Search

This chapter provides an overview of the state of knowledge of complement component C4. Details on its physicochemical properties, its structure and its functional role in the complement pathways; its sites of expression and regulation thereof; its genomic organisation; and its polymorphisms are covered. The chapter also summarises insights from the X-ray crystal structures of human C4 and its proteolytic split products C4B and C4D. Since in humans there are two isotypes of C4, C4A and C4B, displaying different covalent binding properties and genomic features, such as gene copy number variation, how these result in either under expression or overexpression of a particular C4 isotype is discussed. Additionally, the human disease correlates of both C4 isotypic deficiency states and overexpression states are discussed in the context of possible mechanisms derived from our current knowledge of C4 structure and function. Finally, the chapter presents key insights derived from mutant animals, including gene-targeted C4 knockout mice.

Complement regulation leads to the generation of complement split products (CSPs) such as complement component (C)4d, a marker for disease activity in autoimmune syndromes or antibody-mediated allograft rejection. However, the physiologic role of C4d has been unknown. By screening murine thymoma BW5147 cells expressing a cDNA library generated from human monocyte-derived dendritic cells with recombinant human C4d, we identified Ig-like transcript (ILT)4 and ILT5v2 as cellular receptors for C4d. Both receptors, expressed on monocytes, macrophages, and dendritic cells, also interacted with the CSPs C3d, C4b, C3b, and iC3b. However, C4d did not bind to classic complement receptors (CRs). Interaction between cell surface-resident ILT4 and soluble monomeric C4d resulted in endocytosis of C4d. Surprisingly, binding of soluble ILT4 to C4d covalently immobilized to a cellular surface following classic complement activation could not be detected. Remarkably, C4d immobilized to a solid phaseviaits intrinsic thioester conferred a dose-dependent inhibition of TNF-α and IL-6 secretion in monocytes activatedviaFc-cross-linking of up to 50% as compared to baseline. Similarly, C4d conferred an attenuation of intracellular Ca(2+)flux in monocytes activatedviaFc-cross-linking. In conclusion, ILT4 represents a scavenger-type endocytotic CR for soluble monomeric C4d, whereas attenuation of monocyte activation by physiologically oriented C4d on a surface appears to be dependent on a yet to be identified C4d receptor.-Hofer, J., Forster, F., Isenman, D. E., Wahrmann, M., Leitner, J., Hölzl, M. A., Kovarik, J. K., Stockinger, H., Böhmig, G. A., Steinberger, P., Zlabinger, G. J. Ig-like transcript 4 as a cellular receptor for soluble complement fragment C4d.

In physiological settings, the complement protein C3 is deposited on all bacteria, including invasive pathogens. However, because experimental host-bacteria systems typically use decomplemented serum to avoid the lytic action of complement, the impact of C3 coating on epithelial cell responses to invasive bacteria remains unexplored. Here, we demonstrate that following invasion, intracellular C3-positive Listeria monocytogenes is targeted by autophagy through a direct C3/ATG16L1 interaction, resulting in autophagy-dependent bacterial growth restriction. In contrast, Shigella flexneri and Salmonella Typhimurium escape autophagy-mediated growth restriction in part through the action of bacterial outer membrane proteases that cleave bound C3. Upon oral infection with Listeria, C3-deficient mice displayed defective clearance at the intestinal mucosa. Together, these results demonstrate an intracellular role of complement in triggering antibacterial autophagy and immunity against intracellular pathogens. Since C3 indiscriminately associates with foreign surfaces, the C3-ATG16L1 interaction may provide a universal mechanism of xenophagy initiation.

Degenerative disc disease (DDD) is associated with spinal pain often leading to long-term disability. However, the non-chondrodystrophic canine intervertebral disc is protected from the development of DDD, ostensibly due to its retention of notochordal cells (NC) in the nucleus pulposus (NP). In this study, we hypothesized that secretome analysis of the NC-rich NP will lead to the identification of key proteins that delay the onset of DDD. Using mass-spectrometry, we identified 303 proteins including components of TGFβ- and Wnt-signaling, anti-angiogeneic factors and proteins that inhibit axonal ingrowth in the bioactive fractions of serum free, notochordal cell derived conditioned medium (NCCM). Ingenuity Pathway Analysis revealed TGFβ1 and CTGF as major hubs in protein interaction networks. In vitro treatment with TGFβ1 and CTGF promoted the synthesis of healthy extra-cellular matrix proteins, increased cell proliferation and reduced cell death in human degenerative disc NP cells. A single intra-discal injection of recombinant TGFβ1 and CTGF proteins in a pre-clinical rat-tail disc injury model restored the NC and stem cell rich NP. In conclusion, we demonstrate the potential of TGFβ1 and CTGF to mitigate the progression of disc degeneration and the potential use of these molecules in a molecular therapy to treat the degenerative disc.

The interaction of complement receptor 2 (CR2)--which is present on B cells and follicular dendritic cells--with its antigen-bound ligand C3d results in an enhanced antibody response, thus providing an important link between the innate and adaptive immune systems. Although a cocrystal structure of a complex between C3d and the ligand-binding domains of CR2 has been published, several aspects of this structure, including the position in C3d of the binding interface, remained controversial because of disagreement with biochemical data. We now report a cocrystal structure of a CR2(SCR1-2):C3d complex at 3.2 angstrom resolution in which the interaction interfaces differ markedly from the previously published structure and are consistent with the biochemical data. It is likely that, in the previous structure, the interaction was influenced by the presence of zinc acetate additive in the crystallization buffer, leading to a nonphysiological complex. Detailed knowledge of the binding interface now at hand gives the potential to exploit the interaction in vaccine design or in therapeutics directed against autoreactive B cells.

