DE19805371A1 - New semaphorin L proteins - Google Patents

Abstract

A semaphorin L protein (I) comprising a N-terminal signal peptide, a characteristic semaphorin domain and a C-terminal region comprising an immunoglobulin-like domain and a transmembrane domain, is new. Also claimed are: (i) derivatives of (I); (ii) nucleic acids encoding (I) and its derivatives; and (iii) antibodies that recognise the human semaphorin L epitope corresponding to amino acids 179-378 or 480-666 of a 666 amino acid sequence.

Description

The invention relates to new semaphorins, which are characterized by a special Characterize domain structure and its derivatives, nucleic acids (DNA, RNA, cDNA) which code for these semaphorins and their derivatives as well as the Using the same.

Semaphorins were first developed by Koldokin {Kolodkin et al. (1993) Cell 75: 1389- 1399} as members of a conserved gene family.

In the meantime, the genes or parts of the genes of further semaphorins have been cloned and partially characterized. So far, a total of 5 human (H-Sema-III, H-Sema-V, H-Sema-IV, H-Sema-B and H-Sema-E) {Kolodkin et al. (1993); Roche et al. (1996) Oncogenes 12: 1289-1297; Sekido et al. (1996) Proc. Natl. Acad. Sci. USA 93: 4120-4125; Xiang et al. (1996) Genomics 32: 39-48; Hall et al. (1996) Proc. Natl. Acad. Sci. USA 39: 11780-11785; Yamada et al. (1997) (GenBank assignment no. AB000220)}, 8 murine (mouse genes; M-Sema A to M-Sema-H) {Püschel et al. (1995) Neuron 14: 941-948; Messerschmidt et al. (1995) Neuron 14: 949-959; Inigaki et al. (1995) FEBS Letters 370: 269-272; Adams et al. (1996) Mech. Dev. 57: 33-45; Christensen et al. (1996) (Genbank assignment no. Z80941, Z93948)}, 5 gallide (chicken) (Collapsin-1 to -5) {Luo et al. (1993); Luo et al. (1995) Neuron 14: 1131-1140}, and rat genes (R-Sema-III) {Giger et al. (1996) J. Comp. Neurol. 375: 378-392}, zebrafish, insects (fruit fly (Drosophila melanogaster: D-Sema-I and D-Sema-II), beetles (Tribolium confusum: T-Sema-I), grasshopper (Schistocerca americana: G-Sema- I)) {Kolodkin et al. (1993)}, and nematodes (C.elegans: Ce-Sema) {Roy et al. (1994) (GenBank Association No. U15667)}. Furthermore, two poxviruses (Vaccinia (ORF-A39) and Variola (ORFA39 homolog)) {Kolodkin et al. (1993)} and the Aleclaphine Herpesvirus Type 1 (AHV-1) (AHV-Sema) {Ensser and Fleckenstein (1995) Gen. Virol. 76: 1067} genes homologous to semaphorins.

An overview of the semaphorins identified so far in different Species gives Table 1. In Table 1 are the names of the semaphorins that used synonyms, the species from which the respective semaphorin is isolated and, as far as known, data on the domain structure of the encoded protein and for chromosomal localization (column 4 in Table 1), the Assignment number under which the sequence of the gene in gene databases, e.g. B. in an EST (expressed sequence tags) database, EMBL (European Molecular Biology Laboratory, Heidelberg) or NCBI (National Center for Biotechnology Information, Maryland, USA) and the corresponding reference who published this data (column 5 in Table 1).

All gene products of the previously known semaphorin genes have an N-terminal signal peptide, at the C-terminal end of which there is a characteristic Sema domain with a length of approximately 450 to 500 amino acids. Within the Sema domain there are highly conserved amino acid motifs and a number of highly conserved cysteine residues. The gene products differ in the C-terminal sequences following the Sema domain, which are composed of one or more domains. For example, in these C-terminal amino acid sequences they have transmembrane domains (TM), immunoglobulin-like domains (Ig) (constant part of the immunoglobulin), cytoplasmic sequences (CP), processing signals (P), for example with the consensus sequence (RXR), where R is for the Amino acid arginine and X stands for any amino acid and / or hydrophilic C-termini (HPC). Based on the different domain structure in the C-terminus, the semaphorins can be divided into 5 different subgroups (I to V):

I secreted, without further domain (e.g. ORF-A49)
II Ig secreted (without transmembrane domain) (e.g. AHV sema)
III Ig, TM, CP membrane anchored with cytoplasmic sequence (e.g. CD100)
IV Ig, (P), HPC secreted with hydrophilic C-terminus (e.g. H-Sema-III, M-Sema-D, Collapsin-1)
V Ig, TM, CP membrane anchored with C-terminal 7 thrombospondin motif (e.g. M-Sema-F and -G).

A receptor or extracellular ligand for semaphorins has not yet been found described. In connection with semaphorin-mediated effects were intracellular, heterotrimeric GTP-binding protein complexes are described. As a In chickens, so-called CRMP- Proteins (Collapsin response mediator protein) identified, which presumably Are part of the semaphorin-induced intracellular signaling cascade {Goshima et al. (1995) Nature 376: 509-514}. The CRMP62, for example, has Homology to unc-33, an essential for directed axon growth Nematode protein. A human protein with 98% amino acid identity too CRMP62 is also known {Hamajima et al. (1996) Gene 180: 157-163}. In Several CRMP-related genes have also been described in rats {Wang et al. (1996) Neurosci. 16: 6197-6207}.

The secreted or transmembral semaphorins convey repulsive signals for growing nerve buds. They play a role in the development of the central nervous system (CNS) and are mainly found in muscle and Nerve tissue expressed {Kolodkin et al. (1993); Luo et al. (1993) Cell 75: 217-227}.

In addition to the CNS, a clear expression of M-Sema-G could also be expressed on cells of the lymphatic and hematopoietic systems are observed, in contrast to the closely related M-Sema-F {Furuyima et al. (1996) J. Biol. Chem. 271: 33376- 33381}.

Two other human semaphorins have recently been identified, H-Sema-IV and H- Sema-V in a region on chromosome 3p21.3, the deletion with different forms of bronchial carcinoma is associated. H-Sema-IV {Roche et al. (1996), Xiang et al. (1996), Sekido et al. (1996)} is at the amino acid level about 50% identical to M-Sema-E, while H-Sema-V {Sekido et al. (1996)} that  is direct homologue to M-Sema-A (86% amino acid identity). Because these genes (H- Sema-IV and -V) in the context of DNA sequencing projects of the deleted 3p21.3 loci were found is the complex intron-exon structure of these two Known genes. The two genes are in different neurons and not neuronal tissues expressed.

Also recently, the cellular surface molecule CD100 (human), expressed and induced on activated T cells, identified as semaphorin (also listed in Table 1). It supports interaction with B cells via the CD40 receptor and the corresponding CD40L ligand. CD100 is a Membrane-anchored glycoprotein dimer of 150kd (kilodalton). there has been a Association of the intracytoplasmic C-terminus of CD100 with one more unknown kinase {Hall et al. (1996)}. That was CD100's first and so far the only semaphorin whose expression in cells of the immune system could be demonstrated.

