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Biomedical subjects

R B Sim

Publications and source records attributed to R B Sim.

At least 55 records · Page 3Linked to original sources
PubMed1997

Interactions between human complement components factor H, factor I and C3b.

Using a microtitre plate assay, direct binding between complement factors I and H was demonstrated, and ligand blotting indicated that factor H interacts with the heavy chain of factor I. Similarly, direct C3(NH3)-factor I and C3(NH3)-factor H binding was characterized [where C3(NH3) is a form of C3 that is cleaved by factor I in the presence of factor H]. Both factor H and factor I interacted with both chains of C3(NH3) in ligand blotting. Binding reactions between all three pairs of components were highly dependent on ionic strength, and showed similar pH optima. Binding assays with all three components present led to the following conclusions. (a) Binding sites for C3(NH3) and factor I on factor H do not overlap, and binding of factor I and C3(NH3) to soluble factor H promotes the weak factor I-C3(NH3) interaction. (b) Anomalies arise with immobilized factor H, which may be artefactual or may reflect the physiological situation. (c) Similarly, binding sites on factor I for C3(NH3) and for factor H do not overlap, and binding of factor H and C3(NH3) to factor I promotes direct factor H-C3(NH3) interactions. Based on these results, a model of the interactions between factor H, factor I and C3(NH3) leading to the processing of C3(NH3) is proposed.

Cations, Divalent↗
PubMed1997

Role of the distal hinge region of C1-inhibitor in the regulation of C1s activity.

A synthetic peptide corresponding to residues 448-459 of C1-inhibitor (C1-inh) binds to C1s, is a non-competitive inhibitor of C1s activity and prevents formation of an SDS-stable C1s-C1-inh complex. Substitutions of residues Q452, Q453 or F455 in this peptide resulted in loss of C1s binding and inhibitory activity of the peptide. NMR analysis of the peptide showed an area of well-defined structure from E450 to F455. The side chains of Q452, Q453 and Q455 were exposed to the solvent and therefore available for C1s binding. The defined structure in the peptide is compatible with our computer model of the serpin domain of C1-inh.

Complement C1 Inactivator Proteins↗
PubMed1997

Expression of a biologically active recombinant mouse IL-1 receptor antagonist and its use in vivo to modulate aspects of the acute phase response.

Recombinant mouse IL-1receptor antagonist protein (rmIL-1ra) was expressed in Escherichia coli. In vivo administration of rmIL-1ra, in a casein-induced murine model of acute inflammation, completely abolished the hepatic induction of the mRNAs specifying serum amyloid A1 (A-SAA1) and A-SAA2 for up to 12 h, indicating that hepatic A-SAA mRNA synthesis is totally IL-1 driven. A-SAA protein, however, was present in the serum of rmIL-1ra-treated casein-stimulated mice (although at lower levels than in untreated casein-stimulated mice) at 12 h indicating that extrahepatic A-SAA synthesis is driven in part by factors acting independently of IL-1. Hepatic mRNA levels of the other mouse acute phase reactants (APRs), serum amyloid P component, C-reactive protein, alpha1-acid glycoprotein, and C3 were also induced with casein after 12 h, as were serum protein levels of SAP and C3. These inductions were only partially inhibited by rmIL-1ra, indicating that hepatic expression of the latter APRs (unlike that of A-SAA) is driven partly by IL-1 and partly by factors acting independently of IL-1. Hepatic mRNA levels of the negative APRs apolipoprotein A-I and serum albumin were down-regulated 12 h after casein stimulation. rmIL-1ra partially restored serum albumin mRNA levels but not apo A-I mRNA levels, indicating differential regulation of these negative APRs. The rmIL-1ra will be useful in studies of IL-1-mediated gene regulation in murine models of inflammation.

Acute-Phase Reaction↗
PubMed1997

Complement evasion by Echinococcus granulosus: sequestration of host factor H in the hydatid cyst wall.

Hydatid disease, which affects humans and various livestock species, is caused by the larval stages of the cestode parasite Echinococcus granulosus. The disease involves the growth of a fluid-filled cyst in the host's internal organs. The hydatid cyst wall is generally thought to preclude the access of host immune cells to the cyst, while allowing passage of macromolecules. Thus, evasion of humoral effector mechanisms, in particular the complement system, may be crucial to parasite survival. Evasion of complement might involve inhibition of the C3b deposition step, which can in principle be achieved through parasite-derived or host-derived and parasite-bound regulatory molecules. In this work, extracts from bovine hydatid cysts were analyzed for the presence of factor H-like function, namely the cofactor activity for the breakdown of amidated bovine C3 by bovine factor I. Both this activity and antigenically detectable bovine factor H were found in most extracts. The cofactor activity was retained by an anti-bovine factor H Ab column; as no factor H-cross-reactive parasite molecules were detected in the active extracts, the activity was indeed due to host factor H. The pattern of extraction of host factor H from the parasite tissue indicated that at least part of it was selectively bound to the cyst wall, and that a strong charge component was involved in the binding. Our results indicate that the selective concentration of host factor H from plasma/interstitial fluid may contribute to complement evasion by E. granulosus.

