has also been shown to contain somatomedin B sequences at its amino terminus. We conclude that S-protein and vitronec- tin are identical and discuss the ...
The EMBO Journal vol.4 no.12 pp.3153-3157, 1985
Molecular cloning of S-protein, a link between complement, coagulation and cell-substrate adhesion
Dieter Jenne' and Keith K.Stanley2 lInstitute of Medical Microbiology, Justus-Liebig-University in Giessen, Schubertstrasse 1, 6300 Giessen, and 2European Molecular Biology Laboratory, Meyerhofstrasse 1, Postfach 10.2209, 6900 Heidelberg, FRG Communicated by R.Cortese
cDNA clones coding for human S-protein have been isolated using monoclonal antibodies to screen a cDNA library in pEX. These clones are shown to be authentic S-protein clones on the basis of sequence, composition and immunological criteria. The complete open reading frame sequence for S-protein has been determined and shows it to be a single polypeptide chain of 459 amino acids preceded by a cleaved leader peptide of 19 residues. No evidence was found for polymorphism of Sprotein suggesting that different molecular weight forms arise by proteolytic degradation. Of the first 44 amino-terminal residues 42 are identical with the so-called somatomedin B peptide suggesting that S-protein is the somatomedin B precursor. Striking homology is found in the rest of the sequence with the serum spreading factor, vitronectin, which has also been shown to contain somatomedin B sequences at its amino terminus. We conclude that S-protein and vitronectin are identical and discuss the relevance of this finding to the coagulation and complement pathways. Key words: cell spreading/SC5b-9 complex/somatomedin B/ thrombin/vitronectin Introduction S-protein is found at high concentrations in human plasma (140-700 jig/ml, Podack and Miiller-Eberhard, 1979; Jenne et al., 1985b; Dahlback and Podack, 1985) and is able to bind to protein complexes in the terminal stages of both the complement and coagulation pathways. Stable complexes of S-protein with the terminal complement components have been observed after C5 is activated via the alternative pathway (Kolb and MullerEberhard, 1975; Bhakdi and Roth, 1981), in C8- or C9-depleted serum (Podack et al., 1977; Bhakdi and Roth, 1981), and with detergent-solubilised C5b-9 (Bhakdi and Tranum-Jensen, 1982b). In all these situations the C5b-7 complex is generated in the absence of a target lipid bilayer. Binding of S-protein to this fluid phase C5b-7 may prevent its attachment to cell membranes, and although C8 and a few molecules of C9 can still bind (Kolb and Miiller-Eberhard, 1975), the cytolytic terminal complex containing polymerised C9 is not formed (Podack et al., 1984; Dahlback and Podack, 1985). SC5b-9 complex formation has also been inferred during complement attack on some bacteria (Joiner et al., 1982) and in systemic lupus erythematosus (Falk et al., 1985). If the S-protein is dissociated from the fluid phase complex by detergent or proteolytic treatment apolar surfaces become exposed and the resulting complexes aggregate (Bhakdi et al., 1979; Podack and Miller-Eberhard, 1980; Bhakdi and TranumJensen, 1982a). The functions of S-protein may therefore be the solubilisation of fluid phase C5b-9 complexes, the protection of (©) IRL Press Limited, Oxford, England
bystander cells against lysis by fluid phase C5b-7 and the inhibition of C9 polymerisation during fluid phase assembly. S-protein may also have a physiological role in the coagulation pathway since S-protein can be observed in a complex with thrombin in serum (after coagulation), but not in plasma (Podack and Muller-Eberhard, 1979). This complex has been shown to be a stable trimolecular complex containing antithrombin HI in addition (Jenne et al., 1985a). S-protein can modulate the activity of thrombin by annulling the heparin-dependent activation of the thrombin inhibitor, antithrombin Il (Preissner et al., 1985), and by a direct reduction of antithrombin IH inhibition of thrombin (Jenne et al., 1985a). Here we report the molecular cloning and cDNA sequence of S-protein. Comparison of this sequence with partial peptide sequence data of the serum spreading factor called 'vitronectin' (Holmes, 1967; Hayman et al., 1982, 1983; Barnes and Silnutzer, 1983) shows that the two proteins are identical.