Under the question "Transforming genes of rhadinoviruses" complete genome of the Alcelaphinen herpesvirus type 1 (AHV-1) and cloned sequenced {Ensser et al. (1995)}. AHV-1 is the causative agent of the malignant Catarrh fever, a condition associated with lymphoproliferative syndrome mostly fatal disease of various ruminants. The analysis was on one end of the viral genome with an open reading frame, but significant homology to a gene from vaccinia virus (ORF-A39 ≘ VAC-A39 in Ensser et al. (1995) J. Gen. Virol: 76: 1063-1067), which belongs to the gene family of Semaphorine was found. While the AHV-1 semaphorin (AHV-Sema) has a well-preserved semaphorin structure, the poxvirus Genes (ORF-A39 and ORF-A39 homologous, see Table 1) C-terminally truncated, i.e. H. with them the conserved Sema domain is only incomplete.

A database comparison of the AHV scheme found with dbEST (EST (expressed sequence tags) database (db)) each provided 2 EST sequences of 2 independent cDNA clones from human placenta (assignment numbers H02902, H03806 (clone 151129), assignment numbers R33439 and R33537 (clone 135941)).  

These had significantly higher homology with the AHV-1 semaphorin than with the neural semaphorins described so far.

The present invention relates to semaphorins with a new, previously unknown and unexpected domain structure, which a biochemical function in the immune system (immunomodulating Semaphorins). The semaphorins according to the invention are called semaphorins from Type L (Sema-L) designated. They contain an N-terminal signal peptide, a characteristic Sema domain and a in the C-terminal region of the protein Immunoglobulin-like domain and a hydrophobic domain, which is a potential Represents transmembrane domain.

The amino acid sequence of the signal peptide can be less than 70, preferably less than 60 amino acids and more than 20, preferably more than 30 Have amino acids, a length of about 40 to 50 is particularly preferred Amino acids. In a special embodiment of the invention Signal peptide 44 amino acids in length, i. H. between amino acids 44 and 45 is a cleavage site for a signal peptidase.

The Sema domain can be from 300 to 700 or more in length, preferably from have about 400 to 600 amino acids. Sema domains with a are preferred Length from 450 to 550 amino acids, preferably from about 500 amino acids. In A special embodiment of the invention joins the Sema domain to the signal peptide, the Sema domain preferably extending up to Amino acid 545 stretches.

The immunoglobulin-like domain can be about 30 to 110 or long have more amino acids, lengths between 50 and 90 are preferred, particularly preferably about 70 amino acids.

The transmembrane domain can have a length of about 10 to 35, preferably of have about 15 to 30, particularly preferably from about 20 to 25, amino acids.  

The invention relates to type L semaphorins from different species, in particular from vertebrates, for example from birds and / or fish, preferably from mammals, for example from primates, rats, rabbits, Dog, cat, sheep, goat, cow, horse, pig, particularly preferably from humans and mouse. The invention also relates to corresponding semaphorins Microorganisms, in particular from pathogenic microorganisms, for example from bacteria, yeast and / or viruses, e.g. B. from retroviruses, in particular human pathogenic microorganisms.

One embodiment of the invention is a corresponding human semaphorin (H-Sema-L), which is a signal peptide, a Sema domain, an immunoglobulin has a similar domain and a transmembrane domain. A special one Embodiment is the semaphorin, which according to the amino acid sequence Table 4 is given.

Corresponding semaphorins are a further embodiment of the invention in other species that have an amino acid identity in the area of the Sema domain greater than 40%, preferably greater than 50%, particularly preferably greater than 60% in Reference to the Sema domain of H-Sema-L (amino acids 45 to 545 of the sequence in Table 4). From more closely related species (e.g. primates, mouse) the corresponding semphorins can do amino acid identities greater than 70%, preferably greater than 80%, particularly preferably greater than Have 90%.

Such an embodiment of the invention is a corresponding one Mouse semaphorin (murine semaphorin (M-Sema-L)). For example, contains this is the partial amino acid sequence according to Table 5 (murine semaphorin (M-Sema-L)).

The invention also relates to corresponding semaphorins, the one Amino acid identity (over the total length of the amino acid sequence of the protein considered) of only about 15 to 20% in little related species (phylogenetic far apart), preferably 25 to 30%, particularly preferably 35 to  40% or higher identity with respect to the total amino acid sequence of Have H-Sema-L according to Table 4.

The genes coding for type L semaphorins have a complex exon Intron structure. These genes can, for example, have between 10 and 20 exons, preferably about 11 to 18, particularly preferably 12 to 16 exons and one have the corresponding number of introns. But you can also do the same Number of exons and introns have, like the gene of H-Sema-L (13 or 15 exons, preferably 14 exons). A particular embodiment of the invention relates the gene from H-Sema L. This gene is preferably from 8888 to 10,000 or more nucleotides. The human semaphorin gene preferably contains the nucleotide sequence given in Table 14 or the nucleotide sequence, which are stored in the GenBank database under the access number AF030697 has been. These nucleotide sequences contain at least 13 introns. Furthermore the human semaphorin gene has an additional 5 'end Sequence area. This area may contain further coding Sequences, e.g. B. one or two more introns.

Attempts to chromosomal localization of human semaphorin type L revealed that the corresponding gene is located at position 15q22.3-23. Accordingly, the gene for M-Sema L was located at position 9A3.3-B.

As a result of the complex intron-exon structure, the primary transcript of the Semaphorin mRNA can be spliced differently, causing different Splice variants of the semaphorins are created. The ones from these splice variants Translated proteins are derivatives of the semaphorins according to the invention. she correspond in their amino acid sequence and also largely in their Domain structure of the described semaphorins of the type according to the invention L, however, are shortened compared to these. For example, splice variants, which completely or partially lack the transmembrane domain. A Semaphorin derivative, which has no or no complete transmembrane domain, but containing a signal peptide can be secreted and so act locally or over large distances outside the cell,  for example to other cells. Another splicing variant can, for example no longer contain a sequence which codes for a signal peptide and, if appropriate also no sequence that codes for a hydrophobic amino acid sequence that represents a potential transmembrane domain. One consequence would be that this Semaphorin derivative is neither built into the membrane nor secreted (be it because of secretory vesicles). Such a semaphorin derivative can be intracellular processes, for example involved in signal transduction processes be. In this way, the same basic molecules (claimed Semaphorins) and the derivatives derived from them (e.g. Splicing variants) regulates and / or diverse intra- and extracellular processes be coordinated.

A special embodiment of the invention relates to a semaphorin derivative, which are derived from the type L semaphorins according to the invention, but which contain no or no complete transmembrane domain.

Another embodiment of the invention relates to semaphorin derivatives that are derived from the type L semaphorins according to the invention, but none Signal peptide included.

The signal peptide can also be split off post-translationally. This will a membrane-bound (with TM domain) or a secreted (splice variant without TM domain) Semaphorin derivative with a shortened domain structure formed. One on this way post-translationally processed semaphorin derivative only contains Sema domain, Ig domain and possibly transmembrane domain. A Signal peptide interface can, for example, directly at the end of the signal peptide lie, e.g. B. 40 to 50 amino acids or further from the amino terminus be localized.

