Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte.

A human erythrocyte membrane glycoprotein of 205,000 mol wt (gp205) has been identified as the C3b receptor of the erythrocyte, polymorphonuclear leukocyte (PMN), B lymphocyte, and monocyte. Initially, gp205 was sought and characterized as a constituent of the human erythrocyte membrane that can impair activation of the alternative complement pathway by inducing loss of function of the properdin-stabilized amplification C3 convertase (C3b,Bb,P) through displacement of Bb from C3b and by promoting cleavage-inactivation of C3b by C3b inactivator. These inhibitory activities of gp205 suggested that this membrane glyeoprotein had an affinity for C3b and prompted an analysis of its possible identity as the C3b receptor of human peripheral blood cells. The F(ab')2 fragment of rabbit IgG anti-gp205 inhibited the formation of rosettes with sheep EC3b of human erythroeytes, B lymphocytes, monocytes and PMN in a dose-response manner; the 50 percent inhibitory doses were 0.13/mug/ml, 0.90 mug/ml, 1.25 mug/ml, and 1.20 mug/ml of F(ab')2, respectively. Anti-gp205 did not impair the formation of rosettes by monocytes and B lymphocytes with sheep EC3bi or with EC3d. Scatchard analysis of the number of specific (125)I-F(ab')(2) anti-gp205 binding sites/cell revealed 950 sites/erythrocyte, 21,000 sites/cell of B lymphocyte preparation, 57,000 sites/PMN, and 48,000 sites/monocyte, indicating that the higher concentrations of antibody that had been required for inhibition of rosette formation by the nucleated cells reflected larger numbers of receptors on these cells. Direct evidence for the identity of gp205 as the C3b receptor of the four cell types was obtained when detergent-solubilized membrane proteins of the surface-radioiodinated cells were reacted with anti- gp205 and the immunoprecipitate was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In each instance, the antigenic material reacting with anti-gp205 represented a single protein with an apparent 205,000 mol wt. Thus, gp205 is the C3b receptor of human erythrocytes, PMN, B lymphocytes, and monocytes.

Tissue-specific phosphorylation of complement receptors CR1 and CR2.

CR1 of neutrophils and monocytes may exist in a resting state, in which it only binds ligand-coated particles, or an activated state, in which it mediates phagocytosis. Because the activated state of CR1 can be induced by the stimulation of protein kinase C with PMA, CR1 was assessed for phosphorylation. Purified human neutrophils, monocytes, eosinophils, tonsilar lymphocytes, SB cells, and erythrocytes were labeled with 32PO4 and incubated with buffer or 100 ng/ml PMA. Membrane proteins were immunoprecipitated and analyzed by SDS-PAGE and autoradiography. CR1, unlike HLA class I heavy chain, was not constitutively phosphorylated by any cell type. PMA induced phosphorylation of CR1 in three phagocytic cell types, but did not induce the phosphorylation of CR3 or FcR. FMLP also induced the phosphorylation of CR1 in neutrophils. In contrast, PMA did not induce phosphorylation of CR1 in tonsilar B lymphocytes, SB cells, or erythrocytes, indicating restriction of this reaction to phagocytic cell types. This may be due to differences in the structure or presentation of the cytoplasmic domain of CR1 in phagocytic vs. nonphagocytic cells. Phosphorylation of CR2, however, did occur in PMA-treated B lymphocytes and SB cells, suggesting that this receptor, rather than CR1, may be involved in regulation of B lymphocyte function.

Properdin factor D: characterization of its active site and isolation of the precursor form.

