Biomedical subjects

Robert B Sim

Publications and source records attributed to Robert B Sim.

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↗

PubMed2007

The impact of glycosylation on the biological function and structure of human immunoglobulins.

Immunoglobulins are the major secretory products of the adaptive immune system. Each is characterized by a distinctive set of glycoforms that reflects the wide variation in the number, type, and location of their oligosaccharides. In a given physiological state, glycoform populations are reproducible; therefore, disease-associated alterations provide diagnostic biomarkers (e.g., for rheumatoid arthritis) and contribute to disease pathogenesis. The oligosaccharides provide important recognition epitopes that engage with lectins, endowing the immunoglobulins with an expanded functional repertoire. The sugars play specific structural roles, maintaining and modulating effector functions that are physiologically relevant and can be manipulated to optimize the properties of therapeutic antibodies. New molecular models of all the immunoglobulins are included to provide a basis for informed and critical discussion. The models were constructed by combining glycan sequencing data with oligosaccharide linkage and dynamics information from the Glycobiology Institute experimental database and protein structural data from "The Protein Data Bank."

Arthritis, Rheumatoid↗

PubMed2006

Human follicular lymphoma cells contain oligomannose glycans in the antigen-binding site of the B-cell receptor.

Expression of surface immunoglobulin appears critical for the growth and survival of B-cell lymphomas. In follicular lymphoma, we found previously that the Ig variable (V) regions in the B-cell receptor express a strikingly high incidence of N-glycosylation sequons, NX(S/T). These potential glycosylation sites are introduced by somatic mutation and are lymphoma-specific, pointing to their involvement in tumor pathogenesis. Analysis of the V region sugars from lymphoma-derived IgG/IgM reveals that they are mostly oligomannose and, remarkably, are located in the antigen-binding site, possibly precluding conventional antigen binding. The Fc region contains complex glycans, confirming that the normal glycan processing pathway is intact. Binding studies indicate that the oligomannose glycans occupying the V regions are accessible to mannose-binding lectin. These findings suggest a potential contribution to lymphoma pathogenesis involving antigen-independent interaction of surface immunoglobulin of the B-cell receptor with mannose-binding molecules of innate immunity in the germinal center.

Binding Sites↗

PubMed2006

Mannan binding lectin and viral hepatitis.

Mannan binding lectin (MBL) is a pattern recognition molecule of the innate immune system that binds to sugars on the surface of invading micro-organisms. Target binding, complement activation and other functions of MBL are dependent on the presence of multiple carbohydrate recognition domains. Several polymorphisms in the promoter and structural regions of MBL2 adversely affect the plasma concentration and oligomeric state of MBL. The possession of mutant alleles has been linked to disease outcome for a variety of bacterial and viral infections. Viral hepatitis is caused by unrelated viruses referred to as hepatitis virus A-E. The disease usually has both acute and chronic phases, the latter leading to cirrhosis and hepatocellular carcinoma. Hepatitis viruses B and C (HBV and HCV, respectively) are a significant cause of morbidity worldwide. HBV encodes envelope glycoproteins termed large, middle, and small that may exist in glycosylated or unglycosylated forms on the virion. An interaction between HBV glycoproteins and MBL has been demonstrated in vitro. Significant associations between MBL levels, determined by MBL2 haplotypes, and HBV persistence and disease progression have been described. HCV encodes two highly glycosylated envelope proteins, E1 and E2, which are potential targets for interaction with MBL. Mutant MBL2 haplotypes have been linked to disease progression and response to therapy in HCV infection. Here we summarise the effect of MBL2 polymorphisms on MBL function and how this may relate to disease outcome in HBV and HCV infection.

Hepacivirus↗

PubMed2006

Human complement factor I glycosylation: structural and functional characterisation of the N-linked oligosaccharides.

