Structure-Guided Identification of C3d Residues Essential for Its Binding to Complement 2 (CD21)

This information is current as Liliana Clemenza and David E. Isenman of September 28, 2021. J Immunol 2000; 165:3839-3848; ; doi: 10.4049/jimmunol.165.7.3839 http://www.jimmunol.org/content/165/7/3839 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Structure-Guided Identification of C3d Residues Essential for Its Binding to 2 (CD21)1

Liliana Clemenza2 and David E. Isenman3

A vital role for complement in adaptive humoral immunity is now beyond dispute. The crucial interaction is that between and follicular dendritic cell-resident complement receptor 2 (CR2, CD21) and its Ag-associated ligands iC3b and C3dg, where the latter have been deposited as a result of classical pathway activation. Despite the obvious importance of this interaction, the location of a CR2 binding site within C3d, a proteolytic limit fragment of C3dg retaining CR2 binding activity, has not been firmly established. The recently determined x-ray structure of human C3d suggested a candidate site that was remote from the site of covalent attachment to Ag and consisted of an acidic residue-lined depression, which accordingly displays a significant electro- negative surface potential. These attributes were consistent with the known ionic strength dependence of the CR2-C3d interaction

and with the fact that a significant electropositive surface was apparent in a modeled structure of the C3d-binding domains of CR2. Downloaded from Therefore, we have performed an alanine scan of all of the residues within and immediately adjacent to the acidic pocket in C3d. By testing the mutant iC3b molecules for their ability to bind CR2, we have identified two separate clusters of residues on opposite sides of the acidic pocket, specifically E37/E39 and E160/D163/I164/E166, as being important CR2-contacting residues in C3d. Within the second cluster even single mutations cause near total loss of CR2 binding activity. Consistent with the proposed oppositely charged nature of the interface, we have also found that removal of a positive charge immediately adjacent to the acidic

pocket (mutant K162A) results in a 2-fold enhancement in CR2 binding activity. The Journal of Immunology, 2000, 165: 3839–3848. http://www.jimmunol.org/

he essential role of C3 in many aspects of the innate im- an impaired primary response, virtually no secondary response, mune response (opsonization, phagocytosis, inflamma- and greatly reduced number and size of germinal centers upon T tion, and complement-mediated cytolysis) has long been immunization with T-dependent Ag (4, 5). Because CR1 and CR2 recognized. More recently, there has been an accumulating body of are differential splice products of the Cr2 in the mouse (6), evidence indicating that C3 also participates in the regulation of the phenotype of the knockout reflects losing both types of recep- the adaptive immune response (reviewed in Ref. 1). This regula- tors. Nevertheless, in view of the similarity of the immune re- tory action is mediated by the interaction of C3dg, an activation sponse profile of the CR1/CR2 knockout mice to the mice in which product of C3, with its complement receptor CR24 (CD21), the binding of ligand to CR2 was blocked by a CR2-specific mAb, or by guest on September 28, 2021 latter being primarily present on the membranes of B cells and by a competing soluble form of CR2, it suggests that minimally an follicular dendritic cells. Several in vivo studies have pointed out interaction between iC3b/C3dg-coated Ag and CR2 is required for the importance of this interaction in the modulation of the Ab- an optimal immune response. The phenotype of the Cr2 gene mediated humoral response. For example, the administration to knockout is in fact very similar to that observed in C3- and C4- mice of a blocking anti-mouse CR2 mAb (2), or of a soluble form deficient mice (7) and to that of CD19-deficient mice (8, 9). The of CR2 (3), at the time of the primary immunization with subop- mechanism through which CR2 is thought to participate in a B cell timal doses of T-dependent Ags-diminished the primary response signaling event involves its association with a pre-existing signal and abolished the secondary response and isotype switching. transduction complex, namely the CD19/CR2/TAPA-1 complex Knockout mice generated by disruption of the Cr2 gene displayed (10). CD19 is considered to be the key molecule in the signaling process, but a direct ligand for it has not been identified. Thus, a model has been proposed that bridges the B cell receptor (BCR) to CD19 via the dual recognition of a C3dg-coated Ag by CR2 and by Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada the Ab component of the BCR complex (11). In support of this Received for publication May 15, 2000. Accepted for publication July 18, 2000. model, it has been found that there was about a 10-fold decrease in The costs of publication of this article were defrayed in part by the payment of page the threshold concentration of anti-BCR required for B cell acti- charges. This article must therefore be hereby marked advertisement in accordance vation in vitro when Abs that simultaneously cross-linked the with 18 U.S.C. Section 1734 solely to indicate this fact. CD19/CR2 complex were also present (12). A study by Dempsey 1 This work was supported by funding from Aventis-Pasteur in the form of a Canadian Universities Research Program Grant and by the Medical Research Council of Canada et al. (13) has provided compelling evidence for the threshold hy- (MT-7081). pothesis in vivo. Specifically, when hen egg lysozyme (HEL) that 2 Current address: Department of Biopathology and Bio-medical Technologies, Uni- had been biosynthetically conjugated to two or three tandem cop- versity of Palermo, Corso Tukory 211, Palermo 90134, Italy ies of C3d (a proteolytic limit fragment of C3dg retaining full 3 Address correspondence and reprint requests to Dr. David E. Isenman, University of ability to bind CR2) was administered to mice in the absence of Toronto, Department of Biochemistry, Medical Sciences Building, Room 5306, To- ronto, Ontario M5S 1A8, Canada. E-mail address: [email protected] any other adjuvant, it was found to be, respectively, 1000- and 10,000-fold more immunogenic than similarly administered HEL 4 Abbreviations used in this paper: CR, complement receptor; DVB, dextrose-Veronal buffer; DGVB, dextrose-gelatin-Veronal buffer; EAC4b2a, EAC423b, EAC423bi, he- alone. Moreover, the IgG response to HEL-C3d3 was 100-fold molysin-sensitized sheep erythrocytes coated respectively with complement compo- higher than that to HEL administered in CFA. Besides providing in nents C4b and C2a, C4b, C2a and , C4b, C2a and iC3b; HEL, hen egg lysozyme; SCR, short consensus repeat; sCR1, soluble CR 1; SGVB, sucrose-gelatin-Veronal vivo evidence for the threshold hypothesis, this study clearly dem- buffer; VBS, Veronal-buffered saline; BCR, B cell receptor. onstrated the potential of using C3d as a molecular adjuvant. A

