The and C4B Isotypic Forms of Human Complement Fragment C4b Have the Same Intrinsic Affinity for 1 (CR1/CD35) This information is current as of October 4, 2021. Liliana Clemenza and David E. Isenman J Immunol 2004; 172:1670-1680; ; doi: 10.4049/jimmunol.172.3.1670 http://www.jimmunol.org/content/172/3/1670 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The C4A and C4B Isotypic Forms of Human Complement Fragment C4b Have the Same Intrinsic Affinity for (CR1/CD35)1

Liliana Clemenza*† and David E. Isenman2*

Several previous reports concluded that the C4b fragment of human C4A (C4Ab) binds with higher affinity to CR1 than does C4Bb. Because the isotypic residues, 1101PCPVLD and 1101LSPVIH in C4A and C4B, respectively, are located within the C4d region, one may have expected a direct binding contribution of C4d to the interaction with CR1. However, using surface plasmon resonance as our analytical tool, with soluble rCR1 immobilized on the biosensor chip, we failed to detect significant binding of C4d of either isotype. By contrast, binding of C4c was readily detectable. C4A and C4B, purified from plasma lacking one of the isotypes, were C1៮s converted to C4Ab and C4Bb. Spontaneously formed disulfide-linked dimers were separated from monomers Downloaded from and higher oligomers by sequential chromatographic steps. The binding sensorgrams of C4Ab and C4Bb monomers as analytes reached steady state plateaus, and these equilibrium data yielded essentially superimposable saturation curves that were well fit by a one-site binding model. Although a two-site model was required to fit the equilibrium-binding data for the dimeric forms of

C4b, once again there was little difference in the KD values obtained for each isotype. Independent verification of our surface plasmon resonance studies came from ELISA-based inhibition experiments in which monomers of C4Ab and C4Bb were equi- potent in inhibiting the binding of soluble CR1 to plate-bound C4b. Although divergent from previous reports, our results are http://www.jimmunol.org/ consistent with recent C4Ad structural data that raised serious doubts about there being a conformational basis for the previously reported isotypic differences in the C4b-CR1 interaction. The Journal of Immunology, 2004, 172: 1670Ð1680.

he immune adherence receptor mediates attachment of main combinations of SCRs 8–10 and SCRs 15–17 of LHR-B - and C4b-opsonized particles to primate erythrocytes and LHR-C, respectively, each became known as functional site and phagocytic cells (1, 2). The protein responsible for 2. It later became clear that site 2 was also capable of binding T 3 this activity, subsequently named complement receptor 1 (CR1), C4b (9, 10), although there is dispute about whether the site 2 consists in its most common variant form of 30 extracellular re- interaction with C4b is weaker (9) or stronger (10) than that of peating modules, alternatively referred to as short consensus repeat C4b with site 1. by guest on October 4, 2021 (SCR) or complement control protein domains (3, 4). Based on In humans, C4 is encoded by two distinct genes, C4A and internal sequence homology, the N-terminal-most 28 domains may C4B. Although the resulting mature heterotrimeric proteins be further subdivided into four long homologous repeats (LHRs), consisting of 93-kDa ␣-, 75-kDa ␤-, and 33-kDa ␥-chains are A, B, C, and D, each one consisting of seven SCR domains. Initial Ͼ99% identical with each other, the C4A and C4B isotypic ligand-mapping studies indicated that C4b binding was localized variants are defined by the presence of one of two hexapeptide to LHR-A, whereas LHR-B and LHR-C each had a binding site for sequences, 1101PCPVLD1106 and 1101LSPVIH1106 for C4A and C3b (5–7). It was further determined that most of the binding C4B, respectively (mature human C4 numbering used through- energy was contributed by the first three SCR domains of each out). These residues are located in the central C4d fragment LHR, although the first four domains were required to yield bind- (ϳ42 kDa) of the ␣-chain ϳ100 aa C-terminal to the thioester- ing activity equivalent to the full-length LHR segment (7, 8). The forming residues, the latter mediating covalent attachment of C4b binding site in SCR domains 1–3 became known as functional nascently activated C4b fragment to the C1-bearing target sur- site 1, and the C3b binding sites in the nearly identical three do- face. Each of the isotypes displays allelic polymorphisms, with the C4d portion of ␣-chain being the most polymorphic segment of the molecule (11). Included in these are residues at four *Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada; and †Department of Biopathology and Biomedical Methodologies, University of Palermo, positions (1054, 1157, 1188, and 1191), which together with Palermo, Italy those in the 1101–1106 isotypic sequences, give rise to the Received for publication July 11, 2003. Accepted for publication November 14, 2003. Rodgers (Rg) and Chido (Ch) blood group alloantigens. Gen- The costs of publication of this article were defrayed in part by the payment of page erally, the Rg-determining polymorphic residues segregate with charges. This article must therefore be hereby marked advertisement in accordance the C4A isotypic residues, while Ch-determining residues seg- with 18 U.S.C. Section 1734 solely to indicate this fact. regate with the C4B isotypic residues, but there are rare excep- 1 This work was supported by Grant MOP-7081 from the Canadian Institutes of Health Research. tions to this rule (12). The most profound functional difference between the isotypes is 2 Address correspondence and reprint requests to Dr. David E. Isenman, Department of Biochemistry, University of Toronto, 1 King’s College Circle, MSB 5306, Toronto, in the nature of the covalent bond formed between the isotypic C4b Ontario, Canada, M5S 1A8. E-mail address: [email protected] fragments and the target surface, specifically an amide bond in the 3 Abbreviations used in this paper: CR1, complement receptor type 1; C4BP, C4b- case of C4Ab and an ester bond in the case of C4Bb (13, 14). This binding protein; Ch, Chido; fI, ; LHR, long homologous repeat; Rg, Rodgers; RU, resonance unit; SCR, short consensus repeat; sCR1, soluble CR1; is in turn controlled by the residue at position 1106, with histidine SLE, systemic lupus erythematosus; SPR, surface plasmon resonance. dictating ester bond formation, whereas virtually any other amino

