localization and functional significance of a polymorphic determinant in the third component of...

Molecular Immunology, Vol. 24, No. 10, pp. 1097-1103, 1987 Printed in Great Britain

0161-5890/87 $3.00 + 0.00 Pergamon Journals Ltd

LOCALIZATION AND FUNCTIONAL SIGNIFICANCE OF A POLYMORPHIC DETERMINANT IN THE

THIRD COMPONENT OF HUMAN COMPLEMENT

NIELS BEHRENDT,*~ OLE C. HANSEN,~ MICHAEL PLOUG,*

VIBEKE BARKHOLT* and CLAUS KOCH$

*Institute of Biochemical Genetics, University of Copenhagen, 2A Oster Farimagsgade, DK-1353 Copenhagen K, Denmark; IProtein Laboratory, University of Copenhagen, 34 Sigurdsgade, DK-2200 Copenhagen N, Denmark; and $Hybridoma Laboratory, State Serum Institute, DK-2300 Copenhagen S,

Denmark

(Received 25 February 1987; accepted 16 April 1987)

Abstract-A polymorphic epitope in the third component of human complement was studied. This allotypic system is distinct from the electrophoretically determined C3 S/F polymorphism and is defined by the recognition of one allotype by a monoclonal antibody. Allotypic protein variants, C3F+ (reactive with this antibody) and C3’- (non-reactive with the antibody), were purified. Deglycosylation studies and N-terminal sequencing of CNBr fragments, reactive with the antibody, revealed that the polymorphic epitope was present in a B chain fragment of mol. wt 20,000. In the intact C3 molecule, this fragment is situated with N-terminus at residue No. 202, using the numbering of the cDNA derived amino acid sequence of human prepro C3. Addition of Fab fragments from the alloselective antibody preferentially inhibited the activity of C3F+ in a haemolytic assay which is selective for the C3 activity in the alternative complement pathway.

INTRODUCTION

The third component of complement, C3, is a multifunctional protein playing a major role in the complement cascade. Several distinct biological reactions can be assigned to specific C3 fragments that are generated as a result of activation of the complement system (Brown et al., 1984; Lambris and Miiller-

Eberhard, 1986). Human C3 exhibits genetic polymorphism. An

allotypic variation that can be demonstrated after Agarose gel electrophoresis gives rise to two common C3 types, C3s (slow) and C3F (fast) (Alper and Propp, 1967). In addition, a genetic variation exists that can be demonstrated by the selective reaction of one C3 type with a monoclonal antibody, HAV 4-l (Koch and Behrendt, 1986). The two polymorphic systems are genetically coupled. The majority of C3’ homozygous individuals do not show reaction with HAV 4-1, whereas, generally, individuals holding the C3F allele do react. On the other hand, HAV 4-l positive C3s

TAuthor to whom correspondence should be sent. Present address: Laboratory of Tumor Virology, the Fibiger Institute, 70 Ndr. Frihavnsgade, DK-2100 Copenhagen 0, Denmark.

Abbreviations: Con A, Concanavalin A; Endo H, endo-/?- N-acetylglucosaminidase H (EC 3.2.1.96); Fab, fragment antigen binding; HPLC, high performance liquid chromatography; PTH, phenylthiohydantoin; SDS- PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; PMSF, phenylmethylsulphonylfluoride; TFA, trifluoroacetic acid; Tris, Tris(hydroxymethyl)- aminomethane.

homozygous individuals have indeed been demonstrated, as well as HAV 4-l negative C3S/C3F hetero- zygotes. These findings indicate that the allotypic variation giving rise to a difference in electrophoretic mobility is not identical to that defined by the antibody. The entire amino acid sequence of human C3, derived from cDNA sequencing, has been published (DeBruijn and Fey, 1985). However, the allotype in question has not been stated, and neither has any polymorphic site as yet been identified.

Population studies, performed before the polymorphism defined by HAV 4-l was known, have shown that presence of the C3F gene is associated with an increase in the disposition to various diseases. These include rheumatoid arthritis (Bronnestam,

1973), atherosclerosis (Sorensen and Dissing, 1975) and Indian childhood cirrhosis (Srivastava and Srivastava, 1985). However, the above-mentioned coupling among the two allotypic systems makes it uncertain which of the two is responsible for these findings.

In the present report, analyses of the two more common genetic C3 variants, i.e. HAV 4-l reactive C3F and HAV 4-l non-reactive C3’, are presented. The designations C3F+ and C3’-, respectively, are used here to indicate these protein variants. The analyses have led to the partial localization of the HAV 4-l binding site in the C3 primary structure. Further, we present data concerning the effect of Fab fragments, derived from HAV 4-l antibody, upon the activity of human C3 in the alternative complement pathway.

