Biomedical subjects

Kenneth B M Reid

Publications and source records attributed to Kenneth B M Reid.

At least 19 recordsLinked to original sources

PubMed2007

Molecular interactions between MASP-2, C4, and C2 and their activation fragments leading to complement activation via the lectin pathway.

Activation of component C3 is central to the pathways of complement and leads directly to neutralization of pathogens and stimulation of adaptive immune responses. The convertases that catalyze this reaction assemble from fragments of complement components via multistep reactions. In the lectin pathway, mannose-binding lectin (MBL) and ficolins bind to pathogens and activate MBL-associated serine protease-2 (MASP-2). MASP-2 cleaves C4 releasing C4a and generating C4b, which attaches covalently to the pathogen surface upon exposure of its reactive thioester. C2 binds to C4b and is also cleaved by MASP-2 to form the C3 convertase (C4b2a). To understand how this complex process is coordinated, we have analyzed the interactions between MASP-2, C4, C2, and their activation fragments and have compared MASP-2-catalyzed cleavage of C4b2 and C2. The data show that C2 binds tightly to C4b but not to C4, implying that C4 and C2 do not circulate as preformed complexes but that C2 is recruited only after prior activation of C4. Following cleavage of C4, C4b still binds to MASP-2 (KD approximately 0.6 microM) and dissociates relatively slowly (koff approximately 0.06 s-1) compared with the half-life of the thioester (<or=0.7 s) (Sepp, A., Dodds, A. W., Anderson, M. J., Campbell, R. D., Willis, A. C., and Law, S. K. (1993) Protein Sci. 2, 706-716). We propose that the C4b.MASP-2 interaction favors attachment of C4b near to the activating MBL.MASP complex on the bacterial surface so that, following recruitment of C2, the proximity of enzyme and substrate (C4b2) combined with more favorable reaction kinetics drive the formation of the C3 convertase, promoting complement activation.

Animals↗

PubMed2006

Existence of different but overlapping IgG- and IgM-binding sites on the globular domain of human C1q.

C1q is the first subcomponent of the classical complement pathway that binds antigen-bound IgG or IgM and initiates complement activation via association of serine proteases C1r and C1s. 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 some structural and functional autonomy. Although a number of studies have tried to identify IgG-binding sites on the gC1q domain, no such attempt has been made to localize IgM-binding site. On the basis of the information available via the gC1q crystal structure, molecular modeling, mutational studies, and bioinformatics, we have generated a series of substitution mutants of ghA, ghB, and ghC and examined their interactions with IgM. The comparative analysis of IgM- and IgG-binding abilities of the mutants suggests that the IgG- and IgM-binding sites within the gC1q domain are different but may overlap. Whereas Arg(B108), Arg (B109), and Tyr(B175) mainly constitute the IgM-binding site, the residues Arg(B114), Arg(B129), Arg(B163), and His(B117) that have been shown to be central to IgG binding are not important for the C1q-IgM interaction. Given the location of Arg(B108), Arg (B109), and Tyr(B175) in the gC1q crystal structure, it is likely that C1q interacts with IgM via the top of the gC1q domain.

Arginine↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2006

Impaired recognition of apoptotic neutrophils by the C1q/calreticulin and CD91 pathway in systemic lupus erythematosus.

OBJECTIVE: A deficiency in a subcomponent of C1q can result in increased susceptibility to autoimmune diseases such as systemic lupus erythematosus (SLE). The monocyte endocytic receptor CD91 is implicated in the endocytosis of apoptotic neutrophils via interactions with C1q and calreticulin. In this clinical study, we studied the binding of C1q to leukocytes and determined whether C1q bound specifically to calreticulin and CD91 on cells undergoing apoptosis in SLE. METHODS: Proximal antibody phage display, calreticulin-transfected cells, and immunocytochemical and confocal techniques were used in a comprehensive analysis of direct binding of C1q to apoptotic neutrophils that were obtained from healthy individuals and from patients with SLE. In addition, apoptotic cellular systems were assessed in vitro. RESULTS: C1q appeared to colocalize to apoptotic blebs on the surface of leukocytes in association with both calreticulin and CD91, as determined by phage display and transfected cell studies. However, C1q did not bind to apoptotic cells isolated from SLE patients, despite the positivity of the cells for both calreticulin and CD91. Surface expression of calreticulin decreased on neutrophils as they aged, but increased on monocytes. In an apoptotic phagocytic assay, the addition of C1q and calreticulin significantly enhanced the phagocytosis of apoptotic cell debris by monocyte-derived cells. CONCLUSION: These observations indicate that neutrophils from SLE patients have a reduced ability to be recognized and removed by the C1q/calreticulin/CD91-mediated apoptotic pathway, despite the presence of main apoptotic recognition partners. This suggests that an additional component, as yet unidentified, acts as a C1q binding partner on apoptotic cells, and this component may be lacking in cells isolated from SLE patients.