The activity of properdin factor D was measured by the generation of the hemolytically active cellular intermediate, EAC43B(D), bearing the C3b-dependent alternate pathway C3 convertase. Treatment of factor D with DFP prevented formation of EAC43B(D); thus, a serine esterase is essential for the generation of the alternate pathway C3 convertase, a situation analogous to the role of C1 in the formation of the classical C3 convertase, C42. The definition of factor D as a serine esterase prompted a search for its proenzyme form, and resulted in the chromatographic isolation from plasma of a single peak of trypsin-inducible factor D activity, distinct from activated factor D. Analytical gel filtration indicated an apparent mol wt of 25,000. This protein from which trypsin elaborated factor D activity, as assessed by the formation of EAC43B(D), the generation of the CoVF-dependent C3 convertase, and the cleavage of factor B in the presence of C3b, was designated "precursor factor D." The DFP resistance of precursor factor D, and the susceptibility of its trypsin-activated form to inactivation by DFP is analogous to the behavior of other plasma serine esterases, including C1.

Properdin: binding to C3b and stabilization of the C3b-dependent C3 convertase.

A function of P in the alternative complement pathway is to prolong the first order decay of the hemolytic sites on EAC43B in a dose-dependent manner. As the number of initial convertase sites is not changed, even when activated properdin (P) increases the t1/2 10-fold or more, P acts to stabilize rather than to uncover additional sites. P binds to EAC43 to generate EAC43P in a reaction that proceeds slightly more rapidly at 15 degrees C than at 0 degrees C, but reaches the same plateau and does not require divalent cations. The presence of P on EAC43P not only stabilizes the convertase subsequently formed on that cell, but, alternatively, permits transfer to convertase sites on other cells with the stability of the recipient intermediate being dependent on the P available for transfer. The capacity of P to bind to C3b and stabilize C3B contrasts with the inhibitory effect of the C3b inactivator on formation of this amplification convertase.

Membrane distribution and adsorptive endocytosis by C3b receptors on human polymorphonuclear leukocytes.

C3b receptors on human polymorphonuclear leukocytes (PMN) were nonrandomly distributed in small clusters on the plasma membranes of these cells when assessed by indirect immunofluorescence at 0 degree C using monospecific rabbit Fab' or F(ab')2 anti-C3b receptor and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat IgG anti-F(ab')2. When PMN were incubated with the bivalent anti-C3b receptor at 37 rather than at 0 degree C, almost no immunofluorescence was observed, which indicates that the C3b receptor-F(ab')2 complexes had been rendered inaccessible to TRITC-IgG anti-F(ab')2. Endocytosis of the anti-C3b receptor ligand was quantitated by measuring the binding 131I-IgG anti-F(ab')2 by PMN that had previously taken up 125I-F(ab')2 anti-C3b receptor at 0 and at 37 degree C, respectively. There was a constant 2: 1 molar ratio of anti-F(ab')2 to anti-C3b receptor with PMN that had been incubated with the first antibody at 0 degree C. In contrast, when increments of F(ab')2 anti-C3b receptor were taken up by the cells at 37 degree C, there was a dose-related decline in this molar ratio to a minimum of 0.2 molecules of anti-F(ab')2 anti-F(ab')2 bound per molecule of PMN-associated anti-C3b receptor. 125I-F(ab')2 anti-C3b receptor taken up by PMN at 37 degree C was also inaccessible to release by proteolytic treatment of the cells with pronase. The rate of endocytosis of 125I-F(ab')2 anti-C3b receptor was rapid as the PMN-bound antibody fragment became inaccessible to 131I-IgG anti-F(ab')2 within 10 min during incubation of the cells at 37 degree C. In contrast to these findings, 125I-Fab' anti-C3b receptor that was taken up by PMN at 37 degree C remained accessible to both 131I-IgG anti-F(ab')2 and to proteolytic release by pronase, which suggests that monovalent interaction of ligand with C3b receptors was not sufficient for induction of endocytosis. The requirement for multivalency was also demonstrated using the C3b-OR, the normal ligand for the C3b receptor. 125I-C3b-OR was specifically bound by PMN but remained on the cell receptor. 125I-C3b-OR was specifically bound by PMN but remained on the cell surface, as determined by its accessibility to pronase, unless it was cross-linked with F(ab')2 anti-C3. Although C3b receptors on PMN do not mediate internalization of adsorptive pinocytosis of soluble ligand indicates their potential for the clearance of C3b-bearing immune complexes without recruitment of other cell surface receptors.

