(CR2)-binding site and a neoantigenic site in the C3d domain of the ...

Proc. Nati. Acad. Sci. USA Vol. 82, pp. 4235-4239, June 1985

Immunology

Mapping of the C3d receptor (CR2)-binding site and a neoantigenic site in the C3d domain of the third component of complement (membrane complement receptors/B lymphocytes/monoclonal antibody 130/synthetic peptides)

JOHN D. LAMBRIS, VISHWAS S. GANU, SHIRISH HIRANI, AND HANS J. MULLER-EBERHARD Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA 92037

Contributed by Hans J. Muller-Eberhard, March 22, 1985

The C3d domain of C3 contains the site that ABSTRACT binds to the C3d receptor (CR2) which is expressed on B lymphocytes. It also contains a neoantigenic determinant that is recognized by monoclonal antibody (mAb) 130 and is expressed when C3b is cleaved to iC3b and subsequently to C3dg or C3d. mAb 130 inhibits the binding of C3d to CR2. In this study, the locations of the CR2-binding site and of the neoantigen recognized by mAb 130 within the C3d domain were investigated. Treatment of human C3d with CNBr generated two major fragments with Mrs of 12,500 and 8600. Binding studies showed that only the Mr 8600 fragment was capable of binding to both CR2 and mAb 130. Amino-terminal sequence analysis of the Mr 8600 fragment and comparison with the amino acid sequence derived from human C3 cDNA [de Bruijn, M. H. L. & Fey, G. H. (1985) Proc. Nati. Acad. Sci. USA 82, 708-712] placed it between residues 1199 and 1274 of the C3 sequence. Several peptides were synthesized according to the derived C3 sequence of amino acid residues 1209-1236. Based on their differential binding to CR2 and mAb 130, we localized the CR2-binding site and mAb 130 neoantigenic site, respectively, to residues 1227-1232 and 1217-1232 of the C3 sequence.

by mAb 130 are located in C3d, which also contains the site for covalent binding of membrane and which remains bound to the target cell or particle after removal of C3c by factor I. Various cleavage products of C3 exhibit diverse biological activities that are relevant to inflammation, host defense, and the immune response in vitro (1-3, 12, 13). Pertinent to the present work are reports showing that C3d (14) and C3dk (15), a closely related fragment, are capable of inhibiting antigen- and alloantigen-induced T-lymphocyte proliferation. Recently, it was shown that C3dk also suppresses mitogeninduced B-lymphocyte growth and interleukin 2 synthesis in mixed lymphocyte cultures (16). Using CNBr fragments of C3d and synthetic peptides, we were able to assign the CR2-binding function of C3d and the reactivity with mAb 130 to two adjacent linear amino acid sequences.

MATERIALS AND METHODS Antibodies. mAb anti-B2 was purchased from Coulter Immunology Laboratories and purified by affinity chromatography on an anti-mouse IgM-agarose column (Zymed Laboratories, Burlingame, CA). mAb 130 was produced with C3b as antigen (10). mAb OKB7 was purchased from Ortho Diagnostics. Isolation of the gp72 Fragment of CR2. The gp72 fragment of CR2 was isolated from spent medium from Raji cell cultures as described (17). Preparation of CNBr Fragments of C3d. These fragments were prepared by addition of a 10-fold (wt/wt) excess of CNBr to C3d (5 mg/ml) in 70% formic acid and incubation of the mixture in the dark for 20 hr at 25°C. The mixture then was diluted 1:20 with water and freeze-dried, and the fragments were isolated by HPLC (Waters Associates) on a reversedphase preparative C4 column (Vydac) equilibrated with 10% acetonitrile containing 0.1% trifluoroacetic acid. Separation was performed at room temperature at a flow rate of 2 ml/min, and the fragments were eluted with an 80-ml gradient of 10-90% acetonitrile. Synthesis of Peptides. Four peptides (see Table 2), corresponding to amino acids 1209-1236, 1227-1236, 1217-1232, and 1211-1225 of the amino acid sequence of C3 were synthesized, according to the solid-phase methods of Mer-

