Interaction of C1q subcomponent with immunoglobulin M

The affinity of human C1q subcomponent for IgM of normal human serum and Waldenstr?m macroglobulins of patients Sew and Zuk were investigated by the polyethylene glycol 6,000 immune complexes precipitation test. This test was calibrated with heat-aggregated gamma-globulin (HAGG); maximum fixation of C1q ranged from 60 to 80% (measured as percentage of radioactivity of the immune complexes precipitate) and was observed when the C1q:HAGG concentration ratio was about 1:250. At the ratio of 1:20 the radioactivity of the precipitate was about 43% of the total. The capacity of polyclonal IgM and Waldenstr?m macroglobulins for C1q fixation is low and variable. The percentage of C1q fixed at the C1q:IgM ratio of 1:20 for polyclonal IgM and Zuk macroglobulin was about 9%, whereas for Sew it was only about 1%.     

Chemical studies on the isolated collagen-like and globular fragment of complement component C1q. Comparative studies on bovine and human C1q

Both the collagen-like and the globular fragments of a subcomponent C1q of the first component of bovine and human complement were highly purified by enzymic digestion followed by gel filtration. Analyses by polyacrylamide gel electrophoresis showed that the former was composed of covalently linked peptide chains with an average molecular weight of 14 000, and that the latter was composed of three non-covalently linked peptide chains each having a molecular weight of approximately 15 000. Great similarities between amino acid compositions of the globular fragments and some similarities between those of the collagen-like fragments were found. Moreover, great similarities of amino acid compositions were found among three non-covalently linked chains of each globular fragment as well as between the corresponding chains of both globular fragments. These results suggested that both the collagen-like and the globular domains on the C1q molecule remained highly conserved in its evolution.     

Platelet C1q receptor interactions with collagen- and C1q-coated surfaces

We recently described specific binding sites for C1q on human blood platelets. Structural similarities between the amino-terminal of C1q and collagen have suggested that receptors for both molecules on platelets might be the same. The present study thus compared the interaction of purified C1q receptors (C1qR) and whole platelets with collagen- and C1q-coated polystyrene surfaces. Surfaces coated with BSA or gelatin served as controls. Purified 125I-labeled C1qR recognized both C1q- and collagen-coated surfaces in a divalent, cation-independent manner. This adhesion was inhibited by polyclonal or monoclonal (II1/D1) anti-C1qR antibodies. Although C1qR adhered preferentially to C1q-coated surfaces, adhesion to bovine and human type I collagen, as well as to human type III and V collagen, was also noted. In parallel studies, 51Cr-labeled platelets bound equally well to collagen- or C1q-coated surfaces, albeit in a magnesium-dependent manner. Partial inhibition of platelet adhesion was observed in the presence of RGDS, despite the inability of RGDS to modify C1qR interaction with C1q or collagen. Moreover, anti C1qR antibodies selectively inhibited platelet adhesion to C1q-coated surfaces, whereas antibodies specific for the GPIa/IIa collagen receptor (6F1) preferentially inhibited platelet collagen interactions. These data support the presence of distinct platelet membrane C1qR, which may cross-react with collagen, and suggest that C1qR are necessary but not sufficient for platelet adhesion to C1q-coated surfaces. Additional divalent cation and/or RGD-sensitive binding sites may participate.     

Enzymatic alteration of C1q, the collagen-like subcomponent of the first component of complement, leads to cross-reactivity with type II collagen

Native serum C1q, the collagenous-like subcomponent of the first component of complement, is not recognized by polyclonal anti-collagen type II antibodies. However, when purified C1q was subjected to limited proteolysis by collagenase it showed antigenic cross-reactivity with collagen type II. The same cross-reactivity was observed with hemolytically active C1q in synovial fluids of patients with rheumatoid arthritis (RA), whereas C1q from synovial fluids of patients with osteoarthritis (OA), villo-nodular synovitis and ankylosing spondylitis was not recognized by this antibody. However, incubation of synovial fluid C1q of OA patients with synovial fluid leucocytes from RA patients led to an alteration of OA-C1q which was now recognized by the anti-collagen type II antibody.     

