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R B Sim

Publications and source records attributed to R B Sim.

At least 73 records · Page 4Linked to original sources
PubMed1994

Collectins: collagenous C-type lectins of the innate immune defense system.

Collectins are humoral lectins found in mammals and birds. They are oligomers whose subunits comprise three polypeptide chains each containing a collagenous section and a C-terminal lectin domain. They are related structurally and functionally to the first component of the classical complement pathway, C1q, and seem to serve important roles in innate immunity through opsonization and complement activation. The lectin domains bind carbohydrates on microorganisms, while the collagenous regions are ligands for the collectin receptor on phagocytes and also mediate C1q-independent activation of the classical complement pathway.

Animals↗
PubMed1994

Collectins, collectin receptors and the lectin pathway of complement activation.

The collectins are a group of soluble multimeric lectins, which contain collagenous segments, and resemble the complement protein C1q in aspects of their structures and functions. This group of proteins, which includes MBP, SP-A, SP-D, conglutinin and CL-43, are known to act as opsonins in various circumstances, and are likely to have roles in innate immunity. The focus of current research is to pursue the hypothesis that the collectins recognize and bind to non-host carbohydrate structures on microorganisms and particles, and participate in the processing or elimination of such material, either by direct interaction with phagocytic cell receptors, or by indirect routes such as complement activation .

Animals↗
PubMed1994

Bovine conglutinin binds to an oligosaccharide determinant presented by iC3b, but not by C3, C3b or C3c.

Bovine conglutinin is a serum lectin that agglutinates erythrocytes preincubated with antibodies and complement. This agglutination occurs through the binding of conglutinin to iC3b, a fragment of the complement component C3. It was reported that conglutinin binds fluid-phase C3b and C3c as well as iC3b. We re-investigated the reactivity of conglutinin towards fluid-phase C3 degradation products. ELISA wells were coated with conglutinin and reacted with C3 split products generated in normal human serum, in factor I-deficient serum, or in factor I-depleted serum. Conglutinin-bound C3 fragments were detected with anti-C3c and anti-C3d antibodies. An increased signal was observed during the activation of complement in normal human serum with the peak response after 1-2 hr, following which the signal decreased, reaching background level after 72 hr. The oligosaccharides on C3c, generated in serum, are thus not recognized by conglutinin. No signal was observed when factor I-deficient serum or factor I-depleted serum was used instead of normal serum. Reconstitution with purified factor I re-established the normal pattern. Examination of the conglutinin-bound C3 molecules by SDS-PAGE and Western blotting with anti-C3c and anti-C3d antibodies revealed bands characteristic for iC3b, and no bands corresponding to C3b or C3c. Reduction of the disulphide bonds prior to the incubation of the activated serum with the conglutinin-coated wells revealed a band of 63,000 MW, characteristic of the N-terminal fragment of the alpha-chain of iC3b. We also investigated the binding to the solid-phase conglutinin of purified C3 and degradation products generated with enzymes. In this case, C3 as well as C3b and C3c were bound, suggesting conformational changes in C3 during purification. In conclusion, when C3 conversion takes place at near physiological conditions, conglutinin interacts specifically with the oligosaccharide on the alpha-chain of iC3b.

Animals↗
PubMed1994

Mannan-binding protein levels in human amniotic fluid during gestation and its interaction with collectin receptor from amnion cells.

Collectins are a group of soluble proteins, each of which has collagenous and non-collagenous globular domains, and may be complement associated and/or have lectin activity. The complement protein C1q is structurally and functionally related to the collectins. Members of the collectin family are mannan-binding protein (MBP), lung surfactant protein A, CL-43, conglutinin and lung surfactant protein D. All except the last of these proteins have been shown to interact with a single, widely distributed receptor, named collectin receptor (also called C1q receptor). It has been proposed that collectins play an important role as opsonins in innate immunity. MBP is a serum protein and has been shown to activate the classical complement pathway. MBP has also been shown to opsonize bacteria and enhance clearance of bacteria by phagocytosis. In this article we demonstrate the presence of MBP in amniotic fluid and the localization of collectin receptor on the surface of amniotic epithelium. MBP levels in amniotic fluid were found to increase sharply from about 32 weeks of gestation. Collectin receptor was purified from a cell line, FL, derived from amniotic epithelium and was shown to interact with MBP. These results suggest that MBP may play a role in the antibody-independent recognition and clearance of pathogens in the amniotic cavity, towards term.

Amino Acid Sequence↗
PubMed1993

Modelling of the serine-proteinase fold by X-ray and neutron scattering and sedimentation analyses: occurrence of the fold in factor D of the complement system.