The complement system plays an important role in eliminating invading pathogens. Activation of complement results in C3b deposition (opsonization), phagocytosis, anaphylatoxin (C3a, C5a) release, and consequently cell lysis. Moraxella catarrhalis is a human respiratory pathogen commonly found in children with otitis media and in adults with chronic obstructive pulmonary disease. The species has evolved multiple complement evasion strategies, which among others involves the ubiquitous surface protein (Usp) family consisting of UspA1, A2, and A2 hybrid. In the present study, we found that the ability of M. catarrhalis to bind C3 correlated with UspA expression and that C3 binding contributed to serum resistance in a large number of clinical isolates. Recombinantly expressed UspA1 and A2 inhibit both the alternative and classical pathways, C3b deposition, and C3a generation when bound to the C3 molecule. We also revealed that the M. catarrhalis UspA-binding domain on C3b was located to C3d and that the major bacterial C3d-binding domains were within UspA1299–452 and UspA2165–318. The interaction with C3 was not species specific since UspA-expressing M. catarrhalis also bound mouse C3 that resulted in inhibition of the alternative pathway of mouse complement. Taken together, the binding of C3 to UspAs is an efficient strategy of Moraxella to block the activation of complement and to inhibit C3a-mediated inflammation.

We recently characterized an interaction between the Staphylococcus aureus immune evasion molecule Staphylococcus aureus binder of Ig (Sbi) and complement C3, an interaction mediated primarily through the binding of C3d(g) to Sbi domain IV. Events related to these studies prompted us to investigate via mutagenesis the binding interface of C3d for Sbi domain IV (Sbi-IV), as well as to revisit the controversial issue of the complement receptor 2 (CR2) binding site of C3d. Specifically, we had shown that Sbi domains III and IV fragment binding to C3dg inhibited the latter’s binding to CR2. Moreover, a published cocrystal structure of C3d bound to complement inhibitory C-terminal domain of extracellular fibrinogen-binding protein (Efb-C), a structural and functional homolog of Sbi-IV, showed Efb-C binding to a region on the concave face of C3d previously implicated in CR2 binding by our mutagenesis data but not confirmed in the CR2(short consensus repeat [SCR]1–2):C3d cocrystal structure. We have now analyzed by surface plasmon resonance the binding of a series of variant C3dg molecules to biosensor-bound Sbi-IV or CR2(SCR1–2). We found that mutations to the concave face acidic pocket of C3d significantly affected binding to both Sbi-IV and CR2, although there was divergence in which residues were most important in each case. By contrast, no binding defects were seen for mutations made to the sideface of C3d implicated from the cocrystal structure to be involved in binding CR2(SCR1–2). The results with Sbi-IV suggest a mode of binding highly similar to that visualized in the Efb-C:C3d complex. The results with CR2 confirm our earlier mapping studies and cast even further doubt on the physiologic relevance of the complex visualized in the C3d:CR2 cocrystal.

Contains fulltext : 79801.pdf (Publisher’s version ) (Open Access) Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy associated with mutations in complement proteins, most frequently in the main plasma alternative pathway regulator factor H (FH). The hotspot for the FH mutations is in domains 19-20 (FH19-20) that are indispensable for FH activity on C3b bound covalently to host cells. In aHUS, down-regulation of cell-bound C3b by FH is impaired, but it is not clear whether this is due to an altered FH binding to surface-bound C3b or to cell surface structures. To explore the molecular pathogenesis of aHUS we tested binding of 14 FH19-20 point mutants to C3b and its C3d fragment, mouse glomerular endothelial cells (mGEnC-1), and heparin. The cell binding correlated well, but not fully, with heparin binding and the cell binding site was overlapping but distinct from the C3b/C3d binding site that was shown to extend to domain 19. Our results show that aHUS-associated FH19-20 mutants have different combinations of three primary defects: impaired binding to C3b/C3d, impaired binding to the mGEnC-1 cells/heparin, and, as a novel observation, an enhanced mGEnC-1 cell or heparin binding. We propose a model of the molecular pathogenesis of aHUS where all three mechanisms lead eventually to impaired control of C3b on the endothelial cell surfaces. Based on the results with the aHUS patient mutants and the overlap in FH19-20 binding sites for mGEnC-1/heparin and C3b/C3d we conclude that binding of FH19-20 to C3b/C3d is essential for target discrimination by the alternative pathway.

Fibrinogen-like protein 2 (FGL2) is a multifunctional protein, which has been implicated in the pathogenesis of allograft and xenograft rejection. Previously, FGL2 was shown to inhibit maturation of BM-derived DC and T-cell proliferation. The mechanism of the immunosuppressive activity of FGL2 remains poorly elucidated. Here, we focus on identification of FGL2-specific receptor(s) and their ability to modulate APC activity and allograft survival. Using flow cytometry and surface plasmon resonance analysis, we show that FGL2 binds specifically to Fc gamma receptor (FcgammaR)IIB and FcgammaRIII receptors, which are expressed on the surface of APC, including B lymphocytes, macrophages and DC. Antibody to FcgammaRIIB and FcgammaRIII, or deficiency of these receptors, abrogated FGL2 binding. FGL2 inhibited the maturation of BMDC from FcgammaRIIB+/+ mice but not from FcgammaRIIB(-/-) mice and induced apoptosis in the FcgammaRIIB+ mouse B-cell line (A20) but not the A20IIA1.6 cell line that does not express FcgammaRIIB. Recombinant FGL2 infused into FcgammaRIIB+/+ (C57BL/6J, H-2b) mice but not FcgammaRIIB(-/-) mice inhibited rejection of fully mismatched BALB/cJ (H-2d) skin allografts. The identification of specific receptor binding has important implications for the pathogenesis of immune-mediated disease and suggests a potential for targeted FGL2 therapy.

