Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Extracts from myelinated and unmyelinated nerves, prepared using Nonidet P-40, contained receptors for C3b/C4b (CR1). Extracts from myelinated nerves inhibited EAC3b rosette formation with peripheral blood leucocytes and agglutinated EAC3b, whereas extract from unmyelinated nerves did not. Rosette formation with EAC3bi or EAC3d was not affected. CR1 in extracts from myelinated nerves also expressed decay-accelerating activity of the alternative pathway C3 convertase and cofactor activity in factor I-mediated cleavage of C3b, whereas CR1 in extract from unmyelinated nerves did not. Monoclonal anti-CR1 antibodies, but not monoclonal anti-CR2 (C3d receptors) or anti-CR3 (C3bi receptors) antibodies inhibited the functional activities. Accordingly, CR1 are the only C3 receptor present in the extracts and only CR1 in myelinated nerve extracts are functionally active. CR1 in both myelinated and unmyelinated nerve extracts had a molecular weight of approximately 190 kDa. The electrophoretic mobility did not change after reduction and the 190 kDa band was stained by concanavalin A, indicating that the CR1 are single-chained glycoproteins. Binding to lentil lectin-Sepharose 4B further sustained the glycoprotein nature of the CR1. Periodic acid abolished functional activities of CR1, whereas trypsin and heat did not, indicating the functional significance of the carbohydrate moiety. That CR1 are functionally active in myelinated nerves, but not in unmyelinated nerves, may therefore be due to differences in the carbohydrate moiety. The cofactor and decay-accelerating activities of CR1 may be of significance in the pathogenesis of demyelinating polyneuropathies by limiting complement activation.
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PMID:Isolation and characterization of complement receptors CR1 from human peripheral nerves. 252 22

We examined the role of non-enzymatic glycosylation in abnormalities of the complement system commonly found in Type 1 diabetes. Fourteen patients were found to have significantly increased levels of glycosylated C3 (p less than 0.001) and C4 (p less than 0.002) with levels of glycosylated C3 being higher than those of C4 (4.1 +/- 2.5% vs 0.8 +/- 0.8%). This correlated with the finding that in vitro, purified C3 was far more susceptible to this modification than C4. Autoradiography of limited trypsin digests of H3-glycosylated iC3 showed several sites were available for reaction. However, high levels of in vitro glycosylation had no significant effect on the haemolytic activity of C3 and C4, or the binding of iC3 to the CR1 receptor on erythrocytes. Further, IgG was similarly unaffected by non-enzymatic glycosylation in its ability to activate the complement pathway, or when present in an immune complex, to be dissociated from it by complement. We concluded that the immune complex-mediated damage implicated in some complications of Type 1 diabetes is unlikely to derive from any loss of complement function due to non-enzymatic glycosylation.
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PMID:Does non-enzymatic glycosylation affect complement function in diabetes? 269 83

A large-scale procedure for the isolation of complement receptor type 1 (CR1, the C3b receptor) from human erythrocytes is described. Two of the four known phenotypes of CR1 are detectable in the isolated material. Amino acid and hexosamine analysis of the A phenotype (Mr 240 000) indicates a polypeptide chain length of about 2030 amino acids and a carbohydrate content of 8%. Both N- and O-linked sugars appear to be present. Trypsin digestion of isolated CR1 shows that it is degraded rapidly and extensively, and no stable products of Mr greater than 25000 are found. The ability of the receptor to bind to solid-phase ligand is destroyed after a single cleavage by trypsin. The capacity of the receptor to act as a cofactor for Factor I-mediated cleavage of soluble C3b is, however, only gradually decreased by proteolysis, and 30% of this activity remains after extensive degradation. The same pattern of loss of binding to solid-phase ligand, with partial retention of interaction with soluble ligand, is also characteristic of the complement proteins Factor H and C4bp, which are functionally related to CR1.
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PMID:Large-scale isolation of complement receptor type 1 (CR1) from human erythrocytes. Proteolytic fragmentation studies. 293 34