1097

1098 NIELS BEHRENDT et al.

MATERIALS AND METHODS

Antibodies, enzymes and chromatographic media

HAV 4-1 antibody was produced as published previously (Koch and Behrendt, 1986). Secondary antibody for immunoblotting experiments (peroxidase-coupled rabbit anti-mouse Ig) was from Dakopatts, code P260. Sephadex G-75 SF, Protein A-Sepharose CL4B and standard mol. wt proteins (LMW-kit) for SDS-PAGE were from Pharmacia Fine Chemicals. Papain for the production of HAV 4-l Fab fragments was from Sigma, lot 18C- 8055. Endo H for deglycosylation studies was from Boehringer Mannheim, lot 10530981-01.

Pur@ation andfragmentation of allotypic C3 variants

C3F+ was purified from a plasma pool obtained from C3F homozygous donors, reactive with HAV 4-l; C3’- was isolated from a pool obtained from non-HAV 4-l reactive C3s homozygotes. Purification of C3 and isolation of the C3 p chain were performed essentially as described by Tack et al. (1981). The C3 fi chain was subsequently precipitated from the SDS solution by the addition of 6 vols of acetone (OC). CNBr cleavage of the p chain and gel filtration of fragments on Sephadex G-75 SF were performed as published (Lundwall et al., 1984) except that two successive treatments with CNBr were performed; the first in 70% formic acid and the second in 70% TFA.

Immunoblotting experiments and protein chemical analyses

Analytical SDS-PAGE was performed according to Laemmli (1970). A modified sample treatment procedure according to Sim and Sim (1981) was used in order to avoid cleavage of the C3a chain, induced by the denaturation conditions. For preparative SDS-PAGE, the modifications of the electrophoresis system and the conditions for electroelution of Coomassie stained bands of Hunkapiller et al. (1983) were employed.

Reaction of C3 fragments with monoclonal antibody was demonstrated after electroblotting of SDS polyacrylamide gels onto nitrocellulose sheets. After treatment with 2% Tween 20 for 2 min, the sheets were incubated with the monoclonal antibody followed by peroxidase-coupled secondary antibody. Incubation and intermediate washing steps were performed in Tris buffered saline, pH 10.2, holding 0.05% Tween 20. Subsequently, peroxidase activity was demonstrated, using H,O, and 3,5,3’,5’-tetra- methylbenzidine.

Deglycosylation of polypeptides was performed in the sample buffer employed for SDS-PAGE (above) including 1 mM PMSF. After addition of Endo H (50 mU/mg polypeptide) the samples were incubated at 37°C for 18 hr. In order to study the progress of the deglycosylation reaction, the samples were analysed by SDS-PAGE followed by a lectin overlay of the slab gel (Burridge, 1978; Chu et al., 1981), using

12SI-labelled Con A. Binding of the lectin was visual- ized by autoradiography.

Automatic microsequencing of protein fragments was performed on a gas phase sequencer (Applied Biosystems, model 470 A), using the program “02NRUN” supplied with the instrument (Hewick et

al., 1981). The released PTH amino acids were analysed by HPLC, using a gradient of 16% methanol to 40% ethanol in 0.04 M ammonium acetate, pH 4.2, on a Waters HPLC system equipped with a Sperisorb ODS II column (125 x 4.6mm).

Inhibition studies

HAV 4-l IgG for the production of Fab fragments was purified from culture supernatant, containing the antibody, using a column of Protein A-Sepharose CL4B. Fab fragments were produced by incubation of the purified antibody with papain at an enzyme to substrate ratio of 1:40 w/w for 4 hr at 37°C and purified on the same column. Complete cleavage was demonstrated by SDS-PAGE. C3 activity in the alternative complement pathway was measured using the AP-C3 assay (Jessen et al., 1983). The inhibitory effect of HAV 4-l Fab fragments was tested by preincubation with purified C3 samples at 37°C for 20min before assay.

RESULTS

Reactivity of HAV 4-l antibody with the PpoIypeptide

chain of C3’+

The reactivity of HAV 4-l antibody towards the c( and fi polypeptide chains of each of the purified C3 variants was analysed. Reduced samples of each variant were subjected to SDS--PAGE followed either by Coomassie staining or by immunoblotting, using HAV 4-l as the primary antibody. The results are shown in Fig. 1, lanes 2 and 5. On the Coomassie stained gel [Fig. l(A)], the electrophoretic mobilities of the a (mol. wt 115,000) and fl (mol. wt 75,000) chains are evident. Identical mobilities were observed when C3F+ and C3s were compared. Comparing this pattern to the nitrocellulose blot [Fig. l(B)], it is evident that HAV 4-l antibody was reactive only with the C3Ff b chain. No reaction occurred with the C3F+ c[ chain or with any of the C3’- polypeptide chains.