Among the recently discovered Staphylococcus aureus immune evasion proteins, Sbi is unique in its ability to interact with components of both the adaptive and innate immune systems of the host. Sbi domains I and II (Sbi-I and Sbi-II) bind IgG. Sbi domain IV (residues 198-266) binds the central complement protein C3. When linked to Sbi-III, Sbi-IV induces a futile consumption of complement via alternative pathway activation, whereas isolated Sbi-IV specifically inhibits the alternative pathway without complement consumption. Here we have determined the three-dimensional structure of Sbi-IV by NMR spectroscopy, showing that Sbi-IV adopts a three-helix bundle fold similar to those of the S. aureus complement inhibitors Efb-C, Ehp, and SCIN. The (1)H-(15)N HSQC spectrum of Sbi-III indicates that this domain, essential for futile complement consumption, is natively unfolded, at least when isolated from the rest of Sbi. Sbi-IV and Sbi-III-IV both bind C3dg with 1:1 stoichiometry and submicromolar affinity. Despite low overall sequence identity, Sbi possesses the same residues as Efb at two positions essential for Efb-C binding to C3d. Mutation to alanine of either of these residues, Arg-231 and Asn-238, abolishes both Sbi-IV binding to C3dg and Sbi-IV alternative pathway inhibition. The almost complete conservation of Sbi-III and Sbi-IV amino acid sequences across more than 30 strains isolated from human and animal hosts indicates that the unique mechanism of Sbi in complement system subversion is a feature of infections of both humans and economically important animals.

Staphylococcal immunoglobulin-binding protein, Sbi, is a 436-residue protein produced by many strains of Staphylococcus aureus. It was previously characterized as being cell surface-associated and having binding capacity for human IgG and β2-glycoprotein I. Here we show using small angle x-ray scattering that the proposed extracellular region of Sbi (Sbi-E) is an elongated molecule consisting of four globular domains, two immunoglobulin-binding domains (I and II) and two novel domains (III and IV). We further show that together domains III and IV (Sbi-III-IV), as well as domain IV on its own (Sbi-IV), bind complement component C3 via contacts involving both the C3dg fragment and the C3a anaphylatoxin domain. Preincubation of human serum with either Sbi-E or Sbi-III-IV is inhibitory to all complement pathways, whereas domain IV specifically inhibits the alternative pathway. Monitoring C3 activation in serum incubated with Sbi fragments reveals that Sbi-E and Sbi-III-IV both activate the alternative pathway, leading to consumption of C3. By contrast, inhibition of this pathway by Sbi-IV does not involve C3 consumption. The observation that Sbi-E activates the alternative pathway is counterintuitive to intact Sbi being cell wall-associated, as recruiting complement to the surface of S. aureus would be deleterious to the bacterium. Upon re-examination of this issue, we found that Sbi was not associated with the cell wall fraction, but rather was found in the growth medium, consistent with it being an excreted protein. As such, our data suggest that Sbi helps mediate bacterial evasion of complement via a novel mechanism, namely futile fluid-phase consumption.

C4b-binding protein (C4BP) is a multimeric serum protein that is a potent regulator of the classical and lectin complement pathways. The binding site for C4b has been localized to complement control protein (CCP) domains 1-3 of the C4BP alpha-chain and, in particular, to a cluster of positively charged amino acids predicted to be at the interface between CCP 1 and CCP 2. To determine the regions of C4b contributing to C4BP binding, we have examined via surface plasmon resonance technology the binding of the C4c and C4dg subfragments of C4b to C4BP. At half-physiologic ionic strength, specific and saturable binding was observed for both C4c and C4dg. C4c exhibited much greater ionic strength sensitivity in its binding than did C4dg. Analysis of the effect on binding of the subfragments to various C4b-binding-defective C4BP mutants, together with cross-competition experiments, suggests that the subsites in C4BP for C4c and C4dg are adjacent, but distinct. Additionally, we observed synergy in subsite filling such that the presence of C4dg enhanced the extent of C4c binding over its basal level, and vice versa. The enhanced binding of C4c in the presence of C4dg was not due to an increase in affinity but rather reflected a 2-3-fold increase in the number of sites capable of binding C4c. This suggests the existence of a conformational equilibrium between high- and low-affinity states in the C4c binding subsite within each C4BP subunit, an equilibrium which is shifted in favor of the high-affinity state by the filling of the C4dg subsite.