Adolescent↗

PubMed2005

Surfactant protein D is proatherogenic in mice.

Surfactant protein D (SP-D) is an important innate immune defense molecule that mediates clearance of pathogens and modulates the inflammatory response. Moreover, SP-D is involved in lipid homeostasis, and pulmonary accumulation of phospholipids has previously been observed in SP-D-deficient (Spd-/-) mice. Atherogenesis involves both inflammation and lipid deposition, and we investigated the role of SP-D in the development of atherosclerosis. SP-D synthesis was localized to vascular endothelial cells. Atherosclerotic lesion areas were 5.6-fold smaller in the aortic roots in Spd-/- mice compared with wild-type C57BL/6N mice on an atherogenic diet. HDL cholesterol (HDL-C) was significantly elevated in Spd-/- mice. Treatment of Spd-/- mice with a recombinant fragment of human SP-D resulted in decreases of HDL-C (21%) as well as total cholesterol (26%), and LDL cholesterol (28%). Plasma TNF-alpha was reduced in Spd-/- mice (45% difference). SP-D was proatherogenic in the mouse model used. The effect is likely to be due to the observed disturbances of plasma lipid metabolism and alteration of the inflammatory process, which underlie the reduced susceptibility to atherosclerosis in Spd-/- mice.

Animals↗

PubMed2005

Role of Ca2+ in the electrostatic stability and the functional activity of the globular domain of human C1q.

C1q is the recognition subunit of the classical pathway of the complement system and a major connecting link between classical pathway-driven innate immunity and IgG- or IgM-mediated acquired immunity. The basic structural subunit of C1q is composed of an N-terminal triple-helical collagen-like region and a C-terminal heterotrimeric globular head domain (gC1q) that is made up of individual A, B, and C chains. Recent crystallographic studies have revealed that the gC1q domain, which is the main target-binding region of C1q, has a compact and spherical heterotrimeric assembly, held together by both electrostatic and nonpolar interactions, with quasi-3-fold symmetry. A characteristic feature of the gC1q domain is the presence of a exposed Ca(2+) located near the apex. We have investigated, using theoretical and experimental approaches, the role of Ca(2+) in the electrostatic stability and target-binding properties of the native C1q as well as recombinant monomeric forms of the C-terminal regions of the A, B, and C chains. Here, we report that Ca(2+) primarily influences the target recognition properties of C1q toward IgG, IgM, C-reactive protein, and pentraxin 3. At pH 7.4, the loss of Ca(2+) leads to changes in the direction of electric moment from coaxial (where the putative C-reactive protein-binding site is located) to perpendicular to the molecular axis (toward the most likely IgG-binding site), which appears important for target recognition by C1q and subsequent complement activation.

Calcium↗

PubMed2005

Surfactant proteins SP-A and SP-D: structure, function and receptors.

Surfactant proteins, SP-A and SP-D, are collagen-containing C-type (calcium dependent) lectins called collectins, which contribute significantly to surfactant homeostasis and pulmonary immunity. These highly versatile innate immune molecules are involved in a range of immune functions including viral neutralization, clearance of bacteria, fungi and apoptotic and necrotic cells, down regulation of allergic reaction and resolution of inflammation. Their basic structures include a triple-helical collagen region and a C-terminal homotrimeric lectin or carbohydrate recognition domain (CRD). The trimeric CRDs can recognize carbohydrate or charge patterns on microbes, allergens and dying cells, while the collagen region can interact with receptor molecules present on a variety of immune cells in order to initiate clearance mechanisms. Studies involving gene knock-out mice, murine models of lung hypersensitivity and infection, and functional characterization of cell surface receptors have revealed the diverse roles of SP-A and SP-D in the control of lung inflammation. A recently proposed model based on studies with the calreticulin-CD91 complex as a receptor for SP-A and SP-D has suggested an anti-inflammatory role for SP-A and SP-D in naïve lungs which would help minimise the potential damage that continual low level exposure to pathogens, allergens and apoptosis can cause. However, when the lungs are overwhelmed with exogenous insults, SP-A and SP-D can assume pro-inflammatory roles in order to complement pulmonary innate and adaptive immunity. This review is an update on the structural and functional aspects of SP-A and SP-D, with emphasis on their roles in controlling pulmonary infection, allergy and inflammation. We also try to put in perspective the controversial subject of the candidate receptor molecules for SP-A and SP-D.

