PubMed Health⌕ Search

Biomedical subjects

R B Sim

Publications and source records attributed to R B Sim.

At least 109 records · Page 6Linked to original sources
PubMed1988

Sequence polymorphism of human complement factor H.

Factor H is a major regulatory protein of the complement system. The complete cDNA coding sequence has been derived from overlapping clones, and a polymorphism at base 1277 has been characterized. In four clones there is a T at nucleotide 1277 and in two others there is a C. This T/C change represents a tyrosine/histidine polymorphism at position 384 in the derived amino acid sequence. Protein sequence studies on peptides generated by trypsin digestion of factor H, purified from pooled plasma from 12 donors, confirmed the presence of both tyrosine and histidine at this position. Tyrosine and histidine were observed in a ratio of 2:1, respectively, and therefore this polymorphism is likely to represent a sequence difference between the two most abundant charge variants, FH1 and FH2, of factor H.

Amino Acid Sequence↗
PubMed1988

Appearance of acceptor-bound C3b on HLA-DR positive macrophages and on stimulated U937 cells; inhibition of Fc gamma-receptors by the covalently fixed C3 fragments.

The appearance and the functional role of acceptor-bound C3b during differentiation of human monocytes into macrophages were studied. Acceptor-bound C3b could be detected by the immune adherence (IA) test parallel to the expression of antigenic determinants specific to mature cells--i.e. on days 4-5 of culture. Consequently, the capacity of these phagocytes to fix C3b covalently via C3b-acceptors (C3bAs) can be considered as one of the signs of their activation/differentiation. All the mature macrophages positive in the IA test were also found to express HLA-DR antigens on their membrane. Using solubilized extracts of stimulated, 35S-cysteine-labelled cells of the human monocytic cell line, U937, we demonstrate that C3 synthesized by these cells can bind to C3bAs of the same cells. Covalently fixed C3 fragments were found to inhibit Fc gamma-receptor-mediated ingestion of immune complexes and also antibody-dependent cellular cytotoxicity of monocyte-derived macrophages.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1987

Complement factor H-binding protein of Raji cells and tonsil B lymphocytes.

Several reports have indicated that Factor H has specific effects on certain cell populations, suggesting that Factor H receptors may exist. Lambris & Ross [(1982) J. Exp. Med. 155, 1400-1411] purified a protein from Raji B-lymphoblastoid cell culture supernatants, using Factor H-Sepharose affinity chromatography. This species appeared to consist of two disulphide-linked components each of Mr 50,000, with an additional 50,000-Mr chain attached non-covalently. The existence of cell-surface Factor H-binding proteins has now been re-investigated with 125I surface-labelled Raji and tonsil B cells. Non-ionic-detergent extracts of the cells, in 0.1% Nonidet P40/10 mM-sodium phosphate buffer, pH 7.4, were incubated with Factor H-Sepharose in the presence of proteinase inhibitors. After the beads had been washed, bound components were eluted with 50 mM-NaCl. A single radioactive species was eluted from the resin, which migrates identically with Factor H (apparent Mr 170,000) in SDS/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions. Biosynthetic radiolabelling studies confirmed that this species was synthesized by Raji cells. Examination of culture supernatants from biosynthetically radiolabelled Raji cells showed again the presence of a single soluble species that bound to Factor H-Sepharose, but this species was of lower Mr (approx. 105,000) than the membrane-derived protein. The soluble form may be produced by proteolysis of the membrane form, or may be of separate origin. The similarity in size of the cell-surface protein to Factor H was initially confusing, but it is distinct from cell-surface Factor H on the basis of three criteria: (1) it is not recognized by anti-(Factor H) monoclonal antibodies MRC OX23 and MRC OX24, nor by polyclonal F(ab')2 anti-(Factor H); (2) it does not bind to Zn2+-chelate resin, whereas Factor H does; (3) cell-surface Factor H present on U937 cells does not bind to Factor H-Sepharose.

B-Lymphocytes↗
PubMed1987

The functions and relationships of Ty-VLP proteins in yeast reflect those of mammalian retroviral proteins.