Formation of a hemolytically active cellular intermediate by the interaction between properdin factors B and D and the activated third component of complement.

The present studies demonstrate that the factor B-dependent C3 convertase can be affixed to an erythrocyte by use of an intermediate bearing C3b and that this convertase brings the hemolytic reaction to completion with an efficiency comparable to that of classical convertase. The evidence that the EAC43 intermediate was lysed by a new pathway includes requirements for factors B and D and cell-bound C3b for subsequent lysis by the terminal components, C3-C9. The linear stoichiometry of the effective molecule titrations of C3b and factor B, and the first-order kinetics displayed by the generation and decay of the factor B-dependent hemolytic site are characteristics consistent with the one-hit theory as initially developed for the classical complement system. The use of hemolytically active cellular intermediates to examine the reactions occurring with C3b and factors B and D has allowed extension of the one-hit theory to this molecular sequence, development of effective molecule titrations, recognition of the analogies to the functional characteristics of the classical C3 convertase, and discrimination of the probable mechanism of terminal complement activation from reactive lysis.

Properdin factor D. II. Activation to D by properdin.

The protein in the properdin pathway responsible for conversion of precursor factor D to D has been isolated and found to be identical with properdin. Sequential ion exchange and gel filtration chromatography demonstrated identity between properdin protein, measured by radial immunodiffusion, and the capacity to activate D to D, assessed by formation of the intermediate, EAC43B(D). Properdin, purified in this manner, was homogeneous on acid polyacrylamide disc gel electrophoretic analysis, with the band of protein corresponding to the position of eluates in the replicate gel capable of activating highly purified D. Demonstration of the homogeneity of purified D by alkaline disc gel electrophoresis, coupled with the linear stoichiometric hemolytic titrations of each factor, indicates that direct interaction between properdin and D generates D. Thus, activation of D by properdin represents a mechanism in the properdin pathway by which D becomes available for formation of the C3b-dependent C3 convertase.

Activation of the alternative complement pathway with rabbit erythrocytes by circumvention of the regulatory action of endogenous control proteins.

Cleavage of C3 by the alternative complement pathway occurs in at least two distinct phases: continuous low grade generation of C3b by the interaction of native C3, B, D, and P, and subsequent amplified cleavage of C3 by the interaction of C3b, B, D, and P which forms the amplification convertase, P,C3b,Bb. Transition to C3b-dependent amplification is necessary to achieve substantial C3 cleavage and is normally limited by the combined action of C3b inactivator (C3bINA) and betalH. An activator of the alternative pathway, such as rabbit erythrocytes (E(r)), provides sites that protect bound C3b and P,C3b,Bb from the action of these regulatory proteins and permits C3b deposited by the low grade fluid phase reaction to assemble a membrane-associated amplification convertase which can deposit additional protected C3b. Under conditions in which the control proteins, C3bINA and beta1H, almost completely inactivated C3b bound to sheep erythrocytes (E(s)), which does not activate the alternative pathway, the function of C3b bound to E(r) was diminished by less than one-fifth. Further, the P- stabilized amplification convertase on E(r) was 10-fold less sensitive to beta1H-mediated decay-dissociation than the convertase on E(s). The addition of E(r) to a regulated mixture of purified C3, B, D, P, C3bINA, and beta1H resulted in amplified inactivation of C3 and B by formation of the amplification convertase on E(r) as indicated by its lysis with subsequent exposure to C3-C9. In contrast, E(s) did not advance the low grade fluid phase inactivation of C3 and B to amplified inactivation and the cell was not converted to an intermediate susceptible to lysis by C3- C9. Since E(r) and E(s) did not differ in their inefficient fixation of C3b generated during an unregulated fluid phase reaction, the activating capacity of E(r) must reside in its protection of bound C3b and P, C3b,Bb from the regulatory proteins rather than in enhanced capacity to bind C3b from the fluid phase. When the reaction is limited to low grade fluid phase turnover, introduction of E(r) but not E(s) results in a 100-fold increase in the deposition of C3b, indicating that surface-dependent activation of the alternative pathway is characterized by efficient deposition of C3b on the initiating surface. Thus, the activating surfaces advance the interaction of the alternative pathway proteins to the amplification phase because of the selective inability of the regulatory proteins to deal with their substrates when deposited on these surfaces and results in a specificity that is not necessarily dependent on adaptive immunity.

