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Bioscience Reports 5, 855-865 {1985) Printed in Great Britain 855 Structural and functional studies on C4b-binding protein, a regulatory c o m p o n e n t of the human complement system L. Ping CHUNG and Kenneth B. M. REID M.R.C. Im munoehemistry Unit, Department of Biochemistry, South Parks Road, Oxford OX1 3QU; U.K. {Received 22 August 1985) The binding and cofactor activities of C4b-binding protein were examined before and after limited proteolysis by pepsin, trypsin and chymotrypsin. The major fragments generated were characterized by amino acid sequencing, thus establishing the precise points of limited proteolysis. These studies allow a tentative assignment of the cofactor activity site to the residues 177-322 of the 549 amino acid long chain of C4b-binding protein but indicated that residues in the region 332-395 are important in the binding activity. Human C4b-binding protein (C4BP) is an important regulatory component of the serum complement system, being involved in the control of the classical pathway enzyme complex C4bC2a. The C4BP displays binding activity by interacting with C4b and thus displacing C2a from the complex (Gigli et al., 1979) and it is also a cofactor in the degradation of C4b by the enzyme factor I (Fujita & Nussenzweig, 1979). Human C4BP is a glycoprotein of approx. 5 5 0 0 0 0 Mr in dissociating and non-dissociating conditions and is composed of probably seven identical, disulphide-linked chains each of 70 000 Mr and 549 amino acids long (Dahlb~ick & M/iller-Eberhard, 1984; Chung et al., 1985a). In the electron microscope the C4BP has a spider-like structure with seven flexible tentacles (each 3 nm • 33 nm) joined at one end to a small central body (Dahlb/ick et al., 1983). However, X-ray solution scattering experiments by synchroton radiation indicated that in solution the molecule is more compact, the tentacles being relatively close together in distinction to the splayed out images seen in the electron micrographs (Perkins et al., 1985). In this paper some correlation between the structure and function of the C4BP molecule has been established by examining the binding and cofactor activities of C4BP before and after limited proteolysis by various enzymes. Materials and Methods Preparation and assay o f c o m p l e m e n t c o m p o n e n t s Cls, C4, factor I and C4BP were prepared as described by Gigli et al. (1976), Bolotin et al. (1977), Hsiung et al. ( 1 9 8 2 ) a n d Chung et al. (1985b) 856 CHUNG & REID respectively. C4i was prepared by the repeated freezing (at --70~ and thawing of native C4 or by treatment of the native C4 with 50 mM methylamine at 37~ for 1 h. The complete inactivation of the purified C4 was demonstrated by the cleavage of the C4i c~i chain by factor I in the presence of C4BP and the fact that the C4i was insensitive to Cls. The C4 haemolytic assays were performed as described by Kerr (19-80). Radioiodination of proteins. The iodination procedure used was adapted from the method of Fraker and Speck (1978). Assay of C4BP cofactor activity. Protease-treated or untreated C4BP was incubated with C4i (0.4 rag/m1) and factor I (0.01-0.02 mg/ml) at 37~ for 1-3 h, followed by SDS-polyacrylamide gel electrophoresis analysis. Prefiminary experiments had shown that it was preferable to follow the degradation of C4i rather than C4b because of the clearer pattern of the degraded fragments on SDS-polyacrylamide gel. The cofactor activity o f the C4BP sample, determined by the degree of degradation of the od chain of C4i, was estimated from the staining intensities of the 80000 M, call fragment (generated after the first cleavage) and the 45 000 Mr C4d fragment (generated after the second cleavage). C4BP binding assay. To test for the binding of intact C4BP (or treated C4BP) to C4i, 50 tal of C4i (0.4 mg/ml) in 50 mM Tris HCI/5 mM EDTA/ 150 mM NaC1, pH 7.4, was incubated in each well of a microtitre plate for 2 h at 37~ After this incubation, a solution of bovine serum albumin (2.5 mg/ml) in the Tris HC1/EDTA/NaC1 buffer was used to wash the wells (three times) and to saturate any non-specific binding sites by incubation at 37~ for 30 min. The wells were then emptied and the C4BP sample (0-10 tag) of untreated or protease-treated C4BP, '2sI-C4BP (approx. 