A "truncated" (i.e., fewer domains) semaphorin L derivative is from other semaphorins that are not derived from type L semaphorins, to be distinguished in that it has a very large (<90%) amino acid identity or an identical amino acid sequence with the semaphorins of type L in the  existing domains.

The semaphorins according to the invention can also be used in other ways be post-translationally modified. For example, they can be one, two, three, four five-, six-, seven-, eight-, nine- ten- or multiple glycosylated (N- and / or O-glycosylated) are present. The amino acid sequences of the semaphorins can then as many or more consensus sequences for potential glycosylation sites have, preferably five such places. An embodiment of the invention affects semaphorins where the glycosylation sites are localized at positions are positions 105, 157, 258, 330 and 602 of H-Sema-L Correspond to amino acid sequence (Table 4).

In addition, the semaphorins can be in the form of their phosphorylated derivatives available. Semaphorins can be the substrates of different kinases, for example, the amino acid sequences can be consensus sequences for protein Kinase C, tyrosine kinase and / or creatine kinases. Can continue the amino acid sequences of the semaphorins consensus sequences for potential Have myristilation sites. At these points, corresponding Semaphorin derivatives must be esterified with myristic acid.

The type L semaphorins according to the invention and their derivatives can be found in Form of monomers, dimers and / or multimers are present, for example can two or more semaphorins or their derivatives via intermolecular Disulfide bridges are interconnected. In addition, you can Form intramolecular disulfide bridges.

Derivatives of the semaphorins according to the invention are also fusion proteins. A Such a fusion protein contains a type L semaphorin or parts the same and in addition a further peptide or protein or a part the same. Peptides or proteins or parts thereof can e.g. B. Epitope tags (e.g. His-Tag (6xHistidine), Myc-Tag, flu-Tag), which e.g. B. to purify the Fusion proteins can be used, or those used to label the Fusion proteins can be used, e.g. B. GFP (green fluorescent protein),  be.

Another object of the invention are DNA and RNA sequences for encode type L semaphorins and / or their derivatives, for example the corresponding genes, the different splicing variants of the mRNA, the corresponding cDNAs and their derivatives, e.g. B. salts of DNA or RNA. Derivatives in the sense of the inventions are sequences or parts of which z. B. changed with molecular biological methods and to the respective Requirements are adapted, for example truncated genes, cDNAs or Chimeras thereof, constructs for expressions and cloning and their Salts.

One embodiment relates to the genomic sequences (genes) of the Type L semaphorins. The invention relates to intron and exon sequences and gene regulatory sequences, for example promoter, enhancer and silencer sequences Sequences.

An object of this embodiment is the gene of H-Sema-L or its Derivatives. An object of the invention is a gene that the nucleotide sequence, the given in Table 14. Another object of the invention is that Gene, which is the nucleotide sequence found in the GenBank database under the Accession number AF030697 is stored, contains.

Another object of this embodiment is the gene of M-Sema-L or its derivatives.

Another object of the invention is the cDNA of H-Sema-L or their Derivatives. A special embodiment is the cDNA of H-Sema-L according to the Amino acid sequence in Table 2. Another object of the invention is cDNA from H-Sema L available in the GenBank database under accession number AF030698 is deposited.

Another object of the invention is the cDNA of M-Sema-L or its  Derivatives. A particular embodiment is the partial cDNA sequence of M-Sema-L according to Table 3 and cDNA sequences which this partial cDNA Sequence included. Another embodiment of the invention relates to the cDNA by M-Sema L, registered in the GenBank database under accession number AF030699 is deposited.

The invention also includes alleles and / or individual forms of expression Genes / mRNAs / cDNAs that differ only slightly from those described here Semaphorin sequences differ and are identical or only marginal altered protein (deviation in the amino acid sequence less than or equal to 10%) encode.

Another object of the invention are plasmids, the DNA for the Type L semaphorins or their derivatives encoded. Such plasmids can, for example, be plasmids with high replication rates which are necessary for a Amplification of the DNA, e.g. B. are suitable in E. coli.

A special embodiment are expression plasmids with which the Semaphorins or parts thereof or their derivatives in prokaryotes and / or eukaryotic expression systems can be expressed. It is both Expression plasmids are constitutive as well as those that have inducible promoters included, suitable.

The invention relates to a method for producing the semaphorins of type L, wherein a nucleic acid sequence coding for a type L semaphorin in an expression vector is cloned and expressed. The expression can preferably in prokaryotic or eukaryotic cells. The semaphorins Type L can also be produced chemically if necessary.

In addition, the semaphorins or their derivatives can be used as fusion proteins are expressed, for example with proteins or peptides, which are a detection of the expressed fusion protein, e.g. B. as a fusion protein with GFP (green fluorescent protein). The semaphorins can also be used as fusion proteins with  two, three or more epitope tags, for example with Myc and / or His (6xHistidine) and / or flu tags are expressed. Correspondingly, plasmids used or manufactured which contain DNA sequences which are suitable for this Encode fusion proteins. For example, semaphorin encoding Sequences are cloned into plasmids that contain DNA sequences required for GFP and / or epitope tags, e.g. B. Myc tag, His tag, flu tag.

Another object of the invention are antibodies that specifically Type L semaphorins, their derivatives or parts thereof bind or recognize. This can be, for example, polyclonal or monoclonal antibodies, e.g. B. in Mouse, rabbit, goat, sheep, chicken, etc. can be made.

A particular embodiment of this object of the invention are Antibodies against the epitopes that correspond to the amino acid sequences of position 179 to 378 or 480 to 666 correspond to the H-Sema-L sequence according to Table 4, are directed. The invention also relates to a method for producing specific anti-semaphorin L antibodies, being used for the production of antigens are used that contain the epitopes mentioned.

These antibodies can be used, for example, to purify the corresponding Semaphorins, e.g. B. from H-Sema-L and its derivatives z. B. via affinity columns or for immunological detection of the proteins, e.g. B. in ELISA, in Western blot and / or used in immunohistochemistry.

The H-Sema L cDNA is 2636 nucleotides in length (Table 2). The gene product of the H-Sema L cDNA has a length of about 666 amino acids (Table 4) and has the typical domain structure of a type L semaphorin. The gene product has an N-terminal signal peptide (amino acids 1 to 44), Sema domain (amino acid 45 to approximately amino acid 545) and Ig (immunoglobulin) domain (approximately amino acids 550 to 620) and a hydrophobic amino acid sequence at the C-terminal end , which represents a potential transmembrane domain. This domain structure has never been described for semaphorins. It is a membrane-associated glycoprotein, probably located on the cell surface, of a new subgroup. Due to this previously unknown domain structure, the semaphorins can now be divided into VI subgroups:

I secreted, without further domain (e.g. ORF-A49)
II Ig secreted (without transmembrane domain) (e.g. AHV sema)
III Ig, TM, CP membrane anchored with cytoplasmic sequence (e.g. CD100)
IV Ig, (P), HPC secreted with hydrophilic C-terminus (e.g. H-Sema-III, M-Sema-D, Collapsin-1)
V Ig, TM, CP membrane anchored with C-terminal 7 thrombospondin motif (e.g. M-Sema-F and -G)
VI Ig, TM membrane anchored (e.g. H-Sema-L).