Solution scattering is a powerful means of determining the overall arrangement of domains in the multidomain proteins of complement. the serine-proteinase domain is central to all proteolytic events during complement activation. As models of this domain, bovine beta-trypsin, trypsinogen, alpha-chymotrypsin and chymotrypsinogen A were studied by neutron and X-ray synchrotron solution scattering. At pH 7, all the X-ray and neutron M(r) values corresponded to monomeric proteins. The X-ray radii of gyration, RG, of beta-trypsin, trypsinogen, alpha-chymotrypsin and chymotrypsinogen A (measured in positive solute-solvent contrasts) were 1.59 nm, 1.78 nm, 1.71 nm and 1.76 nm (+/- 0.05-0.11 nm) in that order. Neutron contrast variation showed that the RG at infinite contrast, RC, for these four proteins were 1.57 nm, 1.70 nm, 1.67 nm and 1.78 nm (+/- 0.03 nm) in that same order. The radial inhomogeneity of neutron-scattering density, alpha, was positive at (5-13) x 10(-5), and corresponds to the preponderance of hydrophilic residues near the protein surface. On trypsinogen activation, a small reduction in the RG value of 0.13 +/- 0.07 nm was just detectable, while the RG of chymotrypsinogen A was unchanged after activation. The RC and alpha values of the four proteins can be calculated by using crystallographic co-ordinates. The reduced RG of beta-trypsin relative to trypsinogen was explained in terms of the removal of the extended N-terminal hexapeptide of trypsinogen. The full X-ray and neutron-scattering curves in positive and negative contrasts agreed well with scattering curves calculated from crystallographic coordinates to a nominal structural resolution of 4.5 nm, provided that the internal structure was considered in neutron modelling, and that the hydration was considered in X-ray modelling. Sedimentation-coefficient data also provide information on the disposition of domains in multidomain proteins. It was found that the hydrated X-ray sphere model could be directly utilized to calculate sedimentation coefficients. X-ray scattering on factor D showed from its RG of 1.78 nm that this is monomeric and very similar in structure to beta-trypsin. The X-ray-scattering curve of factor D was readily modelled using the beta-trypsin crystal structure after allowance for sequence changes. The success of these modellings provides a basis for the constrained modelling of solution scattering data for the multidomain proteins of complement.

Chymotrypsin↗
PubMed1993

Molecular modelling of the domain structure of factor I of human complement by X-ray and neutron solution scattering.

Factor I is a typical multidomain protein of the complement system. It regulates complement activation by proteolytic degradation of C3b or C4b in the presence of factor H, complement receptor type 1, membrane cofactor protein or C4b-binding protein as cofactor. It is constructed from five presumed independently folded domains, namely a factor I module, a CD5-like domain, two low-density-lipoprotein receptor type A domains and a serine-proteinase domain. X-ray and neutron solution scattering was used to study the arrangement of these domains in factor I. Factor I was determined to be monomeric in solution, with an A280(1%,1cm) of 12.3-14.1. Its radius of gyration (RG) was 3.96 nm by X-rays in a high positive solute-solvent contrast, and 3.84 nm by neutrons at infinite solute-solvent contrast. The cross-sectional radius of gyration (RXS) was likewise found to be 1.64 nm by X-rays and 1.55 nm by neutrons. The RG data were not noticeably dependent on the solute-solvent contrast, whereas the RXS data showed a small dependence. The maximum dimension of factor I was determined to be 12.8 nm from the RG and RXS data, and 14-15 nm from the X-ray and neutron distance distribution functions. This length is too short to account for a linear arrangement of the domains in factor I. Small sphere models were developed for factor I in which the largest domain was modelled from the crystal structure for beta-trypsin. The attachment of either an elliptical cylinder or a two-armed V-shaped structure to this domain to represent the remaining four small domains gave good scattering curve-fits for factor I, and were compatible with experimental sedimentation coefficients. The non-extended domain models for factor I imply that the steric accessibility of each domain will be reduced, and this may be important for its functional activity.

Carrier Proteins↗
PubMed1993

Solution structure of a pair of complement modules by nuclear magnetic resonance.