Phagocytic cell molecules that bind the collagen-like region of C1q. Involvement in the C1q-mediated enhancement of phagocytosis

C1q binds to and elicits cellular responses by several cell types, including monocytes, macrophages, neutrophils, B cells, and fibroblasts. The cell-binding domain is located within the collagen-like pepsin-resistant region of the C1q molecule (C1q tails). An affinity matrix of C1q tails coupled to Sepharose was used to select C1q-binding proteins from detergent extracts of surface-iodinated human monocytes, polymorphonuclear leukocytes, and the U937 cells. The major radiolabeled polypeptide eluted specifically from the ligand affinity column had an apparent molecular mass (Mr) of 126,000. Minor iodinated components eluted from Sepharose-tails migrated with Mr of 216,000 and 55,000. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions no change in the migration of any of these polypeptide bands was detected. None of these polypeptides reacted with antibodies directed against the integrins alpha 5 beta 1 (fibronectin receptor) or alpha v beta 3 (vitronectin receptor), LFA-1, or to several other cell adhesion molecules. The Mr 126,000 band was found to contain more than one polypeptide. Lectin binding properties, susceptibility to glycosidases and proteases, and immunoreactivity with the monoclonal antibody L-10, indicated that CD43 (sialophorin/leukosialin) is a component of this band. However, further data show that a monoclonal antibody, generated by immunization with the isolated Clq-binding fractions, recognizes a cell surface sialoglycoprotein distinct from CD43 and inhibits the C1q-mediated enhancement of phagocytosis in monocytes. These latter observations provide the first definitive connection between a specific phagocytic cell surface protein and a known C1q-mediated function. While these proteins contain sialic acid, binding assays and functional assays using neuraminidase-treated cells demonstrate that the functional interaction between C1q and the cell surface is not via sialic acid. The data taken together indicate either that the functional C1q receptor on phagocytic cells is a multi-subunit complex or that multiple proteins can interact with the fragment of C1q containing the cell-binding domain, at least one of which is involved in the C1q-mediated enhancement of phagocytosis.     

Interaction of chromatin with a histone H1 containing swapped N- and C-terminal domains

Although the details of the structural involvement of histone H1 in the organization of the nucleosome are quite well understood, the sequential events involved in the recognition of its binding site are not as well known. We have used a recombinant human histone H1 (H1.1) in which the N- and C-terminal domains (NTD/CTD) have been swapped and we have reconstituted it on to a 208-bp nucleosome. We have shown that the swapped version of the protein is still able to bind to nucleosomes through its structurally folded wing helix domain (WHD); however, analytical ultracentrifuge analysis demonstrates its ability to properly fold the chromatin fibre is impaired. Furthermore, FRAP analysis shows that the highly dynamic binding association of histone H1 with the chromatin fibre is altered, with a severely decreased half time of residence. All of this suggests that proper binding of histone H1 to chromatin is determined by the simultaneous and synergistic binding of its WHD–CTD to the nucleosome.     

Interaction of C1q with IgG1, C-reactive protein and pentraxin 3: mutational studies using recombinant globular head modules of human C1q A, B, and C chains

C1q is the first subcomponent of the classical complement pathway that can interact with a range of biochemically and structurally diverse self and nonself ligands. The globular domain of C1q (gC1q), which is the ligand-recognition domain, is a heterotrimeric structure composed of the C-terminal regions of A (ghA), B (ghB), and C (ghC) chains. The expression and functional characterization of ghA, ghB, and ghC modules have revealed that each chain has specific and differential binding properties toward C1q ligands. It is largely considered that C1q-ligand interactions are ionic in nature; however, the complementary ligand-binding sites on C1q and the mechanisms of interactions are still unclear. To identify the residues on the gC1q domain that are likely to be involved in ligand recognition, we have generated a number of substitution mutants of ghA, ghB, and ghC modules and examined their interactions with three selected ligands: IgG1, C-reactive protein (CRP), and pentraxin 3 (PTX3). Our results suggest that charged residues belonging to the apex of the gC1q heterotrimer (with participation of all three chains) as well as the side of the ghB are crucial for C1q binding to these ligands, and their contribution to each interaction is different. It is likely that a set of charged residues from the gC1q surface participate via different ionic and hydrogen bonds with corresponding residues from the ligand, instead of forming separate binding sites. Thus, a recently proposed model suggesting the rotation of the gC1q domain upon ligand recognition may be extended to C1q interaction with CRP and PTX3 in addition to IgG1.     