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 3840 CR2 BINDING SITE IN C3d subsequent investigation showed that a single molecule of C3b that 25). Of interest, mutagenesis studies have suggested that a similar was conjugated to HEL through the physiologically relevant thio- charge complementarity exists between acidic residues in C3b (26, ester-mediated transacylation mechanism also greatly enhanced 27) and basic residues in known C3b-binding regions of CR1 (28), the immunogenicity of HEL (14). Although providing a second the latter also consisting of multiple SCR motifs. example of the use of complement ligand-receptor interactions as Because of the above reasons, we considered the acidic pocket a molecular adjuvant, this experiment did not distinguish among of C3d to be an attractive candidate for the site mediating the the contributions of CR1, CR2, CR3, and CR4 to the adjuvant interaction with CR2. In the present study we have performed ala- effect as at least a portion of the Ag-conjugated C3b would have nine scanning mutagenesis of all of the residues within, and im- been cleaved to iC3b postimmunization, thus changing its receptor mediately adjacent to, the acidic pocket in C3d and assessed the class binding preference from CR1 to the latter three CRs. CR2 binding ability of the recombinant in two indepen- CR2 is a member of the regulators of complement activation dent binding assays. This structure-guided analysis has proved (RCA) family (15) and depending on splice site usage, its extra- fruitful in allowing us to identify two clusters of C3d residues that cellular region consists of 15 or 16 short consensus repeat (SCR) are crucial for the interaction with CR2. domains. The binding sites for its ligands, C3dg and iC3b, have been localized to SCR domains 1 and 2 (16) and peptide segments Materials and Methods 10-LNGRIS-15 of SCR-1, 84-GSTPYRHGDSVTFA-97 of SCR-2 Buffers, cell media, and purified components (17), and 63-EYFNKYS-69 located between SCRs 1 and 2 (18), have been suggested to be important for iC3b/C3dg binding. The The following buffers were used: Veronal-buffered saline (VBS; 4 mM diethylbarbiturate (Veronal), 0.15 M NaCl, 0.15 mM CaCl2, and 0.5 mM three-dimensional structure of CR2 has not been determined but a Downloaded from MgCl2; pH 7.2); sucrose-gelatin-Veronal buffer (SGVB; low ionic strength model of CR2 SCR-1 and SCR-2 has been produced based on the VBS containing 227 mM sucrose, 30 mM NaCl, and 1 g/L of gelatin); nuclear magnetic resonance structure of SCRs 15–16 of SGVB-EDTA buffer (SGVB containing 10 mM EDTA); dextrose-Veronal (19). In this model the peptide segments considered important for buffer (DVB; 2.5 mM Veronal (pH 7.2), 140 mM glucose, 71 mM NaCl, 0.5 mM MgCl2, and 0.15 mM CaCl2); and dextrose-gelatin-Veronal-buffer iC3b/C3dg binding are largely surface exposed and with some ro- (DGVB; DVB containing 1 g/L gelatin). tation about the interdomain segment, the 10–15 and 84–97 pep- COS-1 cells were maintained in high-glucose DMEM containing 10%

