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R B Sim

Publications and source records attributed to R B Sim.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1993

A calreticulin-like protein co-purifies with a '60 kD' component of Ro/SSA, but is not recognized by antibodies in Sjögren's syndrome sera.

In this study, we used human tonsils for the isolation of the 60 kD component of the Ro/SSA autoantigen, following the method described by Wu et al. (J Immunol Methods 1989; 121:219-24). Western blot analyses were carried out using Ro/SSA-reactive human Sjögren's syndrome sera, to follow the autoantigen through the purification procedure. A 60 kD Ro/SSA component was eluted as a broad peak from a Mono Q column. Within this peak, a much more abundant protein, co-migrating with the Ro/SSA component on SDS-PAGE, was also eluted. The more abundant protein was further purified on a Superose 12 column and its N-terminal sequence was shown to be identical to that of human calreticulin. The 60 kD Ro/SSA autoantigen was also further purified on the Superose 12 column and was eluted as an asymmetric peak, with the majority being eluted at a position corresponding to 60 kD, whereas the calreticulin-like protein was eluted from the same column as an apparent dimer of approximately 120 kD. A panel of five Ro/SSA-reactive human sera reacted with the purified Ro/SSA antigen, but not with the calreticulin-like protein. Therefore, it is clear that the calreticulin-like protein is not a Ro autoantigen and is distinct from the 60 kD Ro/SSA antigen. As the calreticulin-like protein is a much more abundant protein than the 60 kD Ro/SSA component, its co-purification with the autoantigen on ion-exchange and its close migration with the autoantigen on SDS-PAGE may explain why peptide sequences for human calreticulin were derived from apparent 60 kD Ro/SSA antigen preparations.

Amino Acid Sequence↗
PubMed1993

Structure and homology of human C1q receptor (collectin receptor).

In this paper we report partial amino acid sequence for C1q receptor (C1qR). The N-terminal amino acid sequence of isolated C1qR and the sequences of peptides obtained by V8/trypsin digestion show a high degree of similarity to the cDNA-derived amino acid sequence of a human protein which was initially reported as a component of RoSSA and subsequently as calreticulin. This sequence in turn shows homology with Onchocerca volvulus antigen (RAL-1) and B50 murine melanoma antigen. A component of approximately 53,000 MW, isolated from human spleen, was found to have identical mobility on SDS-PAGE to C1qR and identical N-terminal sequence, but a different overall charge. Human antibodies from Sjögren's syndrome patients did not recognize C1qR, but showed positive reaction with the purified 53,000 MW component from spleen. Rabbit antibodies against denatured C1qR, in contrast, recognized both C1qR and the purified 53,000 MW component. The 53,000 MW spleen component thus has an identical N-terminal sequence to calreticulin, and to the reported RoSSA component, and is recognized by antibodies in Sjögren's syndrome sera. The data obtained indicate that C1qR and the reported calreticulin/RoSSA component are similar but not identical molecules, which belong to the same protein superfamily.

Agglutination↗
PubMed1992

Activity, disulphide mapping and structural modelling of the fifth domain of human beta 2-glycoprotein I.

Complexes formed by the interaction of negatively charged phospholipids and beta 2-glycoprotein I (beta 2-I) are the target of autoantibodies in systemic lupus erythematosus. The highly positively charged fifth (C-terminal) domain of human beta 2-I was produced as a fusion protein in an Escherichia coli expression system and was shown to bind to the negatively charged phospholipid, cardiolipin, almost as well as the intact protein. In an attempt to define the 3D structure of this domain, the disulphide linkage pattern was determined and shown to be Cys 1-4, Cys 2-5 and Cys 3-6 in contradiction to an earlier report. In the light of this information, the sequence of the fifth domain of beta 2 I (beta 2-I-5) is readily aligned with that of the 16th repeat of factor H, of which the 3D structure is known, and a model of beta 2I-5 has been built by homology. On the basis of the model we suggest residues that might be the target of profitable site-directed mutagenesis in structure-function studies.

Amino Acid Sequence↗
PubMed1992

Human interleukin-1 receptor antagonist is expressed in liver.

Using PCR and Northern blot analysis, an IL-1 receptor antagonist specific transcript was amplified from HepG2- and liver mRNA. cDNA clones coding for IL-1 receptor antagonist were isolated from a liver cDNA library and sequence comparison revealed complete identity with the secreted, monocytic form of IL-1 receptor antagonist.

Blotting, Northern↗
PubMed1992

Human interleukin-1 receptor antagonist. High yield expression in E. coli and examination of cysteine residues.

The human IL-1 receptor antagonist (IL-1ra) was produced in a high yield E. coli expression system, and was purified in a rapid two-step purification. This recombinant IL-1ra molecule possessed full binding activity to the IL-1 receptor (type I) and totally inhibited IL-1-induced PGE2 production by human dermal fibroblasts. Radioalkylation and analysis of V8-derived IL-1ra peptides indicate that the four cysteines present in the IL-1ra are not disulphide-linked.