Animals↗
PubMed1997

A secondary C1s interaction site on C1-inhibitor is essential for formation of a stable enzyme-inhibitor complex.

This paper examines the location of a secondary binding site for C1s on C1-inhibitor (C1-inh) which is required for the formation of SDS-stable Cls-C1-inh complexes. We used a synthetic peptide (residues 448-459) corresponding to the distal hinge region of C1-inh. This peptide binds to C1s and C1s preincubated with the peptide cleaves C1-inh but does not form a stable C1s-C1-inh complex. Computer modelling of C1-inh shows that residues Q452, Q453 and F455 are surface-exposed and that the secondary binding site may also include residues H291 and F292 which are conserved in serpins.

Animals↗
PubMed1997

The C1q and collectin binding site within C1q receptor (cell surface calreticulin).

C1q receptor (C1qR/collectin receptor/cC1qR) has an almost complete amino acid sequence identity with calreticulin (CRT). C1qR/CRT is located on the surface of many cell types. Binding of C1q to C1q receptor elicits a range of immunological responses. C1qR also interacts with the collectins SP-A, MBL, CL43 and conglutinin via a cluster of charged residues on the collagen tails of the ligands. In order to localise C1q and collectin binding activity within C1qR/CRT, recombinant C1qR/CRT domains [N (residues 18-196), P (197-308) and C (309-417)] were produced. Both the N- and P-domains bound to C1q, demonstrating that the binding site spans the intersection of these domains. Amino acid alignment analysis identified a putative CUB module within this region. This S-domain (residues 160-283) was expressed and showed concentration-dependent binding to immobilised C1q, demonstrating that it contains the C1q binding site. Competitive inhibition studies of the S-domain-C1q interaction revealed that the S-domain binds to C1q collagen tails and to the collectin proteins, SP-A, MBL, CL43 and conglutinin. The C1q and collection binding site on C1qR/CRT has therefore been localised to the S-domain.

Amino Acid Sequence↗
PubMed1997

Antibodies to beta2-glycoprotein I--a specific marker for the antiphospholipid syndrome.

The antiphospholipid syndrome is a disorder characterized by recurrent thrombosis and the presence of antibodies specific to phospholipids. However, the diagnosis of this syndrome is hampered by the lack of a specific laboratory test. In this study an ELISA for the measurement of antibodies to solid-phase beta2-glycoprotein I (beta2-GPI) was established and compared with anticardiolipin antibodies for diagnosis of antiphospholipid syndrome. Significantly elevated levels of antibodies to beta2-GPI were found in all patients with definite antiphospholipid syndrome (median = 91 AU). Marginally elevated levels of antibodies to beta2-GPI were observed in 5% of patients with systemic lupus erythematosus (SLE; median = 4 AU), 1% with stroke (median = 3 AU), 13% with infectious mononucleosis (median = 3 AU), 10% with HIV infection (median = 3 AU) and 8% with VDRL false-positive serology for syphilis (median = 4 AU), but not in patients with rheumatoid factor, syphilis or carotid artery stenosis. In contrast, significantly raised levels of anticardiolipin antibodies were observed in 100% of patients with definite antiphospholipid syndrome, 30% with SLE, 88% with HIV infection, 94% with syphilis, 62% with infectious mononucleosis, 9% with rheumatoid factor-positive sera, 74% VDRL false-positive serology for syphilis, 47% with stroke and 0% with carotid artery stenosis. This solid-phase assay for antibodies to beta2-GPI is highly specific for the antiphospholipid syndrome and represents an advance in the laboratory diagnosis of this disorder.

Antibodies, Anticardiolipin↗
PubMed1997

Degradation of C1-inhibitor by plasmin: implications for the control of inflammatory processes.