The third component, C3, plays a central role in the complement system because its function requires interaction with various other complement proteins and cell surface receptors. The protein and its physiological reaction products have binding sites for 11 different ligands (1-3). In addition, a membrane-binding site is transiently expressed upon C3 activation (4). The physiological activation and degradation of C3 proceeds in the following manner (1, 2, 5). C3 is cleaved by C3 convertase into the fragments C3a (Mr 9000) and C3b (Mr -176,000). A Mr 2000 peptide is then removed from the a chain of C3b by factors H and I, thereby producing iC3b. This fragment is further cleaved by factor I with CR1, the C3b receptor, acting as cofactor. The products are C3c (Mr -140,000) and C3dg (Mr 39,000). C3g can then be removed by tryptic enzymes, giving rise to C3d (Mr 35,000). C3b contains the binding sites for factors B, H, and I; properdin; C5; CR1; and C4-binding protein and, in its metastable form, for cell membranes (1, 4, 6). The latter site is intimately related to the internal thioester of C3 (7), which is located in the d domain and allows C3b to become covalently bound to targets of complement attack (8). iC3b contains sites for CR3, the iC3b receptor, and for conglutinin (9), and C3dg and C3d exhibit a site for CR2, the C3d receptor. C3d expresses an antigenic determinant, detectable by monoclonal antibody (mAb) 130 (10), which is also expressed by iC3b and C3dg but not by C3 or C3b (11). Thus, both the CR2 binding site and the neoantigenic site detected

rifield (18), by use of a BioSearch peptide synthesizer. Peptides with an amidated COOH-terminus were prepared with a p-methylbenzhydrylamine resin (MBHA resin) (19). The derivatized L amino acids used in the synthesis were those recommended by Barany and Merrifield (20). The removal of resin-bound peptide was accomplished with 10 ml of HF and 1 ml of anisole per g of peptide-resin conjugate (20). The peptides were purified by fast protein liquid chromatography (FPLC) on a C8 reversed-phase column (Pharmacia)

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Abbreviations: C3, third component of complement; CR1, CR2, and CR3, receptors for the C3b, C3d, and iC3b fragments of C3, respectively; mAb, monoclonal antibody. 4235

4236

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Proc. Natl. Acad. Sci. USA 82 (1985)

1.2

60

0.8

40

0.4

20

0

5 11

10

15

25

20

Time,

30

35

40

min

FIG. 1. Separation of the CNBr fragments of C3d by HPLC. Two milligrams of C3d were incubated with 20 mg of CNBr in 70% formic acid for 20 hr at 250C. After freeze-drying, the sample was dissolved in 50%o acetic acid and injected into a reversed-phase C4 column equilibrated with 10%o acetonitrile containing 0.1% trifluoroacetic acid. The C3d fragments were eluted with an 80-ml gradient of 10-90%o acetonitrile.

and analyzed, after hydrolysis, in a Beckman automated amino acid analyzer. Amino Acid Sequence Analysis. Edman degradation was performed in a Beckman Model 890D sequencer employing the Quadrol 0.1 M program (21). Phenylthiohydantoins were identified by HPLC with a Zorbax ODS column equilibrated with 0.01 M sodium acetate buffer (pH 4.5), employing an acetonitrile gradient (22). Ligand-mAb 130 Binding Studies. The binding of mAb 130 to C3d fragments or synthetic peptides was determined by an ELISA. Microtiter plates were coated overnight at 4°C with 50 ,l of serially diluted C3d fragments or synthetic peptides. Wells were washed, saturated with phosphate-buffered saline containing 1% (wt/vol) bovine serum albumin (BSA), and then incubated for 30 min at 22°C with 0.5 ,g of mAb 130. Bound mAb was detected with peroxidase-conjugated goat anti-mouse Ig. Ligand-CR2 Binding Studies. Three different assays were employed: (i) Purified C3d fragments or the synthetic peptides were bound to coumarin-stained, 0.9-,m diameter microspheres (Covalent Technology, Redwood City, CA) and tested for their ability to bind to Raji cells as described

C3d or fragment, pmol FIG. 2. Binding of mAb 130 to C3d fragments. Various amounts of C3d (o) or its M, 8600 (A) or 12,500 (O) fragment were fixed to ELISA plates. After saturation of unoccupied sites on the plates with 1% bovine serum albumin, 50 ,ul of mAb 130 (10 ,g/ml) was added, and the bound mAb 130 was detected with peroxidase-labeled goat anti-mouse Ig.

(23). Specificity of binding was determined by treatment of Raji cells with either mAb anti-B2, mAb OKB7, or fluidphase C3d fragments. (it) Synthetic peptides were labeled with 125I (New England Nuclear) by the chloramine-T method (24) and tested for their ability to bind to Raji cells. Cells (106) in 100 ,.l of phosphate-buffered saline containing 1% bovine serum albumin were incubated for 30 min at 370C with different amounts of 125I-labeled ligand, either alone or in the presence of a 100-fold excess of unlabeled ligand. Duplicate 40-,ul samples were layered on 300 1Ld of a 1:1 mixture of dioctyl phthalate and dibutyl phthalate, and the cells were pelleted by centrifugation at 8000 x g for 2 min in a Beckman B microcentrifuge. Specific binding of ligand was determined by subtracting the amount bound in the presence of unlabeled ligand from the amount bound in its absence. (iii) The CNBr fragments of C3d were fixed to microtiter plates as described above and then reacted with 125I-labeled gp72 for 30 min at 25°C. Bound radioactivity was detected by cutting out the microtiter wells and determining the radioactivity in each.

