cctgcataaa aattcccatc gctccgcca 2795093PRTDanio rerio 50Met Val Arg Val Ser Asp Ala Leu Val Thr Leu Val Thr Leu Cys Cys1 5 10 15Val Leu Lys Gly Thr Val Gly Gly Tyr Gly Pro Ala Ser Pro Ala Ser 20 25 30Pro Ala Ser Lys Ala Cys Asp Cys His Pro Val Gly Ala Ala Gly Lys 35 40 45Thr Cys Asn Gln Thr Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr 50 55 60Gly Ile Thr Cys Asn Arg Cys Ala Asn Gly Tyr Gln Gln Ser Arg Ser65 70 75 80Pro Ile Ala Pro Cys Ile Lys Ile Pro Ile Ala Pro Pro 85 905158PRTDanio rerio 51Lys Ala Cys Asp Cys His Pro Val Gly Ala Ala Gly Lys Thr Cys Asn1 5 10 15Gln Thr Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr Gly Ile Thr 20 25 30Cys Asn Arg Cys Ala Asn Gly Tyr Gln Gln Ser Arg Ser Pro Ile Ala 35 40 45Pro Cys Ile Lys Ile Pro Ile Ala Pro Pro 50 5552343PRTMus musculus 52Met Ala Gly Cys Pro Val Leu Arg Val Pro Thr Leu Phe Leu Ile Leu1 5 10 15Leu Leu Phe Pro Glu Leu His Thr Ala Gly Thr Leu Ala Ser Gly Ser 20 25 30Ser Ala Arg Asn Leu Pro Glu Thr His Ser His Leu Pro Ser Ser Ala 35 40 45Leu Trp Val Ser Gln Ala Ser His His Gly Arg Arg Gly Leu Gly Lys 50 55 60Lys Asp Arg Gly Pro Gly Arg Pro Ser Arg Ala Gln Glu Gly Ala Val65 70 75 80Val Thr Ala Thr Lys Gln Ala Ser Gln Met Thr Leu Gly Gln Pro Pro 85 90 95Ala Gly Leu Leu Gln Asn Lys Glu Leu Leu Leu Gly Leu Thr Leu Pro 100 105 110Tyr Pro Glu Lys Glu Ala Arg Ser Pro Ala Trp Glu Arg Val Lys Lys 115 120 125Arg Gly Arg Glu His Lys Arg Arg Arg Asp Arg Leu Arg Leu His Arg 130 135 140Gly Arg Ala Ala Ile Arg Gly Pro Ser Ser Leu Met Lys Lys Val Glu145 150 155 160Pro Ser Glu Asp Arg Met Leu Glu Gly Thr Met Glu Glu Ser Ser Thr 165 170 175Ser Leu Ala Pro Thr Met Phe Phe Leu Thr Met Thr Asp Gly Ala Thr 180 185 190Pro Thr Thr Glu Glu Ser Arg Ile Leu Pro Val Thr Ser Leu Arg Pro 195 200 205Gln Thr Gln Pro Arg Ser Asp Gly Glu Val Met Pro Thr Leu Asp Met 210 215 220Ala Leu Phe Asp Trp Thr Asp Tyr Glu Asp Leu Lys Pro Glu Val Trp225 230 235 240Pro Ser Ala Lys Lys Lys Glu Lys His Trp Ser His Phe Thr Ser Asp 245 250 255Gly Asn Glu Thr Ser Pro Ala Glu Gly Asp Pro Cys Asp His His Gln 260 265 270Asp Cys Leu Pro Gly Thr Cys Cys Asp Leu Arg Glu His Leu Cys Thr 275 280 285Pro His Asn Arg Gly Leu Asn Asn Lys Cys Phe Asp Asp Cys Met Cys 290 295 300Met Glu Gly Leu Arg Cys Tyr Ala Lys Phe His Arg Asn Arg Arg Val305 310 315 320Thr Arg Arg Lys Gly Arg Cys Val Glu Pro Glu Thr Ala Asn Gly Asp 325 330 335Gln Gly Ser Phe Ile Asn Ile 34053349PRTGallus gallus 53Met Ala Ala Ser Ser Thr Phe Phe Ser Pro Ser Leu Phe Leu Cys Val1 5 10 