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 1671 acid will result in amide bond formation (15–17; mechanism re- Because the isotypic residues reside within the C4d region of the viewed in Ref. 18). The nucleophilic preferences of the respective molecule, one might expect a contribution of this region to the isotypic C4b fragments have been invoked to explain the greater binding interaction with CR1 in a way that reflects the isotype- transacylation efficiency of C4Ab to amino group-rich immune defining sequence differences. Specifically, the isotypic residues complexes vs the greater transacylation efficiency of C4Bb to car- may either be involved in direct contact with the receptor, or they bohydrate-rich cellular target surfaces (14, 15, 19). The higher may affect at a distance the conformation of a contact region in transacylation efficiency of C4Ab to immune complexes has in another part of the C4d molecule. With respect to the latter pos- turn been used to explain the greater ability of native C4A, com- sibility, the presence of an additional proline (P1101) in the C4A pared with C4B, to inhibit immune precipitate formation in serum isotypic sequence appeared to be a promising candidate to mediate or in serum-free mixtures containing Ag, IgG Ab, C1, and C4 (20, such a conformational effect. However, the recently determined 21). Other functional properties, including cleavage rate by C1៮s, x-ray crystal structure of human C4Ad fragment did not support classical pathway C3 convertase subunit activity, regulation by either one of these explanations (40). First, the loop containing the complement factor I (fI) in the presence of C4b-binding protein isotype-defining residues is in very close proximity to the site of (C4BP), and ability to act as a transacylation acceptor for C3b in covalent attachment, and as such, these residues are unlikely to be the formation of the classical pathway C5 convertase, were found accessible for direct contact by CR1. Second, although there cur- not to differ substantially between C4A and C4B (13, 14, 22). rently is no structure for C4Bd, based on the superimposability of However, there have been three reports concluding that C4Ab (or, the C4Ad structure on that of C3d, which is C4B-like in its isotypic in one case, ammonia-treated C4A, a thioester-cleaved C4b-like segment and thus lacks a proline in the first position, we have species) displayed higher binding to CR1 than did C4Bb, or am- previously argued that in all likelihood its backbone conformation monia-treated C4B (23–25). The study by Reilly and Mold (25) will be indistinguishable from that of C4Ad (40). This would then Downloaded from used isotypic forms of covalent dimers of human C4b fragment, in render problematic the long-range conformational explanation for which the cross-link was via the liberated thioester cysteine, to the isotype-dependent differences in the binding of C4b to CR1. actually measure equilibrium-binding constants. They determined There remained the possibility that the CR1-binding differences that dimers of C4Ab displayed a ϳ4-fold higher affinity for CR1 reflect contact differences with the major Ch/Rg epitopes defined than did dimers of C4Bb. by residues 1157, 1188, and 1191. As mentioned above, these One reason for the interest in the functional properties of C4, normally segregate with the isotype-defining residues and, as vi- http://www.jimmunol.org/ and in particular of each isotype, is the apparent protective effect sualized in the C4Ad structure, they should be highly accessible of C4 with respect to susceptibility to the autoimmune disease (40). In view of the various questions raised by the structure of systemic lupus erythematosus (SLE; reviewed in Ref. 26). In hu- C4Ad about the basis for reported CR1-binding differences of mans, there is a 75% prevalence of SLE with complete C4 defi- C4Ab and C4Bb, as well as the possible relevance of this differ- ciency, this being second only to C1q deficiency in which the ential affinity to our understanding of the etiology in humans of prevalence is 93%. By contrast, there is no correlation of SLE with SLE, we decided to re-examine this issue. Specifically, we have C3 deficiency, and the correlation with C2 deficiency is relatively used both surface plasmon resonance (SPR) and ELISA tech- weak both in terms of frequency and severity of the symptoms. niques to determine whether there is indeed an intrinsic affinity by guest on October 4, 2021 Although in humans complete deficiency states of C4 are quite difference between C4Ab and C4Bb for a soluble recombinant rare, in healthy white individuals, homozygous C4A and C4B de- form of CR1 (sCR1). Additionally, we have assessed the re- ficiencies occur with an estimated frequency of 4 and 1%, respec- spective abilities of isolated C4c and isotypic variants of C4d tively (26). Although not seen in every SLE patient cohort (e.g., fragments to bind to sCR1. Refs. 27 and 28), there have been multiple reports spanning two decades of an increased frequency of complete, or even partial, C4A, but not C4B, deficiency states in SLE patient cohorts as Materials and Methods compared with healthy controls (29–37). The association of C4A Purified proteins null alleles with increased risk for SLE transcended ethnic bound- Human rsCR1 was a gift from Avant Immunotherapeutics (Needham, aries, and thus was not readily attributable to the association of the MA), and human C1៮s was purchased from Advanced Research Technol- C4A null allele to an extended MHC haplotype. ogies (San Diego, CA). C4A and C4B were isolated, as previously de- There are two nonmutually exclusive hypotheses for explaining scribed (41), from plasma lacking one of the two isotypes. Because these the strong association of early classical pathway component defi- proteins had been stored at Ϫ70°C for long periods of time, they contained a significant amount of thioester-hydrolyzed C4(H O) material that would ciencies and SLE. First, C4 may have a role in the induction of 2 not be cleavable by C1៮s (42). Accordingly, these materials were repurified self-tolerance against autoantigens (38, 39). Second, C4 has a role on a Mono-Q HR 10/10 FPLC column (Amersham-Pharmacia, Baie in the disposal of immune complexes and apoptotic bodies, the D’Urfe´, Quebec, Canada), essentially as described by Hessing et al. (43), latter being loaded with precisely the autoantigens that one sees a procedure that is able to separate native C4 from C4(H2O). The purified Abs against in SLE patients (26). The deposition of C4b fragment proteins were converted to C4Ab and C4Bb, respectively, by overnight digestion at 37°C with 1:100 w/w C1៮s. The digestion mixture was then onto the target both provides the subunit for classical pathway C3 chromatographed on a Mono-Q HR 10/10 FPLC column, as described convertase formation and acts as a direct ligand for CR1, both of previously, as a means of separating monomer C4b from spontaneously these events being important for the complement-facilitated dis- formed disulfide-linked dimer C4b (43). Briefly, using a flow rate of 1 posal process. At present, nothing is known about the relative ml/min and a starting buffer consisting of 50 mM NaCl, 20 mM Tris-HCl, transacylation efficiency of C4Ab vs C4Bb to apoptotic bodies. and 2 mM EDTA, pH 7.4, the proteins were eluted from the Mono-Q column with a 50 ml linear gradient to a final concentration of 0.5 M NaCl. However, both the greater ability of C4Ab relative to C4Bb to bind C4b dimers and monomers were collected as separate fractions and then covalently to IgG, and thereby inhibit large and insoluble immune dialyzed against PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.2). complexes from forming (20, 21), as well as the reported 3- to Following their respective concentration using Biomax Ultrafree-4 10-kDa 4-fold higher affinity of C4Ab for CR1 (25) can be envisaged as cutoff spin concentrators (Millipore, Bedford, MA), each fraction was sub- jected to gel filtration on an FPLC Superose 6 HR 10/30 column (Amer- preferentially facilitating the clearance of C4Ab-opsonized autoan- sham-Pharmacia) equilibrated with PBS containing 0.02% NaN3 and run- tigens. This, in turn, has been invoked as an explanation for the ning at a flow rate of 0.3 ml/min. Before use in BIAcore experiments, the SLE association with C4A, but not C4B deficiency states. proteins were dialyzed into 10 mM HEPES, 75 mM NaCl, and 3 mM 1672 ANALYSIS OF CR1 BINDING TO C4Ab AND C4Bb

