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Biomedical subjects

R B Sim

Publications and source records attributed to R B Sim.

At least 19 recordsLinked to original sources
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↗
PubMed1991

Oligomeric domain structure of human complement factor H by X-ray and neutron solution scattering.

Factor H is a regulatory component of the complement system. It has a monomer Mr of 150,000. Primary structure analysis shows that the polypeptide is divided into 20 homologous regions, each 60 amino acid residues long. These are independently folding domains and are termed "short consensus repeats" (SCRs) or "complement control protein" (CCP) repeats. High-flux synchrotron X-ray and neutron scattering studies were performed in order to define its solution structure in conditions close to physiological. The Mr of factor H was determined as 250,000-320,000 to show that factor H is dimeric. This structure is maintained at concentrations between 1 and 11 mg/mL in the pH range 5-9. Zn2+ ions are an inhibitor of C3b cleavage by factor I, a reaction in which factor H acts as a cofactor. Additions of Zn2+ to factor H caused it to form oligomers containing 4-10 monomers. The radius of gyration RG of native factor H by X-rays or by neutrons in 0% or 100% 2H2O buffers is not measurable but is greater than 12.5 nm. Two cross-sectional radii of gyration RXS-1 and RXS-2 were determined as 3.0-3.1 and 1.8 nm, respectively. Analyses of the cross-sectional intensities show that factor H is composed of two distinct subunits. The RXS-1 corresponds to the cross-sectional properties of both subunits and exhibits an unusual radiation dependence on the X-ray flux. Since RXS-2 is close to the corresponding RXS of C4b binding protein (91% of which is formed from SCR/CCP domains), it is inferred that the SCR/CCP domains of factor H and C4b binding protein have similar solution structures. The use of hydrodynamic spheres to reproduce literature sedimentation coefficients of 5.5-5.6 S showed that these were compatible with a V-shaped arrangement of two rods (36 spheres each, length 87 +/- 5 nm) joined at an angle of 5 degrees. The use of a similar arrangement of 244 spheres arranged in two rods (length 77 nm) to fit the experimental X-ray and neutron scattering curves showed that the two rods are joined at an angle of 5 degrees. This model corresponds to an actual RG of 21-23 nm. The separation between each SCR/CCP in factor H is close to 4 nm. In the solution structure of factor H, the SCR/CCP domains are in a highly extended conformation.

Complement C3b↗
PubMed1991

Secondary structure of a complement control protein module by two-dimensional 1H NMR.

The complement control protein (CCP) module (also known as the short consensus repeat) is a consensus sequence of about 60 amino acid residues which is thought to fold independently. It occurs over 140 times in more than 20 extracellular mosaic proteins including 12 proteins of the complement cascade. An isolated CCP module, the 16th repeat from human complement factor H, has been expressed in a yeast vector and shown to fold with the same pattern of disulfide bond formation as is seen in the native protein. Two-dimensional 600-MHz 1H NMR spectra of this module have been recorded at pH 3.3 and 6.0 and analyzed to permit determination of secondary structure in solution. The CCP module comprises two predominantly extended segments (Glu1-His13 and Ala17-Glu27), two segments of double-stranded antiparallel beta-sheet (Gly14-Val16 paired with Tyr31-Cys33 and Gly38-Asp40 paired with Ser57-Ile59), and a short piece of triple-stranded beta-sheet (Glu27-Thr30, Ile44-Leu48, and Lys51-Ser53). Turns occur at Asp22, Gly36, and Glu50, while Gly41-Ala43 appear to form a looped-out segment or bulge. This structure is compared with a secondary structure prediction made on the basis of an alignment scheme of 101 sequences for CCP modules [Perkins, S. J., Haris, P. I., Sim, R. B., & Chapman, D. (1988) Biochemistry 27, 4004-4012]--the experimentally determined secondary structure bears an overall resemblance to the predicted one but differs in the number and position of turns. Some of those amino acid residues which are highly conserved throughout the range of CCP modules appear to play a role in stabilizing the global fold.