We have identified the major structural core proteins of Ty virus-like particles (Ty-VLPs) and shown that they are generated by proteolytic cleavage of the primary translation product of TYA, p1. This precursor protein is therefore functionally similar to the gag precursor of retroviruses. Cleavage is mediated by a Ty-encoded protease located at the 5' region of TYB and is accompanied by a change in particle morphology. p1 contains sufficient information for the assembly of a pre-Ty-VLP complex, which does not require the presence of either Ty protease or reverse transcriptase. The results indicate that the requirements and pathway of Ty-VLP formation reflect the initial stages of mammalian retroviral assembly and further support the idea of a common origin for Ty elements and retroviruses.

Amino Acid Sequence↗
PubMed1987

Characterization of primary amino acid sequence of human complement control protein factor I from an analysis of cDNA clones.

A cDNA clone of the mRNA coding for the human complement system control protein Factor I has been isolated. The predicted amino acid sequence obtained from the DNA sequence demonstrates a protein consisting of a heavy chain (Mr 35,400) linked to a light chain (Mr 27,600), both of which contain three sites for N-linked glycosylation. The light chain has clear homology with other serine proteinases, most notably in the region of the catalytically active and structurally important amino acids and shares some of the features characteristic of the plasminogen activators. The heavy chain has a clear 'mosaic' nature typical of the plasma serine proteinases; in particular it contains class A and class B LDL (low-density lipoprotein) receptor repeats with conserved cysteine residues similar to those found in other complement proteins.

Amino Acid Sequence↗
PubMed1987

Sequence analysis of a cDNA clone encoding the C-terminal end of human complement factor H.

Peptide sequencing of the complement system regulatory protein, factor H, permitted the synthesis of a mixed sequence oligonucleotide probe. Human liver cDNA libraries were screened and factor H-specific clones selected. No full-length clone was obtained, but the largest available clone, R2a, was found to encode the C-terminal 657 amino acids of factor H. The derived amino acid sequence consists of 10 contiguous internally homologous segments, each about 60 amino acids long. Sequences homologous to these are found in several other complement and non-complement proteins. Such sequences are likely to represent a particular type of tertiary structure subunit.

Amino Acid Sequence↗
PubMed1987

Structure and specificity of complement receptors.

Fifteen to 16 cell surface proteins which interact with soluble components of the complement system have now been identified. Most of these--CR1, CR2, CR3, "CR4", DAF, HSV-1 c glycoprotein, Gp 45-70, p150,95, cell-surface Factor H, and a 90 kD protein--interact with C3 or C4 and their degradation products. Other receptors for C1q, Factor H, C5a, and the C5b-9 complex have been identified. Receptors for additional complement proteins such as Factor B or its fragments are likely to exist. Complement receptors have a wide tissue distribution and have major roles in controlling the turnover of the complement system, and regulating behaviour and growth of leukocytes.

Animals↗
PubMed1987

Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients.

Deposition of amyloid in pancreatic islets is a common feature in human type 2 diabetic subjects but because of its insolubility and low tissue concentrations, the structure of its monomer has not been determined. We describe a peptide, of calculated molecular mass 3905 Da, that was a major protein component of amyloid-rich pancreatic extracts of three type 2 diabetic patients. After collagenase treatment, an extract containing 20-50% amyloid was solubilized by sonication into 70% formic acid and the peptide was purified by gel filtration followed by reverse-phase high-performance liquid chromatography. We term this peptide diabetes-associated peptide, as it was not detected in extracts of pancreas from any of six normal subjects. Diabetes-associated peptide contains 37 amino acids and is 46% identical to the sequences of rat and human calcitonin gene-related peptide, indicating that these peptides are related in evolution. Sequence identities with conserved residues of the insulin A chain were also seen in a 16-residue segment. On extraction, the islet amyloid is particulate and insoluble like the core particles of Alzheimer disease. Their monomers have similar molecular masses, each having a hydropathic region that can probably form beta-pleated sheets. The accumulation of amyloid, including diabetes-associated peptide, in islets may impair islet function in type 2 diabetes mellitus.

Aged↗
PubMed1986

Molecular modelling of human complement component C3 and its fragments by solution scattering.