3 0 0 0 0 c.p.m, in less than 0.1 tag of protein) and bovine serum albumin (2.5 mg/ml) in a total vol of 35 tal was added and incubated for 90 rain at 37~ After washing the wells three times with BSA/Tris HC1/EDTA/NaC1 the plate was dried, and the wells were cut out and placed in tubes for gamma counting. The binding activity of t h e C4BP sample was estimated by its inhibitory effect on the binding of 12sI-C4BP to the C4i coated microtitre wells. Limited proteolysis o f C4BP. Trypsin treatment. The trypsin-treated C4BP was prepared by incubating purified C4BP (0.4-0.6 mg/ml) in 50 mM TrisHC1/0.15 M NaC1, pH 7.4, with trypsin at a substrate/enzyme ratio of 250/1 (w/w) at 37~ for 4 h. The digestion was stopped by the addition of 1 mM DFP or a three-fold excess of soy bean trypsin inhibitor and the sample stored at 4~ before use. For amino acid sequencing the tryptic fragments (from a digest designed to yield only T1 and T3) were dissolved in an SDS/urea/Tris buffer and separated on a BRL preparative SDS-polyacrylamide gel apparatus. After extensive dialysis and freeze drying, the purified fragments were then subjected to automated N-terminal amino acid sequencing. Chymotrypsin treatment. The 4 8 0 0 0 Mr and 1 6 0 0 0 0 M r chymotryptic fragments used in the eofactor assay were prepared fi'om chymotrypsindigested C4BP samples by gel filtration on a Sephacryl S-300 column (2.5 cm • 80 cm) equilibrated with 25 mM Tris HC1/2 mM EDTA/0.15 M NaC1 pH 7.4 at room temperature. C4BP (20 ml of 0.5 mg/ml) was incubated with chymotrypsin (at a substrate/enzyme ratio of 100/1, w/w) at 37~ for 3.5 h C4b-BINDING PROTEIN STRUCTURE AND ACTIVITY 857 to give a good yield of the 48 000 Mr fragment or for 6 h to obtain a good yield of the 160 000 Mr fragment. In both cases the chymotryptic activity was stopped by 1 mM DFP and the sample applied to Sephacryl S-300. For sequencing studies, native C4BP (10 mg) was incubated with chymotrypsin (at a substrate to enzyme ratio of 400/I, w/w) at 37~ for 75 min in 50 mM Tris HC1/100 mM NaC1, pH 7.4. DFP (1 mM) was added to stop the reaction and the sample was dialysed at 4~ against 0.2 M NH4HCO 3. The dialysate was then freeze dried, reduced and alkylated (Chung et al., 1985b). The fragments were separated on a Sephacryl $300 column (1.5 cm X 90 cm) equilibrated in 6 M guanidine HC1/20 mM Tris HC1, pH 7.4. Pepsin treatment. Pepsin-digested C4BP used in the cofactor and binding activity assays was prepared by incubating C4BP (0.3 mg/ml) in 0.1 M sodium acetate/0.1 M NaC1, pH 4.8, with pepsin (at a substrate/enzyme ratio of 25/1, w/w) at 37~ for 2.5 h. The pH of the digest was adjusted to 7.1 and the solution made 1 mM with respect to DFP. In all the experiments performed on the pepsin-treated C4BP, a C4BP sample dialysed in the pH 4.8 buffer and subsequently adjusted to pH 7.1 was used as an extra control. To prepare pepsin-treated C4BP for amino acid sequence analysis, 12 mg of C4BP (0.5 mg/ml) in 0.1 M sodium acetate/0.1 M NaC1, pH 4.2, was incubated with pepsin (at a substrate/enzyme ratio of 75/1, w/w) at 37~ for 4 h. After raising the pH to 7.1 and adding DFP (to 1 mM) the sample was dialysed, freeze dried, reduced and alkylated. The peptic fragments P1 and P2 were fractionated on a Sephacryl S-300 column run in 6M guanidine HC1/20 mM Tris HC1, pH 7.4, and then