Allergens↗

PubMed2005

Collectin surfactant protein D binds antibodies and interlinks innate and adaptive immune systems.

Innate immune collectins, such as surfactant protein D (SP-D), contain fibrillar collagen-like regions and globular carbohydrate-recognition domains (CRDs). SP-D recognizes carbohydrate arrays present on microbial surfaces via its CRDs, agglutinates microbes and enhances their phagocytosis. In contrast, adaptive immune proteins such as immunoglobulins (Igs) recognize pathogens via binding to specific antigens. Here we show that: SP-D binds various classes of immunoglobins, including IgG, IgM, IgE and secretory IgA, but not serum IgA; the globular domains of SP-D bind both the Fab and Fc domains of IgG; SP-D recognizes IgG via calcium-dependent protein-protein interactions, aggregates IgG-coated beads and enhances their phagocytosis by murine macrophage RAW 264.7 cells. Therefore, we propose that SP-D effectively interlinks innate and adaptive immune systems.

Animals↗

PubMed2005

Mannose-binding lectin recognizes peptidoglycan via the N-acetyl glucosamine moiety, and inhibits ligand-induced proinflammatory effect and promotes chemokine production by macrophages.

Peptidoglycan (PGN) is the major cell wall component (90%, w/w) of Gram-positive bacteria and consists of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharide repeating arrays that are cross-linked by short peptides. We hypothesized that PGN is a ligand for pathogen-associated pattern-recognition proteins. Mannose-binding lectin (MBL) and serum amyloid component P are two carbohydrate-binding innate immune proteins present in the blood. In this study we show that human MBL, but not serum amyloid component P, binds significantly to PGN via its C-type lectin domains, and that the interaction can be more effectively competed by GlcNAc than by MurNAc. Surface plasmon resonance analyses show that native MBL binds immobilized PGN with high avidity. Competition experiments also show that both native MBL and MBL(n/CRD), a 48-kDa recombinant trimeric fragment of MBL containing neck and carbohydrate recognition domains, have higher affinity for GlcNAc than for MurNAc. Protein arrays and ELISA show that PGN increases the secretion of TNF-alpha, IL-8, IL-10, MCP-2, and RANTES from PMA-stimulated human monocytic U937 cells. Interestingly, the presence of MBL together with PGN increases the production of IL-8 and RANTES, but reduces that of TNF-alpha. Our results indicate that Gram-positive bacterial is a biologically relevant ligand for MBL, and that the collectin preferentially binds to the GlcNAc moiety of the PGN via its C-type lectin domains. MBL inhibits PGN-induced production of proinflammatory cytokines while enhancing the production of chemokines by macrophages, which suggests that MBL may down-regulate macrophage-mediated inflammation while enhancing phagocyte recruitment.

Acetylglucosamine↗

PubMed2005

Susceptibility of mice genetically deficient in the surfactant protein (SP)-A or SP-D gene to pulmonary hypersensitivity induced by antigens and allergens of Aspergillus fumigatus.

Lung surfactant protein A (SP-A) and D (SP-D) are innate immune molecules which are known to interact with allergens and immune cells and modulate cytokine and chemokine profiles during host hypersensitivity response. We have previously shown therapeutic effects of SP-A and SP-D using a murine model of lung hypersensitivity to Aspergillus fumigatus (Afu) allergens. In this study, we have examined the susceptibility of SP-A (AKO) or SP-D gene-deficient (DKO) mice to the Afu allergen challenge, as compared with the wild-type mice. Both AKO and DKO mice exhibited intrinsic hypereosinophilia and several-fold increase in levels of IL-5 and IL-13, and lowering of IFN-gamma to IL-4 ratio in the lungs, suggesting a Th2 bias of immune response. This Th2 bias was reversible by treating AKO or DKO mice with SP-A or SP-D, respectively. The AKO and DKO mice showed distinct immune responses to Afu sensitization. DKO mice were found more susceptible than wild-type mice to pulmonary hypersensitivity induced by Afu allergens. AKO mice were found to be nearly resistant to Afu sensitization. Intranasal treatment with SP-D or rhSP-D (a recombinant fragment of human SP-D containing trimeric C-type lectin domains) was effective in rescuing the Afu-sensitized DKO mice, while SP-A-treated Afu-sensitized AKO mice showed several-fold elevated levels of IL-13 and IL-5, resulting in increased pulmonary eosinophilia and damaged lung tissue. These data reaffirm an important role for SP-A and SP-D in offering resistance to pulmonary allergenic challenge.