15Leu Val Leu Ile Asp Ile Thr Leu Ala Val Ser Leu Asp Thr Asp Met 20 25 30Lys Leu Lys Ser Glu Asn Asn Asn His Leu Gln Asn Gln Glu Thr Trp 35 40 45Pro Gln Gln Pro Arg Ser Gly His His His Lys His Gly Leu Ala Lys 50 55 60Lys Gly Arg Val Leu Ala Leu Pro Val Arg Gly Gln Pro Ala Gly Glu65 70 75 80Glu Ala Leu Arg Val Gly Ser Gly Ala Pro Ala Met Glu Glu Leu Val 85 90 95Pro Leu Gly Gln Pro Ala Ala Leu Lys Gln Asp Lys Asp Lys Asp Val 100 105 110Phe Leu Gly Phe Glu Leu Pro His Ala Glu Arg Glu Asn Gln Ser Pro 115 120 125Gly Ser Glu Arg Gly Lys Lys Gln Asn Arg Glu Gln Arg Arg His Ser 130 135 140Arg Arg Asp Arg Leu Lys His His Arg Gly Lys Thr Ala Val Gly Pro145 150 155 160Ser Ser Leu Tyr Lys Lys Pro Glu Ser Phe Glu Gln Gln Phe Gln Asn 165 170 175Leu Gln Ala Glu Glu Ala Thr Ser Pro Thr Pro Thr Val Leu Pro Phe 180 185 190Thr Ala Leu Asp Leu Val Val Ser Thr Glu Glu Pro Pro Val Leu Pro 195 200 205Ala Thr Ser Pro Arg Ser Gln Ala Arg Leu Arg Gln Asp Gly Asp Val 210 215 220Met Pro Thr Leu Asp Met Ala Leu Phe Asp Trp Thr Asp Tyr Glu Asp225 230 235 240Leu Lys Pro Glu Met Trp Pro Ser Ala Lys Lys Lys Glu Lys Arg Arg 245 250 255Ser Lys Ser Ser Asn Gly Gly Asn Glu Thr Ser Ser Ala Glu Gly Glu 260 265 270Pro Cys Asp His His Leu Asp Cys Leu Pro Gly Ser Cys Cys Asp Leu 275 280 285Arg Glu His Leu Cys Lys Pro His Asn Arg Gly Leu Asn Asn Lys Cys 290 295 300Tyr Asp Asp Cys Met Cys Thr Glu Gly Leu Arg Cys Tyr Ala Lys Phe305 310 315 320His Arg Asn Arg Arg Val Thr Arg Arg Lys Gly Arg Cys Val Glu Pro 325 330 335Glu Ser Ala Asn Gly Glu Gln Gly Ser Phe Ile Asn Val 340 3455421PRTFelis catus 54Glu Val Met Pro Thr Leu Asp Met Ala Leu Phe Asp Trp Thr Asp Tyr1 5 10 15Glu Asp Leu Lys Pro 205521PRTRattus norvegicus 55Glu Val Met Pro Thr Leu Asp Met Ala Leu Phe Asp Trp Thr Asp Tyr1 5 10 15Glu Asp Leu Lys Pro 205621PRTMus musculus 56Glu Val Met Pro Thr Leu Asp Met Ala Leu Phe Asp Trp Thr Asp Tyr1 5 10 15Glu Asp Leu Lys Pro 205721PRTEquus caballus 57Glu Val Met Pro Thr Leu Asp Met Ala Leu Phe Asp Trp Thr Asp Tyr1 5 10 15Glu Asp Leu Lys Pro 205821PRTBos taurus 58Glu Val Met Pro Thr Leu Asp Met Ala Leu Phe Asp Trp Thr Asp Tyr1 5 10 15Glu Asp Leu Lys Pro 205921PRTSus scrofa 59Glu Val Met Pro Thr Leu Asp Met Ala Leu Phe Asp Trp Thr Asp Tyr1 5 10 15Glu Asp Leu Lys Pro 206021PRTCanis lupus 60Glu Val Met Pro Thr Leu Asp Met Ala Leu Phe Asp Trp Thr Asp Tyr1 5 10 15Glu Asp Leu Arg Pro 206121PRTGallus gallus 61Asp Val Met Pro Thr Leu Asp Met Ala Leu Phe Asp Trp Thr Asp Tyr1 5 10 15Glu Asp Leu Lys Pro 206221PRTTakifugu rubripes 62Glu Val Met Pro Thr Leu Asp Met Thr Leu Phe Asp Trp Thr Asp Tyr1 5 10 15Glu Asp Met Lys Pro 206321PRTXenopus tropicalis 63Asp Val Met Pro Thr Leu Asp Met Thr Leu Phe Asp Trp Thr Asp Tyr1 5 10 15Glu Asp Met Lys Pro 206448PRTDanio rerio 64Cys Asp Cys His Pro Val Gly Ala Ala Gly Lys Thr Cys Asn Gln Thr1 5