prepared for automated amino acid sequencing. Results and Discussion Trypsin-treated C4BP The present results confirm and extend those of Reid and Gagnon (1982), which showed that trypsin-treated C4BP retained its cofactor activity and behaved in an identical fashion to the untreated molecule in non-dissociating conditions on Sephacryl S-300 and in non-reducing conditions on SDSPAGE electrophoresis (Fig. 1a). Under reducing conditions on SDS-PAGE, each of the 70 000 Mr chains was shown to be split initially into two major fragments, T1 of 40 000 M r and T3 of 30 000 Mr (Fig. lb), and with longer digestion times, or a higher trypsin concentration, the 40 000 Mr T 1 fragment was further digested to give a 36 000 Mr T2 fragment. Both forms of C4BP, the C4BP-H and C4BP-L (Fig. la), were split by trypsin to the same extent. Similar fragments to T1, T2 and T3 can be generated by the use of plasmin and are observed during the purification of C4BP if a plasmin inhibitor is not used during the purification procedure. Trypsin-treated C4BP still retained its cofactor activity although its efficiency was approx. 5 times lower than that of untreated C4BP (Fig. 2). However, as shown in Fig. 3, the binding of I~sI-C4BP to the C4i coated on microtitre wells was not inhibited significantly on the addition of trypsintreated C4BP while untreated C4BP gave good inhibition. The difference 858 CHUNG & REID Fig. 1. L i m i t e d p r o t e o l y s i s of C4BP by t r y p s i n . C4BP (0.4 m g / m l ) was i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s of t r y p s i n at 37~ f o r 15 m i n t o 4 h. T h e e n z y m e was i n a c t i v a t e d b y a d d i t i o n o f a 3-fold excess of soy b e a n t r y p s i n i n h i b i t o r f o l l o w e d b y an e q u a l v o l u m e o f gel l o a d i n g b u f f e r ( S D S / u r e a / T r i s ) a n d h e a t i n g at 100 ~ for 10 m i n . T h e samples were e l e c t r o p h o r e s e d o n (a) 3.5% ( w / v ) S D S - P A G E u n d e r n o n - r e d u c i n g c o n d i t i o n s a n d (b) on 15% ( w / v ) S D S - P A G E u n d e r r e d u c i n g c o n d i t i o n s . T h e s u b s t r a t e / e n z y m e ratios a n d t i m e of digestion f o r e a c h t r a c k are 1 - n o e n z y m e , 4 h ; 2 - 2 0 0 / 1 , 4 h ; 3 - 3 0 0 / 1 , 4 h ; 4 4 0 0 / 1 , 4 h ; 5 - 2 0 0 / 1 , 1 h ; 6 - 3 0 0 / 1 , 1 h ; 7 - 4 0 0 / 1 , 1 h ; 8 - 2 0 0 / 1 , 15 m i n ; 9 - 3 0 0 / 1 , 15 r a i n ; 10 - 4 0 0 / 1 , 15 m i n . C4b-BINDING PROTEIN STRUCTURE AND ACTIVITY 859 Fig. 2. Cofactor activity assay of trypsin-treated C4BP. Samples were run on an 8.5% (w/v) SDS-PAGE in reducing conditions. Tracks A, C4i, B and I refer to untreated C4BP, C4i, trypsin-treated C4BP and factor I respectively. C4i (0.4 mg/ml), factor I (0.01 mg/ ml) and C4BP (untreated or trypsin-digested) at 250 to 1 #g/ml (as indicated by the number above each track) were incubated at 37 ~ for 2 h. The positions of the c~i (uncleared), ~il (generated after first cleavage) and C4d (generated after second cleavage) chains are shown. The intensities of the Coomassie Blue stained ~il and C4d fragments were used as a measure of cofactor activity. b e t w e e n the binding activities o f the t r e a t e d and u n t r e a t e d C4BP was over 100-fold (Fig. 3). T h e N - t e r m i n a l a m i n o acid s e q u e n c e o b t a i n e d for T1 m a t c h e d t h a t o f the N - t e r m i n u s o f the i n t a c t C4BP, indicating t h a t the 40 000 M r t r y p t i c fragm e n t was derived f r o m t h e N-terminal end o f the 70 000 M r chain o f C4BP. A d o u b l e a m i n o acid s e q u e n c e was o b t a i n e d for T3 as detailed below: Derived a m i n o acid sequence from c D N A studies Fragment T3 Sequence 1 Sequence2 325 335 345 G E I T Q H R K S R P H N H CV Y F Y G D E KSRPHNHCVYFAGDE SRPP NHCVYFAGDE B o t h s e q u e n c e s 1 and 2 m a t c h e d w i t h the s e q u e n c e derived f r o m the c D N A studies ( C h u n g et al., 1985a) b u t the yields o f P T H a m i n o acids f r o m s e q u e n c e 1 were a p p r o x i m a t e l y twice t h a t o f s e q u e n c e 2, indicating t h a t the g e n e r a t i o n o f the t r y p t i c f r a g m e n t s T1 and T3 was due to splitting b y 860 CHUNG & REID [] " -0 C D 0 -Q " Ik 9 Non-trypsindigestedC4BP i TrypsindigestedCt,BP ~ ~ . 