Factor I (fI) is a key serine protease that modulates the complement cascade by regulating the levels of C3 convertases. Human fI circulates in plasma as a heavily N-glycosylated (25-27% w/w) heterodimer composed of two disulphide linked chains, each carrying three N-linked oligosaccharide chains. It had been suggested that the oligosaccharides may have both structural and functional roles in the interactions with the natural substrate and the cofactor during a catalysis. The N-linked glycans of each fI chain were characterised in detail and the analysis revealed a similar composition of the glycan pools with both chains heavily sialylated. Disialylated structures were in excess over monosialylated ones: 55% over 40% for the heavy chain and 62% over 35% for the light chain. The dominant type of glycan identified on both chains was A(2)G(2)S(2), a biantennary structure with chains terminating in sialic acid linked to galactose. The glycan characterisation facilitated a strategy for the partial deglycosylation of the enzyme. Assessment of the proteolytic activities of the native and partially deglycosylated forms of fI showed that both forms of the enzyme have very similar proteolytic activities against C3(NH(3)) indicating that the charged glycans of fI do not influence the fI-cofactor-substrate interactions.

Amino Acid Sequence↗

PubMed2006

Increased complement classical and mannan-binding lectin pathway activities in schizophrenia.

Schizophrenia is a severe mental disorder, with worldwide prevalence of 1-1.5%. Immunological research in schizophrenia indicates that infectious or autoimmune processes might play a role in the etiopathogenesis. The complement system is a major mediator of innate immune defence against infection and contributes to many functions of the immune system including inflammation, opsonization and cell lysis. Mannan-binding lectin (MBL) activates the complement system via the lectin pathway. Inherited MBL deficiency, common in most human populations, predisposes to infectious and autoimmune diseases. We measured total complement activity (CH50), C4 activity (C4 CH50), MBL level and the activities of MBL-associated serine proteases, MASP-1 and MASP-2 in sera of 45 schizophrenic patients and in 62 healthy volunteers. We found that schizophrenic patients and healthy volunteers have statistically similar MBL levels and MASP-1 activity. However, MBL-bound MASP-2 activity and therefore MBL and MASP-2-mediated complement activation capacity is increased in schizophrenic patients compared with healthy volunteers (P<0.01). The increase was accompanied by increased CH50 (P<0.02) and C4 CH50 (P<0.02). Our results support the idea that complement system alterations may be involved in schizophrenia.

Complement C4↗

PubMed2006

His-384 allotypic variant of factor H associated with age-related macular degeneration has different heparin binding properties from the non-disease-associated form.

A polymorphism in complement factor H has recently been associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly. A histidine rather than a tyrosine at residue position 384 in the mature protein increases the risk of AMD. Here, using a recombinant construct, we show that amino acid 384 is adjacent to a heparin-binding site in CCP7 of factor H and demonstrate that the allotypic variants differentially recognize heparin. This functional alteration may affect binding of factor H to polyanionic patterns on host surfaces, potentially influencing complement activation, immune complex clearance, and inflammation in the macula of AMD patients.

Alleles↗

PubMed2006

Functional significance of factor H binding to Neisseria meningitidis.

Neisseria meningitidis is an important cause of septicemia and meningitis. To cause disease, the bacterium must successfully survive in the bloodstream where it has to avoid being killed by host innate immune mechanisms, particularly the complement system. A number of pathogenic microbes bind factor H (fH), the negative regulator of the alternative pathway of complement activation, to promote their survival in vivo. In this study, we show that N. meningitidis binds fH to its surface. Binding to serogroups A, B, and C N. meningitidis strains was detected by FACS and Far Western blot analysis, and occurred in the absence of other serum factors such as C3b. Unlike Neisseria gonorrhoeae, binding of fH to N. meningitidis was independent of sialic acid on the bacterium, either as a component of its LPS or its capsule. Characterization of the major fH binding partner demonstrated that it is a 33-kDa protein; examination of insertion mutants showed that porins A and B, outer membrane porins expressed by N. meningitidis, do not contribute significantly to fH binding. We examined the physiological consequences of fH bound to the bacterial surface. We found that fH retains its activity as a cofactor of factor I when bound to the bacterium and contributes to the ability of N. meningitidis to avoid complement-mediated killing in the presence of human serum. Therefore, the recruitment of fH provides another mechanism by which this important human pathogen evades host innate immunity.

Amino Acid Sequence↗

PubMed2006

Mannan binding lectin and its interaction with immunoglobulins in health and in disease.