MARCO is a class A scavenger receptor capable of binding both gram-negative and -positive bacteria. Using the surface plasmon resonance technique, we show here that a recombinant, soluble form of MARCO, sMARCO, binds the major gram-negative and -positive bacterial surface components, lipopolysaccharide and lipoteichoic acid. Yet, the interaction of these two polyanions with sMARCO is of much lower affinity than that of polyinosinic acid, a polyanionic inhibitor of bacterial binding to MARCO. To further elucidate the ligand-binding functions of MARCO, we performed a phage display screen with sMARCO. The screening resulted in the enrichment of only a handful of phage clones. Contrary to expectations, no polyanionic peptides, but only those with a predominantly hydrophobic nature, were enriched. One peptide, VRWGSFAAWL, was displayed on two-thirds of the phages recovered after four rounds of screening. The VRWGSFAAWL phage-sMARCO interaction had significantly slower dissociation kinetics than that between sMARCO and lipopolysaccharide or lipoteichoic acid. Further work with this phage, and the second most enriched phage, displaying the peptide RLNWAWWLSY, demonstrated that both peptides bind to the SRCR domain of MARCO, and that they probably bind to the same site. Data base searches suggested that the VRWGSFAAWL peptide represents complement component C4, but we could not convincingly confirm this suggestion. A study with chimeric scavenger receptors indicated that even minor sequence changes in the MARCO scavenger receptor cysteine-rich (SRCR) domain can have profound effects on the binding of the prototypic scavenger receptor ligand, acetylated low density lipoprotein. As shown by differential binding of glutathione S-transferase-VR-WGSFAAWL, these differences were very likely due to conformational changes. These findings led to experiments that demonstrated a crucial role of the SRCR domain for acetylated low density lipoprotein binding in MARCO. Thus, our results strengthen the notion that the SRCR domain is the major ligand-binding domain in MARCO. Furthermore, they suggest that the domain may contain multiple ligand-binding sites.

The human monoclonal antibody 2F5 is a potent neutralizer of most clades of HIV-1 and possesses protective effects against viral transmission. It recognizes the linear epitope ELDKWAS of the viral envelope protein gp41. As structural information about epitope recognition may help to develop an HIV-1 vaccine we initiated crystallographic analyses of mAb 2F5 and its epitope complex. We now report the preparation of the corresponding Fab fragments, complexation with the epitope peptide, and crystallization of free mAb 2F5 Fab as well as the peptide complex. Both crystal forms are well suited for high-resolution structural analysis.

A vital role for complement in adaptive humoral immunity is now beyond dispute. The crucial interaction is that between B cell 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 electronegative 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. 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.

SHP-1 is a protein-tyrosine phosphatase with two Src homology 2 (SH2) domains. These SH2 domains determine which proteins SHP-1 associates with, but they also autoregulate the activity of the catalytic domain. In this report, we find that the murine SHP-1 transcript is processed to yield a series of alternatively spliced in-frame transcripts, the majority of which exclude exons encoding one or the other SH2 domain. We have examined the corresponding protein isoforms in several ways. First, our measurements of V(max) and K(m) under different conditions indicate that the SH2 variants have elevated activity because of lessened autoregulation. Second, to ascertain whether regulation by the SH2 domains reflects intra- or intermolecular effects, we analyzed the state of SHP-1 by high performance liquid chromatography and sucrose density gradient centrifugation. Our results showed that SHP-1 is a monomer and, thus, is regulated in an intramolecular manner. Third, our analyses detected shape differences between SHP-1 and the active splice variant protein deleted of the amino-terminal SH2 domain; i.e. SHP-1 was globular and resistant to proteolytic digestion, while the splice variant protein was "rod-shaped" and more susceptible to proteolytic digestion.

Mapping approaches employing blocking antibodies and synthetic peptides have implicated the 727-767 segment at the NH2 terminus of C3b alpha'-chain as contributing to the interactions with factor B, factor H, and CR1. Our previous mutagenesis study on the NH2-terminal acidic cluster of this segment identified residues Glu-736 and Glu-737 as contributing to the binding of C3b to factor B and CR1 but not factor H. We have now extended the charged residue mutagenic scan to cover the remainder of the segment (738-767) and have assessed the ability of the C3b-like C3(H2O) form of the mutant molecules to interact with factor H, CR1, and membrane cofactor protein (MCP) using a cofactor-dependent factor I cleavage assay as a surrogate binding assay. We have found that the negatively charged side chains of Glu-744 and Glu-747 are important for the interaction between C3(H2O) and factor H, a result in general agreement with an earlier synthetic peptide study (Fishelson, Z. (1991) Mol. Immunol. 28, 545-552) which implicated residues within the 744-754 segment in H binding. The interactions of the mutants with soluble CR1 (sCR1) revealed two classes of residues. The first are residues required for sCR1 to be an I cofactor for the first two cleavages of alpha-chain. These are all acidic residues and include the Glu-736/Glu-737 pair, Glu-747, and the Glu-754/Asp-755 pairing. The second class affects only the ability of sCR1 to be a cofactor for the third factor I cleavage and include Glu-744 and the Lys-757/Glu-758 pairing. The dominance of acidic residues in the loss-of-function mutants is striking and suggests that H and CR1 contribute basic residues to the interface. Additionally, although there is partial overlap, the contacts required for CR1 binding appear to extend over a wider portion of the 727-767 segment than is the case for factor H. Finally, none of the mutations had any effect on the interaction between soluble MCP and C3(H2O), indicating that despite its functional homology to H and CR1, MCP differs in its mode of binding to C3b/C3(H2O).

The covalent binding of C3 to target molecules on the surfaces of pathogens is crucial in most complement-mediated activities. When C3 is activated, the acyl group is transferred from the sulfhydryl of the internal thioester to the hydroxyl group of the acceptor molecule; consequently, C3 is bound to the acceptor surface by an ester bond. It has been determined that the binding reaction of the B isotype of human C4 uses a two-step mechanism. Upon activation, a His residue first attacks the internal thioester to form an acyl-imidazole bond. The freed thiolate anion of the Cys residue of the thioester then acts as a base to catalyze the transfer of the acyl group from the imidazole to the hydroxyl group of the acceptor molecule. In this article, we present results which indicate that this two-step reaction mechanism also occurs in C3.