The non-glycosylated, non-processed form of H-Sema-L has a calculated one Molecular weight of about 74.8 kD (74823 daltons) (calculated with PeptideSort, GCG program package). The isoelectric point is calculated as pH = 7.56. A possible signal peptide interface is between amino acids 44 and 45 (Table 3; calculated with Signal P (http.//www.cbs.dtu.dk/services/Signal P), one program based on neural networks for the analysis of Signal sequences {Nielsen H. et. al. (1997) Protein Engineering 10: 1-6)). This results in for the processed protein (without signal peptide) a molecular weight (MW) of 70.3 kD (70323 Dalton) and an isoelectric point of pH = 7.01.

The genomic structure is also largely clear. The H-Sema-L gene has 13 or 15 or more exons, preferably 14 exons and 12 or 14 introns, preferably 13 introns. Because of this complex exon-intron structure different splice variants possible. The mRNA of the transcribed H-Sema-L- The Northern blot contains genes mainly in placenta, gonads, thymus and Spleen. There was no mRNA in neuronal tissue or in muscle tissue proven. An indication of a specifically regulated expression in Endothelial cells are present.  

Alternative splicing can also be used to create forms of H-Sema-L intracytoplasmic sequences arise that play a role in the intracellular Play signal transduction, similar to e.g. B. with CD100. Would also be possible secreted forms of H-Sema-L resulting from alternative splicing, analogously to the viral AHV scheme.

Nucleotide and amino acid sequence analyzes were performed using the GCG program Packages (Genetics Computer Group (1991) Program manual for the GCG package, version 7, 575 Science Drive, Wisconsin, USA 53711), FASTA (Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85, 2444-2448) and BLAST-Program (Gish and States (1993) Nat. Genet. 3, 266-272; Altschul et al. (1990) J. Mol. Biol. 215, 403- 410) is used. These programs were also used for sequence comparisons GenBank (version 102.0) and Swiss Prot (version 34.0) are used.

Post-translational modifications such as glycosylation and myristilation of H-Sema-L are also possible. Consensus sequences for N-glycosylation sites were found using the Prosite program (GCG program package) at positions 105, 157, 258, 330, 602 of the amino acid sequence of H-Sema-L (according to Table 4), those for myristilation at the positions 114, 139, 271, 498, 499, 502, 654 (consensus sequence: G ∼ (E, D, R, K, H, P, F, Y, W) x (S, T, A, G, C , N) ∼ (P)). In addition, the amino acid sequence of H-Sema-L contains several consensus sequences for potential phosphorylation sites by different kinases. Therefore it can be assumed that H-Sema-L can be the substrate of different kinase, e.g. B. phosphorylation sites for creatine kinase 2 , protein kinase C and tyrosine kinase.

Predicted creatine kinase 2 phosphorylation sites (consensus sequence Ck2: (S, T) x2 (D, E)) (Prosite, GCG) at positions 119, 131, 173, 338, 419, 481 of Amino acid sequence.

Predicted protein kinase C phosphorylation sites (consensus sequence PkC: (S, T) x (R, K)) (Prosite, GCG) at positions 107, 115, 190, 296, 350, 431, 524, 576 of the amino acid sequence.

Predicted Tyrosine Kinase Phosphorylation Sites (consensus sequence: (R, K) x {2,3} (D, E) x {2,3} Y) (Prosite, GCG) at position 205 of the amino acid sequence.  

The consensus sequences are given in the one-letter code for amino acids.

An "RGD" motif characteristic of integrins (arginine-glycine-aspartic acid) can be found at position 267.

The glycosylation sites are well conserved between the AHV viral scheme, H-Sema-L and (as far as known) M-Sema-L.

Di- or multimerization of H-Sema-L is possible and has been done in others Semaphorins such as CD100 are described {Hall et al. (1996)}. The CD100 molecule is also a membrane-anchored glycoprotein dimer of 150kd. However, CD100 is not closely related to the human semaphorin (H-sema L).

The partial cDNA sequence of M-Sema-L is 1195 nucleotides in length. This sequence encodes a 394 amino acid protein. This 394 Amino acids correspond to amino acids 1 to 396 of H-Sema-L. The Signal peptide in M-Sema-L extends over amino acids 1 to 44 (just like in H-Sema-L). The Sema domain begins at amino acid 45 and extends to the end or probably beyond the end of the sequence according to Table 4 out.

Multiple alignments were carried out using the Clustal W program (Thompson et al. (1994)). These alignments were further processed manually with the help by SEAVIEW (Galtier et al. (1996) Comput. Appl. Biosci 12, 543-548). The Phylogenetic distances were measured with Clustal W (Thompson et al. (1994)) certainly.

A comparison of the protein sequences of the known and the new semaphorins and a phylogenetic analysis of these sequences shows that the genes be classified according to their phylogenetic relationship. Flows here of course the C-terminal domain structure of the corresponding semaphorin Subtypes as a deciding factor, which is why semaphorins are the same Subgroups are usually phylogenetically more closely related than  Semaphorins of different subgroups. Which one has influence Species the semaphorin was isolated, i. H. whether the appropriate species are phylogenetically closely related or not.

A phylogenetic analysis (see FIG. 3) of the known semaphorin amino acid sequences (complete sequences and / or partial sequences, the amino acid sequences being used for H-sema-L and M-Sema-L according to Tables 4 and 5, for all other sequences , the sequences stored under the accession numbers or the coding amino acid sequences derived from these sequences)) by means of the program CLUSTALW {Thompson JD et al. (1994) Nucleic Acids Res. 22: 4673-4680} shows that the amino acid sequences of H-Sema-L and M-Sema-L are closely related phylogenetically and form their own phylogenetic group. H-Sema L and M-SemaL, in turn, are phylogenetically most closely related to AHV-Sema and Vac-A39. They are much more closely related to each other than to any other semaphorin known to date. The analysis also shows that other semaphorins are closely related in phylogenetic terms and form separate groups within the semaphorins. For example, the semaphorins that are secreted, e.g. B. H-Sema-III-IV, -V, and -E into a phylogenetic group. This subfamily also includes their homologs in other species, while the human (transmembrane) CD100 with the corresponding mouse homolog (M-Sema G2) and with collapsin-4 falls into a phylogenetic group.