tide segments can be configured to form a contiguous patch (17). heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 http://www.jimmunol.org/ An electrostatic surface potential rendition of the modeled CR2 ␮g/ml streptomycin (complete DMEM). The murine myeloma cell line J558L-pSNRCR2, which secretes the fusion protein (CR2) -IgG1 (Ref. 3; domains indicated that the basic residues within these latter two 2 provided by Dr. Henry Marsh, Avant Immunotherapeutics, Needham, segments were part of two prominent electropositive patches (20). MA), was maintained in complete DMEM supplemented with 50 ␮M The identification of a site(s) within C3d that mediates the in- 2-ME and 0.6 mg/ml active G418-sulfate (Geneticin, Life Technologies, teraction with CR2 has been the subject of some controversy, as Burlington, Ontario, Canada). Raji B lymphoblastoid cells were main- has been the issue of whether there are supplementary sites in iC3b tained in complete RPMI 1640 medium. Complement components C1 (29), C2 (30), C4 (31), C3 (31), factor I in addition to the site(s) in C3d. In 1985, Lambris et al. (21), based (32) and factor H (33) were purified from human plasma as previously largely on synthetic peptide mimetic studies, proposed that a C3d described, and C3dg was purified from Mg2ϩ-activated human plasma as segment having the sequence 1199-EDPGKQLYNVEA-1210 described (34). Native C3 was converted to iC3b as described in (35). by guest on September 28, 2021 (mature C3 numbering) played a major role in the interaction with IgG fractions of rabbit anti-human C3c (Sigma, St. Louis, MO) and CR2. Subsequent studies from this group also suggested that in rabbit anti-human C3d (Serotec Canada, Toronto, Ontario, Canada) were radioiodinated by the lactoperoxidase procedure (36). addition to the C3d contacts, there were additional contacts for CR2 in the C3c part of iC3b (22). However, in 1995 our laboratory Site-directed mutagenesis and expression of recombinant C3 demonstrated that very extensive mutagenesis of the 1199–1210 All mutants were generated by the overlap-extension PCR method (37) in segment of human C3 led to minimal effects on the binding of a modified version (pSV-C3(LC)) of the expression plasmid pSV-C3 con- either iC3b or C3dg to CR2, suggesting no more than a minor role taining the full-length cDNA of human C3 (38). pSV-C3 was modified to for this segment of C3d in mediating binding to CR2 (23). Addi- introduce an AgeI restriction site at nucleotide 3713 and to delete a BglII tionally, our work, like that of an earlier study by Kalli et al. (24), site at nucleotide 4220 of the C3 cDNA sequence. In neither case did the base substitutions alter the amino acids encoded by the nucleotide se- failed to detect a substantial difference between the respective quence. As delimited by the sense and anti-sense flanking primers used for CR2-binding activities of iC3b and C3dg. the overlap-extension PCR, the target region for mutagenesis spanned nu- In the absence of any further clear candidate sites in C3d to test cleotides coding for amino acid residues 925 through 1371 of C3 (mature for CR2 binding, we turned our attention toward obtaining a struc- C3 numbering), which includes within it the nucleotides encoding the en- ture of C3d in the hope that the structure might suggest a candidate tire C3d fragment (residues 980 to 1281). The overlap-extension PCR frag- ments of mutants located upstream of the AgeI site were restricted with SalI site to guide further mutagenesis work. The structure of human and AgeI to produce a 670-bp fragment; for mutants located downstream of C3d has recently been published (20) and reveals the molecule to the AgeI site the overlap-extension fragments were restricted with AgeI and be a highly helical ␣-␣ barrel presenting two distinct surfaces, a BglII to produce a 507-bp fragment. The restricted fragments were then convex surface bearing the thioester residues and, at the opposite exchanged with the corresponding segments in wild-type pSV-C3(LC). The mutations were confirmed by strand denaturation dideoxy DNA se- side, a concave surface. Whereas one might expect that the resi- quencing using a T7 polymerase sequencing (Amersham Pharmacia dues located on the convex end to be partially occluded because of Biotech, Canada). the attachment to the target Ag, the concave surface is fully ac- Wild-type or mutant plasmid DNAs (5 ␮g) were transfected into COS-1 cessible to the solvent, and thus potentially available for receptor cells by our previously described modification of the DEAE-dextran interactions. In addition, the rim of the cavity on the concave sur- method (39, 40) in 100-mm plates. Culture supernatants (about 8 ml) were harvested after 5 days, dialyzed against VBS and concentrated about 5-fold face is surrounded by acidic residues that form a V-shaped entity using Biomax concentrators (Millipore, Bedford, MA). Recombinant C3 providing a surface with a prominent negatively charged electro- protein concentration in the concentrated supernatants was determined by static potential. The involvement of such an electronegative patch a C3-specific ELISA, using plates coated with 10 ␮g/ml rabbit anti-C3c Ab in forming ionic bonds with a complementary surface on CR2 (Sigma), a goat anti-C3 sandwiching Ab (Quidel, San Diego, CA), and finally an alkaline phosphatase-conjugated anti-goat IgG for detection. Pu- would be consistent not only with the electropositive surface on rified human C3 was used to obtain a standard curve. CR2 revealed by the modeling, but also with the well established Metabolic labeling of transfected COS-1 cells was performed 48 h post- ionic strength dependence of the iC3b/C3dg-CR2 interaction (23, transfection as described previously (23). The Journal of Immunology 3841

Preparation of iC3b-coated erythrocytes ton Dickinson) and analyzed as a function of cell number using the CellQuest software package (Becton Dickinson). The preparation of sheep erythrocytes bearing varying amounts of wild- type or mutant iC3b was done via minor modifications of our previously described procedure (23). Briefly, 0.25 ml EAC4b2a (109 cells/ml) in Results and Discussion SGVB-EDTA buffer were incubated with variable amounts of concentrated As mentioned in the Introduction, we considered the acidic resi- transfection supernatant (from 0.175 ml to 1.5 ml at a concentration of due-lined pocket on the concave surface of the C3d molecule to be 1–1.5 ␮g/ml) for2hat37°C. The resulting EAC423b were converted to a candidate CR2 binding site because of its accessibility, because EAC423bi cells by incubating with 4 ␮g factor H and 0.4 ␮g factor I for 3 h at 37°C, followed by washing. of the known ionic strength dependence of the CR2-C3d interac- The relative number of iC3b molecules/cell was determined by evalu- tion, and because a molecular model of the binding domains of ating the binding of 2.5 ␮g 125I-labeled anti-C3c Ab to 50 ␮l containing CR2 indicated the presence of a fairly extensive electropositive 5 ϫ 107 EAC423bi cells as previously described (26). The nonspecific surface (20). To test the validity of our candidate binding site, we 125 binding component was determined by incubating the I-labeled anti-C3c have performed an alanine mutagenic scan on all of the C3d res- with an equal number of EAC4b2a cells. The conversion of red cell-bound recombinant C3b to iC3b was mon- idues, both charged and neutral, that have surface-exposed side itored in experiments using metabolically labeled culture supernatants as chains lying within or just adjacent to the boundaries of the neg- the source of recombinant C3 for building the EAC423bi cells. As de- atively charged pocket visualized in the electrostatic surface ren- scribed in detail in an earlier study (26), membranes of the EAC423bi cells dition of the molecule (20). These mutations were performed were solubilized in 0.75% SDS, treated with 1 M hydroxylamine to break the ester linkages between the 35S-iC3b and red cell surface molecules, and within the context of the intact human C3 molecule, and following then the released labeled molecules were analyzed by SDS-PAGE autoflu- transient expression in COS-1 cells of the mutant C3 cDNAs, the