Solution scattering experiments using both X-rays and neutrons are reported for human complement component C3 and up to six other glycoprotein fragments that are derived from C3. The X-ray and neutron molecular masses and neutron matchpoints are in agreement with the known primary sequence of C3. The X-ray radius of gyration RG of C3 is 5.2 nm and is similar for the related forms C3u, C3(a + b) and C3b. The X-ray cross-sectional radius of gyration RXS of C3b is however less than that of C3, C3u and C3(a + b). The major fragments of C3b, namely C3c and C3dg, were studied. The RG of C3c is 4.7 nm and for C3dg is 2.9 nm. C3c and C3dg do not interact when they coexist in solution in equimolar amounts. When C3u is cleaved into iC3u, the RG of iC3u increases to 5.9 nm and its RXS decreases, showing that C3c and C3dg behave as independent entities within the parent glycoprotein. Analyses of the neutron RG and RXS values by contrast variation techniques confirm the X-ray analyses, and show no evidence for significant hydrophobic or hydrophilic domains within C3 or any of its fragments. Shape analyses show that C3, C3c and C3dg are elongated particles. Debye models were developed using the scattering curve out to Q = 1.6 nm-1. These show that C3 and C3c resemble oblate ellipsoids while C3dg resembles a prolate ellipsoid. C3dg lies on the long edge of C3c within C3. The dimensions of the models are 18 nm X 2 nm X 10 nm for C3, 18 nm X 2 nm X 7 nm for C3c and 10 nm X 2 nm X 3 nm for C3dg. These models are compatible with analyses of the scattering curve RG and RXS values, data from sedimentation coefficients, and images of C3 and C3c seen by electron microscopy.

Complement C3↗
PubMed1986

Loss of complement receptor type 1 (CR1) on ageing of erythrocytes. Studies of proteolytic release of the receptor.

Complement receptor type 1 (CR1) is a glycoprotein of Mr about 250 000 present on erythrocytes and other cell types. CR1 acts as a cofactor in the factor I-mediated breakdown of complement fragment C3b to form iC3b. Using an assay of cofactor activity, a wide variation in mean CR1 levels between erythrocytes from individual donors is observed. CR1 levels also decrease on ageing of erythrocytes in vivo, and again the rate of loss is widely variable between individuals. However, variable loss of CR1 during ageing of erythrocytes is likely to make only a minor contribution to the observed variation in mean CR1 levels. CR1 is very sensitive to proteolysis, and random proteolytic removal of CR1 from erythrocytes is likely to be an important factor in loss of CR1 on ageing of red cells in vivo. In vitro, mild trypsin treatment, plasmin or thrombin digestion of erythrocytes results in the loss of the factor I cofactor activity from the cell surface, and appearance of this activity in the supernatant. We conclude that an active fragment of CR1 is released from the cell surface on proteolysis. Subsequent prolonged trypsin treatment destroys most of the activity of this fragment. Proteolytic removal of CR1 from red cells may account not only for loss on ageing of cells, but also for the acquired CR1 deficiencies observed by others in systemic lupus erythematosus.

Erythrocyte Aging↗
PubMed1986

Ligand binding by the p150,95 antigen of U937 monocytic cells: properties in common with complement receptor type 3 (CR3).

U937 cells (a monocytic cell line) grown in the presence of phorbol myristate acetate were surface labeled with 125I and the iC3b-binding proteins isolated by affinity chromatography on iC3b-Sepharose in the presence of divalent cations. Three polypeptides of 170, 150 and 95 kDa were found to bind specifically to iC3b-Sepharose. The polypeptides of 170 and 95 kDa were identified as the alpha and the beta subunits of CR3 by immunoprecipitation with OKM1 monoclonal antibody. The 150-kDa polypeptide was not immunoprecipitated by antibodies to the alpha subunit of CR3 or LFA-1. However, the 150-kDa polypeptide, together with the 95-kDa polypeptide, was immunoprecipitated with an anti-beta subunit-specific antibody IB4, which immunoprecipitates LFA-1, CR3 and p150,95. These results indicated that the 150-kDa polypeptide is the alpha subunit of the p150,95 antigen. The binding of p150,95 and CR3 to iC3b-Sepharose is specific as neither binds to C3u-Sepharose. A monoclonal antibody, 3.9, which immunoprecipitated the 150 and a 95-kDa polypeptide from U937 cells was characterized as being directed against the alpha-subunit of the p150,95 antigen. Phorbol myristate acetate-stimulated U937 cells from rosettes with EAC3b and EAiC3b but not with EAC3d cells. Monoclonal antibody 3.9 does not inhibit either type of rosetting, but we were unable to exclude a role for p150,95 in adherence of iC3b-coated particles. Since there is no rosetting with C3d-bearing particles it is unlikely that p150,95 is a receptor for C3d, a role for p150,95 which has been suggested by others (Wright, S.D., Licht, M.R. and Silverstein, S.C., Fed. Proc. 1984. 43: 413).