With regard to the total amino acid sequences, the observed ones lie Homologies within the phylogenetic groups between about 90% and 80% Amino acid identity with respect to very closely related genes such as e.g. B. H and M-Sema-E or -III / D and a little less than 40% with little related genes of Semaphorins. The observed one lies within the Sema domain Amino acid identity a few percent higher, and due to its large share in Total protein (50-80% of the protein belongs to the Sema domain) Amino acid sequence significantly affects overall identity. H-Sema-L, calculated on the total protein, is 46% identical to AHV-Sema,  on the other hand, if the Sema domain is considered alone, the Amino acid identity 53%. This is higher than e.g. B. between relatives M-Sema-B and -C (37% identity in relation to the total protein, 43% identity in In terms of the Sema domain), similar to M-Sema-A and -E (43% total protein, 53% Sema domain). The amino acid identity between the partial M-Sema-L Sequence (Table 6) and H-Sema-L (Table 5) is in the range of the Sema domain at 93%, so that it can be assumed that this is the correspondingly homologous mouse gene.

Corresponding semaphorins to H-Sema-L and M-Sema-L in other species can have an amino acid identity greater than 40% im within the Sema domain Have reference to H-Sema-L. In the closely related vertebrates (mammals, Birds) can even find amino acid identities over 70%.

It is a new subfamily of semaphorins with larger ones Amino acid identity to the viral AHV scheme as to the previously known human or murine semaphorins, and with one for human semaphorins previously unknown C-terminal structure. These new semaphorins (members of the subfamily) are characterized by the fact that they are based on their domains structure fall into subgroup IV and / or the same phylogenetic group fall like H-Sema-L and M-Sema-L and / or in relation to the whole Amino acid sequence to H-Sema-L an amino acid identity of at least 30 to 40%, preferably 50 to 60%, particularly preferably 70 to 80% or one have a greater identity and / or in relation to the Sema domain Amino acid identity with H-Sema-L of at least 70%, preferably greater than 80%, particularly preferably have greater than 90%.

Type L semaphorins also come with a different type of biochemical Function too. A new function of these semaphorins is the modulation of the Immune system.

The closest relative of H-Sema L is the viral AHV semaphorin (AHV-Sema). This is of a similar size, but unlike the H-Sema L, it has none Transmembrane domain. The AHV-Sema is believed to be from virus-infected cells  secreted to the H-Sema L equivalent receptor (semaphorin of type L in Blue Wildebeest) in the natural host (Blue Wildebeest), and thus the attack of the To escape the immune system. It also functions as a repulsive agent (Chemorepellent) conceivable for cells of the immune system.

The biochemical function of the new type L semaphorins and their derivatives can be regarded as generally immunomodulating and / or inflammatory. On the one hand, they can

• A) as molecules that inhibit the immune response either locally, e.g. B. as Transmembrane protein on the surface of cells or even larger ones Distances, e.g. B. if by processing (z. B. proteases) or alternative splicing are secreted, e.g. B. by diffusion in the tissue, their Develop effect as a chemorepellent and / or immunosuppressant. For example, the expression of these new semaphorins of type L e.g. B. on the surface of the cells of the vascular endothelia the leukocyte Prevent attachment and its migration through the vessel wall. The New semaphorins can play a role in maintaining Barrier effects, e.g. B. to prevent infections in particular "important" or exposed organs, for example for maintenance the blood-brain barrier, the placental circulation and / or others immunologically privileged places (e.g. pancreatic islands) and / or at Protection against autoimmune diseases. In addition, the new semaphorins and / or their derivatives can be found in different tissues also on repulsive signals, for example for Immune system cells (e.g. leukocytes) as protection against accidental Activation of defense mechanisms may be involved.
• B) Furthermore, the new semaphorins and / or their derivatives Functions as accessory molecules. On the cell surface they can be expressed, for example, by the interaction with cells of the Immune system involved in the activation of defense mechanisms be.

This creates several uses for the new ones Type L sempahorins and their derivatives and those for these proteins coding nucleic acids.

Function A): It is an immunosuppressive and / or anti-inflammatory principle: numerous potential applications lie in the areas of organ transplantation, inflammation therapy, immunotherapy and gene therapy.

For example, using the semaphorin-encoding DNA or derivatives the same non-human, transgenic animals are produced.

An application of these animals is in the inhibition of Graft rejection in transgenic models for organ transplants. For example, transgenic animal organs protected against rejection for xenotransplantation. This should e.g. B. together with other transgenes (e.g. complement regulators such as DAF or CD59) are possible be.

Another application is in the production of non-human knock-out Animals, for example from knock-out mice: by knock-out of the mouse gene M-Sema-L can e.g. B. further functions of the gene can be found. they provide also represent potential model systems for inflammatory diseases if the mice are viable without a semaphorin gene. Should M-Sema-L for immunomodulation important, such mice are to be expected.

Furthermore, non-human knock-in animals, for example mice, can be produced become. Here, for. B. M-Sema-L by normal / changed H-Sema-L or changed M-Sema-L (e.g. integration of the new semaphorin subtypes under the Control of constitutive and / or inducible promoters) replaced. Such Animals can e.g. B. for the search for further functions of the new semaphorins, e.g. B. functions of the human gene or derivatives of these genes serve or Identification and characterization of immunomodulating agents used become.

Use for the production of e.g. B. recombinant immunosuppressive, others  soluble proteins or peptides that differ from the amino acid sequence of the Type L semaphorins, e.g. B. from H-Sema-L or the corresponding Nucleic acids, e.g. B. derive genes; also agonists with a structural similarity are possible. These immunosuppressive agents / agonists can Autoimmune diseases and inflammatory diseases and / or Organ transplants are used.

Gene therapy with type L semaphorins, e.g. B. with sequences for H-Sema-L or their derivatives encode using viral or non-viral methods. Use at Autoimmune diseases and inflammatory diseases, the transduction of Organs as well as before / during / after transplants to prevent Graft rejection.

One embodiment is a method in which the new semaphorins and / or the DNA coding for these semaphorins and derivatives thereof, in particular H-Sema-L, DNA encoding H-Sema-L and derivatives thereof in one Method for screening active substances, in particular for identification and Characterization of immunomodulating agents.

Function B): H-Sema-L is an accessory molecule that is on the cell surface is expressed and in the interaction with cells, e.g. B. the immune system, e.g. B. as accessory molecule in the activation of signaling pathways, is involved. A viral one Gene or the gene product of a viral or other pathogenic gene z. B. microbiological origin could e.g. B. as a competitive inhibitor of this accessory molecule. An application for the new semaphorins lies with this function also in the area of organ transplantation, Inflammation therapy, immunotherapy and / or gene therapy.

For example, the semaphorins according to the invention can be used in one process for the screening of antagonistic agents / inhibitors that are used then z. B. can be used to block the semaphorin receptor. Soluble and / or secreted H-Sema-L antagonists / inhibitors can for example chemical substances or the new semaphorins or Derivatives of the same (e.g. parts / shortened forms of the same, for example without Membrane domain or as Ig fusion proteins or peptides derived therefrom, which are suitable for blocking the corresponding receptor). To this  Specific antagonists and / or inhibitors identified in this way can For example, act competitively and in the inhibition of rejection z. B. in transgenic models for organ transplants and autoimmune diseases, inflammatory diseases and organ transplants are used. DNA which codes for the semaphorins according to the invention or whose with the aid of Derivatives generated by molecular biological methods can be used, for example, for Production of non-human, transgenic animals can be used. A Overexpression of H-Sema-L can increase in these transgenic animals Susceptibility to autoimmune diseases and / or inflammatory diseases to lead. The transgenic animals are therefore suitable for screening new, specific, immunomodulating agents.