10 15Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr Gly Ile Thr Cys Asn 20 25 30Arg Cys Ala Asn Gly Tyr Gln Gln Ser Arg Ser Pro Ile Ala Pro Cys 35 40 456548PRTHomo sapiens 65Cys Asp Cys His Pro Val Gly Ala Ala Gly Lys Thr Cys Asn Gln Thr1 5 10 15Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr Gly Ile Thr Cys Asn 20 25 30Arg Cys Ala Lys Gly Tyr Gln Gln Ser Arg Ser Pro Ile Ala Pro Cys 35 40 45661029DNAMus musculus 66atggcagggt gccccgtcct cagggtcccc acgctgttcc tgatcctcct gctgtttcca 60gagctccaca cggcaggcac ccttgcatct ggatcctctg cccggaacct gccggagacc 120cactcccacc tccccagctc tgcactgtgg gtgtcccagg caagccatca tggccgtcgg 180ggcctgggga agaaagacag gggcccagga aggcctagcc gggcccagga gggggctgtg 240gtcactgcta ccaagcaggc ttcccagatg acactcggac agccccctgc tggccttctg 300cagaataagg agctgcttct ggggctgact ttgccctacc ccgagaagga ggcccggtct 360cccgcttggg agagggtgaa gaaacgtggc agagaacaca agagacgcag ggaccgtctg 420cgactgcacc gaggccgagc tgccatccgt ggccccagct ccctcatgaa gaaggtggaa 480ccctctgaag accggatgct ggagggtacc atggaggagt cttccactag cctggccccc 540accatgttct tcctgaccat gacagacggt gccacgccta ctacagaaga gtcccggatc 600ctgcctgtca cgtccttgcg gccccagaca cagcccaggt ctgacgggga ggtgatgccc 660acactggaca tggccttatt tgactggacg gattatgaag acttaaagcc agaggtctgg 720ccttctgcaa agaagaaaga gaaacactgg agtcatttta ccagtgatgg taacgagacc 780tcgccagctg agggggatcc gtgtgaccat caccaggatt gcttgccagg aacttgctgt 840gacctccggg aacatctctg cacaccccac aaccgcggcc tcaacaacaa atgtttcgac 900gactgcatgt gcatggaagg gctgcgttgc tatgccaaat tccaccggaa ccgcagggtc 960actcggagga aggggcgctg cgtggaaccg gagacagcca acggggacca gggatctttc 1020atcaacatc 1029671047DNAGallus gallus 67atggcagctt cttccacctt cttctctccg tctcttttcc tgtgtgtgct ggttcttatt 60gacatcaccc ttgccgtctc cctggacact gacatgaagc tcaaaagtga gaacaacaac 120caccttcaaa accaagagac gtggcctcag cagcccagga gtgggcacca ccacaagcat 180ggcttggcca agaaagggag ggtccttgcc ctgcctgtta gagggcagcc agctggggaa 240gaggccctcc gagtgggcag tggagctcca gccatggaag agctggtgcc acttggccag 300ccagcagcgc tgaaacagga taaggataag gatgtgttcc tgggctttga gctcccacac 360gctgagcggg agaatcagtc ccctgggtct gagaggggaa agaagcagaa ccgagagcag 420cgacggcaca gccgcaggga caggctgaaa