In humans there are five classes of immunoglobulins (Igs), IgG, IgM, IgA, IgE and IgD, all of which are glycoproteins. The Igs are the major secretory product of the adaptive immune system, and they bind to antigens via variable sequences on their Fab regions. The binding to antigen results in neutralization or agglutination of bound material and also initiates effector functions via the Fc regions, such as opsonisation and activation of the classical complement pathway through binding of C1q. Mannan binding lectin (MBL), the 'recognition' molecule of the lectin pathway of complement activation, is homologous in structure to C1q, and binds in a calcium-dependent manner to terminal mannose and GlcNAc residues which have been identified on the oligosaccharides N-linked to the Igs. MBL binds agalactosylated glycoforms of IgG (IgG-G0), polymeric forms of IgA and certain glycoforms of IgM which have a high incidence of GlcNAc-terminating glycans. This interaction provides a route of clearance of immune complexes from the serum, and a mechanism of complement activation to Ig-coated pathogens. In disease, MBL contributes to inflammation in Rheumatoid Arthritis, a condition in which serum IgG-G0 concentrations can increase significantly compared to healthy individuals. MBL has recently been demonstrated to bind Ig in the B cell receptor complex which expresses abnormal variable region glycosylation, in follicular lymphoma.

Animals↗

PubMed2006

Recognition of Candida albicans by mannan-binding lectin in vitro and in vivo.

Mannan-binding lectin (MBL) is a component of the innate immune system. The goal of the present study was to evaluate binding of MBL to Candida albicans in vitro and in vivo and to assess the impact of MBL treatment on host resistance. The results showed a variable and often discontinuous pattern of binding to individual yeast cells. MBL bound to cells grown at 37 degrees C but not to cells grown at 23 degrees C. The putative MBL ligand was constitutively present on yeast cells grown at 23 degrees C, but the ligand was masked on such cells, such that MBL could not bind. C. albicans yeasts and hyphae in infected tissue bound MBL. Finally, parenteral administration of MBL increased resistance of mice to hematogenously disseminated candidiasis. These results suggest that MBL is an important component of innate resistance to candidiasis and that MBL therapy may be a means to prevent disseminated candidiasis in high-risk patients.

Animals↗

PubMed2006

Molecular organization of human Ficolin-2.

Human Ficolin-2 (L-Ficolin) is an oligomeric serum protein consisting of a collagen-like stalk and fibrinogen-like recognition domains. The protein binds to arrays of sugars present on different microorganisms, enhances phagocytosis and promotes activation of the lectin complement pathway. So far the detailed oligomeric structure and composition of human Ficolin-2 has not been determined. Recombinant human Ficolin-2 was expressed in Chinese hamster ovary cells and its structure and biological functions were investigated by gel filtration, sucrose density gradient ultracentrifugation, mass spectrometry and surface plasmon resonance spectroscopy. It was revealed that Ficolin-2 has a high molecular weight due to extensive disulfide bridge formation. It was able to bind to different ligands, interact with mannose-binding lectin associated serine proteases and activate the complement system. Mass values of 807 and 403 kDa were determined corresponding to a 24-mer and a 12-mer of 34.4 kDa polypeptides. However, the 24-mer was unstable and the 12-mer is likely the major functional form of the protein. Our results are consistent with the view that Ficolin-2 is built up by a mixture of stable homodimers and homotrimers. Based on our findings we propose a model in which disulfide bridges located in the N-terminal region of the polypeptides explain the oligomerization pattern of human Ficolin-2.

Animals↗

PubMed2006

Structural model for the mannose receptor family uncovered by electron microscopy of Endo180 and the mannose receptor.

The mannose receptor family comprises four members in mammals, Endo180 (CD280), DEC-205 (CD205), phospholipase A(2) receptor (PLA(2)R) and the mannose receptor (MR, CD206), whose extracellular portion contains a similar domain arrangement: an N-terminal cysteine-rich domain (CysR) followed by a single fibronectin type II domain (FNII) and 8-10 C-type lectin-like domains (CTLDs). These proteins mediate diverse functions ranging from extracellular matrix turnover through collagen uptake to homeostasis and immunity based on sugar recognition. Endo180 and the MR are multivalent transmembrane receptors capable of interacting with multiple ligands; in both receptors FNII recognizes collagens, and a single CTLD retains lectin activity (CTLD2 in Endo180 and CTLD4 in MR). It is expected that the overall conformation of these multivalent molecules would deeply influence their function as the availability of their binding sites could be altered under different conditions. However, conflicting reports have been published on the three-dimensional arrangement of these receptors. Here, we have used single particle electron microscopy to elucidate the three-dimensional organization of the MR and Endo180. Strikingly, we have found that both receptors display distinct three-dimensional structures, which are, however, conceptually very similar: a bent and compact conformation built upon interactions of the CysR domain and the lone functional CTLD. Biochemical and electron microscopy experiments indicate that, under a low pH mimicking the endosomal environment, both MR and Endo180 experience large conformational changes. We propose a structural model for the mannose receptor family where at least two conformations exist that may serve to regulate differences in ligand selectivity.