1 iili o.lo's i ..... . . . . . . ,ug CLBPadded lb Fig. 3. Binding activity of trypsin-treated C4BP. The binding activity of trypsin-treated C4BP was assayed by comparing its ability to inhibit the binding of 12SI-C4BP to C4i coated microtitre plates with that of untreated C4BP. The amount of radioactivity bound (mean of two determinations) was plotted against the amount of C4BP untreated (o) or trypsin-treated (u), added to each well. trypsin at arginine 331 and partial splitting at lysine 332. An interesting feature which emerged from this sequencing study was that the cDNA sequence studies predicted that residue 343 is tyrosine yet the amino acid sequencing showed that the major amino acid present at that position was alanine. Thus this is evidence for a third polymorphic site in the C4BP molecule in addition to those observed at positions 44 and 309 (Chung et al., 1985a). Chymotrypsin-treated C4BP Nagasawa et al. ( t 9 8 2 , 1983) reported that C4BP could be split by mild chymotrypsin treatment to a 4 8 0 0 0 M r fragment which contained the cofactor active site and a 160 000 Mr fragment. After reduction of disulphide bonds the 160 000 M~ fragment was shown to be composed o f probably 7 disulphide-linked chains of approx. 25 000 Mr. It was established by amino acid sequence studies (Nagasawa et al., 1983; Dahlbgck & M(iller-Eberhard, 1984; Chung et al., 1 9 8 5 a ) t h a t the 4 8 0 0 0 M r and 2 5 0 0 0 M r fragments were derived from the N-terminal and C-terminal portions of the 70 000 Mr C4BP chain respectively, and thus the 25 000 M r fragments form a disulphidelinked C-terminal core o f 160 000 M r . In this study a more detailed time course o f chymotrypsin treatment with low enzyme concentrations showed that a lightly digested form o f the molecule in which the 48 000 Mr fragment was disulphide-bonded to the C4b-BINDING PROTEIN STRUCTURE AND ACTIVITY Fig. 4. Limited proteolysis of C4BP by chymotrypsin and pepsin. C4BP (0.7 mg/ml) was incubated with chymotrypsin (substrate/enzyme ratio 50/1, 100/1, 150/1,200/1, w/w in tracks 1, 2, 3 and 4 respectively) at 30~ for 12 h and electrophoresed on 4% (w/v) non-reduced (a) and 8% (w/v) reduced (b) SDS-PAGE. Tracks in 5(a) and 5(b) are nondigested C4BP. Pepsin digestion of C4BP is shown for samples of C4BP incubated with pepsin for 3 h at 37 ~ at pH 4.2 at substrate/enzyme ratios of 50/1, 75/1, 100/1 and 150/1 and then electrophoresed under (c) non-reducing conditions on 4.5% (w/v) SDS-PAGE and (d) reducing conditions on 8.5% (w/v) SDS-PAGE. Track T contained trypsin-digested C4BP. 861 862 CHUNG & REID 25 000 Mr fragment was produced after the initial cleavage by chymotrypsin. As shown in Fig. 4 (a and b), the liberation of the 48 000 Mr fragment from the disulphide-linked core fragments correlated with the further spfitting of the 48 000 Mr and 25 000 Mr fragments. Since chymotryptic digestion progressively removes an N-terminal 48 000 Mr fragment from each of the 70 000 Mr C4BP chains, analysis of the chymotrypsin treated C4BP on non-reduced SDS-PAGE gives an estimate of the number of chains. The value obtained was seven, which is consistent with that reported by Dahlb~ick and Miiller-Eberhard (1984), who had further shown directly by electronmicroscopy studies that the 48 000 Mr and 160 000 M r fragments were the 'tentacles' and 'core' respectively