Suppression of signal transducer and activator of transcription 3-dependent B lymphocyte terminal differentiation by BCL-6.

Lymphocytes usually differentiate into effector cells within days after antigen exposure, except in germinal centers where terminal differentiation is delayed while somatic hypermutation creates high-affinity antibody mutants. Here we investigate whether arrest of terminal differentiation can be mediated by BCL-6, a transcriptional repressor that is expressed by germinal center B cells and is required for this phase of B cell development. We find that BCL-6 suppresses the differentiation of transformed and primary B cells to plasma cells by inhibiting the signal transducer and activator of transcription 3-dependent expression of the major regulator of plasma cell development, the B lymphocyte-induced maturation protein (Blimp-1). This function of BCL-6 as a repressor of B lymphocyte differentiation may also underlie the association between chromosomal translocations of its gene and B cell lymphomas.

Downregulated expression of SHP-1 in Burkitt lymphomas and germinal center B lymphocytes.

We wish to identify developmental changes in germinal center B cells that may contribute to their rapid growth. SHP-1 is an SH2 domain-containing phosphotyrosine phosphatase that negatively regulates activation of B cells and other cells of hematopoietic lineages. We have found that in all 13 EBV-negative and 11 EBV-positive Burkitt lymphomas with a nonlymphoblastoid phenotype, the mean concentration of SHP-1 was reduced to 5% of that of normal B and T cells. The possibility that this diminished expression of SHP-1 was related to the germinal center phenotype of Burkitt lymphomas was supported by the low to absent immunofluorescent staining for SHP-1 in germinal centers, and by the inverse relationship between the concentration of SHP-1 and the expression of the germinal center marker CD38 on purified tonsillar B cells. In CD38-high B cells, SHP-1 concentration was 20% of that of mantle zone B cells from the same donor. This reduction in SHP-1 is comparable to that of cells from motheaten viable mev/mev mice in which there is dysregulated, spontaneous signaling by cytokine and antigen receptors. Therefore, germinal center B cells may have a developmentally regulated, low threshold for cellular activation.

A subpopulation of B220+ cells in murine bone marrow does not express CD19 and contains natural killer cell progenitors.

Bone marrow of both normal and rearrangement-deficient mice contains a small population of B220(CD45R)+ cells, which do not express the B lineage marker CD19. Instead, part of this population coexpresses the surface marker CD43 and lacks or expresses very low levels of heat stable antigen (HSA) and BP-1, thus representing a part of Hardy's fraction A (B220(+)-CD43+HSA-, BP-1-) of B lineage development. However, some 20-40% of these B220(+)-CD19- cells also coexpress the NK1.1 surface molecule and do not express genes like VpreB or B29 restricted to the B cell lineage. These cells respond to recombinant interleukin 2 in vitro, and develop into killer cells that can lyse the prototypic NK target tumor cell, YAC-1, as well as syngeneic normal lipopolysaccharide or concanavalin A blasts, providing they lack the surface expression of major histocompatibility complex class I molecules. The implications of these findings for studies on B lymphopoiesis are discussed. It is suggested that the CD19-specific monoclonal antibody is more reliable, as in humans, than B220(CD45R) to detect B lineage cells in mice.

Structure of the human B lymphocyte receptor for C3d and the Epstein-Barr virus and relatedness to other members of the family of C3/C4 binding proteins.