Hydrogen-Ion Concentration↗

PubMed2006

Interaction of mannan binding lectin with alpha2 macroglobulin via exposed oligomannose glycans: a conserved feature of the thiol ester protein family?

The serum collectin mannan-binding lectin (MBL) binds to oligomannose and GlcNAc-terminating glycans present on microorganisms. Using a commercial affinity chromatography resin containing immobilized MBL we screened human and mouse serum for endogenous MBL-binding targets. We isolated the serum protease inhibitor alpha(2) macroglobulin (alpha2M), a heavily glycosylated thiol ester protein (TEP) composed of four identical 180-kDa subunits, each of which has eight N-linked glycosylation sites. alpha2M has previously been reported to interact with MBL; however, the interaction was not characterized. We investigated the mechanism of formation of complexes between alpha2M and MBL and concluded that they form by the direct binding of oligomannose glycans Man(5-7) occupying Asn-846 on alpha2M to the lectin domains (carbohydrate recognition domains) of MBL. The oligomannose glycans are accessible for lectin binding on both active alpha2M (thiol ester intact) and protease-cleaved alpha2M (thiol ester cleaved). We demonstrate that MBL is able to interact with alpha2M in the fluid phase, but the interaction does not inhibit the binding of MBL to mannan-coated surfaces. In addition to alpha2M, two other members of the TEP family, C3 and C4, which also contain oligomannose glycans, were captured from human serum using the MBL resin. MBL binding may be a conserved feature of the TEPs, dating from their ancestral origins. We suggest that the inhibition of proteases on the surface of microorganisms by an ancestral alpha2M-like TEP may generate "arrays" of oligomannose glycans to which MBL or other lectins can bind. Binding would lead to opsonization or activation of enzyme systems such as complement.

Animals↗

PubMed2006

Carbohydrate-independent recognition of collagens by the macrophage mannose receptor.

Mannose receptor (MR) is the best characterised member of a family of four endocytic molecules that share a common domain structure; a cysteine-rich (CR) domain, a fibronectin-type II (FNII) domain and tandemly arranged C-type lectin-like domains (CTLD, eight in the case of MR). Two distinct lectin activities have been described for MR. The CR domain recognises sulphated carbohydrates while the CTLD mediate binding to mannose, fucose or N-acetylglucosamine. FNII domains are known to be important for collagen binding and this has been studied in the context of two members of the MR family, Endo180 and the phospholipase A2 receptor. Here, we have investigated whether the broad and effective lectin activity mediated by the CR domain and CTLD of MR is favoured to the detriment of FNII-mediated interaction(s). We show that MR is able to bind and internalise collagen in a carbohydrate-independent manner and that MR deficient macrophages have a marked defect in collagen IV and gelatin internalisation. These data have major implications at the molecular level as there are now three distinct ligand-binding sites described for MR. Furthermore our findings extend the range of endogenous ligands recognised by MR, a molecule firmly placed at the interface between homeostasis and immunity.

Animals↗

PubMed2005

Heterogeneity of MBL-MASP complexes.

In order to study aspects of the stoichiometry and composition of human MBL-MASP complexes in the population, MBL-MASP complexes were bound from sera of 152 healthy individuals onto mannan-coated microtitre plates. Bound mannan-binding lectin (MBL) was measured by ELISA, and the enzyme activities of MBL-bound MASP-1 and MASP-2 were measured by an amidolytic assay and a C4 fixation assay, respectively. MASP-1 activity correlated with MBL concentration, as did MASP-2 activity (in both cases: p<0.0001). This is expected since MASP-1 and MASP-2 are bound to the mannan via MBL. However, when MASP activities were normalised to MBL concentration (i.e. MASP-1 activity/[MBL], MASP-2 activity/[MBL]) MASP-1 activity was inversely correlated with MASP-2. This means on average that high MASP-1 correlates with low MASP-2 and vice-versa, and confirms the hypothesis that native MBL-MASP complexes on average do not have fixed MBL-(MASP-1)-(MASP-2) stoichiometry. The findings are consistent with separate populations of MBL-MASP-1 complexes and MBL-MASP-2 complexes, the concentrations of which show wide inter-individual variation.