RESULTS Localization of the CR2- and the mAb 130-Binding Sites to an Mr 8600 CNBr Fragment of C3d. Five CNBr fragments of C3d were isolated by reversed-phase HPLC (Fig. 1). NaDodS04/PAGE analysis showed that fragments IV and V had Mrs of 8600 and 12,500, respectively. Analysis of the five fragments (I-V) by ELISA with mAb 130 revealed that only the Mr 8600 fragment (IV) contained the antigenic determinant recognized by this antibody (Fig. 2). Since mAb 130 was found to inhibit the binding of particlebound C3d to cell-surface CR2, we tested whether the Mr Table 1. Binding of microspheres coated with C3d fragments and synthetic peptides to Raji cells % binding in the presence of Buffer* or Microspheres murine IgG mAb OKB7 mAb anti-B2 coated with 40 C3d 89 0 Fragments ND ND 0 Mr 12,500 5 0 77 M, 8600 Synthetic peptides 0 38 0 P28 P15 P16 BSA

0 22 0

ND 0 ND

ND 0 ND

BSA, bovine serum albumin; ND, not done. *Buffer: 3.5 mM barbital, pH 7.2/3.2% dextrose/1% bovine serum albumin/2 mM EDTA/0.2% sodium azide.


Immunology: Larnbris et al.

4237

1001

E

E

E co

0

x

E 0 .)

-00

C) 0

0.

in

.a_

'a

C3

3 0 10 20 30 1251-labeled gp72 added, cpm

40 x

10-4

FIG. 3. Binding of the gp72 fragment of CR2 and C3d fragments. Fifty microliters of various dilutions of I251-labeled gp72 was applied to ELISA plates precoated with either C3d (e), or the M, 8600 fragment (A), or the M, 12,500 fragment (E). Bound 1251 was quantitated by cutting out the microtiter wells and determining the radioactivity in each.'

8600 fragment also contained the CR2-binding site of C3d. It found that the Mr 8600 fragment bound to microspheres caused rosette formation with Raji cells that was inhibited by fluid-phase C3d and by mAbs anti-B2 and OKB7 (Table 1). Both monoclonal antibodies are known to bind to CR2 (25, 26). No rosette formation was observed when the Mr 12,500 fragment or bovine serum albumin was used to coat the microspheres. In a second test, the binding of the isolated and radiolabeled gp72 of CR2 to C3d fragments fixed to microtiter plates was measured. Only the Mr 8600 fragment bound gp72 (Fig. 3). Identification of the Primary Structure of the Mr 8600 CNBr Fragment. The sequence of the NH2-terminal six residues was obtained by Edman degradation and found to be GlyArg-Leu-Lys-Gly-Pro. By comparing this sequence with the amino acid sequence derived from the C3 cDNA sequence (27) and considering the location of methionine residues within the C3d domain, the Mr 8600 fragment was identified as representing amino acid residues 1199-1274 of the C3 sequence. Residues 1198 and 1275 are methionyl residues. Localization of the CR2- and mAb 130-Binding Sites to Linear Sequences of C3d by Use of Synthetic Peptides. To localize further the binding sites for CR2 and mAb 130, the amino acid sequence of the Mr 8600 fragment was analyzed by the hydrophilicity program developed by Kyte and Doolittle (28) and by the method of Chou and Fasman (29) to assess the probable secondary structure. According to these analyses, the amino acid sequence 1209-1236 is particularly was

[Unlabeled ligand], -log M FIG. 4. Inhibition of binding of 'l25-labeled P28 to Raji cells by C3d and synthetic peptides. Raji cells were incubated with 1 pmol of l25l-labeled P28 and various concentrations of the indicated unlabeled ligands at 370C for 30 min.