We studied Fc receptor and C3b receptor (CR1) function on U937 cells, a human monocyte cell line. C3b was incorporated into stable soluble heat aggregates of 125I-IgM (A-IgM) and 125I-IgG (A-IgG) by using functionally pure classical pathway components. C3b incorporation was verified by the ability of aggregates to bind to human red cells and by cosedimentation of 125I and 131I during ultracentrifugation. Cell uptake and degradation of A-IgG X C3b was increased up to twofold compared with A-IgG not containing C3b molecules. However, A-IgG X C3b bound to CR1 after Fc receptors were blocked with nonradiolabeled A-IgG were also not endocytosed and catabolized. Moreover, A-IgM X C3b was bound but not degraded by U937 cells. As expected, uptake of A-IgM without C3b was negligible. CR1-mediated binding of A-IgM X C3b was specifically inhibited both by a murine monoclonal antibody against the human CR1 that blocks C3b binding and by C3b oligomers generated by trypsin activation of C3, but not by monoclonal antibodies against the iC3b receptor (CR3). We conclude that CR1 on U937 cells cause increased binding of A-IgG, and this increased binding leads to increased Fc-mediated endocytosis and catabolism of model immune complexes. However, binding of soluble ligands by CR1 alone, even when binding is multivalent, does not lead to endocytosis and degradation of soluble ligands bearing C3b.
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PMID:Binding and catabolism of aggregated immunoglobulins containing C3b by U937 cells. 293 11

The effect of complement fragments C3b and C4b, on the triggering of antigen-specific human T-cell clones by Epstein-Barr virus-transformed human lymphoblastoid B cells (LCL) when these fragments are covalently coupled to the antigen tetanus toxin (TT) is described. TT was chemically cross-linked to purified C3b [(TT-C3b)n], C4b [(TT-C4b)n] or bovine serum albumin [(TT-BSA)n] as a control. T-cell activation was quantified by tritiated thymidine incorporation and 51Cr release. (TT-C3b)n and (TT-C4b)n induced proliferative responses comparable to (TT-BSA)n but at 18-25 and 4-6 lower concentrations, respectively. This enhancing effect required the covalent cross-linking of the complement fragments to the antigen and involved intracellular processing of the latter by LCL. Antigen presentation was similarly enhanced when measuring the cytotoxic activity of a helper T-cell clone against LCL previously pulsed with (TT-C3b)n or (TT-C4b)n compared with (TT-BSA)n. Binding studies, carried out on LCL using TT radiolabelled with 125I before cross-linking, indicated that (TT-C3b)n and (TT-C4b)n gave three- to four-fold more binding than (TT-BSA)n. Addition of antibodies against CR1 and CR2 or proteolytic removal of these complement receptors with trypsin inhibited by about 60% the enhancing effect of TT-bound C3b and C4b in both binding and functional assays. These results indicate that binding of C3b or C4b to antigen enhances antigen-specific proliferative and cytotoxic responses of T cells by targeting opsonized antigen onto complement receptors CR1 and CR2 of LCL. The putative significance of these findings in terms of regulation of immune responses by complement is discussed.
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PMID:Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones. 297 31