caccacagag ggaagactgc cgttgggcca 480agctccctgt ataagaaacc tgaaagcttc gagcaacagt ttcaaaacct ccaggcagag 540gaagcaacca gcccgacccc caccgtgctt cccttcactg cactggatct ggtcgtttcc 600acagaagagc ctcctgttct tccagccacg tcgccgcggt cacaggcccg cctcaggcaa 660gatggggatg tgatgcccac cctagatatg gcactctttg actggacaga ttatgaggac 720ctcaaaccag aaatgtggcc gtcagctaaa aagaaagaga aacgccgcag taagagctcc 780aatggtggaa atgaaacctc atcggcagaa ggagagccgt gtgaccacca ccttgactgc 840ctcccaggct cttgctgtga cttgcgtgag cacctctgca aaccacacaa tcgaggcctt 900aacaacaaat gctacgatga ctgtatgtgc acagaagggc tacgctgtta tgccaaattc 960caccggaacc gaagagtgac ccgaaggaaa gggcgctgtg tggagcctga gtcggccaat 1020ggagagcagg gatcattcat taatgtt 1047681338DNAArtificial Sequencefusion protein (zebrafish signal sequence)- (human Netrin1) 68atggtggctc ctggcttgtg tcaactcttc attctacccg ggctcagcat gttcgcgggc 60caggcggcgc agcccgatcc ctgctcggac gagaacggcc acccgcgccg ctgcatcccg 120gactttgtca atgcggcctt cggcaaggac gtgcgcgtgt ccagcacctg cggccggccc 180ccggcgcgct actgcgtggt gagcgagcgc ggcgaggagc ggctgcgctc gtgccacctc 240tgcaacgcgt ccgaccccaa gaaggcgcac ccgcccgcct tcctcaccga cctcaacaac 300ccgcacaacc tgacgtgctg gcagtccgag aactacctgc agttcccgca caacgtcacg 360ctcacactgt ccctcggcaa gaagttcgaa gtgacctacg tgagcctgca gttctgctcg 420ccgcggcccg agtccatggc catctacaag tccatggact acgggcgcac gtgggtgccc 480ttccagttct actccacgca gtgccgcaag atgtacaacc ggccgcaccg cgcgcccatc 540accaagcaga acgagcagga ggccgtgtgc accgactcgc acaccgacat gcgcccgctc 600tcgggcggcc tcatcgcctt cagcacgctg gacgggcggc cctcggcgca cgacttcgac 660aactcgcccg tgctgcagga ctgggtcacg gccacagaca tccgcgtggc cttcagccgc 720ctgcacacgt tcggcgacga gaacgaggac gactcggagc tggcgcgcga ctcgtacttc 780tacgcggtgt ccgacctgca ggtgggcggc cggtgcaagt gcaacggcca cgcggcccgc 840tgcgtgcgcg accgcgacga cagcctggtg tgcgactgca ggcacaacac ggccggcccg 900gagtgcgacc gctgcaagcc cttccactac gaccggccct ggcagcgcgc cacagcccgc 960gaagccaacg agtgcgtggc ctgtaactgc aacctgcatg cccggcgctg ccgcttcaac 1020atggagctct acaagctttc ggggcgcaag agcggaggtg tctgcctcaa ctgtcgccac 1080aacaccgccg gccgccactg ccattactgc aaggagggct actaccgcga catgggcaag 1140cccatcaccc accggaaggc ctgcaaagcc tgtgattgcc accctgtggg tgctgctggc 1200aaaacctgca accaaaccac cggccagtgt ccctgcaagg acggcgtgac gggtatcacc 1260tgcaaccgct gcgccaaagg ctaccagcag agccgctctc ccatcgcccc ctgcataaag 1320atccctgtag cgccgccg 133869446PRTArtificial Sequencefusion protein (zebrafish signal sequence)- (human Netrin1) 69Met Val Ala Pro Gly Leu Cys Gln Leu Phe Ile Leu Pro Gly Leu Ser1 5 10 15Met