This DNA can also be used for the production of non-human knock-out animals, for example knock-out mice in which the mouse gene M-Sema-L is turned off, used. Such knock-out animals can be used for that Search for other biochemical functions of the gene to be used. she also represent potential model systems for inflammatory diseases if the Mice are viable without the M-Sema-L gene.

This DNA can also be used to produce non-human knock-in animals, for example, mice can be used. The M-Sema-L gene is thereby an altered M-Sema-L gene / cDNA or an altered, e.g. B. mutated semaphorin type L gene / cDNA of another species, e.g. B. from H-Sema-L replaced. Such transgenic animals can be used to search for additional functions of the semaphorins according to the invention can be used.

The invention also relates to the use of type L semaphorins and their Derivatives, as well as the genes / cDNAs coding for these proteins and their Derivatives and / or active ingredients identified with the help of these semaphorins Manufacture of drugs. For example, drugs can be made that can be used in gene therapy and the agonists and / or antagonists of expression of the semaphorins of type L, for example from H-Sema-L. This can be done using viral and / or non-viral methods be used. These drugs can e.g. B. in autoimmune diseases and inflammatory diseases, organ transplants before and / or during  and / or used after the transplant to prevent rejection become.

Use of the new semaphorins, especially H-Sema-L and Genes / cDNAs thereof in methods for screening new drugs. Modified proteins and / or peptides that differ from H-Sema-L and / or M-Sema-L can derive to search for the corresponding receptor and / or its Antagonists or agonists in functional tests, for example using Expression constructs from H-Sema-L and homologues can be used. The genes coding for the new semaphorins, cDNAs and their derivatives can also be used as aids in molecular biology.

The invention relates to the use of a type L or a semaphorin Nucleic acid sequence coding for a type L semaphorin in one Methods for the identification of immunomodulating agents.

The invention also relates to methods for identifying active substances, wherein a Type L semaphorin or a derivative thereof or a nucleic acid sequence, which codes for a type L semaphorin or a derivative thereof is used to identify immunomodulating agents. For example, the Invention a method in which a type L semaphorin under defined Conditions are incubated with an active ingredient to be examined and in parallel second approach without the active ingredient to be investigated, but under otherwise is carried out under the same conditions and then the inhibiting or activating effect of the drug to be investigated is determined. For example, the invention also relates to methods for identifying Active substances, wherein a nucleic acid sequence, which is for a type L semaphorin encoded or a derivative thereof under defined conditions in the presence of an active substance to be examined is expressed and the extent of Expression is determined. If necessary, even in such a procedure two or more approaches carried out in parallel under the same conditions are, but the approaches different amounts of the investigated Active ingredient included.

For example, the active ingredient to be examined can include the transcription and / or the  Inhibit or activate translation.

Test conditions used in the examples:

PCR programs used:

Taq52-60 (with Ampli-Taq ^R polymerase, Perkin Elmer, Weil der Stadt, Germany)
96 ° C / 60s: 1 cycle
96 ° C / 15s-52 ° C / 20s-70 ° C / 60s: 40 cycles
70 ° C / 60s: 1 cycle

Taq60-30
96 ° C / 60s: 1 cycle
96 ° C / 15s-60 ° C / 20s-70 ° C / 30s: 35 cycles
70 ° C / 60s: 1 cycle

Taq60-60
96 ° C / 60s: 1 cycle
96 ° C / 15s-60 ° C / 20s-70 ° C / 60s: 35 cycles
70 ° C / 60s: 1 cycle

Taq62-40
96 ° C / 60s: 1 cycle
96 ° C / 15s-62 ° C / 20s-70 ° C / 40s: 35 cycles
70 ° C / 60s: 1 cycle.

Reaction conditions used for PCR with Taq polymerase:
50µl reaction batches with 100-200ng template, 200µM dNTP, 0.2-0.4 µM per primer, 2.5U Ampli-Taq ^R , 5µl of the 10x reaction buffer supplied.

Programs used for:

• 1. XL62-6 (with Expand-Long Template PCR System ^R , Boehringer Mannheim, Germany)
  94 ° C / 60s: 1 cycle
  94 ° C / 15s-62 ° C / 30s-68 ° C / 6min: 10 cycles
  94 ° C / 15s-62 ° C / 30s-68 ° C / (6min + 15s / cycle): 25 cycles
  68 ° C / 7min: 1 cycle
• 2. XL62-12 (with Expand-Long Template PCR System ^R , Boehringer Mannheim, Germany)
  94 ° C / 60s: 1 cycle
  94 ° C / 15s-62 ° C / 30s-68 ° C / 12min: 10 cycles
  94 ° C / 15s-62 ° C / 30s-68 ° C / (12min + 15s / cycle): 25 cycles
  68 ° C / 7min: 1 cycle.

Reaction conditions for PCR with Expand-Long Template PCR System 50µl reaction batches with 100-200ng template, 500µM dNTP, 0.2-0.4 µM each Primer, 0.75µl enzyme mix, 5µl of the supplied 10x reaction buffer No. 2.

example 1

Starting from sequences of the AHV scheme (Ensser and Fleckenstein (1995), J. General Virol. 76: 1063-1067) PCRs and RACE-PCRs were carried out. As The starting material for this was human cDNA from placenta tissue, to which RACE amplification adapters were ligated (Marathon ™ cDNA Amplification Kit, Clontech Laboratories GmbH, Tullastraße 4, 69126 Heidelberg, Germany). First, using a specific primer (No. 121234 + No. 121236, Table 6) PCR fragment with a length of about 800 bp (base pairs) amplified (PCR Program: (Taq60-60)). This was cloned and sequenced (Taq-Dye-Deoxy- Terminator sequencing kit, Applied Biosystems, Forster: City, CA, USA) Brunnenweg 13, Weilderstadt). Sequencing of the PCR product revealed one Sequence that has high homology to the DNA sequence of AHV-Sema, identical to the sequence of the two ESTs.

A PCR fragment of 600 bp was mixed with the primer pair (No. 121237 + No. 121239, Table 6) identified. It turned out to be clones with DNA sequences acted on the same gene.  

Example 2

The 800bp PCR fragment from Example 1 was radioactively labeled (random priming according to the method of {Feinberg (1983) Anal. Biochem. 132: 6-13}, with ³² P-α-dCTP) and as a probe for a multi- Tissue Northern blot (Human Multiple Tissue Northern Blot II, Clontech, Heidelberg, Germany), which contains mRNA samples from the tissues spleen, thymus, prostate, testis, ovaries, small intestine, large intestine and leukocytes (PBL). The expression of an mRNA with a length of about 3.3 kb was clearly shown in the spleen and gonads (testes, ovaries), and weaker in the thymus and intestine. Hybridization of a master blot (dot blot with RNA from numerous tissues (Human RNA Master Blot ™, Clontech) confirmed this result and also showed a strong expression in placenta tissue.