Amino Acid Sequence↗
PubMed1992

Solution structure of the fifth repeat of factor H: a second example of the complement control protein module.

Modules which share the same consensus sequence are assumed to have common structural features, at the secondary and tertiary level. In order to test the extent of such similarities, it is necessary to examine the structures of several examples from each module family. Recently, the first three-dimensional structure of a complement control protein (CCP) module (the 16th repeat of human factor H, H16) was determined using a combination of two-dimensional NMR and simulated annealing [Norman, D.G., Barlow, P.N., Baron, M., Day, A.J., Sim, R.B., & Campbell, I.D. (1991) J. Mol. Biol. 219, 717-725]. Using the same techniques, the three-dimensional structure of a second CCP module (the 5th repeat of human factor H, H5) has now been determined. The primary sequence of H5 contains 17 residues which are identical and in equivalent position to those in H16. Thirteen of these 17 are part of the consensus sequence. The similarities between the secondary structure of H5 and that of H16 are extensive. This implies that the consensus sequence dictates a particular secondary structure. The tertiary structure of H5, a compact hydrophobic core wrapped in beta-strand and sheet, bears much overall resemblance to that of H16. However, there is a deletion in the first strand of H5, and an insertion in a loop, resulting in slightly shorter overall length. This is associated with a rearrangement of residues within the hydrophobic core. The side chain of the highly conserved Tyr29, which occupies a central position within the core of H16, lies on the periphery of the core of H5.

Amino Acid Sequence↗
PubMed1992

In vitro biosynthesis of complement factor I by human endothelial cells.

We have studied the secretion of the complement regulatory protein factor I by human umbilical vein endothelial cells (HUVEC). Northern and Western blot analysis and biosynthetic labeling experiments indicate that HUVEC secrete factor I at very low levels in basal conditions and that this secretion is significantly enhanced by interferon-gamma. Analysis of the proteolytic inactivation of C3b by HUVEC supernatants show that factor I is secreted in a functional form and can promote the specific proteolytic inactivation of C3b to iC3b. Together with previous studies establishing the secretion of complement factor H by HUVEC, this work demonstrates that the endothelial cell is able to secrete in its environment two complement regulatory proteins, factor I and factor H, which can mediate the degradation of C3b to iC3b. The secretion of factor I by HUVEC provides a useful in vitro model to analyze the modulation of this secretion and may be relevant to the local deposition of iC3b at the surface of the endothelium during the inflammatory reaction.

Cells, Cultured↗
PubMed1992

Interaction of C1q receptor with lung surfactant protein A.

Earlier we reported the purification of C1q receptor (C1qR) from U937 cells and human tonsil lymphocytes (Malhotra, R. and Sim, R. B., Biochem. J. 1989. 218: 625) and showed that C1qR interacts with the ligands C1q, mannose-binding protein, conglutinin and lung surfactant protein A (SP-A) (Malhotra, R., Thiel, S., Reid, K. B. M. and Sim, R. B., J. Exp. Med. 1990. 172: 955). C1qR was characterized as an acidic glycoprotein, which, when solubilized, exists as a dimer of Mr 115,000 under non-denaturing conditions. In this article we provide evidence for binding of radioiodinated SP-A to U937 cells and show that binding of radioiodinated SP-A to U937 cells is specific, saturable, salt dependent and is inhibited by purified C1qR and by C1q. The interaction of SP-A with U937 cells was found to up-regulate the surface expression of C1qR. Incubation of SP-A with U937 cells at 37 degrees C for 80 min was found to increase the receptor number per cell. Increase in receptor number was inhibited in the presence of sodium azide and monensin. Incubation of cells with calcium ionophore A23187 induced increased surface expression in the absence of SP-A. The results indicate that interaction of SP-A with U937 cells triggers the expression of an intracellular pool of C1qR.

Amino Acid Sequence↗
PubMed1992

The human IL-1 receptor antagonist gene (IL1RN) maps to chromosome 2q14-q21, in the region of the IL-1 alpha and IL-1 beta loci.

The IL-1 receptor antagonist (IL-1RN) is a protein that binds to IL-1 receptors and inhibits the binding of IL-1 alpha and IL-1 beta. As a consequence, the biological activity of these two cytokines is neutralized in physiological and pathophysiological immune and inflammatory responses. In this study, using a panel of somatic rodent-human cell hybrids, we show that the gene for the human interleukin-1 receptor antagonist (IL1RN) maps to the long arm of chromosome 2. Previously, we described a length variation polymorphism within the second intron of the IL-1RN gene (Steinkasserer et al., 1991, Nucleic Acids Res. 19: 5095). Segregation of this, together with an IL-1 alpha polymorphism, was followed in a panel of five CEPH families. Linkage analysis permitted the mapping of the IL-1RN gene to band q14-q21 in the region for the IL-1 alpha and IL-1 beta loci. This study supports the view that an early gene duplication event resulted in the creation of an interleukin-1 gene family.