Phe Ala Gly Gln Ala Ala Gln Pro Asp Pro Cys Ser Asp Glu Asn 20 25 30Gly His Pro Arg Arg Cys Ile Pro Asp Phe Val Asn Ala Ala Phe Gly 35 40 45Lys Asp Val Arg Val Ser Ser Thr Cys Gly Arg Pro Pro Ala Arg Tyr 50 55 60Cys Val Val Ser Glu Arg Gly Glu Glu Arg Leu Arg Ser Cys His Leu65 70 75 80Cys Asn Ala Ser Asp Pro Lys Lys Ala His Pro Pro Ala Phe Leu Thr 85 90 95Asp Leu Asn Asn Pro His Asn Leu Thr Cys Trp Gln Ser Glu Asn Tyr 100 105 110Leu Gln Phe Pro His Asn Val Thr Leu Thr Leu Ser Leu Gly Lys Lys 115 120 125Phe Glu Val Thr Tyr Val Ser Leu Gln Phe Cys Ser Pro Arg Pro Glu 130 135 140Ser Met Ala Ile Tyr Lys Ser Met Asp Tyr Gly Arg Thr Trp Val Pro145 150 155 160Phe Gln Phe Tyr Ser Thr Gln Cys Arg Lys Met Tyr Asn Arg Pro His 165 170 175Arg Ala Pro Ile Thr Lys Gln Asn Glu Gln Glu Ala Val Cys Thr Asp 180 185 190Ser His Thr Asp Met Arg Pro Leu Ser Gly Gly Leu Ile Ala Phe Ser 195 200 205Thr Leu Asp Gly Arg Pro Ser Ala His Asp Phe Asp Asn Ser Pro Val 210 215 220Leu Gln Asp Trp Val Thr Ala Thr Asp Ile Arg Val Ala Phe Ser Arg225 230 235 240Leu His Thr Phe Gly Asp Glu Asn Glu Asp Asp Ser Glu Leu Ala Arg 245 250 255Asp Ser Tyr Phe Tyr Ala Val Ser Asp Leu Gln Val Gly Gly Arg Cys 260 265 270Lys Cys Asn Gly His Ala Ala Arg Cys Val Arg Asp Arg Asp Asp Ser 275 280 285Leu Val Cys Asp Cys Arg His Asn Thr Ala Gly Pro Glu Cys Asp Arg 290 295 300Cys Lys Pro Phe His Tyr Asp Arg Pro Trp Gln Arg Ala Thr Ala Arg305 310 315 320Glu Ala Asn Glu Cys Val Ala Cys Asn Cys Asn Leu His Ala Arg Arg 325 330 335Cys Arg Phe Asn Met Glu Leu Tyr Lys Leu Ser Gly Arg Lys Ser Gly 340 345 350Gly Val Cys Leu Asn Cys Arg His Asn Thr Ala Gly Arg His Cys His 355 360 365Tyr Cys Lys Glu Gly Tyr Tyr Arg Asp Met Gly Lys Pro Ile Thr His 370 375 380Arg Lys Ala Cys Lys Ala Cys Asp Cys His Pro Val Gly Ala Ala Gly385 390 395 400Lys Thr Cys Asn Gln Thr Thr Gly Gln Cys Pro Cys Lys Asp Gly Val 405 410 415Thr Gly Ile Thr Cys Asn Arg Cys Ala Lys Gly Tyr Gln Gln Ser Arg 420 425 430Ser Pro Ile Ala Pro Cys Ile Lys Ile Pro Val Ala Pro Pro 435 440 4457026PRTDanio rerio 70Met Val Ala Pro Gly Leu Cys Gln Leu Phe Ile Leu Leu Leu Ile Thr1 5 10 15Leu Ser His Thr Leu His Ser Ser Glu Gly 20 257123PRTDanio rerio 71Met Val Ala Pro Gly Leu Cys Gln Leu Phe Ile Leu Leu Leu Ile Thr1 5 10 15Leu Ser His Thr Leu His Ser 20721740DNAHomo sapiens 72atgcctggct ggccctgggg gctgctgctg acggcaggca cgctcttcgc cgccctgagt 60cctgggccgc cggcgcccgc cgacccctgc cacgatgagg ggggtgcgcc ccgcggctgc 120gtgccaggac tggtgaacgc cgccctgggc cgcgaggtgc tggcttccag