EDTA, pH 7.4, and then surfactant P-20 (BIAcore, Piscataway, NJ) was transformations having the following forms for the one-site (Equation 3) added to a concentration of 0.01%. and two-site (Equation 4) models, respectively (46): C4c was obtained from purified pooled C4 (i.e., containing a mixture of ⌬RU both C4 isotypes) that was digested first with C1៮s, as above, and subse- ϭ ϫ ⌬ Ϫ ϫ ⌬ ͓ ͔ KA RUmax KA RU (Equation 3) quently with factor I (1:30 w/w) and C4BP (1:50 w/w) for 18 h at 37°C. analyte Factor I (44) and C4BP (45) were purified from human plasma, as de- ⌬RU ϭ 0.5 ϫ ͕K ͑⌬RU Ϫ ⌬RU͒ ϩ ͑K ͑⌬RU Ϫ ⌬RU͒ ϩ scribed previously. C4c was then purified by the same two-step chromato- ͓analyte͔ A1 max 1 A2 max 2 graphic procedure described above for C4b and exchanged by dialysis into ͱ͕ ͑⌬ Ϫ ⌬ ͒ Ϫ ͑⌬ Ϫ ⌬ ͖͒2 ϩ ⌬ ⌬ ͖ 10 mM HEPES, 75 mM NaCl, and 3 mM EDTA, pH 7.4 buffer, and for KA1 RUmax 1 RU KA2 RUmax 2 RU 4KA1KA2 RUmax 1 RUmax 2 BIAcore experiments, surfactant P-20 was added to 0.01%. (Equation 4) Recombinant C4Adg and C4Bdg fragments, differing only with respect to their 1101–1106 isotype-specific residues, were constructed and ex- where for Equation 4, the numerical subscripts refer to the equilibrium pressed in Escherichia coli, as previously described (40). Cell lysates were association constant (KA) and maximal response units signal change at ⌬ loaded onto a 100-ml DEAE-Sephacel column (Amersham-Pharmacia) saturation ( RUmax) associated with each class of binding site. Kinetic data equilibrated with 10 mM Na phosphate, 25 mM NaCl, 2 mM EDTA, and were analyzed globally using the BIAevaluation 3.0 software package. The 0.1 mM DTT, pH 7.1 (buffer A). Following extensive washing, the C4dg binding model used for the C4b and C4c monomer sets of sensorgrams was ϩ 7 7 protein was eluted by increasing the NaCl concentration of the buffer to the conformational change model: A B AB AB* (analyte (A) binds to 150 mM. DEAE-purified protein was dialyzed back into buffer A and ligand (B); complex AB changes to AB*, which cannot dissociate directly to AϩB). The binding model used for the C4b dimer curves was the par- loaded onto a Mono-Q HR 10/10 FPLC column equilibrated in this buffer. allel binding model for a two-site heterogeneous binding model: Elution was with a linear gradient to a final buffer containing 0.5 M NaCl AϩB17AB1; AϩB27AB2 (the binding curve obtained is the sum of the at a flow rate of 2 ml/min and a gradient duration of 20 min. The respective two independent reactions). C4Adg and C4Bdg proteins were then further purified by gel filtration on Downloaded from an FPLC Superdex 200 HR10/30 column (Amersham-Pharmacia) equili- ELISA-based inhibition-binding assay brated in 10 mM HEPES, 75 mM NaCl, and 3 mM EDTA, containing 0.1 ELISA plates were coated with 100 ␮lof10␮g/ml mixed-isotype C4b mM DTT. fragment. In a preliminary experiment in which variable concentrations of The purity of all proteins was assessed by SDS-PAGE. The extinction ϳ 1% sCR1 were added to the C4b-coated wells, we established that 50% of coefficients (E280 nm) used were 8.2 for C4Ab/C4Bb and C4c and 13.6 for maximal binding occurred at a concentration of 1 ␮g/ml sCR1. For the C4Adg/C4Bdg. inhibition experiments, this concentration of sCR1 was preincubated for

1 h at room temperature with varying concentrations of C4Ab/C4Bb mono- http://www.jimmunol.org/ mer (from 200 to 3.125 ␮g/ml) in a total volume of 100 ␮l. Uncomplexed SPR measurements sCR1 in each preincubation mix was then allowed to bind to plate-bound C4b (1 h, room temperature), and its binding was detected by using the The respective interactions of C4Ab and C4Bb with sCR1 were analyzed anti-CR1 mAb E11 (BD PharMingen, Mississauga, Ontario, Canada) as the by SPR technology on a BIAcore X instrument (BIAcore). The experi- primary Ab and an anti-mouse IgG conjugated to alkaline phosphatase as ments were performed at room temperature in a half physiologic ionic the secondary Ab (Jackson ImmunoResearch Laboratories, West Grove, strength buffer (10 mM HEPES, 75 mM NaCl, 3 mM EDTA, and 0.01% PA). The assay was performed in 1/3 ionic strength PBS (10 mM Na

surfactant P-20, pH 7.4). sCR1 was diluted in 10 mM Na acetate, pH 5.5, phosphate, 50 mM NaCl, and 0.02% NaN3, pH 7.2). at a concentration of 20 ␮g/ml and immobilized on flow cell 1 of a CM5 chip (BIAcore) by using the amine-coupling kit from BIAcore. Flow cell Results 2, to which mouse IgG (Sigma-Aldrich, St. Louis, MO) was immobilized SPR assessment of the interaction of the C4d and C4c fragments by guest on October 4, 2021 using the same coupling conditions, was used as a control surface. A chip with CR1 with 6800 resonance units (RU) on the sCR1 ligand surface and 6350 RU on the control surface was used to test the binding of C4Ab, C4Bb, and C4c Given that the C4d fragment forms an independently folded do- fragments for most of the experiments shown in this study. A chip with main entity (40) and the indications from the literature (23–25) that 4900 RU on the sCR1 ligand surface and 4400 RU on the control surface the C4d-localized isotypic segment in some way affects the inter- was used to test the binding of the isotypic C4dg fragments. The various C4 action of the parent C4b molecule with CR1, we first wished to fragment protein analytes were used immediately after their last purifica- determine whether C4d on its own displayed any binding activity tion step. Binding was measured by injecting over the ligand-coupled chip several concentrations of analyte at a flow rate of 30 ␮l/min for 90–100 s. for CR1, and if so, whether there were isotype-dependent differ- Following 180 s of buffer flow for dissociation, the sensor chip was re- ences in binding activity. For these experiments, we have used generated with 1 M NaCl. The interaction of the C4dg fragments with rC4d fragments corresponding in length to the physiologic fI sCR1 was also studied by reversing the immobilized ligand-analyte com- cleavage-derived product (residues 938-1317), but, by analogy bination. Specifically, two different chips were generated by immobilizing with the C3 fragment nomenclature, we have named them C4Adg ␮ C4Adg and C4Bdg to the respective sample flow cells (50 g/ml in 10 mM and C4Bdg. This is to distinguish them from the N-terminally trun- Na acetate, pH 4.7, ϳ1900 RU), and the control flow cells were in this case simply sham activated and then ethanolamine deactivated. cated C4d portion of the molecule that is visible in the x-ray struc- Equilibrium phase-binding data were fit to one- and two-site models of ture and that corresponds in length to the C3d proteolytic limit the Langmuir binding isotherm using the nonlinear regression program fragment of fI-derived C3dg. We tested several concentrations of MacCurveFit 1.5 (K. Raner Software, Victoria, Australia). The equations C4Adg or C4Bdg analyte (1–12 ␮M) for binding to biosensor- describing these models were respectively: coupled sCR1, but found no binding signal above that seen on the control IgG-coupled flow cell. Shown in Fig. 1 are representative ⌬RU ϫ ͓analyte͔ ⌬ ϭ max experiments performed at 9.5 ␮M for each of C4Adg and C4Bdg. RU ϩ ͓ ͔ (Equation 1) KD analyte Because within each CR1 molecule there are potentially three binding sites for C4b, and because the interaction of CR1 with an ⌬ ϫ ͓ ͔ ⌬ ϫ ͓ ͔ RUmax 1 analyte RUmax 2 analyte array of C4b molecules on a target is an avidity situation, we ⌬RU ϭ ϩ KD1 ϩ ͓analyte͔ KD2 ϩ ͓analyte͔ wished to determine whether binding of sCR1 would be observed to an array of C4dg molecules, as this better mimics the physio- (Equation 2) logic avidity situation. Accordingly, we reversed the analyte-li- gand combination from the first experiment and tested the binding where for Equation 2, the numerical subscripts refer to the equilibrium of several concentrations of sCR1 to biosensor-coupled C4Adg or dissociation constant (KD) and maximal resonance units signal change at ⌬ saturation ( RUmax) associated with each class of binding site. The steady C4Bdg. Once again, however, no specific binding was observed in state plateau-derived binding curves were also displayed as Scatchard either case (results not shown). The Journal of Immunology 1673