BACKGROUND: A correct balance between protease and inhibitor activity is critical in the maintenance of homoeostasis; excessive activation of enzyme pathways is frequently associated with inflammatory disorders. Plasmin is an enzyme ubiquitously activated in inflammatory disorder, and C1-inhibitor (C1-Inh) is a pivotal inhibitor of protease activity, which is particularly important in the regulation of enzyme cascades generated in plasma. The nature of the interaction between plasmin and C1-Inh is poorly understood. MATERIALS AND METHODS: C1-Inh was immunoadsorbed from the plasma of normal individuals (n = 21), from that of patients with systemic lupus erythematosus (n = 18) or adult respiratory distress syndrome (n = 9), and from the plasma and synovial fluid of patients with rheumatoid arthritis (n = 18). As plasmin is a putative enzyme responsible for C1-Inh was examined using SDS-PAGE. In addition, peptides cleaved from C1-Inh by plasmin were isolated and sequenced and the precise cleavage sites determined from the known primary sequence of C1-Inh. Homology models of C1-Inh were then constructed. RESULTS: Increased levels of cleaved and inactivated C1-Inh were found in each of the inflammatory disorders examined. Through SDS-PAGE analysis it was shown that plasmin rapidly degraded C1-Inh in vitro. The pattern of C1-Inh cleavage seen in vivo in patients with inflammatory disorders and that produced in vitro following incubation with plasmin were very similar. Homology models of C1-Inh indicate that the majority of the plasmin cleavage sites are adjacent to the reactive site of the inhibitor. CONCLUSIONS: This study suggests that local C1-Inh degradation by plasmin may be a central and critical event in the loss of protease inhibition during inflammation. These findings have important implications for our understanding of pathogenic mechanisms in inflammation and for the development of more effectively targeted therapeutic regimes. These findings may also explain the efficacy of anti-plasmin agents in the treatment of C1-Inh deficiency states, as they may diminish plasmin-mediated C1-Inh degradation.

Adult↗
PubMed1996

Localisation of the C1q binding site within C1q receptor/calreticulin.

C1q receptor (C1qR/collectin receptor) is located on many cell types. Binding of C1q to these cells elicits numerous responses. Protein sequencing has shown that C1qR is almost identical to calreticulin (CaR), an abundant multifunctional protein. Radioiodinated C1qR and CaR bind to C1q with identical characteristics. Three recombinant C1qR/CaR domains (N-, C-terminal domains and central P-domain) were expressed using the Thiofusion system, and used to study the interaction with C1q. Both the N- and P-domains were implicated in C1q binding. A region, termed the S-domain, spanning the N and P intersection was expressed, and showed concentration-dependent binding to C1q, demonstrating that the C1q binding site lies within this region.

Amino Acid Sequence↗
PubMed1996

Megalin-mediated endocytosis of transcobalamin-vitamin-B12 complexes suggests a role of the receptor in vitamin-B12 homeostasis.

Kidney cortex is a main target for circulating vitamin B12 (cobalamin) in complex with transcobalamin (TC). Ligand blotting of rabbit kidney cortex with rabbit 125I-TC-B12 and human TC-57Co-B12 revealed an exclusive binding to megalin, a 600-kDa endocytic receptor present in renal proximal tubule epithelium and other absorptive epithelia. The binding was Ca2+ dependent and inhibited by receptor-associated protein (RAP). Surface plasmon resonance analysis demonstrated a high-affinity interaction between purified rabbit megalin and rabbit TC-B12 but no measurable affinity of the vitamin complex for the homologous alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor related protein (LRP). 125I-TC-B12 was efficiently endocytosed in a RAP-inhibitable manner in megalin-expressing rat yolk sac carcinoma cells and in vivo microperfused rat proximal tubules. The radioactivity in the tubules localized to the endocytic compartments and a similar apical distribution in the proximal tubules was demonstrated after intravenous injection of 125I-TC-B12. The TC-B12 binding sites in the proximal tubule epithelium colocalized with megalin as shown by ligand binding to cryosections of rat kidney cortex, and the binding was inhibited by anti-megalin polyclonal antibody, EDTA, and RAP. These data show a novel nutritional dimension of megalin as a receptor involved in the cellular uptake of vitamin B12. The expression of megalin in absorptive epithelia in the kidney and other tissues including yolk sac and placenta suggests a role of the receptor in vitamin B12 homeostasis and fetal vitamin B12 supply.

Animals↗
PubMed1996

Prediction from sequence comparisons of residues of factor H involved in the interaction with complement component C3b.

The amino acid sequence of the region of bovine factor H containing the C3b binding site has been derived from sequencing overlapping cDNA clones. A cDNA sequence encoding 669 amino acids was obtained. Like human and mouse factor H the sequence can be arranged into a number of internally homologous units (CPs), each of which is about 60 amino acids long and is based on a framework of four conserved cysteine residues. Bovine factor H is of the same molecular mass as human and mouse factor H, and is therefore likely to be composed of 20 contiguous CPs. Comparisons with human and mouse factor H indicate that the partial bovine sequence encodes CPs 2-12 inclusive of bovine factor H. Bovine factor H binds to human ammonia-treated C3 (causing thiolester cleavage) [C3(NH3)] and promotes the cleavage of human C3(NH3) in the presence of bovine factor I. Other studies indicate that CPs 2-5 of human factor H encompass the C3b binding and factor I cofactor activity site. Multiple sequence alignments of human factor H, mouse factor H (which also interacts with human C3b) and bovine factor H with CP modules whose structures have been determined experimentally, have been used to predict residues in the hypervariable loops of CPs 2-5 and to identify residues of potential importance in human C3 binding and factor I cofactor activity. Leu-17 and Gly-20 of CP 2, Ser-17, Ala-19, Glu-21, Asp-23 and Glu-25 of CP 3 and Lys-18 of CP 4 are all conserved between the three species. It may be that CPs 3 and 4 interact with C3(NH3) directly, whilst CPs 2 and 5 maintain the correct orientation for CPs 3 and 4 to interact.