Results Identification and sequencing of S-protein cDNA A mixture of five monoclonal antibodies raised against S-protein purified from SC5b-9 complexes (Jenne et al., 1985b) was used to screen a subcloned human liver cDNA expression library in the bacterial expression vector, pEX (Stanley and Luzio, 1984). From 30 000 independent clones in the library we obtained five clones which expressed antigenic determinants of S-protein (Figure 1). These were colony purified and found to contain cDNA inserts which cross-hybridised. All the clones contained large open reading frames assessed by the size of the hybrid proteins on Western blots showing that the S-protein antigenic determinants were unlikely to be expressed from cDNA fragments fused to the expression vector in missense reading frames. The Si
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Fig. 1. S-protein cDNA clones. (a) Clones SI to S5 were obtained by expression screening of a human liver cDNA library in pEX. Clones S108 and S203 were obtained by DNA hybnrdisation screening of a parent library in pKT218. (b) Shows the assembled structure of the S-protein cDNA with restriction enzyme sites as described in the text. The open box represents the open reading frame. (c) Shows the overlapping M13 clones used to sequence the cDNA.
3153
D.Jenne and K.K.Stanley
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Fig. 2. The nucleotide and amino acid sequence of S-protein. Random fragments of clones S 108 and the ends of S203 were sequenced in M 13 and assembled by computer. The deduced amino acid sequence is shown in single letter code beneath the DNA sequence together with the sequence of fragments of the proteins somatomedin B (a) and vitronectin (b - d). Fragments (b) and (d) were generated by cyanogen bromide cleavage while fragment (c) was cleaved using acid (Suzuki et al., 1984). Underlined asparagine residues are possible sites for attachment of N-linked oligosaccharide, and the dashed lines show the amino-terminal sequences of S-protein fragments previously determined (Dahlback and Podack, 1985).
clone with the longest cDNA insert, S3, was chosen as a DNA probe to screen the parent cDNA library in pKT218 (Woods et al., 1982) by hybridisation. The 13 clones obtained in this way were analysed by digestion with Pvull and Pstl and then by hybridisation with a PvuH-BamHl fragment derived from the 5' end of S3. The clone having the longest fragment at the 5' end, S 108, was sequenced on both DNA strands in M 13 by the shotgun procedure of Sanger et al. (1977). This cDNA clone was also capable of expressing S-protein antigenic determninants when subcloned into pEX (see Figure 3), showing that the antigenic determinants of S-protein were not present at the boundary between the fl-galactosidase of the expression vector and the expressed cDNA fragment. In order to obtain the 5' end of the S protein cDNA it was necessary to performn a second screening of the library in pKT2 18 using the PstI/StuI fragment from the 5' end of S108 as a probe. Among 12 clones isolated, one, S203, was estimated to be 100 bp bigger than S108 by restriction enzyme mapping. The additional sequence data from this clone completed the coding sequence of the S-protein shown in
Figure 2. The cDNA sequence of S-protein is 1604 bp long including region at the 5' end and a stretch of 109 bases containing the polyadenylation signal AAUAAA at the 3' end. The open reading frame codes for 478 residues, of which the first 19 appear to be a cleaved leader peptide since they are very hydrophobic in character, and end with an alanine residue which is a suitable cleavage site for signal peptidase. The predicted amino acid composition of the mature protein agrees well with that determined experimentally in several different laboratories (Table I). In addition the amino termiinal dipeptide of the mature protein agrees with the published amino-terminal sequence of S-protein (Dahliback and Podack, 1985). In order to distinguish different epitopes encoded by the cDNA clones we subcloned the two halves of clone S5 digested with NaeI into pEX and tested the individual expressed fragments with the monoclonal antibodies. The amino-terminal portion of this clone bound monoclonal antibodies 1-4, while the carboxyterminal hybrid protein bound only monoclonal antibody 5 a 6 1-bp untranslated