FIGURE 1. Lack of interaction of recombinant C4Adg and C4Bdg with sensor-bound sCR1. A concentration of 9.5 ␮M of analyte was injected for 90 s over a sensor chip. sCR1 was coupled to the experimental flow cell (4900 RU), and human IgG was coupled to the control flow cell (4400 RU). The C4dg

curves are shown as dashed lines; the control curves as dotted lines; and the net curves, obtained by subtracting the respective control RU signals from the Downloaded from experimental RU curves, are shown in solid black. The arrows indicate the start and end points of the analyte injection.

In view of the failure of isolated C4dg to bind to CR1, we next dimer and C4Ab contains only a trace amount (as well as a trace asked whether the complementary fI-generated degradation frag- amount of C4c). The relative paucity of ␣Ј-dimer band(s) upon ment of C4b, namely C4c, could on its own bind to CR1. As can reduction indicates that the vast majority of the dimers are disul- be seen in Fig. 2, injection of variable concentrations of C4c ana- fide linked, as opposed to ester or amide linked, which would http://www.jimmunol.org/ lyte (see Fig. 3 for SDS-PAGE analysis of the C4c material) over an sCR1-coupled flow cell resulted in a dose-dependent and spe- cific binding response. Because a steady state plateau was not reached for any of the concentrations of analyte tested, we were unable to perform a direct analysis of the equilibrium-binding data. Global kinetic analysis of the binding of C4c to sensor-bound sCR1 using the BIAevaluation 3 software package did not yield a satisfactory fit to a simple 1:1 binding model. However, an accept- by guest on October 4, 2021 able fit(␹2 Ͻ 2) was obtained using a model in which a confor- mational change followed an initial binding event, and from these kinetic parameters one could extract a value of 1.6 ␮Masanes-

timated KD for the overall reaction (see Fig. 2 for further details). SPR assessment of the interaction of isotypic variants of C4b monomers and dimers with CR1 Although the isolated C4dg fragments do not have detectable bind- ing affinity for CR1 on their own, this region may still contribute to the binding of the parent C4b fragment, potentially in an iso- type-dependent manner. We therefore next analyzed the binding of FIGURE 2. Analysis of the interaction of C4c with sensor-bound sCR1. monomeric and dimeric forms of C4Ab and C4Bb to biosensor Sensorgram overlays generated by injecting several concentrations of C4c chip-coupled sCR1. As reported previously by Hessing et al. (43) (indicated at the right of the curves in decreasing order from the conditions for mixed isotype human C4, we observed that activation of either of the uppermost curve) over a chip coupled with 6800 RU of sCR1. Here, ៮ C4A or C4B by C1s resulted in 10–20% of the C4b material un- and in subsequent BIAcore data panels, the curves shown represent the net dergoing spontaneous disulfide-mediated dimerization. This most binding after the subtraction of the combined nonspecific binding and bulk likely occurs via the cysteine residue liberated upon the thioester refractive index change on the control channel that had been coupled with hydrolysis that accompanies the proteolytic cleavage event. In ac- 6300 RU of human IgG. The interaction was analyzed by global kinetic cordance with the published procedure, the monomer and disul- analysis using the conformational change model in the BIAevaluation 3.0 fide-linked dimer populations were separated by ion exchange on software package. The fitted curves are indicated with solid lines through the experimental time points. The conformational model is of the form: an FPLC Mono-Q column, and Fig. 4 shows the elution profile for ៮ C1s-treated C4A. Also shown in this figure are the FPLC gel fil- kd1 kd2 tration profiles for each of the monomer and dimer peaks from the A ϩ B N AB N AB* (Equation 5) Mono-Q column. The elution profiles for the C4B-derived material ka1 ka2 were very similar (data not shown). The SDS-PAGE analysis for where ka1 and kd1 are the forward and reverse rate constants for the en- the purified monomer and dimer forms of C4Ab and C4Bb is counter complex and ka2 and kd2 are the forward and reverse rate con- shown in Fig. 3. It can be seen on the nonreduced gel that the stants for the conformational change. The overall equilibrium KD is given dimer fractions contain relatively small amounts of contaminating by the formula (kd1/ka1) ϫ (kd2/ka2) and was 1.6 ␮M in this case. The monomer, and the level of contaminant is the same for both iso- individual rate constant values are as follows: ka1 ϭ 9.2 ϫ 104 MϪ1 sϪ1, types. The C4Bb monomer fraction appears to be totally devoid of kd1 ϭ 0.24sϪ1, ka2 ϭ 6.8 ϫ 10Ϫ3 sϪ1, kd2 ϭ 4.3 ϫ 10Ϫ3 sϪ1. 1674 ANALYSIS OF CR1 BINDING TO C4Ab AND C4Bb Downloaded from http://www.jimmunol.org/