Amino Acid Sequence↗
PubMed1996

Epitope mapping of C1 inhibitor autoantibodies from patients with acquired C1 inhibitor deficiency.

We report six patients with acquired C1 inhibitor (C1-inh) deficiency associated with serum C1-inh autoantibodies and circulating cleaved (96 kDa), functionally inactive C1-inh. In three patients, all of whom had IgG-kappa paraproteins in their sera, the Abs were IgG-kappa. In the remaining three patients, the Abs were IgM (2 kappa, 1 lambda). These data suggest that all the Abs were monoclonal. The autoantibodies recognized two synthetic peptides (peptides 2 and 3), which spanned the reactive center of C1-inh. Binding to peptide 3 (residues 448-459) was greater than to peptide 2 (residues 438-449), suggesting that the epitope recognized by the autoantibodies was expressed principally by peptide 3. Both peptides inhibited the binding of the autoantibodies to C1-inh. None of the autoantibodies recognized peptide 1 (residues 428-440), and this peptide did not inhibit the binding of the autoantibodies to C1-inh. The use of substituted peptides suggested that residues Q452 and Q453 made significant contributions to the epitope, and computer modeling studies showed their side chains to be surface exposed in the intact molecule. However, computer modeling also showed that none of the side chains of the polar residues in peptide 2 were sufficiently close to Q452 and Q453 to be able to contribute to a shared epitope. As peptide 2 could inhibit the binding of C1-inh autoantibodies to peptide 3 and vice versa, we conclude that an autoepitope also exists in peptide 2. Computer modeling and the use of substituted peptides suggested that the sequence LLVF (residues 446-449) in peptide 2 is structurally similar to the sequence QQPF (residues 452-455) in peptide 3. We therefore conclude that there are two potential epitopes in the intact C1-inh molecule that are capable of binding to C1-inh autoantibodies.

Aged↗
PubMed1996

Heterozygous and homozygous factor H deficiency states in a Dutch family.

Factor H, a 150-kD protein, is an important down-regulating protein of the alternative pathway of the complement system. Presently, only 15 persons, representing seven families, have been described with homozygous factor H deficiency. Deficiency of this protein, inherited as an autosomal recessive trait and resulting in uncontrolled breakdown of C3, results in depletion of components of the alternative pathway (factor B, properdin) and of the terminal pathway (C5), and is associated with the onset of bacterial infections, glomerulonephritis and systemic lupus erythematosus (SLE). The proband of the family in this study suffered from subacute cutaneous lupus erythematosus and had had meningococcal meningitis due to serogroup X. She had a complete factor H deficiency at the protein level as determined by Western blotting. Among 21 relatives of the proband studied, encompassing three generations, 10 had low factor H levels, including the two children of the proband, indicating a heterozygous factor H deficiency state. In serum samples of the proband and 11 relatives prospectively studied, a strong correlation of factor H levels with C3, C3 haemolytic activity, factor B and properdin levels (P < 0.0001) was found. Alternative pathway protein levels were significantly lower (Mann-Whitney test; Z values 3.6-2.7) in sera from the four heterozygous relatives studied than in sera from the seven non-deficient relatives. In addition, a defect of the 37/42-kD H-related protein was found in the proband and two of 21 relatives, compared with four of 40 controls. A defect of the 24/29-kD H-related protein was present in one of 21 relatives studied and in none of the 40 controls.

Complement Factor H↗
PubMed1996

Comparison of complement activation in vitro by different Echinococcus granulosus extracts.

In the present study we have investigated and compared in vitro the specific complement (C) activating activity of three metacestode preparations of Echinococcus granulosus. Extracts from hydatid cyst fluid (HCF-ext), protoscoleces (PSC-ext) and hydatid cyst membrane (HCM-ext) activated human C producing C3 conversion and generating the C5b6 complex and the terminal C complex (TCC). HCM-ext showed much lower C activating activity than PSC-ext and HCF-ext. Moreover, its ability to generate C5b6 and TCC was lower than its ability to convert C3. On the other hand, PSC-ext and HCF-ext proved to be good C activators when their specific C activating activities were compared with that of inulin. However, PSC-ext produced lower levels of TCC than those produced by HCF-ext, in spite of the fact that both produced practically the same levels of C3d and C5b6. These results may be consistent with the existence of several mechanisms of C modulation involved in the defence of the parasite against host C damage.

Animals↗