cacgtgcggg 180cggccggcca ctcgggcctg cgacgcctcc gacccgcgac gggcacactc ccccgccctc 240cttacttccc cagggggcac ggccagccct ctgtgctggc gctcggagtc cctgcctcgg 300gcgcccctca acgtgactct cacggtgccc ctgggcaagg cttttgagct ggtcttcgtg 360agcctgcgct tctgctcagc tcccccagcc tccgtggccc tgctcaagtc tcaggaccat 420ggccgcagct gggccccgct gggcttcttc tcctcccact gtgacctgga ctatggccgt 480ctgcctgccc ctgccaatgg cccagctggc ccagggcctg aggccctgtg cttccccgca 540cccctggccc agcctgatgg cagcggcctt ctggccttca gcatgcagga cagcagcccc 600ccaggcctgg acctggacag cagcccagtg ctccaagact gggtgaccgc caccgacgtc 660cgtgtagtgc tcacaaggcc tagcacggca ggtgacccca gggacatgga ggccgtcgtc 720ccttactcct acgcagccac cgacctccag gtgggcgggc gctgcaagtg caatggacat 780gcctcacggt gcctgctgga cacacagggc cacctgatct gcgactgtcg gcatggcacc 840gagggccctg actgcggccg ctgcaagccc ttctactgcg acaggccatg gcagcgggcc 900actgcccggg aatcccacgc ctgcctcgct tgctcctgca acggccatgc ccgccgctgc 960cgcttcaaca tggagctgta ccgactgtcc ggccgccgca gcgggggtgt ctgtctcaac 1020tgccggcaca acaccgccgg ccgccactgc cactactgcc gggagggctt ctatcgagac 1080cctggccgtg ccctgagtga ccgtcgggct tgcagggcct gcgactgtca cccggttggt 1140gctgctggca agacctgcaa ccagaccaca ggccagtgtc cctgcaagga tggcgtcact 1200ggcctcacct gcaaccgctg cgcgcctggc ttccagcaaa gccgctcccc agtggcgccc 1260tgtgttaaga cccctatccc tggacccact gaggacagca gccctgtgca gccccaggac 1320tgtgactcgc actgcaaacc tgcccgtggc agctaccgca tcagcctaaa gaagttctgc 1380aagaaggact atgcggtgca ggtggcggtg ggtgcgcgcg gcgaggcgcg cggcgcgtgg 1440acacgcttcc cggtggcggt gctcgccgtg ttccggagcg gagaggagcg cgcgcggcgc 1500gggagtagcg cgctgtgggt gcccgccggg gatgcggcct gcggctgccc gcgcctgctc 1560cccggccgcc gctacctcct gctggggggc gggcctggag ccgcggctgg gggcgcgggg 1620ggccgggggc ccgggctcat cgccgcccgc ggaagcctcg tgctaccctg gagggacgcg 1680tggacgcggc gcctgcggag gctgcagcga cgcgaacggc gggggcgctg cagcgccgcc 174073577PRTHomo sapiens 73Met Ser Met Phe Ala Ala Gln Thr Ser Pro Pro Asp Pro Cys Tyr Asp1 5 10 15Glu Asn Gly His Pro Arg Arg Cys Ile Pro Asp Phe Val Asn Ala Ala 20 25 30Phe Gly Lys Glu Val Arg Ala Ser Ser Thr Cys Gly Lys Thr Pro Ser 35 40 45Arg Tyr Cys Val Val Thr Glu Lys Gly Asp Glu Arg His Arg Asn Cys 50 55 60His Thr Cys Asp Ala Ser Asp Pro Lys Lys Asn His Pro Pro Ala Tyr65 70 75 80Leu Thr Asp Leu Asn Asn Pro His Asn Leu Thr Cys Trp Gln Ser Asp 85 90 95Asn Tyr Leu Gln Tyr Pro Gln Asn Val Thr Leu Thr Leu Ser Leu Gly 100 105 110Lys Lys Phe Glu Val Thr Tyr Val Ser Leu Gln Phe Cys Ser Pro Arg 115 120 125Pro Glu Ser Met Ala Ile Phe Lys Ser Met Asp Tyr Gly Lys Ser Trp 130 