FIGURE 3. SDS-PAGE of the purified C4b and C4c fragments used for BIAcore analysis. The proteins were analyzed on an 8% gel under nonre- ducing conditions (top) and a 10% gel under reducing conditions (bottom). Molecular mass markers are indicated on the left. by guest on October 4, 2021 represent thioester-mediated transacylation products. The material depicted on these gels is representative of the samples used in our SPR measurements, these measurements being performed as soon as possible after the Superose 6 chromatography. We did, how- ever, note that upon storage at 4°C, dimer formation occurred in the purified monomer fraction and that this appeared to be more prevalent for the purified C4Ab monomers. Depicted in Fig. 5A are the sensorgram overlay curves obtained for the interaction of the C4Ab and C4Bb monomer analytes in- jected over sensor-coupled sCR1 using an analyte concentration range of 0.06–1 ␮M. As can be seen readily for the first five incremental concentrations, in which the analyte concentrations were perfectly matched, the two isotypes of C4b monomer be- haved in a very similar manner. Both displayed fast association and fast dissociation kinetics and, as for C4c, the satisfactory global fitting of the kinetic data required the use of a conforma- tional model. As the sensorgrams reached a steady state plateau in all cases, we were able to directly analyze the equilibrium data by both nonlinear regression analysis of the saturation curves (Fig. 5B) and Scatchard transformation of the data (Fig. 5C). These analyses showed that the data for both C4Ab and C4Bb monomers were well fit by a simple 1:1 Langmuir binding model and that the two saturation curves were essentially superimposable. The Scat- ៮ chard transformations similarly yielded nearly identical linear FIGURE 4. Purification of monomers and dimers of C4b. C1s-digested C4A or C4B was first chromatographed on a Mono-Q column to separate plots that were indicative of simple homogeneous binding. Al- monomers from spontaneously formed dimers (top panel). The proteins though for both C4Ab and C4Bb the lowest concentration point were eluted with a 50 ml NaCl gradient, as indicated. Monomer (middle deviated somewhat from linearity and may reflect a degree of bio- panel) and dimer (bottom panel) fractions were further purified on a chemical heterogeneity in the binding reaction (see Discussion), Superose 6 gel filtration column. The boundaries of the collected ma- the deviation could also be artifactual, as this part of a Scatchard terial for each peak are indicated. The data shown are for C4A, but were plot is the most sensitive to deviations from ideal behavior as a similar for C4B. The Journal of Immunology 1675 Downloaded from

FIGURE 5. BIAcore analysis of the binding of purified monomers of C4Ab and C4Bb to biosensor-bound sCR1. A, Sensorgram overlay plots of the net binding data as a function of analyte concentration, with these being indicated in descending order on each set of overlay curves. The sCR1-coupled chip used was the same as for the experiment displayed in Fig. 2. The solid lines through the experimental points represent the fitted data obtained by global http://www.jimmunol.org/ kinetic analysis using the conformational change model, as described in Fig. 2. The KD values determined from the kinetic analysis were 0.26 and 0.31 ␮M for C4Ab and C4Bb monomers, respectively. B, Steady state analysis of the interaction of C4Ab and C4Bb monomers with sensor-bound sCR1. ⌬RU values determined from the steady state plateau region were plotted as a function of analyte concentration, and the data were fit by nonlinear regression according to the single site Langmuir binding model described by Equation 1 of Materials and Methods. The KD and RUmax values derived from these fits are given in Table I. C, Scatchard transformation of the steady state binding data. The lines drawn represent insertion into Equation 3 of Materials and Methods the parameters obtained from the nonlinear fits shown in B.

result of small errors in the measurements. As can be seen in Table pendent classes of binding site. Although we realize it to be some- by guest on October 4, 2021 ⌬ I, both isotypes of monomeric C4b displayed virtually identical KD what of an approximation, we have taken the RU values at the values of ϳ0.9 ␮M, and the total binding capacity, as defined by end of the injection phase to represent pseudo-steady state plateau ⌬ the respective RUmax values, was similarly close. values, as this allowed a comparison of equilibrium phase func- A previous report that compared the binding of the isotypic tional affinity constants for the dimer forms of C4Ab and C4Bb for forms of human C4b with red cell-associated CR1 used chemically CR1. Consistent with the kinetic analysis, the minimal model that cross-linked dimers of C4b (bismaleimidohexane cross-linking via could fit the pseudo-steady state saturation data was that having the thioester cysteine) to take advantage of the avidity effect, and two classes of binding sites (Fig. 6B). This is most clearly indi- therefore enhanced readout signal, that dimerization provides (25). cated by the inverted hyperbola shape of the Scatchard-trans- Accordingly, we also measured the binding of our spontaneously formed and fitted data (Fig. 6C). Nevertheless, the values of the formed disulfide-linked dimers of C4Ab and C4Bb to biosensor- equilibrium dissociation constants extracted for each class of site coupled sCR1. As can be seen from the overlay plots shown in Fig. are very similar for the two C4 isotypes, as is their fractional ⌬ 6A, the C4Ab and C4Bb dimers once again showed very similar representation of RUmax (see Table I for a summary of all the concentration-dependent binding behavior. This time, the best ki- equilibrium-binding data). Specifically, ϳ13% of the binding sites ϳ netic fit was obtained with a parallel binding model to two inde- are of high affinity and have a KD of 10 nM, but the bulk of the

Table I. BIAcore equilibrium state analysis of the interaction of sCR1 with C4Ab and C4Bb

Monomers Dimers

1-site modela 2-site modelb

␮ ⌬ 2c ␮ d ⌬ e d ⌬ e 2c KD, M RUmax R KD1, M RUmax1 (fraction RUmax) KD2,nM RUmax2 (fraction RUmax) R C4Ab 0.93 Ϯ 0.15 261 Ϯ 25 0.993 0.33 Ϯ 0.08 247 Ϯ 18 (0.86 Ϯ 0.06) 8.4 Ϯ 4.4 39 Ϯ 10 (0.14 Ϯ 0.03) 0.999 C4Bb 0.94 Ϯ 0.14 281 Ϯ 23 0.990 0.39 Ϯ 0.15 291 Ϯ 39 (0.88 Ϯ 0.12) 11.7 Ϯ 7.7 39 Ϯ 15 (0.12 Ϯ 0.05) 0.999

a Parameters fit according to Equation 1 of Materials and Methods; error estimates of the fit are indicated. b Parameters fit according to Equation 2 of Materials and Methods; error estimates of the fit are indicated. c Correlation coefficient of the fit. d For the 2-site binding model, reported KD values are based on concentrations of the C4b covalent dimer species and would be 2-fold higher if calculated based on the concentration of monomeric subunit. e ⌬ ⌬ Projected RU change at saturation for each component of the binding, with the fractional numbers in parentheses being the individual component RUmax divided by the sum of these for the two components. 1676 ANALYSIS OF CR1 BINDING TO C4Ab AND C4Bb Downloaded from

FIGURE 6. BIAcore analysis of the binding of purified dimers of C4Ab and C4Bb to biosensor-bound sCR1. A, Sensorgram overlay plots of the net binding data as a function of analyte concentration, with these being indicated in descending order on each set of overlay curves. The concentrations of http://www.jimmunol.org/ analyte used are based on the dimer molecular mass of 380 kDa. The sCR1-coupled chip used was the same as for the experiment displayed in Fig. 2. The solid lines through the experimental points represent the fitted data obtained by global kinetic analysis using the parallel binding to two classes of binding site model. B, Steady state analysis of the interaction of C4Ab and C4Bb dimers with sensor-bound sCR1. ⌬RU values derived from the end of the analyte injection phase were plotted as a function of analyte concentration, and the data were fit by nonlinear regression according to the two-site Langmuir binding model described by Equation 2 of Materials and Methods. The KD and fractional RUmax values derived from these fits are given in Table I. C, Scatchard transformation of the steady state binding data. The lines drawn represent insertion into Equation 4 of Materials and Methods the parameters obtained from the nonlinear fits shown in B. by guest on October 4, 2021 ϳ ␮ binding is of lower affinity, having a KD of 0.4 M. Because the we will argue that the higher binding of C4Ab observed was most analyte concentrations were calculated on the basis of a dimer likely due to effects that conferred on it a functional affinity/avidity molecular mass of 380 kDa, if one calculates it on the basis of C4b advantage in a particular experimental system. subunits present, this latter KD value becomes very similar in mag- nitude to what we had observed for monomeric C4b binding to biosensor-coupled sCR1.