The hybridization was carried out for 16 hours under stringent conditions (5xSSC, 50 mM Na phosphate pH 6.8, 50% formamide, 100 pg / ml yeast RNA) at 42 ° C carried out. The blots were washed stringently (65 ° C, 0.2XSSC, 0.1% SDS) and a Fuji BAS2000 Phosphoimager ™.

Example 3

A cDNA library from human spleen, cloned in the bacteriophage lambda gt10 (Human Spleen 5 'STRETCH PLUS cDNA, Clontech) was searched with this probe and a lambda clone was identified. The cDNA with a length of 1.6 kb inserted in this clone was amplified by means of PCR (Expand ™ Long Template PCR System, Boehringer Mannheim GmbH, Sandhofer Strasse 116, 68305 Mannheim), the vector-specific primers No. 207608 + No. 207609 (Table 6) were used (flanking the EcoRI cloning site) and the PCR fragment obtained was sequenced. This clone contained the 5 'end of the cDNA and extended the known cDNA sequence even after 3'. Based on the new partial sequences of the cDNA, new primers for RACE-PCR were developed (No. 232643, No. 232644, No. 233084, Table 6). Together with an improved thermal cycler technology (PTC-200 from MJ-Research, Biozym Diagnostik GmbH, 31833 Hess. Oldendorf) with significantly better performance data (heating and cooling rate) a 3'-RACE PCR product was amplified, whereby the primer no. 232644 resp No. 232643 and AP1 were used and cloned into the vector pCR2.1 (Invitrogen, De Schelp 12, 9351 NV Leek, The Netherlands). The 3 'RACE PCR product was sequenced and the 3' end of the cDNA was identified in this way. RACE amplification after 5 '(primer no. 131990 or no. 233084 and AP1) extended the 5' end of the cDNA by a few nucleotides and confirmed the amino terminus of the H-Sema-L found in the identified lambda clone.

Example 4

Starting from a short murine EST (assignment no. AA260340) and one derived primer No. 260813 (Table 6) and the H-Sema-L specific Primer No. 121234 (Table 6) was applied by PCR (conditions: Taq52-60) DNA fragment with a length of about 840 bp murine cDNA amplified and in the Vector pCR2.1 cloned. The gene containing this DNA fragment was M-Sema-L called. A cDNA library with the M-Sema-L DNA fragment obtained from mouse spleen (Mouse Spleen 5 'STRETCH cDNA, Clontech) examined, where several clones have already been identified.

PCR (Taq60-30) with primers No. 260812, No. 260813 from murine, endothelial cDNA provided a PCR fragment with a length of 244 base pairs. The PCR Results showed significant basal expression in murine Endothelial cells are present, which after stimulation with the cytokine interferon-y and lipopolysaccharides declines.

Example 5

Chromosomal localization studies were performed using fluorescence in situ hybridization (FISH) performed. To do this, metaphase chromosomes of humans and mice, based on a human blood sample or the BINE 4.8 mouse cell line (Keyna et al. (1995) J. Immunol. 155, 5536-5542) (Kraus et al. (1994) Genomics 23, 272-274). The slides were made with RNase  and treated with pepsin (Liehr et al. (1995) Appl. Cytogenetics 21, 185-188). For the Hybridization was 120 mg of human nick-translated semaphorin sample or 200 mg of a corresponding mouse sample was used. The hybridization was each in the presence of 4.0 µg COT1-DNA and 20 µg STD at 37 ° C (3 days) in a humidified chamber.

The slides were washed with 50% formamide / 2 × SSC (3 times 5 min at 45 ° C) and then washed with 2 × SSC (3 times for 5 min at 37 ° C.) and the biotinylated sample with the FITC-Avidin system (Liehr et al. (1995)). The slides were evaluated using a fluorescence microscope. There were 25 Metaphases / sample evaluated, with each experiment performed twice has been.

It can be seen that H-Sema L is localized on chromosome 15q23. Chromosomal the locus for the Bardet-Biedl-Sydrom and Tay-Sachs are adjacent Disease (hexosaminidase A).

Example 6

The genomic intron-exon structure of the H-Sema L gene is largely enlightened.

Genomic DNA fragments were amplified from 250 mg of human genomic DNA isolated from PHA-stimulated peripheral lymphocytes (blood): shorter fragments were ampli Taq ^R (Perkin Elmer), longer fragments with the Expanded Long Template PCR System ^R ( Boehringer Mannheim) amplified.

Until now, almost the entire genomic locus of the H-Sema-L can be cloned and characterized. Overall, more than 8888 bp of the genomic sequence can be determined and so the intron exon Structure of the gene to be largely elucidated.  

Example 7 Expression cloning

Since there is no complete clone of the semaphorin gene from the Lambda gt10 cDNA library could be isolated, and a complete clone could not be obtained by PCR either the coding region of the cDNA was overlapped in 2 Subfragments using PCR (XL62-6) with the help of primer no. 240655 and no. 121339 for the N-terminal DNA fragment, and primer no. 240656 (contains HindIII and Pmel interfaces) and No. 121234 for the C-terminal DNA fragment amplified. The DNA fragments (subfragments) obtained were inserted into the vector pCR21 cloned. The two subfragments were completely sequenced and finally the complete H-Sema-L CDNA by inserting a 0.6kb C-terminal SstI-HindIII restriction fragment into the one with the restriction enzymes SstI and HindIII cut plasmid containing the N-terminal DNA fragment, manufactured. From this plasmid pCR2.1-H-Sema-L (according to the sequence in Table 7) the complete gene was identified using the EcoRI site (in pCR2.1) and HindIII Cut out the interface (in primer no. 240656, table 6) and into one correspondingly cut, constitutive expression vector pCDNA3. 1 (-) MycHisA (Invitrogen) ligated. From the resulting recombinant plasmid pCDNA3. 1 (-) H- Sema-L-MycHisA (according to the sequence in Table 8) became the EcoRI-Apal fragment (without Myc-His tag) cut out and ligated into the inducible vector pIND (Ecdyson-Inducible Mammalian Expression System, Invitrogen), who also previously was cut with EcoRI-Apal. The recombinant plasmid was made with pIND-H-Sema-L-EA (sequence according to Table 9). An EcoRI pmel Fragment (with Myc-His tag) from pCDNA3.1 (-) H-Sema-L-Myc-HisA (sequence according to Table 9) was pIND into a vector cut with EcoRI-EcoRV used. The recombinant plasmid was treated with pIND-H-Sema-L-EE (sequence referred to in Table 10).

A fusion gene from H-Sema-L with Enhanced Green Fluorescent Protein (EGFP) was made by ligation of the PCR amplified EGFP reading frame (from the vector pEGFP-C1 (Clontech), with the aid of the primer no. 243068 + no. 243069, Taq52-60) into the Pmel site of the plasmid pCDNA3.1 (-) H-Sema-L- MycHisA thus the plasmid pCDNA3. 1 (-) H-Sema-L-GFP-MycHisA (sequence  according to Table 11) was obtained.