of the unusual C4BP molecule. Cofactor assays performed on the purified chymotryptic fragments confirmed the findings of Nagasawa et al. (1982) that the 4 8 0 0 0 Mr fragment retained the cofactor activity of C4BP while none could be detected in the 160 000 Mr fragment. No binding assays were performed using the chymotryptic fragments; however, Fujita et al. (1985) have shown quite clearly that both the 48 000 Mr and the 160 000 Mr fragment had lost the ability to bind to C4b. N-terminal sequence analysis of the 25 000 Mr chymotrypsin fragment showed that chymotrypsin initially splits the 70 000 Mr chain principally at the Tyr-Ser bond at position 395-396. This point of splitting is 30 amino acids upstream from the Trp-Ser bond at position 425-426 which was the major site of splitting reported by Dahlbgck and Miiller-Eberhard (1984)in their study of the limited proteolysis of C4BP by chymotrypsin. The difference between the two studies is probably due to the use of different digestion conditions. Limited proteolysis by pepsin When C4BP was treated with pepsin and examined in reducing conditions on SDS-PAGE the 70 000 Mr chain was shown to be split into a 55 000 Mr (P1) fragment which was further rapidly degraded into a 50000Mr (P2) fragment (Fig. 4c and d). No fragment of 15-20 000 Mr, which might have been expected to be present after the cleavage of the 70 000 Mr C4BP chain into the 50-55 000 Mr fragment, was observed on SDS-PAGE or gel filtration, and it was concluded that the 'missing' 15-20000 Mr fragment had been extensively digested by pepsin. Under non-reducing conditions the pepsin-treated C4BP behaved as a diffuse band with an Mr of approx. 400 000. The conversion of the 550 000Mr C4BP doublet into the 400 000 Mr band observed on the non-reduced gel correlated with the conversion of the 7 0 0 0 0 Mr chain into P1/P2 fragments on the reduced gel, which showed that the peptic fragments are disulphide-linked to each other and therefore likely to represent the C-terminal portion of the chain (as was confirmed by the sequencing studies). The cofactor and binding assays performed using pepsin-digested C4BP (containing approximately equal amounts of the P1 and P2 fragments) showed that the protease-treated C4BP had an identical activity to that of the untreated C4BP. The P1 and P2 fragments were isolated from the reduced and alkylated pepsin-treated C4BP by fractionation on a Sephacryl S-300 column run in 6 M guanidine HC1. Although P1 and P2 were not completely separated, 863 C4b-BINDING PROTEIN STRUCTURE AND ACTIVITY NH2~. Trypsin IChymo- / COox/" S,S A - important in cofactor activity B -important in binding activity Cofactor Activity Binding Activity Major Sites of Limited Proteolysis Treatment SDS-PAGE Non-reduced Reduced Untreated 550000 70000 + + Trypsin 550000 400001 30000 + - Arg 331 Lys 332 Chymotrypsin 160000 48000 25000 48000 + -22 - Tyr 395 Trp 425 Pepsin 400000 550003 + + Leu 161 Gly 177 i. Further cleaved to 36000 2. F u j i t a et al (1985) 3. Further cleaved to 50000 Fig. 5. Summary of the major sites of limited proteolysis by trypsin, pepsin and chymotrypsin on C4BP along with the cofactor and binding activities of the protease-treated C4BP. 864 CHUNG & REID pools enriched in each fragment were obtained. The amino acid sequence data from each pool showed that the major pepsin cleavage points, which yield P1 and P2, are at the Leu-Leu bond (position 161-162) and the Gly-Val bond (position 177-178) respectively, i.e. 155 CDPRF 160 165 170 SLLGHASISCTVENETIGVWR t P1 175 180 P2 The asparagine residue at position 173 was not detected in the identification of the PTH-amino acids in the automated sequencing, indicating that it may be attached to N-linked carbohydrate. This would be consistent with the fact that the P1 fragment contains only 17 amino acids more than P2 and yet there was approx. 