ELISA-based inhibition assay measuring the fluid-phase interaction of sCR1 with C4Ab and C4Bb monomers As a means of verifying our SPR results via an independent ap- proach, we have used an ELISA-based inhibition assay in which the binding of sCR1 to plate-bound mixed-isotype C4b was eval- uated after preincubation with variable concentrations of mono- meric C4Ab or C4Bb as fluid-phase competitors. This assay format was chosen because any masking effect on C4b isotypic ligand preference brought about by the covalent coupling of sCR1 to the biosensor chip would not be a factor in this fluid-phase competi- tion assay. As shown in Fig. 7, and in accord with the SPR data, monomers of C4Ab and C4Bb were equipotent in their inhibitory activity.

Discussion In contrast to several previous reports in the literature (23–25), we find that there is no difference in the intrinsic binding ability of the FIGURE 7. ELISA-based inhibition assay of the binding of sCR1 to isotypic forms of human C4b fragment to human CR1. As will be plate-bound mixed isotype C4b by fluid-phase, monomeric C4Ab and elaborated upon below, the basic conflict between our results and C4Bb. The dashed line at the top of the figure indicates the absorbance in the earlier ones is not about the correctness of the earlier data, but the absence of fluid-phase inhibitors, and the background absorbance ob- rather that the higher binding to CR1 observed for C4Ab was being tained in the absence of added sCR1, but with all other ELISA detection ascribed to an inherent property of its contact site for CR1. Instead reagents present, is indicated by the dotted line at the bottom of the figure. The Journal of Immunology 1677

Intrinsic affinity generally refers to the equilibrium-binding con- of both C4Ab and C4Bb did show enhanced binding to biosensor- stant between a monovalent ligand and an acceptor molecule hav- coupled sCR1 due to avidity effects, but in this instance too the ing a single class of binding site for that ligand. So long as the magnitude of the effect was essentially the same for each C4 iso- acceptor sites are sufficiently separate so that there is no steric type. The high affinity component was ϳ100-fold greater than the interference between them when ligand is bound, multivalency of affinity observed with C4b monomers (Table I). This is commen- the acceptor molecule will in principle not affect the observed af- surate with the approximations made by others when they com- finity of the monovalent ligand. Although to the best of our knowl- pared the relative ability of unlabeled monomers and dimers of edge there is no definitive biophysical proof on this point, it has C4b, or C4(CH3NH2), to inhibit the binding of their labeled been generally assumed that a single C4b molecule acts as a mono- dimeric counterparts to the endogenous CR1 on human red cells valent ligand for CR1. CR1, however, has three potential binding (9, 47). What was somewhat unexpected was that higher avidity sites for C4b (9, 10), one in LHR-A (site 1) and one in each of component accounted for a relatively small fraction (ϳ13%) of the

LHR-B and LHR-C (site 2). There is evidence in the literature that total binding, whereas the majority of the binding had a KD com- a dimeric form of C4(CH3NH2), a thioester-cleaved C4b-like mol- mensurate with binding of C4b monomers (Table I). Either the ecule, can bind to sCR1 in solution in a monogamously bivalent procedure used to couple sCR1 to the biosensor chip had made manner, that is to two of the three potential C4b binding sites on bivalent attachment a sterically unfavorable event, or the time the same CR1 molecule (47). There is, however, dispute about course of the BIAcore experiment (100 s) is too short for a more whether binding of C4b to site 1 has a relative affinity ϳ3-fold substantial amount of bivalent binding to occur. With respect to the higher than that observed for site 2 (9) or whether the binding of latter point, although it was done at a lower temperature than our C4b to site 2 is, if anything, ϳ2-fold stronger than that to site 1 experiment (0°Cvs22°C), it has previously been reported that (10). In our experiments in which monomeric C4Ab and C4Bb binding of C4b dimers to red cell CR1 displayed very slow kinet- Downloaded from were injected over an sCR1-coupled biosensor chip, the data were ics, taking 90 min to reach equilibrium (9). well fit to a model having a single class of binding site. If there Inferences drawn from the structure of C4Ad, and its compari- were significant contribution of a second class of binding site son with the structure of C3d (40), had essentially predicted what whose affinity differed by 3-fold or more from that of the first class, we have observed experimentally, namely that the isotypic se- there should be more curvature in the Scatchard transformation of quence differences should not influence the respective intrinsic the data (Fig. 5C) than what was observed. Given that the reported CR1-binding affinities of C4Ab and C4Bb. The crux of the argu- http://www.jimmunol.org/ differences in binding strength in either direction of the two sites ment was that the backbone segments in and around the thioester, are relatively small (9, 10), and also that for monovalent ligand as well as the three-dimensionally proximal isotype-defining se- there is a 2:1 ratio of site 2 to site 1 present in full-length sCR1, we quences, were completely superimposable. Yet, the C4A-specific believe that the most likely interpretation of the essentially homo- sequence PCPVLD was at least as dissimilar from the correspond- geneous nature of the binding data observed is that there are no ing C3d segment DAPVIH as it was from the C4B-specific iso- major differences in affinity for C4b between sites 1 and 2. Thus, typic sequence LSPVIH. In particular, the extra proline of the ϳ ␮ the observed KD of 0.9 M for both C4Ab and C4Bb binding to C4A-specific sequence was without conformational consequence. intact sCR1 most likely reflects a weighted average affinity of col- Although there is no structure available for C4Bd, it is highly by guest on October 4, 2021 lective binding to sites 1 and 2. However, because Reilly et al. (9) unlikely to be different at the level of backbone structure from that found that dimers of C4b bound to cells transfected with cDNA of C4Ad. Consequently, there is no conformational basis for the encoding full-length CR1 with the same affinity as to cells trans- isotype-defining segment influencing a remote CR1 contact site in fected with a cDNA encoding only site 1, and that in their hands C4d. Furthermore, direct contact of the isotypic residues with CR1 this affinity was 3-fold higher than to cells bearing only site 2, we is probably precluded by their proximity to the covalent attach- cannot totally rule out the possibility that the homogeneous bind- ment site. We know from previous serotyping (48) that the C4A ing that we observe is dominated by binding to site 1 only. and C4B proteins that we used were of the common A3 and B1 Because we were concerned that the surface effects inherent in allotypes, and that they had also been typed as being RgϩChϪ and the SPR technology might be obscuring subtle differences in in- RgϪChϩ, respectively. Although the Ch/Rg epitope-determining trinsic affinity of the isotypic forms of C4b for CR1, we also con- residues at 1157, 1188, and 1191 are surface exposed in the C4d ducted an ELISA-based competition experiment that allowed one to structure, and are well away from the site of covalent attachment, monitor the binding of monomeric C4Ab and C4Bb to sCR1 in so- the equivalence of the intrinsic affinities of C4Ab and C4Bb for lution. In this instance, too, however, the respective binding behaviors CR1 that we find shows that these residues also do not directly of C4Ab and C4Bb to sCR1 were indistinguishable, thereby confirm- affect this interaction. ing the results obtained using the BIAcore instrumentation. We believe that the difference between our results and the three It is worth mentioning that in choosing to work with monomeric previous reports claiming that C4Ab displayed a higher affinity for C4Ab and C4Bb so that we monitored intrinsic binding ability in CR1 than did C4Bb can be attributed to issues related to the char- both the BIAcore and competition ELISA, the technical compro- acterization of the ligands. In the study by Gatenby et al. (23), mise that we had to make was to work at subphysiologic ionic radioiodinated IgG-containing Ag-Ab complexes were opsonized strength. In this way, it was possible to achieve a meaningful de- in the presence of purified C1៮ with either C4A or C4B before their gree of binding site saturation for what is well established to be a binding to CR1-bearing human red cells was determined. Although highly salt-sensitive interaction (10, 24). Although it is reasonable substantially higher binding of the radioiodinated immune com- to have some concern about extrapolating our results to physio- plexes to CR1 was observed when C4A was the opsonin, the study logic ionic strength, we note that in one of the earlier studies that made no correction for what was certain to be a higher deposition reported higher sCR1 binding of a thioester-cleaved form of C4A of C4Ab, than C4Bb, to the same amount of immune complex. relative to C4B, the authors stated that the binding difference was Based on the experiments of Kishore et al. (19), for the same actually accentuated at low ionic strength (24). amount of IgG transacylation target and constant C1៮, C4A Spontaneously formed disulfide-linked dimers of C4b, or even transacylates onto IgG H chain at a 3- to 4-fold higher efficiency