orography under reducing conditions. culture supernatants served as a source of rC3 for deposition onto Downloaded from C3 convertase-bearing sheep red cells (EAC42). The deposited Preparation of C3dg-coated erythrocytes C3b molecules were then converted to iC3b by treatment of the Wild-type and mutant C3dg-coated erythrocytes were prepared as de- cells with excess H and I and these EAC423bi cells were then used scribed above for the conversion of EAC423b to EAC423bi except that in rosetting assays with Raji cells that bear CR2, but no other CRs instead of using factor H as the I cofactor, soluble CR1 (sCR1; provided by Dr. Richard Smith while at SmithKline Beecham, Harlow, U.K.) was used on their surfaces. We have previously confirmed in rosetting as- for this purpose (5 ␮g/3 ϫ 108 EAC423b) as sCR1, but neither factor H nor says that CR2 on Raji cells binds iC3b and C3dg with similar http://www.jimmunol.org/ soluble membrane cofactor protein, is an efficient cofactor for the so-called avidities (23). Because conversion of the red cell-deposited C3b to third factor I-mediated cleavage converting iC3b to C3c and C3dg (27). C3dg either requires considerably more H and I than is required for Because the C3dg fragment remains cell-associated, whereas the C3c frag- ment does not, the success of the conversion was in each case ascertained conversion of C3b to iC3b, or it requires the use of sCR1 as an I by first measuring the binding of 125I-anti-C3c at the EAC423b stage and cofactor, for reasons of reagent economy, in most of our experi- subsequently to the washed cells following the treatment with sCR1 and ments we have measured the effect of our mutations on the iC3b- factor I. In all cases, binding of the 125I-anti-C3c was reduced to back- CR2 interaction, but have interpreted the results within the context ground levels following treatment with sCR1 and factor I, whereas specific of the structure of C3d. Further justification of this approach comes binding of a 125I-labeled rabbit anti-C3d to the cells was retained. from a rosette inhibition assay using as fluid-phase competitors

Rosetting of iC3b-coated erythrocytes to CR2-bearing Raji cells purified iC3b and C3dg. It can be seen in Fig. 1 that on a molar by guest on September 28, 2021 Raji cells (5 ϫ 104) were incubated with 4 ϫ 106 EAC423bi in 50 ␮lof basis these two ligands were equipotent in inhibiting rosette for- the low ionic strength buffer, SGVB, for 30 min at 37°C with gentle ro- mation between iC3b-coated red cells and CR2-bearing Raji cells. tation. Cells were fixed with 0.2% glutaraldehyde for 5 min before quench- The molecular locations of the 17 residues targeted for mutation ing with 20 mM Tris-HCl (pH 8.2). Rosette formation was evaluated in in this study are shown in Fig. 2, together with a listing of the 27 duplicate by assessing the percentage of Raji cells bearing four or more erythrocytes. Binding specificity was determined by incubating Raji cells with EAC42 and also by preincubating Raji cells with 4 ␮g/ml of OKB7, an IgG2b functional site blocking anti-human CR2 mAb, (provided by Dr. P. Rao, Ortho Diagnostics Systems, Raritan, NJ) before the addition of iC3b-coated erythrocytes. The IgG2b anti-CR3 mAb OKM1 (provided by Dr. W. Reed, University of North Carolina, Chapel Hill, NC) was used as a nonspecific Ab control. Rosetting of C3dg-coated erythrocytes to Raji cell CR2 was conducted exactly as described for the iC3b-coated erythrocytes. Rosette inhibition by purified iC3b and C3dg was performed as above for the OKB7 blocking mAb experiments except that instead of OKB7, the Raji cells were preincubated with various concentration of purified iC3b or C3dg before the addition of EAC423bi cells made with sufficient purified C3 to yield ϳ80–90% rosette formation in the absence of inhibitor. Binding of soluble CR2 to iC3b-coated erythrocytes Culture supernatants from J558L-pSNRCR2 cells were dialyzed against

DVB. The amount of secreted (CR2)2-IgG1 was determined by ELISA, using plates coated with a rabbit Ab to mouse IgG (Jackson ImmunoRe- search, West Grove, PA) and a sandwiching rabbit Ab to mouse IgG that is conjugated to alkaline phosphatase (Jackson ImmunoResearch). Purified mouse IgG (Sigma) was used to obtain a standard curve. For the binding assay, 50 ␮l culture supernatant containing 4 ␮g/ml ϫ 6 (CR2)2-IgG1, were incubated overnight at 4°C with 5 10 iC3b-coated erythrocytes. Binding specificity was established by preincubating (CR2)2- IgG1 with 40 ␮g/ml OKB7 (or OKM1 as an isotype-matched negative control) at 37°C for 1 h. Erythrocytes were then washed with DGVB and FIGURE 1. Comparison of the ability of purified iC3b and C3dg (and binding was detected by using a fluorescein-conjugated donkey Ab to C4 as a negative control) to inhibit rosette formation between iC3b-coated mouse IgG (Jackson ImmunoResearch). After1hofincubation at 4°C, red cells and CR2-bearing Raji cells. The solid line at the top indicates the cells were washed and resuspended in 1 ml DGVB. Cell-bound fluores- level of rosette formation in the absence of inhibitor. The range of duplicate cence intensity was determined using a FACScalibur flow cytometer (Bec- rosette counts within the assay is indicated by the error bars. 3842 CR2 BINDING SITE IN C3d Downloaded from http://www.jimmunol.org/ FIGURE 2. A list of the 27 single and combination mutants analyzed in this study (A) and their location (B) as depicted on a space-filling model of the concave surface of C3d (20). The figure was produced using the program RasMol 2.6 and the numbering system is that employed in the C3d structure’s Brookhaven file 1C3D. Acidic, basic, and neutral side chain-containing amino acids are respectively colored red, blue, and yellow. The orientation is the same as the figure showing the electrostatic surface potential of this face of the molecule (figure 3 of Ref. 20).