Amino Acid Sequence↗
PubMed1991

An angle-variable three-dimensional pulsed field gel electrophoresis system.

A three-dimensional pulsed field electrophoretic method based on the simultaneous application of fixed and cyclically alternating polarity fields at a right angle is described. Requiring only minimal electronic hardware it provides highly homogeneous field conditions over a large gel area and the versatility to vary the pulse vector angle. The electrophoretic parameters critical to achieve fast high resolution separation over a wide range of molecular sizes have been optimized and applied to megabase-size chromosomal DNA molecules. The empirical relationships between pulse time, field strength conditions, and resolution limits derived allow selection of coordinated experimental conditions for the separation of specific DNA size ranges.

Buffers↗
PubMed1991

C1: molecular interactions with activating systems.

The molecular events controlling complement activation have been gradually unravelled over the past three decades, stimulated by improved isolation procedures and a better understanding of the roles of individual proteins. In this review, Bob Sim and Ken Reid examine the interactions between C1q and its numerous ligands in the initiation of the classical pathway cascade.

Animals↗
PubMed1990

Human leukocyte C1q receptor binds other soluble proteins with collagen domains.

A receptor binding to the C1q subcomponent of complement has been reported by many workers. In this paper we report for the first time that C1q receptor binds not only to C1q, but also to three other structurally similar ligands, namely mannan binding protein (MBP), conglutinin, and lung surfactant protein (SP-A). All these ligands have been reported to enhance removal of species bound to their globular domain from blood (MBP, conglutinin, C1q) or lung (SP-A) through phagocytosis. One of the possible roles for ligand-receptor binding may be initiation of phagocytosis.

Binding, Competitive↗
PubMed1990

Molecular modeling of human complement component C4 and its fragments by X-ray and neutron solution scattering.

The solution structures of human complement component C4 and five derived fragments, C4u, C4(a + b), C4b, C4c, and C4d, were analyzed by synchrotron X-ray and neutron scattering. The X-ray radii of gyration RG for C4, C4u, and C4(a + b) in H2O buffers are similar at 5.23-5.28 nm, and likewise the cross-sectional radii of gyration RXS are similar at 2.48-2.52 nm. Molecular mass calculations using X-rays and neutrons show unexpectedly that C4c is dimeric; however, all the other forms are monomeric. C4c2 has an X-ray RG of 5.18 nm and an RXS of 2.89 nm. Neutron contrast variation gives RG values at infinite contrast of 4.87-4.93 nm for C4 and C4u, 4.79 nm for C4b, 4.94 nm for C4c2, and 2.69 nm for C4d. The RXS values at infinite contrast are 2.23-2.25 nm for C4 and C4u, 1.89 nm for C4b, and 2.62 nm for C4c2. These data show that a large conformational change occurs on going from C4 to C4b, but not on going from C4 to C4u, and this is attributed to the presence of the C4a moiety in C4u. Comparisons of the C4 and C4u scattering curves show that these are very similar out to a nominal resolution of 4 nm. Scattering-curve models were developed to account for the neutron scattering curves of C4, C4c2, and C4d in 2H2O buffers. The C4c monomer could be represented by a lamellar ellipsoid of size 8 nm x 2 nm x 18 nm. C4d was found to be 4 nm x 2 nm x 9 nm. The combination of these structures gave good accounts of the neutron data for C4, C4b, and C4c2 to resolutions of 5-6 nm. The C4 model was obtained by placing the long axis of C4d parallel to that of C4c such that the cross section is extended. C4b was best modeled by repositioning C4d relative to C4c such that this cross section becomes more compact. The C4 and C4b models are compared with possible structures for the C1 component of complement to show the importance of the surface accessibility of the protease domains and short consensus repeat domains in C1 for C4 activation.

Blood Donors↗