Human complement receptor type 2 (CR2) is the B lymphocyte receptor for C3d and the Epstein-Barr virus. This protein is also a member of a family of C3b/C4b binding proteins that regulate complement activation, comprise tandemly repeated 60-75 amino acid sequences, and whose genes map to band q32 on chromosome 1. Overlapping cDNA clones encoding the entire human CR2 protein have been isolated from a human tonsillar cDNA library. The derived amino acid sequence of 1,032 residues encodes a peptide of 112,716 mol wt. A signal peptide was identified, followed by 15 copies of the short consensus repeat (SCR) structure common to the C3/C4 binding protein family. The entire extracellular portion of the protein comprised SCRs, thus, the ligand binding sites both for C3d and the EBV protein gp350/220 are positioned within this structure. Immediately following the final SCR was a transmembrane sequence of 24 amino acids and a cytoplasmic region of 34 amino acids. One of five cDNA clones isolated contained an additional SCR, providing evidence for alternative mRNA splicing or gene products of different human alleles. The CR2 cDNAs were used to isolate CR2-specific genomic phage. The entire CR2 coding sequences were found within 20 kb of human DNA. Analysis of the CR2 cDNA sequence indicated that CR2 contained internally homologous regions and suggested that CR2 arose by duplication of a primordial gene sequence encoding four SCRs. Comparison of the CR2 peptide sequence with those of other members of the gene family has identified many regions highly homologous with human CR1, fewer with C4bp and decay accelerating factor, and very few with factor H, and suggested that CR2 and CR1 arose by duplication of the same ancestral gene sequence. The homology between CR2 and CR1 extended to the transmembrane and cytoplasmic regions, suggesting that these sequences were derived from a common membrane-bound precursor.

Surface-associated heparin inhibits zymosan-induced activation of the human alternative complement pathway by augmenting the regulatory action of the control proteins on particle-bound C3b.

Discrimination by the human alternative pathway between activating and nonactivating particles occurs after deposition of C3b by the continuous low-grade interaction of the alternative pathway components in biologic fluids and is dependent on the modulation by surface constituents of the interaction of bound C3b with the control proteins, beta 1H, and C3b inactivator (C3bINA). When heparin glycosaminoglycan was coupled to activating particles, such as zymosan or Sepharose, by cyanogen bromide activation, their capacity to activate the human alternative pathway was inhibited. The loss of alternative pathway-activating capacity was directly correlated to the number of heparin molecules bound/zymosan particle, whether the ratio was varied by increasing the amounts of heparin in the initial coupling reactions or by treating a fully inhibited particle with incremental concentrations of heparinase. Analysis by linear regression of the inhibitory effect of each procedure (r = 0.97, r = 0.98, respectively) for adjusting the number of heparin molecules/particle revealed that the dose-response relationships were identical and that complete inhibition occurred with greater than 12 X 10(8) molecules of heparin/zymosan particle. The coupling of heparin to zymosan did not impair the uptake of C3b from the fluid-phase interaction of C3, B, and D, and did not alter the capacity of bound C3b to associate with B so as to permit its inactivation by D. Although the regulatory proteins present in normal serum chelated with EDTA or presented as a combination of purified C3bINA and beta 1H were relatively inefficient in inactivating C3b function on an activating particle of the alternative pathway such as zymosan or zymosan-cyanogen bromide, the control proteins rapidly inactivated C3b on a nonactivating particle wuch as a sheep erythrocyte or zymosan with coupled heparin. The increased numbers of C3b sites susceptible to inactivation by C3bINA in the presence of beta 1H were significantly correlated to the number of molecules of heparin/particle. By linear regression analysis of the correlation (r = 0.99) the number of heparin molecules/particle required to promote total inactivation of bound C3b by purified control proteins was 13.8 X 10(6). This molecular analysis suggests that the action of heparin coupled to an activating particle of the alternative pathway is to promote the interaction between particle-bound C3b and the regulatory proteins, thereby preventing particle-associated amplified C3 cleavage. It is noteworthy that both surface constituents known to maintain a particle as a nonactivator of the alternative pathway, sialic acid and N-sulfated mucopolysaccharide, act by facilitating the inactivation by regulatory proteins of the function of particle-bound C3b.