mutants that were analyzed. The numbering system used refers to under our standard conditions to convert the deposited C3b to the C3d residue number because this most readily allows interpre- iC3b. Following hypotonic lysis and recovery of the membrane tation with reference to the C3d structure (add 971 to convert to ghosts bearing ester-linked adducts of 35S-labeled iC3b (or C3b if by guest on September 28, 2021 mature C3 numbering). In addition to the 17 single-residue mu- the conversion had not worked), hydroxylamine was added to tants constructed, acidic residues within each of the 36–39 and break the ester linkages and the chain structure of the released 160–167 segments were mutated in several combinations. In two 35S-labeled protein was assessed by SDS-PAGE autofluorography. cases, mutants D36N/E37Q/E39Q and D163N, the effects of isos- The results of this experiment are shown in Fig. 3. As a reference teric amide substitutions were also examined, and R49 was re- point, the hydroxylamine-released chains of the wild-type protein placed by both alanine and the more isosteric residue methionine. are shown both before and after treatment with factors H and I With the exception of the R49A mutant, which was expressed at (right side of upper panel). Whereas the former shows predomi- Ͻ100 ng/ml, the expression levels of all of the other mutant C3 nantly bands migrating at the positions of ␣Ј-chain and ␤-chain, molecules fell within the range (250–450 ng/ml) observed for the the latter shows the chains characteristic of iC3b, namely ␤, ␣Ј-67, wild-type molecule in the numerous replicate transfections con- and a doublet of ␣Ј-43/␣Ј-40, where the doublet represents the ducted in the course of this study. Because the more isosteric R49 respective products of the first and second factor I-mediated cleav- M mutant was expressed at normal levels, this permitted the eval- ages. It has long been assumed, and now has been directly dem- uation of the role of this residue in mediating CR2 binding. The onstrated that it is the first factor I-mediated cleavage that corre- only other anomaly noted was for the isosteric amide substitution lates with the functional status of the molecule (43). It can further mutant D163N that, although secreted at wild-type levels, was be seen in Fig. 3 that all of the mutants examined in our study only poorly deposited on the C3 convertase-bearing red cells. In displayed wild-type-like behavior with respect to extent of depo- contrast, the D163A mutant was normal with respect to both its sition and conversion to their respective iC3b forms. secretion level and deposition efficiency. Rosetting of wild-type and mutant iC3b-coated red cells to CR2- Characterization of the C3-bearing red cells bearing Raji cells Because it is known that there is a binding site for factor H within Aliquots of C3 convertase-bearing red cells were treated with var- C3d (41, 42), and further that red cells on which the deposited C3b ious amounts of recombinant C3-containing supernatants to create was not converted to iC3b would show very much impaired ability sets of cells with various amounts of iC3b on their surfaces. Within to form CR2-dependent rosettes (23), it was important to establish an experiment, the relative amount of iC3b deposited on each that none of the mutations that we introduced prevented the factor group of red cells was determined by measuring the binding of H- and I-mediated conversion of the cell-associated C3b to iC3b. 125I-labeled anti-C3c to a fixed number of red cells. Fig. 4 presents To examine this issue, culture supernatants from transfected the rosette formation dose response curves for the various substi- COS-1 cells that had been metabolically labeled with [35S]Met/ tution mutants engineered for this study. Each panel represents a Cys were used to build the red cells bearing the various mutant separate experiment in which the dose-response curve for the wild- C3b molecules. These cells were then treated with factors H and I type molecule acts as an internal reference point. These data are The Journal of Immunology 3843 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Demonstration that the mutant C3b molecules deposited on the C3 convertase-bearing red cells were convertible to iC3b by factors H and I. This experiment was performed using ϳ600 ng of [35S]Met/Cys metabolically labeled rC3 to build the EAC423bi cell under our standard conditions (see Materials and Methods). Hydroxylamine treatment (1 M, pH 9) of the detergent-solubilized membranes of these cells released the ester-linked C3 activation fragments, which were then analyzed by SDS-PAGE (reducing conditions) and autoflurography. As a reference to indicate the migration position of intact ␣Ј-chain (and ␤-chain), the far right of the upper panel shows the results from EAC423b cells made with 35S-labeled recombinant wild-type C3, but not treated with H and I so as to preserve the deposited molecule as C3b. The complete conversion to iC3b of all of the mutants tested in this study is indicated by the absence of intact ␣Ј-chain and the appearance of the ␣Ј-67 and ␣-43/40 doublet that are the characteristic chains of iC3b.