In addition it was investigated whether carbo- hydrates were involved in the antigenic reaction. The carbohydrate of human C3 consists of two asparagine-linked high-mannose oligosaccharides (Hase et al., 1985), which are susceptible to enzymatic cleavage by Endo H under denaturing conditions (Hirani et al., 1986). Consequently, a parallel set of C3 samples was treated with Endo H prior to reduction and analysed on the same gel (Fig. 1, lanes 3 and 4). In these samples, a decrease in the apparent mol. wt was observed for both of the polypeptide chains. Again, the patterns were identical when C3FC and C3’- samples were compared. When the gel was

A polymorphic determinant in C3

A 1 234 5 6

67 000 -

Fig. 1. Reactivity of HAV 4-l antibody towards the polypeptide chains constituting C3F+ and C3s-. SDS-PAGE (7.5% gel) of reduced samples, followed either by Coomassie staining (A) or by immunoblotting, using HAV 4-l (B). Lanes 1 and 6: mol. wt standard proteins, stained with Coomassie G-250. Molecular wts are indicated; lane 2: C3F+, untreated; lane 3: C3F+ after treatment with Endo H; lane 4:

C3s- after treatment with Endo H; lane 5: C3s-, untreated.

incubated with L2SI-labelled Con A it was found that the change in the electrophoretic mobilities was ac- companied by loss of the binding capacity for the lectin (results not shown). On the nitrocellulose blot it is evident that the deglycosylated C3Ff /I chain was still reactive with HAV 4-l antibody. Correspond- ingly, deglycosylation did not lead to reactivity of C3’- towards HAV 4-l.

Analysis of /3 chain CNBr fragments

The fl chains from C3Ff and C3s- were isolated, cleaved with cyanogen bromide and gel filtrated. SDS-PAGE of the total fragment mixture indicated that while exactly the same components could be demonstrated in the two preparations, cleavage had been incomplete as several fragments were larger than those predicted from the published, human C3 primary structure. A series of fragments in the mol. wt range from 12,000 to 40,000 was present. The only HAV 4-l reactive material observed was contained within a single pool from the gel filtration of C3Fc fragments. When this pool was analysed by SDS- PAGE followed by immunoblotting, two fragments

*Throughout this communication, numbering of amino acid residues refers to the human prepro C3 numbering of DeBruijn and Fey (1985).

(mol. wts 20,000 and 17,000) showed positive reaction with HAV 4-1 [Fig. 2(B) lane 21. The reaction was selective as the corresponding C3s- pool did not contain any HAV 4-l reactive fragments [Fig. 2(B) lane 31, though exactly the same compounds were present when comparing the two preparations after amido black staining [Fig. 2(A) lanes 2 and 31.

The HAV 4-l reactive bands from the C3FC pool and the corresponding bands from the C3s- pool were isolated by preparative SDS--PAGE followed by electroelution from the gel. The N-terminal amino acid sequences of the 20,000 mol. wt bands were determined by automatic sequencing, while for the 17,000 mol. wt bands yields were too low to give any sequence information. It came out [Table l(A)] that two components were present in each 20,000 mol. wt band. The same two fragments were present in both preparations. The double sequence data obtained made it possible to identify the components in question by comparison with the published C3 primary structure (outlined in the Discussion section). The fragments identified were those with N-termini at residue Nos 43 and 202, respectively.*

Deglycosylation of CNBr fragments

Of the two fragments identified, only the one with the N-terminus at residue No. 43 holds N-linked

1100

30000 -

20 100 *

14 400 -

NIELS BEHRENDT er al.

A

Fig. 2. Immunoblotting experiment, demonstrating the reactivity of HAV 4-l antibody towards fragments derived from purified C3 variants. SDS-PAGE (1%20% gradient gel) of gel filtration pools holding CNBr fragments of the C3 /I chain was followed by electroblotting of the gel. The nitrocellulose sheet shown in (A) was stained with amido black, while the sheet shown in (B) was analysed for reactivity with HAV 4-1. Lane 1: mol. wt standard proteins with mol. wts as indicated on the figure; lane 2: pool obtained from C3F+; lane 3: pool obtained from C3s-. The 20,000 and 17,000 mol. wt components are indicated

by arrows.