135 140Val Pro Phe Gln Tyr Tyr Ser Thr Gln Cys Arg Lys Met Tyr Asn Lys145 150 155 160Pro Ser Lys Ala Thr Ile Thr Lys Gln Asn Glu Gln Glu Ala Ile Cys 165 170 175Thr Asp Ser His Thr Asp Met His Pro Leu Ser Gly Gly Leu Ile Ala 180 185 190Phe Ser Thr Leu Asp Gly Arg Pro Ser Ala His Asp Phe Asp Asn Ser 195 200 205Pro Val Leu Gln Asp Trp Val Thr Ala Thr Asp Ile Lys Val Thr Phe 210 215 220Ser Arg Leu His Thr Phe Gly Asp Glu Asn Glu Asp Asp Ser Glu Leu225 230 235 240Ala Arg Asp Ser Tyr Phe Tyr Ala Val Ser Asp Leu Gln Val Gly Gly 245 250 255Arg Cys Lys Cys Asn Gly His Ala Ser Arg Cys Val Lys Asp Arg Asp 260 265 270Gly Asn Leu Val Cys Glu Cys Lys His Asn Thr Ala Gly Pro Glu Cys 275 280 285Asp Arg Cys Lys Pro Phe His Tyr Asp Arg Pro Trp Gln Arg Ala Thr 290 295 300Ala Arg Glu Ala Asn Glu Cys Val Ala Cys Asn Cys Asn Leu His Ala305 310 315 320Arg Arg Cys Arg Phe Asn Met Glu Leu Tyr Lys Leu Ser Gly Arg Lys 325 330 335Ser Gly Gly Val Cys Leu Asn Cys Arg His Asn Thr Ala Gly Arg His 340 345 350Cys His Tyr Cys Lys Glu Gly Tyr Tyr Arg Asp Met Ser Lys Pro Ile 355 360 365Ser His Arg Lys Ala Cys Lys Ala Cys Asp Cys His Pro Val Gly Ala 370 375 380Ala Gly Lys Thr Cys Asn Gln Thr Thr Gly Gln Cys Pro Cys Lys Asp385 390 395 400Gly Val Thr Gly Ile Thr Cys Asn Arg Cys Ala Asn Gly Tyr Gln Gln 405 410 415Ser Arg Ser Pro Ile Ala Pro Cys Ile Lys Ile Pro Ile Ala Pro Pro 420 425 430Thr Thr Thr Ala Ser Ser Thr Glu Glu Pro Ser Asp Cys Glu Ser Tyr 435 440 445Cys Lys Ala Ser Lys Gly Lys Leu Lys Ile Asn Met Lys Lys Tyr Cys 450 455 460Lys Lys Asp Tyr Ala Val Gln Val His Ile Leu Lys Ala Asp Lys Ala465 470 475 480Gly Glu Trp Trp Lys Phe Thr Val Asn Ile Ile Ser Val Tyr Lys Gln 485 490 495Gly Glu Ser Arg Ile Arg Arg Gly Asp Gln Phe Leu Trp Val Arg Ala 500 505 510Lys Asp Val Ala Cys Lys Cys Pro Lys Ile Lys Ser Gly Lys Lys Tyr 515 520 525Leu Leu Leu Gly Asn Asp Glu Asp Ser Pro Gly Gln Ser Gly Met Val 530 535 540Ala Asp Lys Gly Ser Leu Val Ile Gln Trp Arg Asp Thr Trp Ala Arg545 550 555 560Arg Leu Arg Lys Phe Gln Gln Arg Glu Lys Lys Gly Lys Cys Lys Lys 565 570 575Ala74501DNAHomo sapiens 74tgcaagtgca atggacatgc ctcacggtgc ctgctggaca cacagggcca cctgatctgc 60gactgtcggc atggcaccga gggccctgac tgcggccgct gcaagccctt ctactgcgac 120aggccatggc agcgggccac tgcccgggaa tcccacgcct gcctcgcttg ctcctgcaac 180ggccatgccc gccgctgccg cttcaacatg gagctgtacc gactgtccgg ccgccgcagc 240gggggtgtct gtctcaactg ccggcacaac accgccggcc gccactgcca ctactgccgg 300gagggcttct atcgagaccc tggccgtgcc ctgagtgacc gtcgggcttg cagggcctgc 