representative of minimally two, and often three, repeat experi- both of them should serve as a contact for the electropositive ments for every mutant examined in this study. The data were also surface contributed by the first two SCR domains of CR2. The plotted using a semilogarithmic scale for the x-axis, which denoted D36/E37/E39 cluster lines one edge of the negatively charged the relative amount of iC3b ligand per red cell (not shown). The depression visible in the electrostatic surface potential render- horizontal displacement of the psuedo-linear part of a given dose- ing of the C3d molecule (see figure 3 in Ref. 20), D163 and response curve from that of the wild-type curve allows one to E166 line the opposite edge, and the carboxylate side chain of estimate the extent of the defect or enhancement in CR2-binding E160 extends down into the depression (Fig. 2). Dealing first introduced by any given mutation. This in turn allowed us to sum- with the D36/E37/E39 cluster (Fig. 4, A and B, and Fig. 5), it marize in Fig. 5 the results of all of these rosette experiments in a can be seen that whereas mutation of D36 on its own is without more convenient bar graph format. effect, both the E37A and E39A mutant molecules show about We initially focused our attention on the two most prominent a 2-fold defect in CR2 binding activity in the rosette assay. The clusters of acidic residues on the concave end of the molecule combination E37A/E39A mutant molecule was more than because the hypothesis we were testing predicted that either or 4-fold compromised in CR2-binding activity and mutation of 3844 CR2 BINDING SITE IN C3d Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Rosette formation between CR2-bearing Raji cells and sheep red cells bearing various amounts of recombinant iC3b. The latter is indicated as cpm 125I-labeled anti-C3c bound per aliquot of 5 ϫ 107 iC3b-bearing red cells. The solid line near the bottom of each panel represents the background rosetting observed in control experiments employing EAC42 cells. The presence in the assay of the CR2-specific, functional site-blocking mAb OKB7 yielded a similar level of background rosetting when EAC423bi cells bearing high levels of wild-type iC3b were employed as the source of CR2 ligand. A recombinant wild-type control dose-response curve was included in all experiments. The error bars indicate the range of duplicate rosette counts (i.e., separate wet mount samplings) within an experiment. Data for the single and multiple alanine-substitution mutants of the 36–39 charged residue cluster are shown in A and B, respectively. Data for the single and multiple alanine-substitution mutants of the charged residues within the 160–167 cluster are shown in C and D, respectively. E–G, The results for the remainder of the mutants tested.

D36A in combination with E37A somewhat magnified the de- se, and at the same time address the issue of side chain stere- fect caused by the mutation of E37A alone. Although the com- ochemistry that becomes more of a factor when analyzing the bination mutant D36A/E39A showed a similar CR2-binding de- results of a triple mutation, the isosteric amide version of this fect as did the single E39A mutant, the D36A/E37A/E39A mutant was engineered. Like its alanine-substituted counterpart, triple mutation, which would effectively remove the negatively the D36N/E37Q/E39Q mutant was also found to be essentially charged rim from that side of the depression, yields a molecule inactive in mediating CR2 binding. Finally, alanine substitu- that displays only about 10% of the wild-type CR2-binding ac- tions were made at E42, an acidic residue that protrudes up at tivity. To further test the importance of the negative charges per the extreme edge of this surface of the molecule, and D292, The Journal of Immunology 3845

FIGURE 5. Bar graph summary of the CR2 binding activities of all of the mutants engineered for this study as determined from the CR2-depen- dent rosette assay. The percentage of wild-type CR2 binding activity of the mutant iC3b mole- cules was calculated from the horizontal displace- ment of the pseudo-linear part of a given dose- response curve from that of the wild-type curve on a semilogarithmic plot (relative amount of iC3b per cell is the log axis). Results from at least two (often three) independent experiments in- volving separate transfections were averaged. The error bars represent the range of activities ob-

served in the replicate measurements. Downloaded from http://www.jimmunol.org/ which extends the negative surface at the other edge of this rim contrast to the almost complete absence of CR2-binding activity of acidic residues (Fig. 2). In each case we found that the re- displayed by the D163A mutant, the double mutant K162A/D163A sultant mutant molecules retained between 80 and 90% of wild- retains much of the CR2-binding activity of the wild-type protein type CR2-binding activity (Figs. 4, E and F, and 5). Collec- (Fig. 4D and 5). It would seem that to overcome the charge re- tively, these data suggest that the side chains of the D36/E37/ pulsion caused by K162, contact with acidic residues D163, E166, E39 cluster make important ionic interactions with CR2 and the and perhaps E160 is necessary. However, in the absence of the contacts with E37 and E39 appear to be the most important K162 positive charge, the ionic contribution of at least D163 to the by guest on September 28, 2021 within this grouping of acidic residues. binding interface with CR2 becomes dispensable. We next turned our attention to the acidic residues along the When mutations to the acidic residues in the 160–167 segment opposite rim of the depression, which encompasses residues 160– were conducted in combination, the molecules were generally de- 167 of the primary sequence (Fig. 2). Also within this surface fective in CR2 binding (Fig. 4D and 5). For example, the residual patch, toward the edge of the cavity rim, is the hydrophobic side binding of the E160A mutant is abrogated in the double mutant chain of I164 and protruding away from the rim, but in between E160A/D163A and the combined mutation to alanine of D163 and acidic residues D163 and E166, is the basic side chain of K162 E166, both of which were largely inactive as single mutants, does (Fig. 2). This region appears to be extremely important for the not result in the acquisition of any substantial new CR2 binding interaction between C3d and CR2 as even single-alanine substitu- activity. The one exception to this general rule is that the combi- tions were sufficient to decrease CR2 binding levels to Ͻ5% of nation of E166A, which was largely inactive as a single mutant, wild-type for D163 and I164 and to ϳ10% of wild-type for E166 with E167A, which as a single mutant retained a near wild-type (Figs. 4, C and E, and 5). D163 was also mutated to its isosteric level of activity, yielded a molecule retaining ϳ30% of wild-type amide, and although the deposition efficiency onto the C3 conver- CR2-binding activity. This result is difficult to explain, but it is tase-bearing cells was inexplicably lower for the D163N mutant possible that in the absence of the mutual charge repulsion be- protein than for any of the other mutants examined, by using more concentrated culture supernatants, sufficient deposition was tween the carboxylates of D163, E166, and E167, the side chain of achieved to ascertain that it was the negative charge per se at the D163 may reorient so as to partially accommodate the loss of the D163 position that was required to mediate binding to CR2 (Fig. ionic contribution of E166 to the binding energy. 5). The mutation to alanine of D160, which lies at the bottom of Fig. 4, E and F, shows the rosette assay results for six additional the cavity, had a somewhat lesser effect, but still reduced CR2 single-residue mutants examined in this study (V97A, N98A, R49 binding 4-fold relative to wild-type. In contrast, E167, which rel- M, K251A, K291A, and Y201A). It can be seen that these muta- ative to the cavity is the most distally located acidic residue in this tions were found to have relatively minor effects on CR2 binding segment, could be mutated to alanine with minimal effect on CR2- activity, the largest effect being seen for the K251A mutant, which binding activity. One of the most interesting results was obtained retains about 60% of wild-type activity. From these data we con- with the K162A mutant protein as this molecule displayed a 2-fold clude that CR2 appears to primarily bridge between contacts in the enhancement in CR2 binding activity. Our interpretation of this D36/D37/D39 cluster and the E160/D163/I164/E166 cluster. Be- result is that the positive side chain of K162 partially inhibits the cause V97 and N98 essentially form a surface bridge between binding to this region of C3d of the electropositive surface con- these two contact clusters, it was somewhat surprising that their tributed by CR2, so its removal results in increased binding of mutation had virtually no effect on CR2-dependent rosette CR2. In keeping with this interpretation, we have found that in formation. 3846 CR2 BINDING SITE IN C3d