Example 8

For the production of H-Sema-L specific antibodies, cDNA fragments of H-Sema-L were integrated into prokaryotic expression vectors, extracted in E. coli and the semaphorin derivatives were purified. The semaphorin derivatives were expressed as fusion proteins with a His-Taq. Accordingly, vectors were used which contain the sequence for a His-Taq and which made it possible to integrate the semaphorin cDNA fragment into the reading frame. An N-terminal 6x histidine tag enables, for example: a purification using nickel chelate affinity chromatography (Qiagen GmbH, Max-Volmer Strasse 4, 40724 Hilden):

• 1. The part of the H-Sema-L cDNA coding for amino acids 179-378 was amplified by PCR with primers No. 150788 and No. 150789 and this DNA fragment in the vector pQE30 (Qiagen), which was previously with the Restriction enzymes BamHI and HindIII had been cut, ligated (Construct pQE30-H-Sema-L-BH (sequence according to Table 12)).
• 2. The section of the coding for the C-terminal amino acids 480-666 H-Semal-L cDNA was obtained with the restriction enzymes SstI and HindIII from the Plasmid pCR 2.1 cut and into the vector pQE31 (Qiagen), which previously had been cut with SstI and HindIII (construct pQE31-H- Sema-L-SH (sequence according to Table 13)).

The correct integration of the sequences in the correct reading frame was checked by DNA sequencing. The fusion proteins, consisting of an N-terminal 6xhistidine Taq and part of the semaphorin H-Sema-L, were purified by means of Ni ²⁺ affinity chromatography. The purified fusion proteins were used to immunize various animals (rabbit, chicken, mouse).

Illustrations Fig. 1 Tissue specific expression of H-Sema L

• A) Multiple-tissue Northern Blot (Clontech, Heidelberg, Germany). From left to right are plotted: 2 µg poly-A-RNA per trace from spleen, Thymus, prostate, testicles, ovaries, small intestine, large intestine mucosa, peripheral (blood) leukocytes. Sizing standards are marked.

The blots were made under stringent conditions with an 800 base pair long H-Sema L probe hybridized.

Fig. 2

Schematic representation of the cloning of the H-Sema L cDNA and the genomic organization of the H-Sema L coding sequences (H-Sema L gene) Above: Localization of the EST sequences (access numbers; location of the EST Sequences is shown relative to the AHV-Sema sequence).

Below: amplified PCR and RACE products as well as the position of the cDNA Clones for localization in the full H-Sema L cDNA and the open reading frame (ORF) for the encoded protein.

Bottom: Relative position of the exons in the H-Sema L gene in relation to the genomic Sequence.

The position of the oligonucleotide primer used is indicated by arrows.

Fig. 3

Phylogenetic tree: obtained by multiple alignment of the listed ones Semaphorin sequences. Due to the grouping of the semaphorins in the  phylogenetic tree can be based on their phylogenetic relationship getting closed.  

Table 2

H-Sema-L cDNA sequence (2636 nucleotides) (SEQ ID NO .: 1)

Table 3

Nucleotide sequence of the M-Sema-L cDNA (partial, 1195 nucleotides) (SEQ ID NO.:2)

Table 4

H-Sema-L amino acid sequence (666 amino acids) (SEQ ID NO .: 3)

Table 5

(Partial) amino acid sequence of M-Sema-L (394 amino acids, corresponds to position 1-396 of H-Sema-L) (SEQ ID NO .: 4)

Table 6

Synthetic oligonucleotides, (Eurogentec, Seraing, Belgium)

Table 7

Nucleotide sequence of the recombinant plasmid pCR2.1-H-Sema-L (SEQ ID NO .: 34)

Table 8

Nucleotide sequence of the recombinant expression plasmid pCDNA3.1 (-) H-Sema-L-MycHisA (SEQ ID NO .: 35)

Table 9

Nucleotide sequence of the recombinant plasmid pIND-H-Sema-L-EA (SEQ ID NO .: 36)

Table 10

Nucleotide sequence of the recombinant plasmid pIND-H-Sema-L-EE (SEQ ID NO .: 37)

Table 11

Nucleotide sequence of the recombinant plasmid pCDNA3.1 (-) H-Sema-L-GfP-MycHisA (SEQ ID NO .: 38)

Table 12

Sequence of the recombinant plasmid pQE30-H-Sema-L-BH (SEQ ID NO .: 39)

Table 13

Sequence of the recombinant plasmid pQE31-H-Sema-L-SH (SEQ ID NO .: 40)

Table 14

(Partial) nucleotide sequence of the human semaphorin L gene. (8888 nucleotides) (SEO ID NO .: 41)

SEQUENCE LOG

Claims (16)

1. Semaphorin containing a characteristic Sema domain, characterized in that the protein has an N-terminal signal peptide and in the C-terminal region an immunoglobulin-like domain and a transmembrane domain, the semaphorin being referred to as type L semaphorin (Sema L) and derivatives of type L semaphorin 2. Semaphorin according to claim 1, characterized in that the protein (human semaphorin (H-Sema L)) the amino acid sequence SEQ ID NO. 3 has. 3. Semaphorin according to one or more of claims 1 and 2, characterized characterized in that the protein in the region of the Sema domain Amino acid identity of at least 40% with respect to the Sema domain of H-Sema L. 4. Semaphorin according to one or more of claims 1 to 2, characterized characterized in that the protein has the partial amino acid sequence SEQ ID NO. 4th contains (murine semaphorin (M-Sema L)). 5. Nucleic acid containing a nucleic acid sequence necessary for a semaphorin type L encoded according to one or more of claims 1 to 4 and Derivatives of the same. 6. The nucleic acid of claim 5, wherein said nucleic acid sequence is one Semaphorin L gene is. 7. Nucleic acid according to one or more of claims 5 and 6, wherein said nucleic acid sequence contains the gene of H-Sema L. 8. The nucleic acid of claim 5, wherein said nucleic acid sequence Contains a type L semaphorin cDNA.   9. The nucleic acid of claim 8, wherein the cDNA is the cDNA of M-Sema L. 10. A method for producing a type L semaphorin according to one or several of claims 1 to 4, characterized in that a nucleic acid Sequence coding for a type L semaphorin into an expression vector is cloned and expressed. 11. The method according to claim 10, characterized in that for the Expression a eukaryotic cell is used. 12. Use of a type L semaphorin or a derivative thereof or a nucleic acid sequence which codes for a type L semaphorin or a derivative thereof for the manufacture of a medicament for treatment or prevention of immunological diseases can be used. 13. Use of a nucleic acid sequence or a derivative thereof according to claim 12 in gene therapy. 14. Use of a type L semaphorin or a nucleic acid Sequence coding for a type L semaphorin in a method for Identification of immunomodulating agents. 15. A method for identifying immunomodulating agents, thereby characterized in that a semaphorin of type L under defined conditions with a drug to be investigated is incubated, in parallel a second batch without the active substance to be investigated, but under otherwise identical conditions is carried out and then the inhibitory or activating effect of the investigating active ingredient is determined. 16. A method for identifying immunomodulating agents, thereby characterized in that a nucleic acid sequence for a semaphorin of the type L codes under defined conditions and in the presence of one to be examined Active ingredient is expressed and the extent of expression is determined.