Herpes simplex virus type 1 (HSV-1) infection induces the appearance of viral analogues of human Fc IgG and C3 receptors on the surface of human cells. The virally induced C3 receptor(s) has been broadly defined as a C3b receptor, but its ligand binding characteristics have not been rigorously defined. In this study, human epidermal cells, A431 cells, and human umbilical vein endothelial cells infected with HSV-1 demonstrated rosetting with sheep erythrocytes (E) coated with IgG (E-IgG) or the complement components C3b (EAC3b) or iC3b (EAC3bi), but not with E-IgM, C4 (EAC14), C3d (EAC3d), or E alone. Rosetting was markedly enhanced by pretreatment of HSV-1-infected cells with neuraminidase. Unlike human C3 receptors, the HSV-1-induced C3 receptor was found to be trypsin resistant. To determine whether HSV-1 induced CR1-like receptors or CR3-like receptors, infected cells were pretreated with EDTA, which is known to inhibit native CR3 function. EDTA failed to prevent rosetting with EAC3bi. Furthermore, blocking studies using monoclonal antibodies against CR1 and CR3 revealed that the anti-CR1 antibody 5C11 consistently blocked EAC3b and EAC3bi rosetting with HSV-1-infected cells in a dose dependent manner, but monoclonal antibodies against CR3 did not. This study indicates that the HSV-1-induced C3 receptor is an analogue of CR1.
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PMID:Characterization of the C3 receptor induced by herpes simplex virus type 1 infection of human epidermal, endothelial, and A431 cells. 302 70

We describe a new method of preparing C3-coated erythrocytes by coupling C3 to thiol-activated erythrocytes. The procedure involves three steps. Firstly, sheep erythrocytes were treated with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to introduce 3-(2-pyridyldithio) propionyl residues into membrane proteins. Secondly, C3 was cleaved with trypsin or CoVF, Bb enzyme to obtain C3b exposing the SH group (C3b-SH). Finally, the C3b-SH was coupled to the thiol-activated erythrocytes (TA-E) through thiol/disulfide exchange to form the TA-EC3b conjugate. E coated with C3d was prepared by treating TA-EC3b with KSCN inactivated serum and plasmin. Studying the rosette formation between TA-EC3b or TA-EC3d and cells expressing C3b (CR1) and C3d (CR2) receptors and the inhibition thereof with anti-CR1 and anti-CR2 antibodies as well as with C3-sheep E membrane protein complexes, we found that TA-EC3b and TA-EC3d bound exclusively to CR1 and CR2, respectively. In addition, TA-EC3b like EAC1423b bound factors B and H as tested by hemolytic and direct binding assays. The advantage of TA-EC3 for complement receptor and hemolytic assays are the simplicity of the preparation method and the general applicability of the TA-EC3.
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PMID:Coupling of C3b to erythrocytes by disulfide bond formation: preparation of EC3b for hemolytic and complement receptor assays. 619 81

In a previous report (M. Barel et al. FEBS Lett., 1981. 136: 111) using radiolabeling methods, we characterized from the membrane of the human B lymphoblastoid cell line Raji, a 140 000-Mr glycoprotein (gp140) carrying a C3b-binding activity with 125I-labeled C3b or Sepharose-bound C3b. The facts of absence on Raji cells of CR1, the C3b receptor purified from human erythrocytes, the observations made by others that H-like activity (the 150 000 Mr C3b binding serum protein) was present in Raji cells and the same molecular weight range of H and gp140, led us to investigate the relationship between both antigens. A rabbit antibody anti-5.4 was prepared against gp140, highly purified from Raji cells. However, anti-H specificities were detected in crude anti-5.4 IgG, while anti-serum H IgG did not react with gp140 antigen. The crude anti-5.4 IgG fraction, anti-gp 140 IgG or F(ab')2 and anti-H specificities present in anti-5.4 IgG, separated by absorption on Sepharose-bound H, and anti-serum H IgG were tested on Raji cells by immunofluorescence techniques, by measuring the inhibition of specific cytotoxic assays and the inhibition of specific binding of soluble or particle-bound C3b to the cell surface and on solubilized antigens by immunoblotting techniques. All the data obtained support that: (a) anti-H specificities are not shared by antibodies bearing anti-gp 140 specificities and their presence in crude anti-5.4 IgG is more likely due to a contamination by H antigen of gp 140 antigen used in the immunization process, and (b) gp 140 antigen is highly expressed on Raji cell surface, whereas H antigen can not be detected under the same conditions. Molecular analysis of gp140 and H antigens confirmed differences between both antigens in molecular weight, trypsin sensitivity and charge properties. All the results presented herein support the notion that gp140 is not identical with the H molecule and that C3b binding to gp140 is not mediated by H. The relationship between gp140 and C3 receptors described by others is discussed.
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PMID:Analysis of gp 140, a C3b-binding membrane component present on Raji cells: a comparison with factor H. 623 77