A subset of the mutant molecules was also examined in the Concluding remarks context of C3dg for their binding activity to CR2 as assessed using We believe that the results of our experiments conclusively show the rosette assay. Our purpose was to ensure that the major defects that two predominantly acidic surface patches provide crucial con- in CR2-binding activity that we had observed in the context of tacts for the interaction with the C3d-binding domains of CR2 (i.e., iC3b were not due to a secondary effect of a particular mutation CR2 SCR domains 1 and 2). Of the two acidic clusters, E37/E39 promoting an unfavorable interaction between the C3d and C3c and E160/D163/E166, which are located on opposite sides of the regions of the molecule such that the CR2 binding site of C3d depression on the concave surface of C3d, the latter appears to be became sterically masked. The results of this experiment are the more important based on the fact that single mutations within shown in Fig. 6. It can be seen that the relative CR2-binding ac- it were sufficient to almost completely abrogate CR2 binding. The tivities observed in the case of the ligand being C3dg largely mir- contribution of the hydrophobic side chain of the adjacent residue rored those obtained using iC3b as the CR2 ligand. The only sub- I164 also appears to be vital. The modeled structure of the C3d- stantial exception noted was that the extent of the defect for the binding domains of CR2 (17, 20) indicates that the adjacent resi- E166A mutant was less than what was observed in the context of dues R89 and H90 are part of one electropositive surface and that iC3b (i.e., 3- to 4-fold as C3dg vs 10-fold as iC3b) and was now R13 is part of another. The significance of these residues is that similar to the defect observed for the E160A mutant. Thus, al- they are contained within the CR2-derived synthetic peptide seg- though for the E166A mutant there appears to be some superim- ments that were shown to be inhibitory to the CR2-C3d interaction posed secondary effect, this is not the case for any of the other (17). Confirmation that ionic contacts are actually formed between mutants showing a CR2-binding defect. Therefore, these results the two acidic patches of C3d identified in this work and the two lend further support to the concept that the CR2-contacting resi- basic patches suggested by the model of the binding domains of Downloaded from dues putatively identified in this study are essentially equally ac- CR2 of course awaits the achievement of the structure of a coc- cessible for CR2 binding regardless of whether the ligand is iC3b rystal of C3d complexed to SCRs 1 and 2 of CR2. or C3dg. It is noteworthy that the negative charges of two of the residues Binding of soluble CR2 to wild-type and mutant iC3b-coated red that we have identified as being key contributors to the binding cells interface, specifically E39 and D163, are not conserved in cobra or trout C3, but they are conserved in mouse C3. Whereas cobra and http://www.jimmunol.org/ Because the rosette assay is highly dependent on avidity effects, trout iC3b/C3d are known not to be able to interact with human i.e., there are many potential points of contact between the ligand- CR2 (44), mouse C3d binds well to human CR2 (45). Additionally bearing red cell and the receptor-bearing Raji cell, it is possible I164, which is conserved in human and mouse C3, is replaced by that this assay would miss the effects of mutations that bring about valine in cobra and trout C3 and E166 is replaced by lysine in relatively minor changes to the intrinsic affinity governing the in- cobra C3. Based on our current work, these substitutions on their teraction between individual iC3b and CR2 molecules. This may own would account for the lack of CR2 binding by trout and cobra be especially true for some of the single-residue mutations exam- C3. However, this does not preclude the possibility of there being ined in the previous section. Although not completely devoid of contacts to other regions of the C3d molecule in which there is by guest on September 28, 2021 avidity effects, an assay measuring the binding of a bivalent sol- further nonconservation of key contact residues among human uble form of CR2 to iC3b-coated red cells should be less prone to CR2-binding and nonbinding species of C3. such avidity “masking” effects because the ligand-receptor con- As mentioned in the Introduction, at one point a peptide segment tacts are maximally bivalent. Accordingly, red cells bearing vari- corresponding to C3d residues 228–239 (1199–1210 mature C3 ous amounts of wild-type or mutant iC3b were prepared and in- numbering) had been thought to be a major contact region for the cubated with a fixed concentration of soluble CR2 in the form of a (CR2)2-IgG fusion protein (3, 23). Binding of the soluble CR2 to the cells was revealed by an FITC-conjugated anti-mouse IgG in conjunction with flow cytometric analysis. The relative amount of iC3b ligand per cell was determined as before using 125I-labeled anti-C3c and background fluorescence levels were determined us- ing EAC42 cells. The results of such assays employing all of the single mutants engineered for this study are shown in Fig. 7. In general, the results were concordant with the rosette assay. In no case did mutants that showed severe defects in the rosette assay not also show severe defects in the soluble CR2 binding assay; how- ever, as expected, the effects of some mutations were more severe in the soluble CR2 binding assay. For