Factor I is an active serine proteinase in plasma that regulates both the classical and alternative complement pathways by cleaving C3b and C4b thereby preventing the assembly of C3 and C5 convertase enzymes. In this study, a full-length human factor I cDNA was cloned into the pMT2 expression vector and the pMT2-fI construct was expressed transiently in COS-1 cells and stably in CHO-K1 cells. The transfected COS-1 cells secreted large amounts of recombinant pro-factor I (85 kD). Co-transfection of COS-1 cells with pMT2-fI and the cDNA expression plasmid for PACE (paired basic amino acid cleaving enzyme), resulted predominantly in the secretion of a proteolytically processed form of recombinant factor I (heavy chain, 47 kD; light chain, 35 kD). Following co-transfection of pMT2-fI and pSVNeo.1 into CHO-K1 cells and selection in medium containing G418, a stably transfected clone was isolated that secreted pro-factor I (85 kd) and proteolytically processed factor I (heavy chain, 48 kD; light chain, 37 kD) in approximately equal amounts. The molecular sizes of the subunit chains of the expressed factor I were generally slightly smaller than those of human plasma factor I. The activity of recombinant factor I present in the culture supernatants of transfected COS-1 and CHO-K1 cells was assayed by its ability to cleave 125I-C3b in the presence of factor H and was found to be low when compared with factor I purified from human plasma. However, since the functional activity of purified factor I was reduced approximately 50% in the presence of conditioned medium from non-transfected cells, it is suggested that the cold C3b present in the factor I-deficient serum used to supplement the culture medium probably competed with the 125I-C3b tracer, thereby decreasing the sensitivity of the assay for the recombinant factor I proteins.

Although human IgG2 is not cytophilic, we have shown previously that an IgG2 antibody expressing the sequence PLLGG (underline = substitution) spanning CH2 domain residues 233-237 (Eu numbering) displayed IgG1-like Fc gamma RI binding activity. In contrast, IgG1 PLLGG exhibited 3-fold less affinity, whereas IgG2 ELLGG was 3-fold more active than native IgG1. These results suggested that additional site(s) conferred enhanced binding properties to the engineered, cytophilic IgG2 variant. These sites were shown to reside in the IgG2 CH2 domain, since the IgG1 CH2 module did not have enhanced activity in a panel of hybrid IgG1/IgG2 antibodies. To map these sites further, human IgG1 and IgG2 constant region gene segments were modified to allow reciprocal COOH-terminal half segment exchanges of CH2 exons. These were cloned into a pSV2neo expression vector bearing a rearranged MOPC 315 heavy chain variable region gene and transfected into a MOPC 315 heavy chain deletion mutant. The dinitrophenol affinity-purified IgGs were radiolabeled and assessed for Fc gamma RI binding activity in direct binding assays using U937 cells. The COOH terminus of the IgG2 CH2 domain was found to contain accessory site(s) since it enhanced the binding properties of both IgG1 PLLGG and native IgG1. In contrast, grafting of the COOH terminus of the IgG1 CH2 domain onto IgG2 PLLGG and IgG2 ELLGG diminished their cytophilic activity. The amino acid responsible for the enhancing properties of the COOH terminus of the IgG2 CH2 domain was shown to be threonine 339, since IgG1 PLLGG/Thr339 displayed increased Fc gamma RI binding affinity. Kinetics studies revealed that this is accomplished through an increase in the forward rate constant of the IgG-Fc gamma RI interaction.

A unique thioester bond, formed between the side chains of neighboring C and Q residues, is present in complement components C3 and C4 and the protease inhibitor alpha 2-macroglobulin. This structure is essential for mediating covalent attachment to target acceptors and also for maintaining these proteins in their native conformation. An examination of the residues in the immediate vicinity of the C and Q reveals a very high degree of sequence similarity among the three proteins which crosses species barriers. The following is the sequence flanking the thioester residues in C3, the highly conserved amino acids being underlined and the the thioester-forming residues being indicated by italics: 1005V-T-P-S-G-C-G-E-Q-N-M-I-G-M-T-P-T1021. Through a site-directed mutagenesis and cDNA expression approach, we have examined the importance of the conserved amino acids in the formation, stability, and function of the thioester bond in C3. The behavior of the mutants fell into three categories. The potential loss in peptide backbone flexibility by the replacement of G1009 by A or S was permissive to thioester formation and function as was replacement of M1015 by the still fairly bulky residue F. In contrast, replacement of M1015 by A resulted in an alpha-chain which was highly unstable toward proteolytic degradation. The third category, which included mutant molecules P1007G, P1020G, E1012Q, and Q1013N, displayed an unusual phenotype in which both the autolytic fragmentation and the hemolytic activity characteristics of thioester-intact molecules were absent. However, like their wildtype counterpart, these molecules retained the ability to be cleaved by C3 convertase (C4b2a), a conformation-dependent property that is normally lost in the conversion of native C3 to thioester-hydrolyzed C3(H2O). Since an identical functional profile was obtained when the thioester was deliberately prevented from forming in the mutant C1010A, we conclude that if a stable thioester fails to form during biosynthesis, at least parts of the mature protein can adopt a more native-like conformation than is the case when the thioester is first formed and then hydrolyzed in the mature protein. In view of these new findings, the interpretation of the previously observed correlation between the loss of thioester integrity and the adoption of a C3b-like conformation must be reassessed.