Allergens↗

PubMed2005

Innate immune collectin surfactant protein D enhances the clearance of DNA by macrophages and minimizes anti-DNA antibody generation.

Dying microbes and necrotic cells release highly viscous DNA that induces inflammation and septic shock, and apoptotic cells display DNA, a potential autoantigen, on their surfaces. However, innate immune proteins that mediate the clearance of free DNA and surface DNA-containing cells are not clearly established. Pulmonary surfactant proteins (SP-) A and D are innate immune pattern recognition collectins that contain fibrillar collagen-like regions and globular carbohydrate recognition domains (CRDs). We have recently shown that collectins SP-A, SP-D, and mannose binding lectin recognize DNA and RNA via their collagen-like regions and CRDs. Here we show that SP-D enhances the uptake of Cy3-labeled fragments of DNA and DNA-coated beads by U937 human monocytic cells, in vitro. Analysis of DNA uptake by freshly isolated mouse alveolar macrophages shows that SP-D, but not SP-A, deficiency results in reduced clearance of DNA, ex vivo. Analysis of bronchoalveolar lavage fluid shows that SP-D- but not SP-A-deficient mice are defective in clearing free DNA from the lung. Additionally, both SP-A- and SP-D-deficient mice accumulate anti-DNA Abs in sera in an age-dependent manner. Thus, we conclude that collectins such as SP-A and SP-D reduce the generation of anti-DNA autoantibody, which may be explained in part by the defective clearance of DNA from the lungs in the absence of these proteins. Our findings establish two new roles for these innate immune proteins and that SP-D enhances efficient pinocytosis and phagocytosis of DNA by macrophages and minimizes anti-DNA Ab generation.

Adjuvants, Immunologic↗

PubMed2005

Induction of DMBT1 expression by reduced ERK activity during a gastric mucosa differentiation-like process and its association with human gastric cancer.

Abnormalities in the expression of DMBT1 (deleted in malignant brain tumors 1) have been implicated in the development of esophageal, gastric and colorectal cancers of the alimentary tract, but the underlying mechanism remains unclear. In the present study, using the gastric cell line AGS, we identified two intracellular signaling molecules protein kinase C (PKC) and extracellular signal-related kinase (ERK). They mediated both the phorbol myristate acetate (PMA) downregulation of DMBT1 expression and the initiation of cell differentiation, which was measured by cell cycle withdrawal and the induction of the tissue-specific marker trefoil factor 1 (TFF1). A time-course study showed that following the PMA activation of ERK kinase, the induction of TFF1 and the reduction of DMBT1 were detected at the same time point. We then demonstrated a minimal level of DMBT1 in proliferating AGS cells seeded at low density, where ERK activity was high. Reduction of ERK activity, either by an ERK inhibitor PD98059 or by high-density seeding, significantly reduced AGS cell growth judged by CFSE labeling. This cellular effect was elicited by cyclin D/p21 (Cip/Waf1) and G(0)/G(1) arrest, and was accompanied by a marked increase in DMBT1-expressing cells. Finally, we showed that siRNA directed against DMBT1 had no effect on the induction of a cell growth arrest marker, gut-enriched Kruppel-like factor (GKLF), but reduced the PMA induction of TFF1. Along with its upregulation coinciding with G(0)/G(1) arrest, and its attenuation in differentiated cells, these results suggest that the transient induction of DMBT1 is apparently specific at an early stage of gastric epithelial differentiation-like process, when it may play a role in cell fate decision. Consistent with such a potential function, we detected frequent abnormalities of the DMBT1 expression in the specimens of human gastric adenocarcinoma.

Adenocarcinoma↗

PubMed2005

Variations in Helicobacter pylori lipopolysaccharide to evade the innate immune component surfactant protein D.