Inhibition of the B cell by CD22: a requirement for Lyn.

Mice in which the Lyn, Cd22, or Shp-1 gene has been disrupted have hyperactive B cells and autoantibodies. We find that in the absence of Lyn, the ability of CD22 to become tyrosine phosphorylated after ligation of mIg, to recruit SHP-1, and to suppress mIg-induced elevation of intracellular [Ca2+] is lost. Therefore, Lyn is required for the SHP-1-mediated B cell suppressive function of CD22, accounting for similarities in the phenotypes of these mice.

Determination of the structural basis for selective binding of Epstein-Barr virus to human complement receptor type 2.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus that selectively infects and immortalizes human B lymphocytes. One determinant of this narrow tropism is human CR2, the only viral receptor within the superfamily of proteins that contain short consensus repeats (SCRs). Human CR2 serves as a receptor for both C3dg and the gp350/220 glycoprotein of EBV, and binds the monoclonal antibody (mAb) OKB7, which blocks binding of both ligands to the receptor. In contrast, although murine CR2 is capable of binding human C3dg and this interaction can be blocked with the mAb 7G6, it does not bind OKB7 or EBV. We have determined the structural basis for absolute specificity of EBV for human CR2 through characterization of a panel of 24 human-murine chimeric receptors, all of which bind human C3dg. The results indicate that preferential binding of EBV to human CR2 is not due to unique amino acids that are capable of binding the virus, but reflects a distinct receptor conformation that can be achieved in murine CR2 with single amino acid substitutions in two discontinuous regions of the primary structure: replacement of proline at position 15 with the corresponding serine from human CR2, and elimination of a potential N-linked glycosylation site between SCR-1 and SCR-2. Furthermore, species-specific binding of EBV, OKB7, and 7G6 can all be manipulated through substitutions among residues 8-15, suggesting that this octapeptide is part of a structural determinant that is critical for binding of both viral and natural ligands to CR2.

Molecular interactions of complement receptors on B lymphocytes: a CR1/CR2 complex distinct from the CR2/CD19 complex.

The complement system augments the humoral immune response to low concentrations of antigen. This effect may be partly mediated by complement receptors on the surface of B lymphocytes that bind immunogenic complexes bearing fragments of C3 and C4. We have shown by immunoprecipitation analysis that the two complement receptors expressed by B lymphocytes, complement receptor 1 (CR1) and CR2, form a detergent-sensitive complex on the surface of tonsillar B lymphocytes and on K562 erythroleukemia cells that were co-transfected with cDNAs encoding CR1 and CR2. The CR1/CR2 complex is distinct from the CR2/CD19 complex and may assist B cell activation by efficiently capturing C3b-containing immunogens and maintaining such immunogens on the B cell after CR1 and factor I-mediated cleavage to iC3b and C3dg. The complement activating immunogen may then trigger signal transduction by the CR1/CR2 complex, the CR2/CD19 complex, or membrane immunoglobulin.

Modulation of the formation of the amplification convertase of complement, C3b, Bb, by native and commercial heparin.

Native rat mast cell macromolecular heparin proteoglycan and commercial hog heparin glycosaminoglycan chains inhibit generation of the amplification convertase, C3b, Bb. The inhibitory action of heparin is not due to chelation of magnesium. Heparin is most active in inhibiting convertase formation on cellular intermediates formed with the lowest C3b input and developed with the highest B concentration, thereby suggesting the receptor site for B on C3b as the point of heparin action. This interpretation is consistent with the demonstration that heparin prevents B utilization during the fluid phase interaction of C3b, B, and D. Inhibition is observed also when C3b,Bb generation takes place on cellular intermediates in the presence of P or C3NeF, which yield stabilized forms of the convertase. 50 times the concentration of heparin required to inhibit convertase generation does not accelerate the decay of the unstabilized or the C3NeF-stabilized convertases and has only a modest effect on the P-stabilized convertase. An additional effect of heparin is to impair beta1H-mediated decay-dissociation of C3b,Bb. The concentration of native or commercial heparin which prevents convertase formation is in the same range as that required for the demonstration of its anti-coagulant and anti-thrombin III cofactor activities. The additional finding that this inhibitory action of heparin can be expressed by the isolated mast cell granule suggests that native heparin may contribute to the modulation of the amplification pathway of complement.