carbohydrate (Hirani et al., 1985; Welinder and led to a decrease in the staining intensity of the 20,000 Svendsen, 1986). Therefore, deglycosylation could be mol. wt band, reflecting the disappearance of one used as a tool to distinguish between the two frag- component from the electrophoretic mobility region ments. The total CNBr fragment mixture from the B in question. Concomitantly, the reactivity of the chain of each C3 variant was treated with Endo H 20,000 mol. wt band towards Con A disappeared and analysed by SDS-PAGE, followed either by [Fig. 3(B)]. By contrast, the parallel immunoblotting incubation with ‘251-Con A or by immunoblotting, experiment [Fig. 3(C)] showed that the antigenicity as using HAV 4-1. The emerging electrophoretic pat- well as the electrophoretic mobility of the HAV 4-l terns are shown in Fig. 3. On Coomassie stained gels reactive fragment were unaffected by the de- [Fig. 3(A)] deglycosylation of the fragment mixture glycosylation reaction.

Table 1. (A) Amino acid sequence obtained from electroeluted 20,ooO mol. wt bands.” (B) Part of the human C3 p chain sequence (DeBruijn and Fey, 1985) derived from

DNA sequencing

A B

Step Residues identified No. Residue No. Residue

42 Met 201 Met 1 Not identified 43 Val 202 Gly 2 Leu,b Gln 44 Lea 203 Gin 3 GlU 45 Glu 204 Trp 4 Ala, Lys 46 Ala 205 LYS 5 Asn,’ Ile’ 41 His 206 Ile 6 Asp, Arg* 48 Asp 207 Arg 7 Ala 49 Ala 208 Ala 8 Gin, Tyr 50 Gin 209 Tyr 9 Gly, Tyr 51 Gly 210 Tyr

10 Asp, Glu 52 Asp 211 Ghl 11 Val, Asn 53 Val 212 Asn

‘Results were identical for the samples derived from C3F+ and C3s- when no difference is stated. The average repetitive yields were 90% (C3F’) and 87% (C3’-). The PTH-derivatives were detected in amounts ranging from 5 to 35 pmol. Occasional occurrence of artefactual peaks, coeluting with PTH-Lys, restricted the identification of this derivative to yields above 10 pmol.

bOnly identified in C3s- fragment sample. cOnly identified in C3F+ fragment sample.

A polymorphic determinant in C3

A B 1 2345 6 1234 5 6

1101

C

23456

94 000 67000

30 000

20 100

14 400

Fig. 3. Analysis of deglycosylated CNBr fragments derived from C3s- and C3F+. The fragments were separated by SDS-PAGE (l&20% gradient gel), whereupon the gel was either: (A) stained with Coomassie G-250, (B) incubated with ‘251-Con A followed by autoradiography or (C) electroblotted onto nitrocellulose followed by incubation with HAV 4-l (C3 fragments) or amido black staining (mol. wt standard proteins). Lanes 1 and 6: mol. wt standard proteins with mol. wts as indicated on the figure. Lane 2: fragments derived from C3 F+ deglycosylated with Endo H; lane 3: fragments derived from C3F+, , untreated; lane 4: fragments derived from C3s-, untreated; lane 5: fragments derived from C3s-, deglycosylated with Endo H. To facilitate comparison, the electrophoretical mobilities of the 20,000 and

17,000 mol. wt bands are indicated by arrows.

Inhibition of C3 activity

HAV 4-l IgG was purified and cleaved with papain. The resulting Fab fragments were isolated and used for inhibition studies. Samples of the purified C3 variant proteins, each containing approx. 8 pg C3, were preincubated with the Fab fragments and tested in the AP-C3 assay. This assay selectively measures the C3 activity in the alternative complement pathway (Jessen et al., 1983). As seen in Fig. 4, preincubation of C3Ft with roughly equimolar amounts of HAV 4-l Fab fragments (l-5 pg) caused a substantial decline in the residual haemolytic activity. The same treatment had only a minor effect on the activity of C3’-. Increasing amounts of the Fab fragments, however, did affect both variants. The same pattern was found during pilot investigations using intact HAV 4-l antibody and purified C3 or HAV 4-l Fab fragments and serum samples.