C4(CH3NH2) (43, 47), have been reported previously, and pre- than does C4B. Additionally, because the transacylation target nu- sumably these form via the thioester cysteine. As expected, dimers cleophiles within the immune complex are going to be different for 1678 ANALYSIS OF CR1 BINDING TO C4Ab AND C4Bb nascent C4Ab and C4Bb, the nature of the ligand clusters formed did not reach a steady state plateau, it was only possible to extract could affect the binding to CR1, which is itself clustered on red binding affinity data though analysis of the kinetics. In our general cells (49). Thus, the higher binding of C4Ab-opsonized complexes experimental design, we were most interested in using the equi- that was observed by Gatenby et al. (23) is most likely accounted librium plateau regions of the sensorgrams as a readout of the for by the well-established covalent binding differences of the two binding C4Ab and C4Bb monomers, as this method of analysis is human C4 isotypes. not influenced by known kinetic artifacts of the BIAcore instru- The study by Reilly and Mold (25) compared the functional mentation. These involve limitations on mass transport to analyte affinity of radiolabeled dimers of C4Ab and C4Bb to CR1 on hu- binding sites within the carboxylated dextran matrix, which leads ϳ man red cells and reported a 4-fold higher affinity for dimers of to an underestimation of kon values, and rebinding of ligand during the C4A isotype. These workers presumably chose to take advan- the dissociation phase of the experiment, which leads to an under- tage of the avidity properties of dimers because their binding as- estimation of koff values. Because for the equilibrium-phase anal- say, which included three wash steps, would not have yielded a ysis one wishes to maximize signal change, the experiments were sufficient binding signal with monomeric ligand. Reilly and Mold conducted at relatively high RU values (6800) of sensor chip-cou- were well aware that the presence of higher molecular mass oli- pled sCR1 and also using relatively high analyte concentrations so gomers would bias their results due to an enhancement of the avid- that binding data extending through at least 60% of the saturation ity effect. Consequently, they put their bismaleimidohexane-cross- curve could be achieved. Unfortunately, these are precisely the linked proteins through a size exclusion chromatography step, conditions that exacerbate the problems in kinetic measurements. which by their estimation reduced the proportion of higher molec- Accordingly, we put little credence into the absolute value of the ular mass aggregates to less than 3%. The gel that they show of

kinetic constants, or the equilibrium constants derived from them. Downloaded from their covalent C4Ab and C4Bb dimers was run under reducing Nevertheless, because C4c and the monomers of C4Ab and C4Bb conditions, which would not break the bismaleimidohexane cross- were analyzed on the same chip, and required the same confor- ␣Ј links between -chains, but would reduce disulfide-linked oli- mational mechanism to kinetically fit the data, it may be valid to gomers. Based on our observations on the unique properties of compare in relative terms the respective kinetically derived equi- C4Ab in forming disulfide-linked oligomers beyond the dimer librium-binding constants. Whereas for C4Ab and C4Bb, the ki- stage, we strongly suspect the presence of such species uniquely ␮