Helicobacter pylori is a common and persistent human pathogen of the gastric mucosa. Surfactant protein D (SP-D), a component of innate immunity, is expressed in the human gastric mucosa and is capable of aggregating H. pylori. Wide variation in the SP-D binding affinity to H. pylori has been observed in clinical isolates and laboratory-adapted strains. The aim of this study was to reveal potential mechanisms responsible for evading SP-D binding and establishing persistent infection. An escape variant, J178V, was generated in vitro, and the lipopolysaccharide (LPS) structure of the variant was compared to that of the parental strain, J178. The genetic basis for structural variation was explored by sequencing LPS biosynthesis genes. SP-D binding to clinical isolates was demonstrated by fluorescence-activated cell sorter analyses. Here, we show that H. pylori evades SP-D binding through phase variation in lipopolysaccharide. This phenomenon is linked to changes in the fucosylation of the O chain, which was concomitant with slipped-strand mispairing in a poly(C) tract of the fucosyltransferase A (fucT1) gene. SP-D binding organisms are predominant in mucus in vivo (P = 0.02), suggesting that SP-D facilitates physical elimination. Phase variation to evade SP-D contributes to the persistence of this common gastric pathogen.

Carbohydrate Sequence↗

PubMed2004

The dimeric and trimeric solution structures of the multidomain complement protein properdin by X-ray scattering, analytical ultracentrifugation and constrained modelling.

Properdin regulates the alternative pathway of the complement system of immune defence by stabilising the C3 convertase complex. It contains six thrombospondin repeat type I (TSR-1 to TSR-6) domains and an N-terminal domain. Properdin exists as either a dimer, trimer or tetramer. In order to determine the solution structure of multiple TSR domains, the molecular structures of dimeric and trimeric properdin were studied by X-ray scattering and analytical ultracentrifugation. Guinier analyses showed that the dimer and trimer have radii of gyration R(G) values of 7.5 nm and 10.3 nm, respectively, and cross-sectional radii of gyration R(XS) values of 1.3 nm and 1.5 nm, respectively. Distance distribution functions showed that the maximum lengths of the dimer and trimer were 25 nm and 30 nm, respectively. Analytical ultracentrifugation gave sedimentation coefficients of 5.1S and 5.2S for the dimer and trimer forms, respectively. Homology models for the TSR domains were constructed using the crystal structure of the TSP-2 and TSP-3 domains in human thrombospondin as templates. Properdin could be represented by seven TSR domains, not six as believed, since the crystal structure determined for TSP-2 and TSP-3 showed that the N-terminal domain (TSR-0) could be represented by a truncated TSR domain with the same six conserved Cys residues found in TSR-1 to TSR-6. Automated constrained molecular modelling revealed the solution conformations of multiple TSR domains in properdin at medium resolution. The comparison of 3125 systematically generated conformational models for the trimer with the X-ray data showed that good curve fits could be obtained by assuming that the linker between adjacent TSR domains possessed limited flexibility. Good trimer models correspond to partially collapsed triangular structures, and extended triangular shapes do not fit the data. The corresponding 3125 models for the dimer revealed a similar outcome in which a partially collapsed TSR structure gave good fits. The models account for the effect of mutations that cause properdin deficiencies, and suggest that the biologically active TSR-4, TSR-5 and TSR-6 domains are exposed for protein-protein interactions. The role of the other TSR domains in properdin may be to act as spacers to make TSR-4, TSR-5 and TSR-6 accessible for function.

Amino Acid Sequence↗

PubMed2004

Nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins A and D and mannose-binding lectin.

Collectins are a family of innate immune proteins that contain fibrillar collagen-like regions and globular carbohydrate recognition domains (CRDs). The CRDs of these proteins recognize various microbial surface-specific carbohydrate patterns, particularly hexoses. We hypothesized that collectins, such as pulmonary surfactant proteins (SPs) SP-A and SP-D and serum protein mannose-binding lectin, could recognize nucleic acids, pentose-based anionic phosphate polymers. Here we show that collectins bind DNA from a variety of origins, including bacteria, mice, and synthetic oligonucleotides. Pentoses, such as arabinose, ribose, and deoxyribose, inhibit the interaction between SP-D and mannan, one of the well-studied hexose ligands for SP-D, and biologically relevant d-forms of the pentoses are better competitors than the l-forms. In addition, DNA and RNA polymer-related compounds, such as nucleotide diphosphates and triphosphates, also inhibit the carbohydrate binding ability of SP-D, or approximately 60 kDa trimeric recombinant fragments of SP-D that are composed of the alpha-helical coiled-coil neck region and three CRDs (SP-D(n/CRD)) or SP-D(n/CRD) with eight GXY repeats (SPD(GXY)(8)(n/CRD)). Direct binding and competition studies suggest that collectins bind nucleic acid via their CRDs as well as by their collagen-like regions, and that SP-D binds DNA more effectively than do SP-A and mannose-binding lectin at physiological salt conditions. Furthermore, the SP-D(GXY)(8)(n/CRD) fragments co-localize with DNA, and the protein competes the interaction between propidium iodide, a DNA-binding dye, and apoptotic cells. In conclusion, we show that collectins are a new class of proteins that bind free DNA and the DNA present on apoptotic cells by both their globular CRDs and collagen-like regions. Collectins may therefore play an important role in decreasing the inflammation caused by DNA in lungs and other tissues.