3154 A
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Molecular cloning of S-protein
1
Table I. Amino acid composition of S-protein and vitronectin
(2)
(3)
Amino acid
(1)
Ala Cys Asp Asn
29.8 9.6
31.9 15.0
28.0 9.6
47.3
52.4
62.9 24.3 38.1 9.6 13.3 22.0 26.2 8.3 35.8 36.7 30.8 19.3 22.0 4.1 22.5
Glu
Gin Phe Gly His Ile Lys Leu Met Pro Arg Ser Thr Val Trp Tyr
(4)
(5)
(6) S 11
55.5
29.8 11.2 43.5
30.8 9.0 50.5
67.0
61.5
62.9
66.1
25.9 42.6 10.5 15.5 27.4 30.7 5.8 39.1 37.4 38.2 20.9 28.1 6.0 23.0
23.9 32.1 10.1 11.9 27.1 37.6 7.8 29.8 27.1 34.9 24.8 23.0 n.d. 14.2
22.5 37.5 17.2 14.2 19.5 30.0 6.7 33.0 33.7 33.0 18.7 21.0 6.0 21.0
22.9 39.0 9.3 13.0 21.8 28.1 5.1 38.5 34.7 28.0 17.4 23.5 n.d. 21.4
28 14 16 34
26 23 37 9 13 20 25 7 35 35 33 19 21 9 23
Comparison of the amino acid composition calculated on the basis of a molecular weight of 52 371 daltons for S-protein (1 -4), vitronectin (5) and the cDNA sequence (6). Values are from (1) Podack and Mulier-Eberhard (1979), (2) Dahlback and Podack (1985), (e) Jenne et al. (1985b), (4) Preissner et al., (1985) and (5) Barnes and Silnutzer (1983). n.d., not determined.
(Figure 3). The epitope for this antibody is therefore located within the last 84 amino acids of the protein. Since the cDNA clones express at least two distinct epitopes of S-protein, and the sequence agrees well with the published amino terminal sequence and composition, we conclude that this sequence codes for authentic S-protein. Proteolytic degradation of S-protein Purified S-protein separates into two major bands on SDS polyacrylamide gels with apparent mol. wts. of - 65 and 75 (Podack and Muller-Eberhard, 1979; Preissner et al., 1985; Dahlback and Podack, 1985; Jenne et al., 1985b). In addition a small peptide of - 12 kd has been reported in SDS gels of reduced preparations of purified S-protein (Podack and Muller-Eberhard, 1979; Dahlback and Podack, 1985) and in plasma where it has been shown to be immune positive (Jenne et al., 1985b; Preissner et al., 1985). Since this fragment is only released after reduction, and it can react with S-protein-specific antibodies, it is most likely derived from the intact molecule by proteolysis. S-protein digested in vitro with trypsin gives rise to a similar pattern of bands on SDS gels (Podack and Muller-Eberhard, 1979). A possible location for a cleavage point is at residue 380, since the sequence at this position has been reported as a contaminating amino-terminal sequence of S-protein (Dahlback and Podack, 1985). The fragment released by cleavage at this position would have a mol. wt. of 9.4 kd. In Figure 3 it can be seen that the 3' end of clone S5 digested with NaeI expresses the epitope of monoclonal antibody 5. Since NaeI cuts the S-protein cDNA such that only four amino acids in addition to the carboxyterminal fragment are expressed, it is very likely that endogenous proteases can cleave S-protein at or near to this site. Another possible origin of heterogeneity is differential splicing within the S-protein mRNA. Although clones S3, S4 and S5 did appear to have slightly longer 3'-PvuH-PstI fragments (Figure