A portion of human complement factor H spanning the 15th (H15) and 16th (H16) of its 20 modules, has been expressed in a yeast vector and subjected to structure determination in solution using two-dimensional 1H-NMR. The structure of H15 is very similar to that already established for the fifth module of factor H and H16, consistent with the view that all such complement control (C-) modules share a common overall topology. In addition, the tertiary structures of the component modules of the H15-16 pair are very similar to those of the modules when expressed individually, implying that each folds entirely autonomously within intact factor H. Aromatic residues in the third turn of H15 and the second turn of H16, together with a leucine residue from the linker region, contribute to a small intermodular interface. Comparatively few nuclear Overhauser effects were observable between protons on different modules. Consequently, a wide range of angles of "twist" (131 (+/- 146) degrees, mean value (+/- 1 standard deviation)), i.e. rotation about the long axis of one module with respect to the other, exists in the family of structures generated on the basis of the experimental data. However, much smaller variations occur in the two, orthogonal, angles (175 (+/- 12) degrees and 103 (+/- 6) degrees) that describe the "tilt". These observations may suggest upper limits on the relative flexibility of the two modules. Models were built to assess the outcome of applying such restrictions to all the neighbours within a string of 20 C-modules, and the resulting structures compare well with factor H as visualized by electron microscopy.

Amino Acid Sequence↗
PubMed1993

Localization of the receptor-binding site in the collectin family of proteins.

Collectin receptor (Clq receptor) has been shown to bind human Clq, mannose-binding protein (MBP), lung surfactant protein A (SP-A) and bovine conglutinin. These ligands have a similar ultrastructure, each consisting of collagenous and globular domains, but do not show a high degree of sequence similarity. For Clq and SP-A, it has been shown that both bind to cell-surface-expressed receptor(s) via their collagenous regions and this is likely to be the case with the other ligands. Within the collagenous region, near the 'bend' region of the collagen triple helix in Clq, MBP and SP-A, a cluster of similar charged residues is observed. This region has been suggested to be associated with receptor binding. A similar region of charge density occurs close to the N-terminus of conglutinin. In this paper we describe a truncated form of conglutinin, which has 55 amino acids missing from the N-terminus and does not bind to the collectin receptor. The results presented here strongly indicate that receptor-ligand interaction is mediated via the N-terminal region of conglutinin, consistent with the earlier proposal for the binding site.

Amino Acid Sequence↗
PubMed1993

Dye-ligand affinity purification of human complement factor B and beta 2 glycoprotein I.

The rapid purification of human factor B using dye-ligand chromatography is described. The 50% ammonium sulphate supernatant of fresh human serum is equilibrated in pH 7.4, 25 mM Tris buffer containing 0.5 mM CaCl2 and 0.5 mM MgCl2, with 25 mM sodium caprylate and chromatographed on Cibacron Blue F3GA-agarose. Caprylic acid binds to the fatty acid binding site of albumin, preventing it from binding to the resin which thus retains a high capacity for binding factor B. Factor B together with the homologous protein beta 2I are eluted from the column by a linear gradient of KCl. Subsequent NaCl gradient FPLC on Hiload S-Sepharose, equilibrated in 10 mM potassium phosphate, 5 mM EDTA, pH 7.0, provides both factor B and beta 2I in homogeneous form.

Chromatography, Affinity↗
PubMed1993

Isolation of a human endothelial cell C1q receptor (C1qR).

C1q binding to endothelial cells has been described previously, but the putative cell surface receptor(s) has not been identified. In the present study, modifications of a reported purification of lymphocyte C1q receptor (C1qR) were used to isolate C1q binding sites from human umbilical vein endothelial cells. Cells were harvested, without protease treatment, at passage 10-17 and lysed with 1% Triton X-100. The lysate was fractionated on Fast-performance liquid chromatography (FPLC) Mono-Q using a linear NaCl gradient, followed by high-performance liquid chromatography (HPLC) ion exchange (TSKgel DEAE-NPR). A major protein was eluted that had the same mobility on sodium dodecyl sulfate-polyacrylamide gels and the same NH2-terminal sequence as lymphocyte C1qR. This protein was expressed on the surface, as judged by surface radioiodination, bound to C1q-coated surfaces, and was recognized by polyclonal antilymphocyte C1qR antibodies. Thus, endothelial cells express a C1q receptor that appears identical to lymphocyte C1qR. The data further support the hypothesis that cell surface C1qRs identified on a variety of somatic and cultured cells are either identical or constitute a family of closely related molecules.

Amino Acid Sequence↗
PubMed1993

Human beta 2-glycoprotein I: molecular analysis of DNA and amino acid polymorphism.

In this study, we describe a two-allelic RsaI restriction fragment length polymorphism identified by Southern blot analysis and by allele-specific polymerase chain reaction amplification for the human beta 2-glycoprotein I (beta 2-I; apolipoprotein H = APOH) gene. This polymorphism, which segregates in a co-dominant fashion, leads to a valine-leucine amino acid exchange at amino acid position 247. The allele frequency has been established in 34 unrelated parents of the Centre d'Etude du Polymorphisme Humain family panel and was found to be 0.76 for valine and 0.23 for leucine. The Val-Leu polymorphism described in this study does not correlate with the four isoelectric focusing alleles previously described, indicating that other variants are responsible for this polymorphism.