Animals↗

PubMed2004

Mutational analyses of the recombinant globular regions of human C1q A, B, and C chains suggest an essential role for arginine and histidine residues in the C1q-IgG interaction.

The first step in the activation of the classical complement pathway by immune complexes involves the binding of the globular domain (gC1q) of C1q to the Fc regions of aggregated IgG or IgM. Each gC1q domain is a heterotrimer of the C-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain. Our recent studies have suggested a modular organization of gC1q, consistent with the view that ghA, ghB, and ghC are functionally autonomous modules and have distinct and differential ligand-binding properties. Although C1q binding sites on IgG have been previously identified, the complementary interacting sites on the gC1q domain have not been precisely defined. The availability of the recombinant constructs expressing ghA, ghB, and ghC has allowed us, for the first time, to engineer single-residue substitution mutations and identify residues on the gC1q domain, which are involved in the interaction between C1q and IgG. Because C1q is a charge pattern recognition molecule, we have sequentially targeted arginine and histidine residues in each chain. Consistent with previous chemical modification studies and the recent crystal structure of gC1q, our results support a central role for arginine and histidine residues, especially Arg(114) and Arg(129) of the ghB module, in the C1q-IgG interaction.

Alanine↗

PubMed2004

C1q and tumor necrosis factor superfamily: modularity and versatility.

C1q is the target recognition protein of the classical complement pathway and a major connecting link between innate and acquired immunity. As a charge pattern recognition molecule of innate immunity, C1q can engage a broad range of ligands via its globular (gC1q) domain and modulate immune cells, probably via its collagen region. The gC1q signature domain, also found in many non-complement proteins, has a compact jelly-roll beta-sandwich fold similar to that of the multifunctional tumor necrosis factor (TNF) ligand family. The members of this newly designated 'C1q and TNF superfamily' are involved in processes as diverse as host defense, inflammation, apoptosis, autoimmunity, cell differentiation, organogenesis, hibernation and insulin-resistant obesity. This review is an attempt to draw structural and functional parallels between the members of the C1q and TNF superfamily.

Amino Acid Sequence↗

PubMed2003

Localization of ligand-binding sites on human C1q globular head region using recombinant globular head fragments and single-chain antibodies.

As a charge pattern recognition molecule, human C1q can bind a range of immunoglobulin and non-immunoglobulin ligands via its carboxy-terminal globular domain and activate the classical complement pathway. Each globular domain has a heterotrimeric organization, composed of the carboxy-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain. Recently, we have found that the recombinant forms of individual ghA, ghB and ghC bind differentially to IgG, IgM, gp41 peptide 601-613 of human immunodeficiency virus-1 (HIV-1), gp21 peptide 400-429 of human T cell lymphotrophic virus-I (HTLV-I), beta-amyloid peptide, and apoptotic cells, suggesting a modular organization of the globular domain. This paper examines the interaction of ghA, ghB and ghC with two known C1q ligands: Klebsiella pneumoniae porin OmpK36 and salivary agglutinin. In addition, we have used a panel of recombinant single-chain antibodies (scFv) specific for ghA, ghB and ghC in order to map sites on the heterotrimeric globular domain which are likely to interact with IgG1, IgG3, IgM, OmpK36, salivary agglutinin and gp41 loop peptide. The combined use of recombinant ghA, ghB, ghC and single-chain antibodies has revealed at least three ligand-binding sites on the globular domain of C1q: one is IgG- and OmpK36-specific, the second (IgM-binding site) is most likely overlapping with IgG/OmpK36 binding site, and the third (the gp41-binding site) seems to be located at the junction between the collagen and globular domains.

Agglutinins↗