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1), we were unable to detect any differences in the restriction fragment lengths of S-protein cDNA after digestion with ApaI which cuts the S-protein cDNA at the extreme ends of the open reading frame (Figure 1). The extra length of S3-5 is probably due simply to longer poly(A) tails. The size of the S-protein polypeptide estimated from the cDNA sequence is 52 371 daltons as compared with reported values between 66 and 89 kd according to the method used (Podack and Muller-Eberhard, 1979). This difference is presumably due to the addition of carbohydrate to the molecule and possibly also due to an unusual secondary structure since S-protein has a high proportion of proline residues. Three possible attachment sites for N-linked oligosaccharide are found in the sequence (underlined in Figure 1). Comparison of S-protein with vitronectin Comparison of the S-protein sequence with the protein sequence data library of the National Biomedical Research Foundation showed a close homology of S-protein with somatomedin B (Fryklund and Sievertsson, 1978). No other significant homologies were detected. Part of the amino-terminal sequence of somatomedin B has also been reported in the serum-spreading factor called vitronectin (Suzuki et al., 1984). We therefore compared the partial amino acid sequence data for this protein with that of the S-protein (Figure 1). In addition to the somatomedin B sequence all other sequenced regions of the vitronectin molecule showed a very similar and sometimes identical sequence. In each case the amino acid residues preceding these sequences agreed with their origin as cyanogen bromide or acid-cleaved fragments. When the amino acid composition of the serum-spreading factor was compared with that of the S-protein, we found a close correspondence (Table I), also suggesting that the two proteins are very similar, if not identical. 3155
D.Jenne and K.K.Stanley
z
Discussion We have described cDNA clones coding for the entire open reading frame of S-protein. These cDNA molecules code for at least two distinct epitopes recognised by a total of five monoclonal antibodies. Since the antigen used to raise these monoclonal antibodies was S-protein dissociated from SC5b-9 complexes, and the same monoclonal antibodies label S-protein complexed to thrombin-antithrombin III from serum (Jenne et al., 1985b), it is beyond reasonable doubt that these cDNA clones code for authentic S-protein. In addition, the amino terminal dipeptide and composition deduced from the cDNA sequence closely agree with the data obtained from S-protein purified from plasma in several different laboratories (Podack and Muller-Eberhard, 1979; Dahlback and Podack, 1985; Jenne et al., 1985b; Preissner et al., 1985). The sequence of S-protein shows an unexpected homology with two other related plasma proteins, somatomedin B and vitronectin. Somatomedin B was originally characterised as a growthhormone-dependent polypeptide with mitogenic activity towards human glial cells (Uthne, 1973) but later its mitogenic activity was accounted for by small contaminations with EGF (Heldin et al., 1981). In this respect it no longer qualifies for the term 'somatomedin'. Of the first 44 amino acids of S-protein, 42 are identical to those of the somatomedin B peptide (Fryklund and Sievertsson, 1978). The two differences are most likely the result of difficulties in the amino acid sequencing (L.Fryklund, personal communication). S-protein is therefore presumably the precursor of somatomedin B, although no independent function has yet been ascribed to the released peptide. Vitronectin (so called because of its affinity for glass surfaces) is a cell spreading factor from serum (Holmes, 1967; Barnes et al., 1980; Hayman et al., 1982) which has been shown to contain the first 30 residues of somatomedin B at its amino terminus (Suzuki et al., 1984) and which is functionally but not immunologically related to fibronectin (Barnes et al., 1980; Hayman et al., 1983). Comparison of the sequence, biochemical and biophysical properties shows that S-protein and vitronectin are indistinguishable. In addition to the 30 amino acid residues of somatomedin B at the amino terminus of vitronectin, three peptide fragments within the molecule have 68 out of 76 residues in common with S-protein. Furthermore the amino acid composition of serum-spreading factor determined by a different laboratory is very similar to that of S-protein (Barnes and Silnutzer, 1983; Table I). Both proteins are present at a similar concentration in plasma (Barnes et al., 1983; Podack and MullerEberhard, 