hydrophilic and the occurrence of a strong P-turn within it is highly probable. Therefore, this peptide (P28) was synthesized, and three smaller peptides (P15, P16, and P10) were each constructed according to a different portion of the P28 sequence (Table 2). The synthetic peptides were bound to microspheres and the conjugates were tested for their ability to bind to the CR2 of Raji cells. Table 1 shows that the synthetic peptides P28 and P16 but not P15 were able to bind to Raji cells and that binding of these peptides was inhibited by mAbs anti-B2 and OKB7. In addition, the direct interaction of the synthetic peptides with cell-surface CR2 was tested by an inhibition test. The results indicate that P28, P16, and P10 but not P15 bound to the CR2 of Raji cells (Fig. 4). The binding of the peptides was inhibited by a 100-fold molar excess of C3d but not of C3c. There was no specific binding of any of the peptides to MOLT-3 cells (30). Binding of mAb 130 was examined by ELISA. The antibody bound to P28 and P16 but not to P10 or P15 (Fig. 5). Thus, the amino acid sequence essential for binding of C3d to CR2 is contained in P10, and the antigenic determinant specific for mAb 130, in P16. DISCUSSION The aim of this study was to localize the CR2-binding function and the reactivity with mAb 130 to discrete amino acid sequences of C3d. A relative proximity of the two sites was presumed because mAb 130 was found to inhibit C3d-CR2 interaction. CR2 is a receptor of B lymphocytes (31, 32) that also occurs on some other leukocytes (33-35). It

Table 2. Localization of CR2- and mAb 130-binding sites to linear sequences of C3d represented by synthetic peptides

Peptide

C3d residues

Sequence*

P28 1209-1236 K F L T T A K D K N R W E D PG K OL Y N V E A T S Y A P15 1211-1225 L T T A K D K N R W E D PG K P16 1217-1232 KN R W E D P G K OL Y N V E A 1227-1236 P10 LY N V E A T S Y A *The single-letter amino acid abbreviations are used. tMicrosphere conjugates and Raji cells. tMeasured by ELISA.

Binding to CR2t mAb 130t + + + +

+ -

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Immunology: Lambris et al.


1.2r ) P28

1.0p0.81-

f

P16

Whereas the precise sequences comprising the two binding sites in C3d remain to be defined, it is clear that some or all of residues 1227-1232 (Leu-Tyr-Asn-Val-Glu-Ala) constitute the CR2 site and that the mAb 130 site is located immediately NH2-terminal to this sequence. The possible contribution of more distant sequences to the conformation and activity of the two sites has not been explored. We wish to thank David Becherer for his excellent technical assistance. This is publication 3755-IMM from the Research Institute of Scripps Clinic. This work was supported by Public Health Service (PHS) Grants A117354, AI21014, CA27489, and HL16411. V.S.G. is supported by PHS Training Grant HL07195. H.J.M.-E. is the Cecil H. and Ida M. Green Investigator in Medical Research, Research Institute of Scripps Clinic.

0.6

0.4

P15

0.2

,>

Plo

25 50 100 200 400 8001600

Synthetic peptide, pmol FIG. 5. Binding of mAb 130 to synthetic peptides, determined by ELISA.

reacts not only with C3d but also with C3dg and iC3b (33).

CR2 has recently been identified as the receptor for Epstein-Barr virus (36), and binding of the virus to the receptor is known to activate B lymphocytes (37). In contrast, C3d (14), C3dk (15, 16), or polyclonal antibody to the gp72 fragment of CR2 (14) inhibits mitogen-, antigen-, or alloantigen-stimulated T-cell proliferation. However, mAb anti-B2, mAb OKB7 (26), or polyclonal anti-CR2 (38) enhances B-cell activity. Thus, the accumulated evidence from various laboratories suggests that the CR2 on B lymphocytes fulfills immunoregulatory functions and that C3-derived ligands are involved in the regulation. After both binding sites were found to be present in the Mr 8600 CNBr fragment of C3d, we synthesized four peptides that were constructed according to hydrophilic sequences of the fragment. These four peptides were tested for binding to CR2 and mAb 130. Evidence was obtained that P28, P16, and P10 can interact with CR2 and that P15 cannot. The specificity of this interaction was suggested by the outcome of inhibition studies. For example, binding of P28 to Raji cells was inhibited by C3d, mAb OKB7, or mAb anti-B2. Comparing the structure of the peptides (Table 2), it is evident that the three positive ones have in common the sequence of amino acid residues 1227-1232, which is not present in P15, the inactive peptide. Thus this sequence of six residues might constitute the actual binding site for CR2, the size of which is comparable to the size of the binding site in C3a for the C3a receptor which has been found to consist of the COOHterminal five residues, -Leu-Gly-Leu-Ala-Arg (13). The two peptides P28 and P16 that are capable of reacting with mAb 130 have in common the sequence of residues 1217-1232. Since the two inactive peptides P15 and P10 overlap with P16 in the NH2 terminus and the COOH terminus, it is probable that the mAb 130-binding site comprises considerably fewer than the 16 residues of P16. mAb 130 was made in the mouse by immunization with human C3b (10). A comparison of mouse (39) and human (27) C3 sequences reveals that the sequence 1218-1236 is identical in both species except for three residues in positions 1222, 1224, and 1225. It is probable, therefore, that these three residues are part of the neoantigenic site.

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