C1q receptor on murine cells

Different cells and cell lines of murine origin were tested for their capacity to bind the C subcomponent C1q by using biotinylated human C1q and streptavidin-FITC. Cytofluorometric analysis of splenocytes and thymocytes shows that the majority of C1q-reactive cells reside in the population of B cells and macrophages. There is a significant difference in the C1q-binding capacity of in vitro activated cells; although more than half of the B cell blasts bind the C subcomponent, T cell blasts are virtually negative. It is shown that pre-B lymphomas and cell lines of myeloid origin bind C1q strongly (90 to 98%), whereas in the case of mature B cell lymphomas, plasmocytomas, and the tested T cell lines, the percentage of C1q binding cells varies from 0 to 56. C1q affinity chromatography of the detergent extracts from P388D1 and WEHI-3 cells followed by SDS-PAGE of the eluted proteins under reducing conditions reveals a band at approximately 80 kDa. Analysis of splenocytes shows two additional minor C1q-binding molecules with apparent molecular masses of 50 and 45 kDa, whereas in the case of B cell blasts three bands of similar density are seen at approximately 95, 50 and 45 kDa. C1q-receptors of murine cells are shown to be antigenically related to their human counterpart, because a polyclonal antibody (266A) raised against the human C1q receptor reacts with them.     

Binding and complement activation by C-reactive protein via the collagen-like region of C1q and inhibition of these reactions by monoclonal antibodies to C-reactive protein and C1q

Ligand-complexed C-reactive protein (CRP), like aggregated or complexed IgG, can react with C1q and activate the classical C pathway. Whereas IgG is known to bind to the globular region and not to the collagen-like region (CLR) of C1q, the site of interaction of C1q with CRP has not been defined. CRP-trimers were prepared by cross-linking and found to bind to C1q and to activate the C system. Heat-aggregated IgG (Agg-IgG) did not block the binding of CRP-trimers to C1q, nor did CRP-trimers block binding of Agg-IgG to C1q, suggesting that CRP and IgG bind at different sites. ELISA and Western blot analysis showed that CRP-trimers bound to the CLR, whereas Agg-IgG bound only to the globular region; similarly, anti-CLR mAb inhibited binding of CRP-trimers to C1q whereas anti-globular region mAb did not. Reactivity with CRP-trimers as well as with Agg-IgG was retained after reduction/alkylation and SDS treatment of C1q. A group of 22 anti-CRP mAb directed against at least six distinct native-CRP epitopes and eight distinct neo-CRP epitopes was tested for ability to inhibit the CRP-CLR interaction; one mAb, anti-native CRP mAb 8D8, with strong inhibitory activity was identified. Fab' of 8D8 blocked binding of CRP-trimers to intact C1q as well as CLR, and also inhibited CRP (CRP-trimers and CRP-protamine complexes) induced C activation, but had no effect on C1q binding or C activation by Agg-IgG. These results indicate that a conformation-determined region on CRP binds to a sequence-determined region on the CLR of C1q in an interaction which leads to C activation. Anti-CRP and anti-C1q mAb that specifically inhibit this interaction are described.     

Interaction of the globular domain of human C1q with Salmonella typhimurium lipopolysaccharide

Gram-negative bacteria can bind complement protein C1q in an antibody-independent manner and activate classical pathway via their lipopolysaccharides (LPS). Earlier studies have implicated the collagen-like region of human C1q in binding LPS. In recent years, a number of C1q target molecules, previously considered to interact with collagen-like region of C1q, have been shown to bind via the globular domain (gC1q). Here we report, using recombinant forms of the globular head regions of C1q A, B and C chains, that LPS derived from Salmonella typhimurium interact specifically with the B-chain of the gC1q domain in a calcium-dependent manner. LPS and IgG-binding sites on the gC1q domain appear to be overlapping and this interaction can be inhibited by a synthetic C1q inhibitor, suggesting common interacting mechanisms.     