The leukocyte integrin complement receptor type III (CR3, CD11b/CD18) binds the C3 cleavage product iC3b. Many other integrins bind their ligands via an Arg-Gly-Asp (RGD) triplet. Both the RGD-containing C3 peptide 1390TRYRGDQDATMS1401 (pro-C3 numbering) and the RGD-like fibrinogen peptide GGAKQAGDV, which binds to the platelet integrin glycoprotein IIb-IIIa, were shown to inhibit the iC3b-CR3 interaction, suggesting that this binding is also RGD-mediated (Wright, S.D., Weitz, J.I., Huang, A. J., Levin, S.M., Silverstein, S.C., and Loike, J.D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7734-7738). However, unlike other integrin-ligand interactions, that of CR3 and iC3b is unaffected by the hexapeptide GRGDSP, and substitutions in the RGD triplet of C3 from other species appear to be tolerated. It was, therefore, proposed (Grossberger, D., Marcuz, A., du Pasquier, L., and Lambris, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1323-1327) that the highly conserved DATMS portion of the inhibitory C3 peptide may have been responsible for its binding. To address these inconsistencies and directly assess the role of the 1390-1401 segment within the complete iC3b molecule in mediating binding to CR3, a human C3 cDNA was altered by site-directed mutagenesis and the expressed recombinant proteins were examined in a CR3-specific assay. Replacement of RGD by AAA did not abolish rosetting of the corresponding iC3b-coated erythrocytes to human CR3-bearing leukocytes. In addition, mutant iC3b molecules in which the positively charged R1391 (corresponding to K in the fibrinogen peptide) and the highly conserved 1397DATMS sequence were replaced by Q and NAAMA respectively, were still bound by CR3. We conclude that the iC3b-CR3 interaction is not mediated by the RGD triplet or its neighboring residues.

The monoclonal antibody 2F5 neutralizes a broad range of human immunodeficiency virus-1 isolates via a conserved epitope on the viral glycoprotein gp41. The conformation of the principal epitope is a type I beta-turn centered on gp41 residues (664)DKW(666); in addition, binding studies indicate that residues N- and C-terminal to this core as well as structurally more distant parts of gp41 also contribute to the interaction. Ab2/3H6 is an anti-idiotypic antibody that inhibits the interaction between 2F5 and gp41 and as such, Ab2/3H6 may, in principle, possess a paratope that mimics the gp41 epitope. To establish the potential of Ab2/3H6 to serve as a guide for the design of vaccine components against human immunodeficiency virus, we investigated the crystal structure of the heterodimeric complex of Ab2/3H6 F(ab) and 2F5 F(ab)'. Ab2/3H6 F(ab) binds to 2F5 F(ab)' via a helix-like protrusion formed by residues (58(H))RYSPSLNTRL(67(H)) of the 2F5 F(ab)' variable domain and proximal to but not overlapping with the gp41 (664)DKW(666) epitope-binding pocket. This defines Ab2/3H6 as an anti-idiotypic antibody of the Ab2gamma class, i.e., an antigen-inhibitable idiotype that does not carry the internal image of the linear primary gp41 (662)ELDKWAS(668) epitope.

The C4B isotype of the fourth component of human complement (C4) displays 3- to 4-fold greater hemolytic activity than does its other isotype C4A. This correlates with differences in their covalent binding efficiencies to erythrocytes coated with antibody and complement C1. C4A binds to a greater extent when C1 is on IgG immune aggregates. The differences in covalent binding properties correlate only with amino acid changes between residues 1101 and 1106 (pro-C4 numbering)--namely, Pro-1101, Cys-1102, Leu-1105, and Asp-1106 in C4A and Leu-1101, Ser-1102, Ile-1105, and His-1106 in C4B, which are located in the C4d region of the alpha chain. To more precisely identify the residues that are important for the functional differences, C4A-C4B hybrid proteins were constructed by using recombinant DNA techniques. Comparison of these by hemolytic assay and binding to IgG aggregates showed that the single substitution of aspartic acid for histidine at position 1106 largely accounted for the change in functional activity and nature of the chemical bond formed (ester vs. amide). Surprisingly, substitution of a neutral residue, alanine, for histidine at position 1106 resulted in an increase in binding to immune aggregates without subsequent reduction in the hemolytic activity. This result strongly suggests that position 1106 is not "catalytic" as previously proposed but interacts sterically/electrostatically with potential acceptor sites and serves to "select" binding sites on potential acceptor molecules.

We have previously shown that IgM-Asn406, a mutant IgM which has asparagine in place of the serine which is normally found at position 406, also has an abnormally glycosylated mu-chain and is defective in complement-dependent cytolysis. Here we show by analyzing cyanogen bromide fragments from normal and mutant mu-chains that the site of abnormal glycosylation is at the neighboring position, Asn402. The cytolytic defect was shown to be due to impaired C1 binding. At physiological ionic strength, the C1 binding defect was estimated to be 12-fold, which correlates well with the measured defect in cytolytic activity; also, the severity of the defect in C1 binding by the mutant protein decreases with decreasing ionic strength. Kinetic studies showed that the difference in affinities is due to a proportional difference in the association rate for C1q. By comparing IgM made in the presence and absence of deoxymannojirimycin, we show further that the defect in cytolytic activity derives mostly from the abnormal oligosaccharide.