5000 Mr difference in their apparent mol.wts, on SDSPAGE, and they could be partially separated by gel filtration. The properties of the fragments generated by limited proteolysis along with the location of the major cleavage sites are given in Fig. 5. The results obtained using trypsin-treated C4BP are similar to the finding that trypsintreated factor H retained its activity on fluid phase C3b but lost the ability to bind or to act as a cofactor in the degradation of C3b on the cell surface (Hong et al., 1982). Both the chymotrypsin- and pepsin-treated C4BP showed cofactor activity at concentrations 10 times lower than the normal plasma C4BP concentration, although the protease-treated C4BP was estimated to be approximately 5 times less efficient on a molar basis than the untreated C4BP in the cofactor assay. The absence of a 15-20 000 Mr peptic fragment after the cleavage of the 70000 Mr C4BP chain into the P1/P2 fragments indicates that there may be multiple pepsin-sensitive sites in the region between residues 1-160. Since the pepsin-treated C4BP still retained its cofactor and binding activities it can be concluded that residues 1-160 are not of primary importance in these functions. This observation is consistent with the finding by Reid and Gagnon (1982) that the N-terminal 40 amino acids of C4BP are probably not important for the cofactor activity. The limited proteotysis results indicate that the cofactor active region of C4BP may lie between the cleavage sites of pepsin and chymotrypsin. However, the electron-microscopy studies of Dahlb~ick et al. (1983) showed that fluid phase C4b appeared to be attached close to the tips of the C4BP tentacles which are about 29 nm from the chymotryptic cleavage site (Dahlbgck & Miiller-Eberhard, 1984) and thus near to the N-terminal portions of the C4BP chains, i.e. apparently N-terminal to the tryptic cleavage site. It is possible, however, that the N-terminal regions of the C4BP chains fold inwards towards the core leaving the putative cofactor active sites at the extremities of the molecule. The present observations allow a tentative assignment of the cofactor site to the residues 177-332 of the 549 amino acid long chains of C4BP but that residues in the region 332-395 are important in the binding activity (Fig. 5). C4b-BINDING PROTEIN STRUCTURE AND ACTIVITY 865 Dedication We dedicate this paper in memory of Professor R. R. Porter in appreciation of his friendship, encouragement and advice over the past sixteen years (K.B.M.R.) and four years (L.P.C.). References Bolotin C, Morris S, Tack B & Prahl J (1977) Biochemistry (Wash.) 16, 2008-2015. Chung LP, Bentley DR & Reid KBM(1985a) Biochem. J. 230, 133-141. Chung LP, Gagnon J & Reid KBM (1985b) Mol. Immunol. 22,427-435. Dahlb~ck B & Miiller-Eberhard HJ (1984) J. Biol. Chem. 259, 11631-11634. Dahlbgck B, Smith CA & Miiller-Eberhard HJ (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3461-3465. Fraker PJ & Speck JC (1978) Biochem. Biophys. Res. Commun. 80,849-857. Fujita T & Nussenzweig V (1979) J. Exp. Med. 148, !044-1051. Fujita T, Kamato T & Tamura N (1985) J. Immunol. 134, 3320-3324. Gigli I, Fujita T & Nussenzweig V (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6596-6600. Gigli i, Porter RR & Sire RB (1976) Biochem. J. 157,541-548. Hong K, Kinoshita T, Dohi Y & Inoue K (1982) J. Immunol. 129,647-652. Hsiung LM, Barclay AN, Brandon MR, Sim E & Porter RR (1982) Biochem. J. 203, 293-298. Kerr MA (1980) Biochem. J. 189, 173-181. Nagasawa S, Mizuguchi KC, Ichihara & Koyama J (1982) J. Biochem. (Tokyo, Japan) 92, 1329-1332. Nagasawa S, Unno H, Ichihara C, Koyama J & Koide T (1983) FEBS Lett. 164, 135-138. Perkins SJ, Chung LP & Reid KBM (1985) Biochem. J., in press. Reid KBM & Gagnon J (1982) FEBS Lett. 137, 75-79.