Interaction of C1q and mannan-binding lectin (MBL) with C1r, C1s, MBL-associated serine proteases 1 and 2, and the MBL-associated protein MAp19

Mannan-binding lectin (MBL) and C1q activate the complement cascade via attached serine proteases. The proteases C1r and C1s were initially discovered in a complex with C1q, whereas the MBL-associated serine proteases 1 and 2 (MASP-1 and -2) were discovered in a complex with MBL. There is controversy as to whether MBL can utilize C1r and C1s or, inversely, whether C1q can utilize MASP-1 and 2. Serum deficient in C1r produced no complement activation in IgG-coated microwells, whereas activation was seen in mannan-coated microwells. In serum, C1r and C1s were found to be associated only with C1q, whereas MASP-1, MASP-2, and a third protein, MAp19 (19-kDa MBL-associated protein), were found to be associated only with MBL. The bulk of MASP-1 and MAp19 was found in association with each other and was not bound to MBL or MASP-2. The interactions of MASP-1, MASP-2, and MAp19 with MBL differ from those of C1r and C1s with C1q in that both high salt concentrations and calcium chelation (EDTA) are required to fully dissociate the MASPs or MAp19 from MBL. In the presence of calcium, most of the MASP-1, MASP-2, and MAp19 emerged on gel-permeation chromatography as large complexes that were not associated with MBL, whereas in the presence of EDTA most of these components formed smaller complexes. Over 95% of the total MASPs and MAp19 found in serum are not complexed with MBL.     

The lymphocyte receptor CD6 interacts with syntenin-1, a scaffolding protein containing PDZ domains

CD6 is a type I membrane glycoprotein expressed on thymocytes, mature T and B1a lymphocytes, and CNS cells. CD6 binds to activated leukocyte cell adhesion molecule (CD166), and is considered as a costimulatory molecule involved in lymphocyte activation and thymocyte development. Accordingly, CD6 partially associates with the TCR/CD3 complex and colocalizes with it at the center of the mature immunological synapse (IS) on T lymphocytes. However, the signaling pathway used by CD6 is still mostly unknown. The yeast two-hybrid system has allowed us the identification of syntenin-1 as an interacting protein with the cytoplasmic tail of CD6. Syntenin-1 is a PDZ (postsynaptic density protein-95, postsynaptic discs large, and zona occludens-1) domain-containing protein, which functions as an adaptor protein able to bind cytoskeletal proteins and signal transduction effectors. Mutational analyses showed that certain amino acids of the most C-terminal sequence of CD6 (-YDDISAA) and the two postsynaptic density protein-95, postsynaptic discs large, and zona occludens-1 domains of syntenin-1 are relevant to the interaction. Further confirmation of the CD6-syntenin-1 interaction was obtained from pull-down and co-immunoprecipitation assays in mammalian cells. Image analyses also showed that syntenin-1 accumulates at CD6 caps and at the IS. Therefore, we propose that syntenin-1 may function as a scaffolding protein coupling CD6 and most likely other lymphocyte receptors to cytoskeleton and/or signaling effectors during IS maturation.     

Interaction of insulin receptor substrate 3 with insulin receptor, insulin receptor-related receptor, insulin-like growth factor-1 receptor, and downstream signaling proteins.