1979; Jenne et al., 1985b), have a similar multiplebanded appearance on SDS polyacrylamide gels (Suzuki et al., 1984; Hayman et al., 1983; Podack and Miuller-Eberhard, 1979; Dahlback and Podack, 1985; Jenne et al., 1985b), and adhere strongly to surfaces (Holmes, 1967; Podack and MillerEberhard, 1979). We conclude therefore that the two molecules are identical. Using the combined information about S-protein and vitronectin we are able to divide the S-protein sequence into five regions with different functional significance. (i) The first 44 residues of S-protein (somatomedin B) contain four disulphide bonds (Fryklund et al., 1974) and has the appearance of an independently folding cysteine-rich domain. No homology was observed between this region and the high cysteine regions of complement component C9, factor IX or wheat germ agglutinin which represent classes of conserved high cysteine structural motifs found in several other plasma and membrane proteins (Stanley et al., 1985a, 1985b). (ii) Immediately following the somatomedin B 3156
domain is the tripeptide Arg-Gly-Asp which is shared by several molecules having cell attachment activity (Hayman et al., 1985; Pierschbacher and Ruoslahti, 1984). Somatomedin B isolated from plasma is sometimes found with the Arg of this tripeptide at its carboxy terminus (L.Fryklund, personal communication) suggesting that it might be the product of S-protein after inactivation of its spreading activity. It will be interesting to determine if thrombin binding or cleavage occurs at this site since thrombin cleaves the same dipeptide (Arg-Gly) in fibrinogen. Previous experiments investigating cleavage of S-protein or vitronectin by thrombin could only show a cleavage at the carboxytenninal end of the molecule which removes both the heparin -binding site [see (iv) below] and the trypsin fragment [see (v) below; Podack and Miuller-Eberhard, 1979; Silnutzer and Barnes, 1984]. (iii) There then follows a long region (residues 48- 347) which contains the three possible sites for attachment of N-linked oligosaccharides. This region is rich in proline residues but shows no amino acid homology with collagen or fibronectin. It is also densely scattered with hydrophobic residues. (iv) The following 32 residues of the molecule contain 14 positive charges, no negative charges, and only two hydrophobic residues. This region has been identified in vitronectin as a heparin-binding site (Suzuki et al., 1984). The ability of S-protein to abolish the heparin stimulation of antithrombin 11 inactivation of thrombin (Preissner et al., 1985) is likely to be due to a direct interaction of heparin with S-protein at this site. This site could also be involved in the binding of S-protein to the terminal components of the complement pathway since it has been observed that poly-lysine can block the inhibition of C5b-7 by serum factors (Lint et al., 1976). (v) Immediately following the heparin-binding region is a site at which S-protein is found to be partially cleaved in plasma and which is rapidly cleaved when the purified protein is treated with trypsin (Podack and Miller-Eberhard, 1979). Endogenous proteolytic cleavage of this carboxy-terminal fragment accounts for the two molecular weight forms of both S-protein and vitronectin (Suzuki et al., 1984). Using five different monoclonal antibodies no evidence was found for multiple forms of S-protein in the cDNA library. Although not all clones were sequenced, the internal restriction fragments from all the clones were indistinguishable suggesting that only one S-protein polypeptide is synthesised in human liver. This does not rule out the possibilty that a different type of vitronectin is made in tissues other than liver. The identity of S-protein and vitronectin leads to new concepts in the control of the complement and coagulation pathways. Fundamental to these is the observation that S-protein (as vitronectin) is present not only in plasma, but also on the surface of many cells, in loose connective tissue (Hayman et