Chromosome Mapping↗
PubMed1992

Assignment of apolipoprotein H (APOH: beta-2-glycoprotein I) to human chromosome 17q23----qter; determination of the major expression site.

Human apolipoprotein H (APOH) is associated with lipoprotein present in plasma. It has been shown that APOH has structural similarities with the regulation of complement activation (RCA) protein superfamily and is involved in phospholipid binding interactions on platelets and as an autoantigen in complex with anionic phospholipids. Nevertheless, additional functional studies are necessary to establish the physiological role of APOH. By hybridizing a cDNA probe for APOH to a panel of somatic cell hybrids, we show that the structural locus for this protein maps to 17q23----qter and is therefore not part of the RCA cluster on chromosome 1. The site of biosynthesis for APOH was established by Northern blot analysis. Hybridization of the APOH cDNA probe to total liver RNA identified a transcript of approximately 1.5 kb.

Apolipoproteins↗
PubMed1991

Biochemical studies on red blood cells from a patient with the Inab phenotype (decay-accelerating factor deficiency).

A 38-year-old Russian woman (KZ) has been identified as the fourth proposita with the Inab blood group phenotype. Like the first two propositi, she has a chronic intestinal disorder and, as shown for the third proposita, her Inab phenotype is demonstrably inherited. KZ's serum contained anti-IFC, which reacted with a red blood cell (RBC) membrane component with an Mr of 70,000, which is decay accelerating factor (DAF). Her RBCs lacked all Cromer-related blood group antigens and DAF. Her RBCs were no more susceptible than normal control RBCs to lysis in acid lysis or in rabbit or human antibody-initiated complement lysis tests. Northern blots of total RNA isolated from KZ's Epstein-Barr virus-transformed lymphoblasts showed a marked reduction of DAF mRNA when compared with normal. Polymerase chain reaction (PCR) amplification of cDNA confirmed this reduced level of DAF mRNA. Sequencing of the PCR product showed a 44-nucleotide deletion in the mRNA close to the short consensus repeats IIIa/IIIb intron/exon boundary. This deletion results in a change in the reading frame that places a termination codon six amino acids after the deletion. The putative translation product would lack a glycosyl phosphatidyl-inositol linkage site and, therefore, would not be membrane-bound in the RBC.

Adult↗
PubMed1991

Complete nucleotide and deduced amino acid sequence of human beta 2-glycoprotein I.

The nucleotide and complete amino acid sequence for the human beta 2-glycoprotein I (beta 2I) was derived by sequencing the cDNA clone pB2I-1. In addition to the 326 amino acid residues of the mature protein this clone codes for a putative leader peptide and contains sequence representing 5' and 3' untranslated regions. When this amino acid sequence was compared with the previously published primary sequence, three major amino acid substitutions were found, two involving cysteine residues. These substitutions lead to a new alignment of the complement control protein (CCP) repeats present in beta 2I and a prediction of the complete disulphide bond organization. Northern-blot analysis indicates that hepatocytes are a major site of biosynthesis for this protein. A transcription signal of about 1.5 kb was detected by using RNA from HepG2 cells.

Amino Acid Sequence↗
PubMed1991

Three-dimensional structure of a complement control protein module in solution.

The complement control protein (CCP) modules (also known as short consensus repeats) are defined by a consensus sequence within a stretch of about 60 amino acid residues. These modules have been identified more than 140 times in over 20 proteins, including 12 proteins of the complement system. The solution structure of the 16th CCP module from human complement factor H has been determined by a combination of 2-dimensional nuclear magnetic resonance spectroscopy and restrained simulated annealing. In all, 548 structurally important nuclear Overhauser enhancement cross-peaks were quantified as distance restraints and, together with 41 experimentally measured angle restraints, were incorporated into a simulated annealing protocol to determine a family of closely related structures that satisfied the experimental observations. The CCP structure is shown to be based on a beta-sandwich arrangement; one face made up of three beta-strands hydrogen-bonded to form a triple-stranded region at its centre and the other face formed from two separate beta-strands. Both faces of the molecule contribute highly conserved hydrophobic side-chains to a compact core. The regions between the beta-strands are composed of both well-defined turns and less well-defined loops. Analysis of CCP sequence alignments, in light of the determined structure, reveals a high degree of conservation amongst residues of obvious structural importance, while almost all insertions, deletions or replacements observed in the known sequences are found in the less well-defined loop regions. On the basis of these observations it is postulated that models of other CCP modules that are based on the structure presented here will be accurate. Certain families of CCP modules differ from the consensus in that they contain extra cysteine residues. As a test of structural consensus, the extra disulphide bridges are shown to be easily accommodated within the determined CCP model.

Amino Acid Sequence↗