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 C1̄s converted to C4Ab and C4Bb. Spontaneously formed disulfide-linked dimers were separated from monomers 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 equipotent in inhibiting the binding of soluble CR1 to plate-bound C4b. Although divergent from previous reports, our results are 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.

C4 fulfills a vital role in the propagation of the classical and lectin pathways of the complement system. Although there are no reports to date of a C4 functional activity that is mediated solely by the C4d region, evidence clearly points to it having a vital role in a number of the properties of native C4 and its major activation fragment, C4b. Contained within the C4d region are the thioester-forming residues, the four isotype-specific residues controlling the C4A/C4B transacylation preferences, a binding site for nascent C3b important in assembling the classical pathway C5 convertase and determinants for the Chido/Rodgers (Ch/Rg) blood group antigens. In view of its functional importance, we undertook to determine the three-dimensional structure of C4d by X-ray crystallography. Here we report the 2.3A resolution structure of C4Ad, the C4d fragment derived from the human C4A isotype. Although the approximately 30% sequence identity between C4Ad and the corresponding fragment of C3 might be expected to establish a general fold similarity between the two molecules, C4Ad in fact displays a fold that is essentially superimposable on the structure of C3d. By contrast, the electrostatic characteristics of the various faces of the C4Ad molecule show marked differences from the corresponding faces of C3d, likely reflecting the differences in function between C3 and C4. Residues previously predicted to form the major Ch/Rg epitopes were proximately located and accessible on the concave surface of C4Ad. In addition to providing further insights on the current models for the covalent binding reaction, the C4Ad structure allows one to rationalize why C4d is not a ligand for complement receptor 2. Finally the structure allows for the visualization of the face of the molecule containing the binding site for C3b utilized in the assembly of classical pathway C5 convertase.

Previous work has indicated a role for the NH2-terminal segment of the C3 α′-chain in the binding interactions of C3b with a number of its protein ligands. In particular, we have identified two clusters of acidic residues, namely, E736 and E737 and to a lesser extent D730 and E731, as being important in the binding of C3b to factor B and complement receptor 1 and the binding of iC3b to complement receptor 3. Whereas human C3 and C4 have an overall sequence identity of 29%, over a segment near the NH2 termini of their respective α′-chains the sequence identity is 56% (70% chemical similarity). Given the functional similarity between the C4b-C2 and C3b-B interactions in the respective formation of the classical and alternative pathway C3 convertases, as well as the sequence conservation of two acidic clusters, we hypothesized that residues 744EED and 749DEDD within the NH2-terminal segment of the C4 α′-chain would mediate in part the binding of C2 to C4b. We tested this hypothesis using three independent approaches. Site-directed mutagenesis experiments revealed that replacing subsets of the charged residues by their isosteric amides within either acidic cluster resulted in molecules having reduced C2 binding activity. Moreover, a synthetic peptide (C4 residues 740–756) encompassing the two acidic clusters was a specific inhibitor of the binding of C2 to red cell-associated C4b. Finally, Ab raised against the above peptide was able to block the interaction between red cell-associated C4b and fluid phase C2. Taken together, these results strongly suggest that the NH2-terminal acidic residue-rich segment of C4 α′-chain contributes importantly to the interaction of C4b with C2.

Activation and covalent attachment of complement component C3 to pathogens is the key step in complement-mediated host defense. Additionally, the antigen-bound C3d fragment interacts with complement receptor 2 (CR2; also known as CD21) on B cells and thereby contributes to the initiation of an acquired humoral response. The x-ray crystal structure of human C3d solved at 2.0 angstroms resolution reveals an α-α barrel with the residues responsible for thioester formation and covalent attachment at one end and an acidic pocket at the other. The structure supports a model whereby the transition of native C3 to its functionally active state involves the disruption of a complementary domain interface and provides insight into the basis for the interaction between C3d and CR2.

It has long been known that mouse C4 has unusually low hemolytic activity relative to the C4 of other mammalian species (e.g. human and guinea pig), the measurements being done in most cases using a C4-deficient guinea pig serum reagent in a one-step assay with EA. This low activity for mouse C4 previously had been attributed to "technical" difficulties such as lability of the protein during blood collection and partial species incompatibilities with guinea pig components. Recently, we presented evidence for the involvement of human C4 beta-chain residues 455-469, a putatively exposed hydrophilic segment, in contributing to a C5 binding site in the C4b subunit of the classical pathway C5 convertase, C4b3b2a. Given that there were five sequence differences between the human and mouse protein within this segment, we hypothesized that these substitutions may have compromised the C5 convertase subunit activity of mouse C4, thereby resulting in its low hemolytic activity. Using a multi-step hemolytic assay which was totally dependent upon C5 cleavage by the classical pathway, we found that mouse C4 was completely devoid of classical pathway C5 convertase subunit activity. We have been able to rule out the most obvious potential species incompatibilities (e.g. between C4mo and C5gp) as being responsible for this lack of activity. Moreover, we found that the low level of hemolytic activity of mouse C4 measured in the one-step assay can be ascribed totally to C5 cleavage, and subsequent terminal component assembly, by the alternative pathway C5 convertase, (C3b)2Bb. However, the assembly of the latter enzyme complex is dependent upon the presence of C3b molecules deposited initially via the classical pathway C3 convertase in which mouse C4b is a subunit. Finally, whereas conversion of human residues 458RP to the mouse-like sequence PL was sufficient to abrogate classical pathway C5 convertase subunit activity in human C4, the five substitutions which "humanized" the 452-466 segment of mouse C4 (corresponding to human residues 455-469) were on their own insufficient to impart this activity to mouse C4. This implies that, in addition to the 455-469 beta-chain segment of human C4, there are other regions of the molecule contributing to C5 binding which are also non-conserved between human and mouse C4.