360gactgtcacc cggttggtgc tgctggcaag acctgcaacc agaccacagg ccagtgtccc 420tgcaaggatg gcgtcactgg cctcacctgc aaccgctgcg cgcctggctt ccagcaaagc 480cgctccccag tggcgccctg t 50175167PRTHomo sapiens 75Cys Lys Cys Asn Gly His Ala Ser Arg Cys Leu Leu Asp Thr Gln Gly1 5 10 15His Leu Ile Cys Asp Cys Arg His Gly Thr Glu Gly Pro Asp Cys Gly 20 25 30Arg Cys Lys Pro Phe Tyr Cys Asp Arg Pro Trp Gln Arg Ala Thr Ala 35 40 45Arg Glu Ser His Ala Cys Leu Ala Cys Ser Cys Asn Gly His Ala Arg 50 55 60Arg Cys Arg Phe Asn Met Glu Leu Tyr Arg Leu Ser Gly Arg Arg Ser65 70 75 80Gly Gly Val Cys Leu Asn Cys Arg His Asn Thr Ala Gly Arg His Cys 85 90 95His Tyr Cys Arg Glu Gly Phe Tyr Arg Asp Pro Gly Arg Ala Leu Ser 100 105 110Asp Arg Arg Ala Cys Arg Ala Cys Asp Cys His Pro Val Gly Ala Ala 115 120 125Gly Lys Thr Cys Asn Gln Thr Thr Gly Gln Cys Pro Cys Lys Asp Gly 130 135 140Val Thr Gly Leu Thr Cys Asn Arg Cys Ala Pro Gly Phe Gln Gln Ser145 150 155 160Arg Ser Pro Val Ala Pro Cys 16576144DNAHomo sapiens 76tgcgactgtc acccggttgg tgctgctggc aagacctgca accagaccac aggccagtgt 60ccctgcaagg atggcgtcac tggcctcacc tgcaaccgct gcgcgcctgg cttccagcaa 120agccgctccc cagtggcgcc ctgt 1447748PRTHomo sapiens 77Cys Asp Cys His Pro Val Gly Ala Ala Gly Lys Thr Cys Asn Gln Thr1 5 10 15Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr Gly Leu Thr Cys Asn 20 25 30Arg Cys Ala Pro Gly Phe Gln Gln Ser Arg Ser Pro Val Ala Pro Cys 35 40 457852PRTHomo sapiens 78Cys Ser Cys His Pro Val Gly Ser Ala Val Leu Pro Ala Asn Ser Val1 5 10 15Thr Phe Cys Asp Pro Ser Asn Gly Asp Cys Pro Cys Lys Pro Gly Val 20 25 30Ala Gly Arg Arg Cys Asp Arg Cys Met Val Gly Tyr Trp Gly Phe Gly 35 40 45Asp Tyr Gly Cys

507948PRTDanio rerio 79Cys Asp Cys His Pro Val Gly Ala Ala Gly Lys Thr Cys Asn Gln Thr1 5 10 15Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr Gly Ile Thr Cys Asn 20 25 30Arg Cys Ala Lys Gly Tyr Gln Gln Ser Arg Ser Pro Ile Ala Pro Cys 35 40 458025DNAArtificial Sequencentn1a morpholino 80atgatggact taccgacaca ttcgt 258125DNAArtificial Sequencentn1b morpholino 81cgcacgttac caaaatcctt atcat 25825PRTArtificial Sequencepeptide linker 82Ser Gly Gly Gly Gly1 5

* * * * *

Current U.S. Class:	1/1
Current CPC Class:	C07K 14/475 20130101; C07K 14/4703 20130101; C07K 14/47 20130101; C07K 16/18 20130101; C07K 2319/30 20130101; C07K 14/461 20130101; A61K 38/00 20130101; C07K 2317/76 20130101
International Class:	C07K 14/47 20060101 C07K014/47; C07K 14/475 20060101 C07K014/475; C07K 16/18 20060101 C07K016/18; C07K 14/46 20060101 C07K014/46