Functional dissection of the CD21/CD19/TAPA-1/Leu-13 complex of B lymphocytes.

The CD21/CD19/TAPA-1 complex of B lymphocytes amplifies signal transduction through membrane immunoglobulin (mIg), recruits phosphatidylinositol 3-kinase (PI3-kinase), and induces homotypic cellular aggregation. The complex is unique among known membrane protein complexes of the immune system because its components represent different protein families, and can be expressed individually. By constructing chimeric molecules replacing the extracellular, transmembrane, and cytoplasmic regions of CD19 and CD21 with those of HLA-A2 and CD4, we have determined that CD19 and TAPA-1 interact through their extracellular domains, CD19 and CD21 through their extracellular and transmembrane domains, and, in a separate complex, CD21 and CD35 through their extracellular domains. A chimeric form of CD19 that does not interact with CD21 or TAPA-1 was expressed in Daudi B lymphoblastoid cells and was shown to replicate two functions of wild-type CD19 contained within the complex: synergistic interaction with mIgM to increase intracellular free calcium and tyrosine phosphorylation and association with the p85 subunit of PI3-kinase after ligation of mIgM. The chimeric CD19 lacked the capacity of the wild-type CD19 to induce homotypic cellular aggregation, a function of the complex that can be ascribed to the TAPA-1 component. The CD21/CD19/TAPA-1 complex brings together independently functioning subunits to enable the B cell to respond to low concentrations of antigen.

Mapping of the C3b-binding site of CR1 and construction of a (CR1)2-F(ab')2 chimeric complement inhibitor.

CR1/CR2 chimeric receptors in which various short consensus repeats (SCRs) of CR1 were attached to CR2 were transiently expressed on COS cells, and assessed for the binding of polymerized C3b (pC3b) and anti-CR2 by immunofluorescence. Of COS cells expressing chimeras containing SCR 1-4, 1-3, 2-4, 1-2, and 2-3 of the long homologous repeats (LHRs) -B or -C, 96%, 66%, 23%, 0%, and 0%, respectively, bound pC3b. K562 cells were stably transfected with wild-type CR1, deletion mutants of CR1, and the CR1/CR2 chimeras, respectively, and assayed for binding of 125I-pC3b. The dissociation constants (Kd) for pC3b of wild-type CR1 and the LHR-BD and -CD constructs were in the range of 1.0-2.7 nM, and of the CR1/CR2 chimeras containing SCRs 1-4, 1-3, and 2-4 of LHR-B or -C were 1.8-2.4, 6-9, and 22-36 nM, respectively. The factor I-cofactor function of the CR1/CR2 chimeras paralleled the C3b-binding function of the constructs. A CR1/immunoglobulin (Ig) chimeric protein was prepared by fusing SCRs 1-4 of LHR-B to the heavy chains of a murine F(ab')2 anti-nitrophenacetyl (NP) monoclonal antibody. The (CR1)2-F(ab')2 chimera, which retained its specificity for NP, was as effective as soluble, full-length CR1 in binding pC3b, serving as a cofactor for factor I-mediated cleavage of C3b, and inhibiting activation of the alternative pathway, indicating that the bivalent expression of these SCRs reconstitutes the alternative pathway inhibitory function of CR1. The feasibility of creating CR1/Ig chimeras makes possible a new strategy of targeting complement inhibition by the use of Ig fusion partners having particular antigenic specificities.