To characterize the region on human IgG1 responsible for its high-affinity interaction with the human Fc gamma receptor class I (Fc gamma RI), we have analyzed the binding properties of a series of genetically engineered chimeric antidinitrophenyl antibodies with identical murine antibody combining sites and hybrid IgG1/IgG2 human constant (C) regions. In addition, we have investigated a panel of reciprocally point-mutated IgG1 and IgG2 chimeric antibodies to identify the amino acid residues that confer cytophilic properties to human IgG1. Our data unambiguously indicate that cytophilic activity of IgG1 is an intrinsic property of its heavy-chain C region 2 (CH2) domain. We report that the entire sequence spanning residues 234-237 (LLGG) is required to restore full binding activity to IgG2 and IgG4 and that individual amino acid substitutions failed to render IgG2 active. Nevertheless, the reciprocal single point mutations in IgG1 either significantly lowered its activity or abolished it completely. Finally, we observed that an IgG2 antibody containing the entire ELLGGP sequence (residues 233-238) was more active than wild-type IgG1. This finding suggests that in addition to the primary contact site identified in the N terminus of the gamma 1 CH2 domain, secondary sites involving residues from the C-terminal half of the domain may also contribute to the IgG1-Fc gamma RI interaction.

The formation of the alternative-pathway C3 convertase has been previously suggested to have an absolute requirement for Mg2+, especially at the level of complex formation between C3b and factor B (B). In the course of defining spectral probes that could be used to monitor the C3b-B interaction (e.g. 1-anilino-8-naphthalene sulfonic acid fluorescence and near-u.v. circular dichroism) we observed that the signal change reporting on this binding was not completely reversed upon addition of excess ethylene-diaminetetraacetic acid (EDTA). Using sucrose gradient ultracentrifugation, we have directly demonstrated a Mg2+-independent C3b-B complex in the fluid phase. B thus bound was not only susceptible to specific proteolytic activation by factor D, but the resulting C3bBb enzyme was able to convert native C3 to C3b. Interestingly, we were unable to detect Mg2+-independent specific binding of 125I-B to C3b which was particle-bound. Using a sensitive hemolytic assay, however, we estimated that the functional activity of B with surface-bound C3b is 80-fold greater in the presence of physiological Mg2+ (0.5 mM) than in 2 mM EDTA. In contrast, the fluid-phase association is estimated to differ less than three-fold under the same conditions. These data demonstrate that the requirement for Mg2+ in the formation of the fluid-phase alternative-pathway C3 convertase is not absolute. Furthermore, they suggest a difference in the stable functional properties of fluid-phase and surface-bound C3b.

The numerous ligand binding properties expressed by the activated forms of C3 (C3b) and C4 (C4b) are best explained as arising from proteolytic-cleavage-induced conformational changes. The studies described above provide direct physical evidence for such conformational transitions in both complement proteins. Significantly, however, a virtually identical conformational end state was also produced by direct nucleophilic scission of the internal thioester bond in C3 and C4 in the absence of any proteolysis. Clearly, it is the integrity of this thiolactone structure that is the determining factor in maintaining the native conformation in C3 and C4. Recent studies suggest a similar conformational role for the thioester in alpha 2-macroglobulin. Proteolytic activation in all three thioester-containing molecules renders this structure more susceptible to nucleophilic or solvolytic attack. Whether this effect is mediated by a peptide-cleavage-induced increase in the electrophilicity of the reactive carbonyl, or simply by increasing the accessibility of the solvent to this structure, is as yet unknown. In the case of C3 and C4, removal of the activation peptide also has a profound effect on the kinetics of the conformational change initiated by the loss of the thioester. This kinetic constraint has made it possible to correlate the acquisition of ligand binding properties with the spectroscopically detectable conformational changes.

An alpha 2-macroglobulin-like protease inhibitor was isolated from the cell-free hemolymph of the american lobster (Homarus americanus) by ion-exchange chromatography and gel filtration. Whereas the undissociated molecule has a molecular weight of 342,000 as determined by ultracentrifugation studies, under reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a subunit molecular weight of 180,000. On the basis of this and other evidence, we conclude that the lobster protein is a dimer consisting of two disulfide-bonded monomers. The purified protein inhibits proteolytic enzymes but protects the esterolytic activity of trypsin toward low molecular weight substrates from inactivation by soybean trypsin inhibitor. The methylamine sensitivity of this activity suggests the presence of an internal thioester bond. This was confirmed by the covalent incorporation of [14C]methylamine, by the formation of Mr 55,000 and 125,000 autolytic cleavage fragments in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and, more directly, by the amino acid sequence of a tryptic peptide containing the putative thioester region. Whereas the N-terminal amino acid sequence (22 residues) of the protein revealed an overall identity of only 18% when compared with the human protein, the sequence of the thioester-containing peptide was highly conserved, both with respect to human alpha 2-macroglobulin and to other proteins having a thioester bond. The protein showed the "slow to fast" conformational change typical in alpha 2-macroglobulins in nondenaturing gel electrophoresis after treatment with trypsin, but not after incubation with methylamine.