Antibodies, Monoclonal↗
PubMed1986

Partial characterization of human complement factor H by protein and cDNA sequencing: homology with other complement and non-complement proteins.

Factor H, a control protein of the human complement system, is closely related in functional activity to two other complement control proteins, C4b-binding protein (C4bp) and complement receptor type 1 (CR1). C4bp is known to have an unusual primary structure consisting of eight homologous units each about 60 amino acids long. Such units also occur in the N-terminal regions of the complement proteins C2 and factor B, and in the non-complement serum glycoprotein beta 2I. Amino acid sequencing, and sequencing of a factor H cDNA clone, show that factor H also contains internal repeating units, and is homologous to the proteins listed above.

Amino Acid Sequence↗
PubMed1986

Release of immune complexes bound to erythrocyte complement receptor (CR1), with particular reference to the role of factor I.

The release of 125I-bovine serum albumin (BSA)-anti-BSA immune complexes (IC) bound to human erythrocyte complement receptors (E-CR1) was studied. IC were complement-solubilized in normal human serum (NHS), and reacted with human erythrocytes at conditions optimal for binding of the IC to E-CR1. E-CR1-bound IC could be released by the addition of NHS or purified factor I. Factor I-deficient or I-depleted serum mediated no release, and addition of purified factor I restored the release. Factor H was not required for the release of IC. The kinetics of IC release was influenced by the NHS concentration, the presence of EDTA, and the time of prior storage of the erythrocytes at 4 degrees C. NHS (1:5 to 1:10) in the presence of EDTA caused nearly maximal release within 10-20 min at 37 degrees C. In the absence of EDTA the NHS-induced IC release was markedly slower. IC released within the first 30 min showed significant rebinding to new E. The release of IC was not associated with loss of the IC binding activity of E-CR1. The NHS-mediated release of IC could be inhibited by rabbit anti-CR1 and by a mixture of protease inhibitors. Release induced by purified factor I was also inhibited by protease inhibitors. The affinity of IC binding to E-CR1 was reduced after cleavage of CR1-bound C3b-IC to iC3b-IC by factor I.

Antigen-Antibody Complex↗
PubMed1985

Structural and functional analysis of the complement component factor H with the use of different enzymes and monoclonal antibodies to factor H.

The action of six different enzymes on the function and structure of Factor H was investigated by use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, haemagglutination, two enzyme-linked immunosorbent assay systems and an assay for Factor I cofactor activity. Six monoclonal antibodies directed against the 38 kDa tryptic fragment of Factor H [which contains the binding site for C3b (a 180 kDa fragment of the third component of complement) and the cofactor activity] were also used to detect cleavage products derived from the same fragment. Elastase, chymotrypsin A4 or trypsin first cleaved Factor H to 36-38 kDa fragments carrying all six monoclonal anti-(Factor H)-binding sites. In parallel, the interaction of Factor H with surface-bound C3b was lost, whereas the cofactor function was preserved. Further cleavage of the 36-38 kDa fragments into two 13-19 kDa fragments (one carrying the MAH4 and MRC OX 24 epitopes, the other the MAH1, MAH2, MAH3 and MRC OX 23 epitopes) destroyed cofactor activity. Pepsin, bromelain or papain rapidly split off a 13-15 kDa fragment of Factor H carrying the MAH1, MAH2, MAH3 and MRC OX 23 epitopes and destroyed all tested functions of Factor H. Ficin cleaved Factor H into disulphide-linked fragments smaller than 25 kDa, but did not affect the functions of the Factor H molecule. The 38 kDa tryptic fragment of Factor H is the N-terminal end of the Factor H molecule, as determined by N-terminal sequence analysis. A model is presented of the substructure of Factor H.

Amino Acids↗