netically derived K values were respectively 0.26 and 0.31 M http://www.jimmunol.org/ within the aggregate portion of their C4Ab dimer preparations, but D (Fig. 5), that for C4c was 1.6 ␮M (Fig. 2), suggesting ϳ5-fold these would only have been detectable on a nonreducing gel. Spe- weaker binding relative to the parent C4b ligand. Given the com- cifically, it has been our experience that C4Ab has a higher pro- plete absence of CR1-binding activity of C4dg on its own, we infer pensity to form spontaneous disulfide-linked dimers than does that the higher affinity of C4b for CR1 represents an indirect scaf- C4Bb. Additionally, on nonreduced, but not on reduced SDS- folding effect whereby one or more contacts between the C4d do- PAGE, the C4Ab dimer fraction from the Mono-Q column con- main and the larger C4c fragment may stabilize the conformation tains an additional higher molecular mass species that is not seen of CR1-contacting segments located within the C4c part of the C4b in the C4Bb dimer fraction (data not shown). Our interpretation of molecule. these observations is that for C4Ab, spontaneous disulfide-medi- The fact that the kinetic data obtained with monomers of C4c, by guest on October 4, 2021 ated oligomerization can occur not only through the thioester cys- C4Ab, or C4Bb could not be fit by a simple 1:1 encounter model, teine (C991), but also through the isotypic region cysteine but rather required invoking a kinetically coupled unimolecular (C1102). This would not only kinetically facilitate more efficient dimerization, but would also allow for the formation of species that conformational change subsequent to the initial binding event, may are larger than dimers. The accessibility of C1102 to the solvent, also be meaningful in terms of the nuclear magnetic resonance and therefore availability for disulfide bond formation, is apparent structure of CR1 site 2 (SCR domains 15–17) and the mapping from inspection of a surface representation of the C4Ad structure onto this structure the location of mutants that are deleterious to (Protein Data Bank file 1HZF). By contrast, cysteine-mediated C3b or C4b binding (52). Whereas such mutations in SCRs 15 and dimerization of C4Bb would stop at the dimer stage because res- 16 are localized on one face of the molecule, those in SCR 17 are idue 1102 in this case is a serine. located on the opposite face of the molecule and require a swivel The study of Gibb et al. (24) used ammonia treatment of C4A about the SCR 16–17 linker segment to align with what is the and C4B to cleave the thioester bond and induce a C4b-like con- presumed ligand contact face contributed by SCRs 15 and 16. If formation. When equal amounts of ammonia-treated C4A and C4B the initial contact of C4b is with the first two domains of either site were coated onto ELISA wells, it was found that the binding of 1 or 2, then the conformational component to the kinetic analysis 125I-labeled sCR1 was ϳ2-fold greater to the C4A material than to may reflect the swivel required to enable the contact with residues C4B. Because the ammonia treatment would liberate the thioester in the third SCR domain contributing to the binding site. cysteine, as well as the isotypic cysteine of C4A, not only would The debate about the possible association of SLE with complete, there be spontaneous disulfide-linked dimers formed somewhat or even partial, C4A deficiency states is at present not settled. It more preferentially for C4A, but as discussed above, higher oli- has been noted (11) that most of the early C4 population studies gomers would only be possible in the case of C4A. Such a cluster relied on C4 protein allotyping in the context of a two-locus per of C4Ab-like molecules may be better suited for capturing the allelic chromosome genetic model. In these studies, the presence sCR1 by binding simultaneously to up to three sites on the same of a null allele was derived from the comparison of the relative CR1 molecule, thus conferring an advantage relative to the wells protein levels of the two isotypes. Recent genetic studies have coated with ammonia-treated C4B. revealed a far more complex picture for the organization of the C4 Although neither C4Adg nor C4Bdg on its own was able to bind gene. In the current model, the C4 gene is part of a module con- to CR1, substantial binding was observed for C4c. This parallels taining the RP-C4-CYP21-TNX genes (RCCX module) that can be the behavior of C3 degradation fragment binding to CR1, namely present in single, double, triple, or even quadruple copies on each that C3c on its own binds to CR1, albeit with weaker affinity than allelic chromosome. In turn, each C4 gene locus may encode either C3b, whereas C3d shows no binding whatsoever to CR1 (50, 51). C4A or C4B protein (11). Therefore, precise C4 genotyping, in- Because the sensorgrams of C4c binding to an sCR1-coupled chip cluding module-specific RFLP and PCR analysis to determine the The Journal of Immunology 1679 exact number of C4 genes (53), is necessary in order not to mis- tidine) converts the functional activity of human complement C4B to C4A. Proc. interpret the overdosage of one isotype as a partial deficiency of Natl. Acad. Sci. USA 87:6868. 16. Sepp, A., A. W. Dodds, M. J. Anderson, R. D. Campbell, A. C. Willis, and the other isotype. Nevertheless, even recent studies that have in- S. K. Law. 1993. Covalent binding properties of the human complement protein cluded the appropriate genotyping at the DNA level continue to C4 and hydrolysis rate of the internal thioester upon activation. Protein Sci. show strikingly conflicting results on the association of C4A de- 2:706. 17. Dodds, A. W., X. D. Ren, A. C. Willis, and S. K. Law. 1996. The reaction ficiency states and SLE (28, 37). Although we cannot resolve this mechanism of the internal thioester in the human complement component C4. controversy, if the selective lack of C4A does indeed contribute to Nature 379:177. the development of SLE in at least some ethnic populations, our 18. Law, S. K., and A. W. Dodds. 1997. The internal thioester and the covalent binding properties of the complement proteins C3 and C4. Protein Sci. 6:263. results suggest that it is not due to differences in the intrinsic bind- 19. Kishore, N., D. Shah, V. M. Skanes, and R. P. Levine. 1988. The fluid-phase ing affinity between C4Ab and C4Bb for CR1. This being said, if binding of human C4 and its genetic variants, C4A3 and C4B1, to immunoglobu- our in vitro observations on the greater propensity of C4Ab to form lins. Mol. Immunol. 25:811. 20. Schifferli, J. A., and J. P. Paccaud. 1989. Two isotypes of human C4, C4A and disulfide-linked dimers, and its unique ability to form higher mo- C4B have different structure and function. Complement Inflamm. 6:19. lecular mass oligomers via its isotypic residue C1102, were to also 21. Paul, L., V. M. Skanes, J. Mayden, and R. P. Levine. 1988. C4-mediated inhi- occur in vivo, the clustering effect of the ligand may confer a bition of immune precipitation and differences in inhibitory action of genetic variants, C4A3 and C4B1. Complement 5:110. higher avidity interaction with CR1 for C4Ab-opsonized targets. In 22. Ebanks, R. O., A. S. Jaikaran, M. C. Carroll, M. J. Anderson, R. D. Campbell, and keeping with an earlier suggestion (25), such a higher functional D. E. Isenman. 1992. A single arginine to tryptophan interchange at ␤-chain affinity for CR1 of C4Ab-opsonized targets could enhance the ef- residue 458 of human complement component C4 accounts for the defect in classical pathway C5 convertase activity of allotype C4A6: implications for the fect due to the inherently higher covalent binding efficiency to location of a C5 binding site in C4. J. Immunol. 148:2803. amino group-rich targets, such as immune complexes, of C4Ab 23. Gatenby, P. A., J. E. Barbosa, and P. J. Lachmann. 1990. Differences between relative to C4Bb. These combined effects may therefore confer an C4A and C4B in the handling of immune complexes: the enhancement of CR1 Downloaded from binding is more important than the inhibition of immunoprecipitation. Clin. Exp. advantage to having high levels of C4A for the disposal of immune Immunol. 79:158. complexes, and perhaps apoptotic bodies, a disposal process that is 24. Gibb, A. L., A. M. Freeman, R. A. Smith, S. Edmonds, and E. Sim. 1993. The thought to be instrumental in the etiology of SLE (26). interaction of soluble human complement receptor type 1 (sCR1, BRL55730) with human complement component C4. Biochim. Biophys. Acta 1180:313. 25. Reilly, B. D., and C. Mold. 1997. Quantitative analysis of C4Ab and C4Bb Acknowledgments binding to the C3b/C4b receptor (CR1, CD35). Clin. Exp. Immunol. 110:310. We are indebted to Dr. John R. Glover of the Department of Biochemistry, 26. Pickering, M. C., M. Botto, P. R. Taylor, P. J. Lachmann, and M. J. Walport. http://www.jimmunol.org/ 2001. Systemic lupus erythematosus, complement deficiency, and apoptosis. Adv. University of Toronto, for generous access to his BIAcore X instrument. Immunol. 76:227. 27. Schur, P. H., D. Marcus-Bagley, Z. Awdeh, E. J. 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