Adult↗

PubMed2005

Immune evasion by a staphylococcal complement inhibitor that acts on C3 convertases.

The complement system is pivotal in host defense but also contributes to tissue injury in several diseases. The assembly of C3 convertases (C4b2a and C3bBb) is a prerequisite for complement activation. The convertases catalyze C3b deposition on activator surfaces. Here we describe the identification of staphylococcal complement inhibitor, an excreted 9.8-kilodalton protein that blocks human complement by specific interaction with C4b2a and C3bBb. Staphylococcal complement inhibitor bound and stabilized C3 convertases, interfering with additional C3b deposition through the classical, lectin and alternative complement pathways. This led to a substantial decrease in phagocytosis and killing of Staphylococcus aureus by human neutrophils. As a highly active and small soluble protein that acts exclusively on surfaces, staphylococcal complement inhibitor may represent a promising anti-inflammatory molecule.

Bacterial Proteins↗

PubMed2005

A true autoactivating enzyme. Structural insight into mannose-binding lectin-associated serine protease-2 activations.

Few reports have described in detail a true autoactivation process, where no extrinsic cleavage factors are required to initiate the autoactivation of a zymogen. Herein, we provide structural and mechanistic insight into the autoactivation of a multidomain serine protease: mannose-binding lectin-associated serine protease-2 (MASP-2), the first enzymatic component in the lectin pathway of complement activation. We characterized the proenzyme form of a MASP-2 catalytic fragment encompassing its C-terminal three domains and solved its crystal structure at 2.4 A resolution. Surprisingly, zymogen MASP-2 is capable of cleaving its natural substrate C4, with an efficiency about 10% that of active MASP-2. Comparison of the zymogen and active structures of MASP-2 reveals that, in addition to the activation domain, other loops of the serine protease domain undergo significant conformational changes. This additional flexibility could play a key role in the transition of zymogen MASP-2 into a proteolytically active form. Based on the three-dimensional structures of proenzyme and active MASP-2 catalytic fragments, we present model for the active zymogen MASP-2 complex and propose a mechanism for the autoactivation process.

Binding Sites↗

PubMed2005

Human serum IgM glycosylation: identification of glycoforms that can bind to mannan-binding lectin.

The glycoprotein IgM is the major antibody produced in the primary immune response to antigens, circulating in the serum both as a pentamer and a hexamer. Pentameric IgM has a single J chain, which is absent in the hexamer. The mu (heavy) chain of IgM has five N-linked glycosylation sites. Asn-171, Asn-332, and Asn-395 are occupied by complex glycans, whereas Asn-402 and Asn-563 are occupied by oligomannose glycans. The glycosylation of human polyclonal IgM from serum has been analyzed. IgM was found to contain 23.4% oligomannose glycans GlcNAc2Man5-9, consistent with 100% occupancy of Asn-402 and 17% occupancy of the variably occupied site at Asn-563. Mannan-binding lectin (MBL) is a member of the collectin family of proteins, which bind to oligomannose and GlcNAc-terminating structures. A commercial affinity chromatography resin containing immobilized MBL has been reported to be useful for partial purification of mouse and also human IgM. Human IgM glycoforms that bind to immobilized MBL were isolated; these accounted for only 20% of total serum IgM. Compared with total serum IgM, the MBL-binding glycoforms contained 97% more GlcNAc-terminating structures and 8% more oligomannose structures. A glycosylated model of pentameric IgM was constructed, and from this model, it became evident that IgM has two distinct faces, only one of which can bind to antigen, as the J chain projects from the non-antigen-binding face. Antigen-bound IgM does not bind to MBL, as the target glycans appear to become inaccessible once IgM has bound antigen. Antigen-bound IgM pentamers therefore do not activate complement via the lectin pathway, but MBL might have a role in the clearance of aggregated IgM.

Acetylglucosamine↗