al., 1983) and in platelets (Barnes et al., 1983). In this respect it is like fibronectin, although the two molecules are distinct and even where they are co-localised they are present at different concentrations. When the endothelial cells lining blood vessels are damaged, fibronectin-like molecules (fibronectin and von Willebrand factor) mediate the adhesion of platelets thus localising the deposition of platelets and fibrin in the vicinity of the lesion (Sixma and Wester, 1977; Giddings, 1983). During this process the transglutaminase activity of factor XIIIa covalently cross-links fibronectin to fibrin and collagen. If S-protein were also exposed at these sites it would be expected that it could play an important role in the localisation of the terminal components of both the complement and coagulation pathways at the site of injury. Clotting could be potentiated both by the local retention of thrombin to the exposed sub-endothelial matrix and by its protection against inactivation by antithrombin III. In a similar way S-protein could
Molecular cloning of S-protein
contribute to the local assembly of terminal complement components or the removal of fluid phase complexes from the circulation under pathological conditions. This prediction is supported by the observation that terminal complement components are deposited at atherosclerotic plaques (Niculescu et al., 1985; De Heer et al., 1985) and also by the striking co-localisation of vitronectin and terminal complement complexes in kidney tissue (Hayman et al., 1983; Falk et al., 1983). Vitronectin is a member of a family of substrate adhesion molecules which include collagen, fibronectin, chondronectin, laminin, thrombospondin and von Willebrand factor (Hynes and Yamada, 1982; Edelman, 1985). It has not until now, however, been ascribed a unique function. We have shown that plasma vitronectin is the same protein as S-protein suggesting that it is a multifunctional protein that can interact with both the terminal components of the complement and coagulation pathways and also with cells and matrix constituents. Materials and methods
Podack,E.R. and Muller-Eberhard,H.J. (1980) J. Immunol., 124, 1779-1783. Podack,E.R., Kolb,W.P. and Miiller-Eberhard,H.J. (1977) J. Immunol., 119, 2024-2029. Podack,E.R., Preissner,K.T. and Muller-Eberhard,H.J. (1984) Acta Pathol. Microbiol. Immunol. Scand. Ser. C, Suppl. 284, 92, 89-96. Preissner,K.T., Wassmuth,R. and Muller-Berghaus,G. (1985) Biochem, J., 231, 349-355. Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467. Silnutzer,J. and Barnes,D.W. (1984) Biochem. Biophys. Res. Commun., 118, 339-343. Sixma,J.J. and Wester,J. (1977) Semin. Hematol., 14, 265. Stanley,K.K. (1983) Nucleic Acids Res., 11, 40774092. Stanley,K.K. and Luzio,J.P. (1984) EMBO J., 3, 1429-1434. Stanley,K.K., Kocher,H.-P., Luzio,J.P., Jackson,P. and Tschopp,J. (1985a) EMBO J., 4, 375-382. Stanley,K.K., Page,M., Campbell,A.K. and Luzio,J.P. (1985b) Mol. Immunol., in press. Suzuki,S., Pierschbacher,M.D., Hayman,E.G., Nguyen,K., Ohgren,Y. and Ruoslahti,E. (1984) J. Biol. Chem., 259, 15307-15314. Uthne,K. (1973) Acta Endocrinol. Suppl., 175, 1-26. Woods,D.E., Markham,A.F., Ricker,A.T., Goldberger,G. and Colten,H.R. (1982) Proc. Natl. Acad. Sci. USA, 79, 5661-5665.
AU methods relating to the cloning and expression of S-protein were as previously described (Stanley, 1983; Stanley and Luzio, 1984).
Received on 2 September 1985
Acknowledgements
Note added in proof
D.J. would like to thank Profs. S.Bhakdi and H.-J. Wellensiek for their support and encouragement during this work.
While this manuscript was in press the sequence of vitronectin cDNA has been published (Suzuki,S., Oldberg,A., Hayman,E.G., Pierschbacher,M.D. and Ruoslahti,E. (1985) EMBO J., 4, 2519-2524). This sequence is included within the S-protein sequence starting at base 71 and is identical except for three omissions (at positions 541, 551, 561) and four substitutions (at positions 736, 1157, 1260 and 1381).
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