Insulin receptor substrates (IRS) mediate biological actions of insulin, growth factors, and cytokines. All four mammalian IRS proteins contain pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains at their N termini. However, the molecules diverge in their C-terminal sequences. IRS3 is considerably shorter than IRS1, IRS2, and IRS4, and is predicted to interact with a distinct group of downstream signaling molecules. In the present study, we investigated interactions of IRS3 with various signaling molecules. The PTB domain of mIRS3 is necessary and sufficient for binding to the juxtamembrane NPXpY motif of the insulin receptor in the yeast two-hybrid system. This interaction is stronger if the PH domain or the C-terminal phosphorylation domain is retained in the construct. As determined in a modified yeast two-hybrid system, mIRS3 bound strongly to the p85 subunit of phosphatidylinositol 3-kinase. Although high affinity interaction required the presence of at least two of the four YXXM motifs in mIRS3, there was not a requirement for specific YXXM motifs. mIRS3 also bound to SHP2, Grb2, Nck, and Shc, but less strongly than to p85. Studies in COS-7 cells demonstrated that deletion of either the PH or the PTB domain abolished insulin-stimulated phosphorylation of mIRS3. Insulin stimulation promoted the association of mIRS3 with p85, SHP2, Nck, and Shc. Despite weak association between mIRS3 and Grb2, this interaction was not increased by insulin, and may not be mediated by the SH2 domain of Grb2. Thus, in contrast to other IRS proteins, mIRS3 appears to have greater specificity for activation of the phosphatidylinositol 3-kinase pathway rather than the Grb2/Ras pathway.     

Interaction between complement subcomponent C1q and bacterial lipopolysaccharides.

The heptose-less mutant of Escherichia coli, D31m4, bound complement subcomponent C1q and its collagen-like fragments (C1qCLF) with Ka values of 1.4 x 10(8) and 2.0 x 10(8) M-1 respectively. This binding was suppressed by chemical modification of C1q and C1qCLF using diethyl pyrocarbonate (DEPC). To investigate the role of lipopolysaccharides (LPS) in this binding, biosynthetically labelled [14C]LPS were purified from E. coli D31m4 and incorporated into liposomes prepared from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) [PC/PE/LPS, 2:2:1, by wt.]. Binding of C1q or its collagen-like fragments to the liposomes was estimated via a flotation test. These liposomes bound C1q and C1qCLF with Ka values of 8.0 x 10(7) and 2.0 x 10(7) M-1; this binding was totally inhibited after chemical modification of C1q and C1qCLF by DEPC. Liposomes containing LPS purified from the wild-strain E. coli K-12 S also bound C1q and C1qCLF, whereas direct binding of C1q or C1qCLF to the bacteria was negligible. Diamines at concentrations which dissociate C1 into C1q and (C1r, C1s)2, strongly inhibited the interaction of C1q or C1qCLF with LPS. Removal of 3-deoxy-D-manno-octulosonic acid (2-keto-3-deoxyoctonic acid; KDO) from E. coli D31m4 LPS decreases the binding of C1qCLF to the bacteria by 65%. When this purified and modified LPS was incorporated into liposomes, the C1qCLF binding was completely abolished. These results show: (i) the essential role of the collagen-like moiety and probably its histidine residues in the interaction between C1q and the mutant D31m4; (ii) the contribution of LPS, particularly the anionic charges of KDO, to this interaction.     

Activation of the first component of human complement, C1, by monoclonal antibodies directed against different domains of subcomponent C1q

Two monoclonal antibodies directed against C1q, and their (Fab)2 and Fab fragments, were used to study the mechanism of C1 activation. Monoclonal antibody 2A10, an IgG2a, was digested by pepsin to yield fully immunoreactive (Fab')2. Monoclonal antibody 1H11, an IgG1, was digested by papain to yield fully immunoreactive, bivalent (Fab)2. Previously 1H11 had been shown to bind to the C1q "heads," whereas 2A10 bound to stalks. Activation of C1 was followed by the cleavage of 125I-C1s in the presence of C1 inhibitor (C1-Inh) at 37 degrees C. Spontaneous activation was minimal at inhibitor concentrations above 0.4 micron (1.3 X physiologic inhibitor concentration); all results were corrected for the spontaneous activation background. Heat-aggregated IgG activated completely in this system and was taken as 100% activation. Monoclonal antibody 2A10 caused precipitation of C1 and slow activation; neither the (Fab')2 nor the Fab' derived from 2A10-caused activation. Probably, aggregates of intact 2A10 and C1 were serving as immune complexes to activate other molecules of C1. In contrast, both 1H11 and its (Fab)2 activated completely and stoichiometrically; that is, maximal activation was achieved at a ratio of one C1q head to one antibody combining site. The monovalent Fab derived from 1H11 bound well to C1q, but no activation of C1 was observed. Thus, bivalent binding of this head-binding monoclonal is required for C1 activation, but not the presence of the antibody Fc portion. Neither 1H11 nor its (Fab)2 fragments caused C1 precipitation; however, the 1H11 did form complexes composed of two C1q cross-linked by multiple 1H11, which were visualized by electron microscopy. The presence of these dimeric complexes correlated well with activation. A model for C1 activation is proposed in which two C1q subcomponents are held together by multiple (Fab)2 bridging C1q heads. The model is roughly analogous to touching opposing pairs of fingers and thumb tips, the two hands representing the two C1q, forming a cage. C1-Inh, which probably binds to C1r through the open end of the C1 cone, is too long asymmetric to be included within the cage. Thus, according to this model, the dimers of C1 are released from the inhibitory action of C1-Inh, and activation proceeds spontaneously and rapidly at 37 degrees C.     