DISCUSSION

A polymorphic site in human C3, recognized by the selective reaction of one allotype with the monoclonal antibody HAV 4-1, was studied. It was established that the polymorphic site is present in the C3 /I chain. A mixture of two CNBr fragments from the /3 chain of C3Ff, one of which reacted with HAV 4-1, was isolated. The two corresponding fragments were present in the C3’- preparation and served as a negative control for the antibody reaction. M.I.M.M. *4,10--G

Automatic sequencing of the fragment mixture allowed the unambiguous identification of the two fragments. The sequences identified by screening of the entire human C3 primary structure were those

01 ’ ’ ’ ’ ’ I 0 5 10 15 20 25 30

pg Fab HAV 4-1

Fig. 4. Inhibition of C3 activity in AP-C3 assay by Fab fragments derived from HAV4-l. The residual haemolytic activities were calculated by comparison with separate standard curves, obtained for each variant. Each deter- mination was performed in triplicate; mean values are

shown. (O-0) C3F+; (0-O) C3’-.

1102 NIEL~ BEHRENDT et uf.

with N-termini at residues Nos 43 and 202, respectively. The sequencing data obtained at steps 8-11 [Table l(A)] served as a basis for this screening, since continuous double sequence data were obtained through these four steps. Among the 16 hypothetical tetrapeptides that could be constructed by combi- nation of these data, only two were present in the published sequence. These were the peptides Gln- Gly-Asp-Val (residue Nos 50-53) and Tyr-Tyr-Glu- Asn (residue Nos 209-212). Table l(B) shows part of the published sequence, holding these tetrapeptides. Consistent with this localization, both of the se- quenced fragments proved to be preceded by a methionine residue in the primary structure of the intact protein. It is evident that the residues identified at steps 2-l 1 in the present sequence analysis completely accounted for the stated, publish~ C3 /? chain sequences with the exceptions of residue No. 47 (His) and No. 204 (Trp). For these two amino acids, however, inherent difficulties exist concerning the recovery and identification of the PTH derivatives. Therefore, in the microsequence analyses reported here, these derivatives were unlikely to be detected. PTH-Asn at step 5 was the only residue of the present sequencing which does not fit with the published sequence. The identification of this PTH derivative was based on a signal close to the lower detection limit (8 pmol recovered) and may have been artefactual in origin; it was identified only in one of the samples.

Too littie material was available to allow the separation of the two 20,000 mol. wt components on a preparative scale. Therefore, it was necessary to distinguish between the fragments by indirect means, taking advantage of the fact that the fragment with the N-terminus at residue No. 43 is glycosylated at residue No. 85 while the other fragment is devoid of N-bound carbohydrate. It was shown that even for the intact fi chain, deglycosylation would lead to a reduction in mol. wt large enough to be clearly detected by SDS-PAGE. However, deglycosylation of the fra~ent mixture did not affect the mol. wt of the HAV 4-l reactive fragment. It was ascertained by the use of ‘2’I-labelled Con A that deglycosylation had indeed occurred. Further, it was shown that the continuous presence of antigenic material with mol. wt 20,000 was not due to trace amounts of glycosylated material being left after the reaction, as glycosylation was not a prerequisite for the antigenic reaction and no antigenic component of reduced mol. wt occurred. It was concluded that the HAV 4-l reactive species was the fragment that is not glycosylated, i.e. the fragment with an N-terminus at residue No. 202. According to the mol. wt, this fragment would be expected to consist of approx. 180 amino acid residues. Referring to the published sequence, it is most likely that the fragment in question has a C-terminus at one of the methionine residues No. 368 or 373.

Binding of Fab fragments derived from HAV 4-l

antibody inhibited the activity of purified C3Ff in a haemolytic C3 assay. Though some inhibition was also found in the case of C3$-, the reaction with C3FC was markedly stronger. Apparently, the selectivity of the antibody is not absolute, although it appeared so in blotting experiments. The reaction with C3’- observed in the inhibition studies was probably due to the fact that the antibody concn used in these experiments was l-2 orders of magnitude higher than that used for incubation of nitrocellulose blots from polyacrylamide gels.

The assay employed is specific for C3 activity in the alternative complement pathway (Jessen et al., 1983), but otherwise it is not yet known which of the numerous binding reactions of C3 or C3 fragments was inhibited by the antibody. Hitherto, no specific biological reaction has been ascribed to the C3 p chain. In the present study, Fab fragments rather than intact Ig were used in order to minimize the degree of steric hindrance towards remote sites on the molecular surface. It would be most interesting to study whether the polymorphic determinant is itself part of a implement binding site, or whether the inhibition observed was due to steric effects, reflecting proximity of the fi chain epitope to a functional site in the folded C3 molecule.

Acknowledgements-This work was supported by grants from the Danish Rheumatism Association and the Danish Medical Research Council. We wish to thank Dr Henning Sorensen for providing the raw material for purification of C3 variant proteins. Solveig R. Jorgensen and Pia Jensen are thanked for expert technical assistance throughout the study.

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A polymorphic determinant in C3 1103

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