example, whereas the D36A and E42A mutations were without effect in both assays, near com- plete CR2 binding impairment was seen in mutants E37A and E39A (Fig. 7A), this contrasting with the 2-fold defect seen in the rosette assay for these latter two mutants. The data in Fig. 7B further confirm the enhancing effect of the K162A mutation noted in the rosette assay. Additionally, the E160A mutation on its own was sufficient to completely abrogate CR2-binding activity mea- FIGURE 6. Rosette formation between CR2-bearing Raji cells and sured by this assay, whereas this mutant showed about 25% re- sheep red cells bearing various amounts of recombinant C3dg. The latter is sidual activity in the rosette assay. The small decreases in CR2 indicated as cpm 125I-labeled anti-C3d bound per aliquot of 5 ϫ 107 C3dg- binding detected by the rosette assay for mutants D292A and bearing red cells. The background level of rosette formation in each ex- K251A were confirmed and mutation of V97, a nonpolar residue periment was determined using EAC42 cells and is indicated by the solid lying immediately adjacent to the critical I164, gave rise to an line at the bottom of the figure. The average of duplicate measurements is ϳ2-fold defect in the soluble CR2 binding assay (Fig. 7D). plotted and the range is indicated by the error bars. The Journal of Immunology 3847 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Flow cytometric measurement of the binding of soluble CR2 (i.e., (CR2)2-IgG fusion protein) to sheep red cells bearing various amounts of recombinant iC3b. The mean fluorescence was plotted as a function of the relative number of iC3b molecules per red cell, as determined by measuring the binding of 125I-labeled anti-C3c to a fixed number of iC3b-coated red cells. Background fluorescence (solid line at bottom of each panel) was determined by measuring the binding of soluble CR2 to EAC42 cells. Only the single substitution mutants were tested in this assay. A, Results for charged residues located in the 36–42 segment. B, Results for charged residues in the 160–167 segment. C and D, Results for the remainder of the mutants tested. The average of duplicate measurements is plotted and the range is indicated by the error bars. interaction with CR2. However, we have previously reported (23) 2. Heyman, B., E. J. Wiersma, and T. Kinoshita. 1990. In vivo inhibition of the that quite extensive substitution of this segment led to a fairly antibody response by complement receptor-specific monoclonal antibody. J. Exp. Med. 172:665. minimal defect in CR2 binding activity and thus at best, it plays a 3. Hebell, T., J. M. Ahearn, and D. T. Fearon. 1991. Suppression of the immune fairly minor role in mediating the C3d-CR2 interaction. Neverthe- response by a soluble complement receptor of B . Science 254:102. less, the coupling of a single copy of a peptide encompassing this 4. Ahearn, J. M., M. B. Fischer, D. Croix, S. Georg, M. Ma, J. Xia, X. Zhou, R. G. Howard, T. L. Rothstein, and M. C. Carroll. 1996. Disruption of the Cr2 segment to the variable region of an Ab was recently reported to locus results in a reduction in B-1a cells and an impaired response to T-dependent have significant molecular adjuvant properties in the production of . Immunity 4:251. anti-Id Abs against the immunizing Ig (46). Given this result, one 5. Croix, D. A., J. M. Ahearn, A. M. Rosengard, S. Han, G. Kelsoe, M. Ma, and M. C. Carroll. 1996. Antibody response to T-dependent antigen requires B cell might predict that a similar approach focusing on the more crucial expression of complement receptors. J. Exp. Med. 183:1857. peptide segments identified in the present work would produce in 6. Kurtz, C. B., E. O’Toole, S. M. Christensen, and J. H. Weis. 1990. The murine an even greater molecular adjuvant effect. We are currently pur- complement receptor gene family. IV. Alternative splicing of Cr2 gene tran- suing such investigations. scripts predicts two distinct gene products that share homologous domains with both human CR2 and CR1. J. Immunol. 144:3581. 7. Fischer, M. B., M. Ma, S. Georg, X. Zhou, J. Xia, O. Finco, S. Han, G. Kelsoe, Acknowledgments R. G. Howard, T. L. Rothstein, et al. 1996. Regulation of the B cell response to T-dependent by classical complement pathway. J. Immunol. 157:549. We thank Russell G. Jones, currently of the Department of Medical Bio- 8. Rickert, R. C., K. Rajewsky, and J. Roes. 1995. Impairment of T-cell-dependent physics, University of Toronto, for his contributions to this study. We also B-cells responses and B-1 cell development in CD19-deficient mice. Nature 376: thank Cheryl Smith, of the Department of Immunology, University of To- 352. ronto, Flow Cytometer Facility for her assistance in carrying out the flow 9. Engel, P., L.-J. Zhou, D. C. Ord, S. Sato, B. Koller, and T. F. Tedder. 1995. cytometric measurements that we have reported. Abnormal B development, activation and differentiation in mice that lack or overexpress the CD19 signal transduction molecule. Immunity 3:39. 10. Matsumoto, A. K., J. Kopicky-Burd, R. H. Carter, D. A. Tuveson, T. F. Tedder, References and D. T. Fearon. 1991. Intersection of the complement and immune systems: a 1. Carroll, M. C. 1998. The role of complement and complement receptors in in- signal transduction complex of the B-lymphocyte-containing complement recep- duction and regulation of immunity. Annu. Rev. Immunol. 16:545. tor type 2 and CD19. J. Exp. Med. 173:55. 3848 CR2 BINDING SITE IN C3d

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