Interaction of epoxide hydrolase with itself and other microsomal proteins

The interactions of rat liver epoxide hydrolase (EC 3.3.2.3) with itself and with cytochromes P-450 and NADPH-cytochrome P-450 reductase were investigated in microsomal preparations and in reconstituted systems in which all of the enzymes are functionally active. Hydrodynamic measurements indicated that purified epoxide hydrolase behaves as a single aggregate of approximately 16 monomeric units and that further aggregation of the protein only occurs in the presence of high concentrations of phospholipid. Neither guanidine-HCl nor the nonionic detergent Lubrol PX was able to completely dissociate the aggregate into monomers. The interactions of epoxide hydrolase with NADPH-cytochrome P-450 reductase and the major forms of cytochrome P-450 isolated from phenobarbital- and 5,6-benzoflavone-treated rats were studied by Soret difference spectroscopy, by perturbation of the fluorescence of NADPH-cytochrome P-450 reductase and fluorescein-labeled epoxide hydrolase, and by CD spectroscopy. The spectra provided evidence that binding of the proteins to each other occurs and some of the results suggest that affinity constants are on the order of 10⁷, m^?1. The spectral perturbations were not observed with other intrinsic membrane proteins. When microsomes were treated with the crosslinking reagent dimethylsuberimidate and solubilized with detergents, epoxide hydrolase could be precipitated with antibodies raised to cytochromes P-450 or NADPH-cytochrome P-450 reductase. Transient times were determined for the conversion of 1-octene to octene-1,2-dihydrodiol in a reconstituted enzyme system and for the conversion of naphthalene to naphthalene-1,2-dihydrodiol in rat liver microscomes and compared to the transient times predicted from the enzymatic rates of hydrolysis of the intermediate epoxides. In all cases the observed transient times were shorter than expected, in support of the view that coupling of epoxide hydrolase with cytochromes P-450 occurs. These results support the view that epoxide hydrolase couples with cytochrome P-450-containing mixed-function oxidase systems and may have relevance to the metabolism of potentially harmful xenobiotics by these enzymes.     

Comparison of autoantibodies to the collagen-like region of C1q in hypocomplementemic urticarial vasculitis syndrome and systemic lupus erythematosus.

Hypocomplementemic urticarial vasculitis syndrome (HUVS) is an apparent autoimmune disorder that resembles SLE. We previously showed that C1q precipitins in HUVS sera are IgG autoantibody to human C1q. We have compared HUVS anti-C1q autoantibody to a similar autoantibody in the serum of some patients with SLE. As with anti-C1q autoantibody in SLE sera, the HUVS autoantibody binds only to the collagen-like region (CLR) of C1q. In both HUVS and SLE, IgG2 is the predominant subclass of IgG autoantibody and IgM autoantibody to C1q is uncommon. In both diseases, anti-C1q autoantibodies bind preferentially to surface-adsorbed C1q or CLR fragments compared to these antigens in solution. Finally, when HUVS or SLE autoantibodies were added to CLR-coated wells already bound, respectively, by SLE or HUVS autoantibodies, no increases in CLR binding were observed, suggesting that HUVS and SLE autoantibodies to C1q bind to the same CLR epitope(s).