Alleles↗
PubMed1993

Pollen grains bind to lung alveolar type II cells (A549) via lung surfactant protein A (SP-A).

Lung surfactant protein A (SP-A) is the most abundant surfactant-associated protein present in the lung. A receptor for SP-A has been shown to be present on A549 alveolar type II cells and on other cell types, including alveolar macrophage. The SP-A receptor on A549 cells has been identified as the collection receptor, or C1q receptor, which binds several structurally-related ligands. SP-A contains C-type lectin domains, but the role of carbohydrate binding by SP-A in physiological and pathological phenomena is not yet established. In this paper we report the binding of SP-A to pollen from Populus nigra italica (Lombardy Poplar), Poa pratensis (Kentucky blue grass), Secale cerale (cultivated rye) and Ambrosia elatior (short ragweed). Saturable and concentration dependent binding of SP-A to pollen grains was observed. Interaction of SP-A with pollen grains takes place through water-extractable components, in which the major species present, in Lombardy polar pollen, are 57 kD and 7 kD (glyco)proteins. The binding of SP-A to pollen grains and their aqueous extracts was calcium ion dependent and was inhibited by mannose, and is therefore mediated by the lectin domain. Binding of SP-A to pollen grains was found to mediate adhesion of pollen grains to A549 cells. The results suggest that pollen grains or other carbohydrate-bearing particles (e.g. microorganisms) could potentially interact with different cell types via the collection receptor (C1q Receptor) in the presence of SP-A.

Calcium↗
PubMed1993

Chromosomal mapping of the human interleukin-1 receptor antagonist gene (IL-1RN) and isolation of specific YAC clones.

Using a panel of somatic rodent-human cell hybrids, we show that the interleukin-1 receptor antagonist gene (IL-1RN) maps to the long arm of human chromosome 2. Linkage studies permitted the regional localization of this gene to band q14-21. This is the same region in which the IL-1 alpha and IL-1 beta genes are localized. Three yeast artificial chromosome (YAC) clones containing the IL-1RN gene were isolated, and these will be used for further characterization of this chromosome 2 region.

Animals↗
PubMed1993

Characterization of Xenopus laevis complement factor I structure--conservation of modular structure except for an unusual insert not present in human factor I.

Factor I (C3b/C4b inactivator) is a regulatory protein of the classical and alternative complement pathways. In this paper, we report the sequence of Xenopus factor I cDNA and the deduced protein structure. The basic structure of human preprofactor I, NH2-heavy chain-cleavage peptide-light chain-COOH, is conserved in the frog. However, the frog heavy chain contains a highly charged segment of 29 amino acids, encoded by a poly dA-rich mRNA insert, which is not found in human factor I. The modular structure of the frog heavy chain was analyzed, and found to differ vis-à-vis previously published analyses of human factor I. We also evaluate the timing of factor I transcription during frog embryogenesis.

Amino Acid Sequence↗
PubMed1993

Interactions in the complement-mediated lysis of blood group AB erythrocytes sensitized simultaneously with anti-A and anti-B monoclonal antibodies.

The lysis of group AB erythrocytes by human complement was studied by different anti-A and anti-B IgM monoclonal antibodies (mabs) in a 51Cr-release assay. The concentration of membrane-bound immunoglobulin was detected by ELISA, and the amount of C1q and C3 bound to sensitized red cells was measured by using purified, 125I-labelled molecules. We have demonstrated that there is an exponential relationship between the concentration of the sensitizing IgM mabs and C1q binding to the sensitized AB cell. The efficiency of binding was related to the number of antibodies bound; thus, anti-A sensitized cells bound 3-6 times more C1q than anti-B sensitized cells did. AB cells, on the other hand, bound similar amounts of C3 whether anti-A or anti-B was present. The lytic efficiencies of the various IgM mabs during short incubation times were different, suggesting that the complement activation rates vary widely with different antibodies on the AB cell membrane. The binding of C1q to an antibody-sensitized target activates a cascade, whose components may migrate away from the sensitizing antibody; interactions between the activation processes generated by the anti-A and anti-B antibodies may thus occur. Choosing appropriate pairs of anti-A and anti-B mabs for the simultaneous sensitization of AB cells has indeed resulted in stimulation in some and inhibition in other combinations of mabs. It is suggested that stimulation is observed when the activated intermediates are produced in excess, whereas inhibition occurs when a shortage of activated intermediates prevents mutual utilization.

ABO Blood-Group System↗