Decay-accelerating factor (DAF), extracted from the stroma of human erythrocytes, was purified to homogeneity and incorporated into the membrane of sheep red cell complement intermediates, where its functional properties were analyzed. Incorporation of DAF into the cell membranes was temperature dependent, took place on pronase- or trypsin-treated erythrocytes, and did not depend on prior deposition of antibody, C1 or C4. Serum lipoproteins (high and low density) effectively inhibited DAF incorporation, but had no effect on the activity of DAF after its association with the cell membrane. The incorporated DAF could not be removed from the red cell surface by repeated washings in the presence of high salt concentration but was solubilized when the stroma were extracted with 0.1% Nonidet P-40. The presence of DAF in the membrane of EA did not affect the deposition of C1 and C4, but as few as 10(2) DAF molecules per cell profoundly inhibited the assembly of C3 and C5 convertases of both the classical and alternative pathways. The DAF inhibitory effect on EAC14 or EAC43 was not overcome by supplying an excess of C2 or factor B, but the alternative pathway C3 convertase could be assembled in the presence of Ni++, or nonphysiological concentrations of Mg++, which enhances the binding affinity of factor B for C3b. The DAF effect on EAC14 or EAC143 was entirely reversed by treating the cells with specific anti-DAF antibodies, showing that DAF did not alter the structure of C4b or C3b. Taken together, the experimental evidence suggests that DAF interacts directly with membrane-bound C3b or C4b and prevents subsequent uptake of C2 and factor B. DAF can function only within the cell membrane. Indeed, the decay dissociation of the C4b2a enzyme on DAF-containing sheep intermediates was not changed by varying the cell concentration. DAF-treated EA had no influence on the decay of nontreated EAC142 present in the same mixture. Moreover, the inhibitory activity of intact human erythrocytes on C4b2a was not blocked by antibodies to DAF, but was abolished by antibodies to the C3b/C4b receptor (CR1). When incorporated into the membrane of rabbit erythrocytes, human DAF inhibited their lysis by human complement. In conclusion, on the basis of these and previous results, it appears that DAF plays a central role in preventing the amplification of the complement cascade on host cell surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes. 623 20

A high proportion of murine resident peritoneal macrophages bear complement receptors 1 and 3 (CR1, CR3) which bind C3b and iC3b components of complement, respectively. By contrast, macrophages derived from bone marrow, blood, and the elicited peritoneal exudate are predominantly CR1+3. To determine if the microenvironment of the normal peritoneal cavity influences CR3 phenotype, we studied the effects of lavage from the cavity on cultures of primary peritoneal exudate macrophages, and on macrophages derived from progenitors in the bone marrow, blood, and peritoneal exudate. The cell-free peritoneal lavage (CFPL), after 24 hr of culture, induced CR3 on primary and culture-derived populations of peritoneal exudate macrophages but had no effect on the CR3 phenotype of macrophages derived from bone marrow or blood. The CR3-inducing activity in CFPL was abolished by heating at 70 degrees C for 30 min and by trypsin, and was not affected by adsorption with EA(IgM)iC3b indicator cells, demonstrating that it is not soluble CR3. Finally, exudate macrophages exposed to CFPL required at least 24 hr before they expressed CR3; such macrophages regenerated CR3 after the receptors were removed by trypsin. The selective effect of the activity in CFPL for peritoneal exudate macrophages indicates that the local microenvironment of the peritoneal cavity can influence the expression of CR3.
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PMID:Selective induction of enhanced complement receptor 3 activity on murine macrophages. 633 30


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