[Complement regulatory factors and immunologic recognition mechanism between species].
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Animals↗
Biomedical subjects
T Seya
Publications and source records attributed to T Seya.
The functions of the ninth component of human complement are sustained by disulfide bonds with different susceptibilities to reduction.
Purified C9 with expected hemolytic and polymerizing activities was found to contain approximately 0.2 mol of sulfhydryl groups/mol of C9. By proteolysis of C9 with labeled SH groups, the SH residues on intact C9 were mapped to Cys-359 and Cys-384 which, presumably, form an intra-domain disulfide bond in the intact molecule. The blocking of these sulfhydryl residues by alkylation, however, had minimal influence on the functions of C9. On the other hand, reduction of C9 by 1 mM dithiothreitol (DTT) (6-fold molar excess over Cys residues) followed by alkylation resulted in a complete block of polymerization activity and a 50% loss of C9 hemolytic activity. In contrast, the ability of C9 to bind EAC1-8 remained largely unaffected. The loss of poly-C9 formation activity correlated with the alkylation of approx. 6 liberated sulfhydryl groups. Hemolytic activity was abolished by treatment with > 5 mM DTT which allowed the liberation of approximately 18 sulfhydryl groups. Most of the DTT-susceptible disulfides were within the C9a fragment (an N-terminal peptide derived by thrombin). Thus, three major functions of C9, EAC1-8 binding, polymerization, and hemolytic activity, are sustained by disulfide bond-dependent conformational motifs with different susceptibility to reducing reagents. The maintenance of the N-terminal C9a region is essential for polymerization, but not EAC1-8 binding activity of C9. Taken together, the results of the present study differentiate in molecular terms several of the functional portions of C9, and stress the significance of intra-chain disulfide linkages in maintaining the structural components necessary for the functions of C9.
Animals↗
Modulation of complement regulatory function and measles virus receptor function by the serine-threonine-rich domains of membrane cofactor protein (CD46).
Three major membrane cofactor protein (MCP) phenotypes with different serine-threonine (ST)-rich regions, namely STc (L-phenotype), STBC (H or U phenotype) and STABC, and the MCP without the ST domain (delta ST) were expressed in Chinese hamster ovary (CHO) cells by transfecting the respective cDNAs. The expressed molecules migrated with a larger molecular mass on SDS/PAGE than those expected from their amino acid sequences. O-Glycanase digestion showed that this was due to O-linked sugar chains. The apparent sugar contents in each ST segment were compatible with their serine and threonine contents in the ST regions. The functional properties of these phenotypes as inhibitors of human complement (C) and receptors of measles virus (MV) were compared. The classical pathway-dependent CHO cell lysis by human C was more effectively suppressed by the expressed delta ST and STC than by the STABC and STBC phenotypes, although the difference was not so prominent. In contrast, alternative C pathway-dependent CHO-cell lysis was most effectively suppressed by the STABC phenotype and was only slightly blocked by the ST-deleted mutant. MV infection occurred with all of the phenotypes, but the infectious dose required to cause the same level of syncytium formation was 100-times higher in large ST (STABC and STBC) than in small ST (STC and delta ST) phenotypes. Thus, the ST domain serves as a functional modulator in MCP: MCP with a large ST domain having high O-linked sugar contents is favourable to the effective suppression of both the alternative C pathway-mediated cytolysis and MV infection, whereas MCP with a small ST domain is favourable to the suppression of the classical C pathway.
Animals↗
Involvement of phosphatidylinositol 3-kinase in Fc gamma receptor signaling.
Wortmannin, a potent and selective inhibitor of phosphatidylinositol (PI) 3-kinase (Okada, T., Sakuma, L., Fukui, Y., Hazeki, O., and Ui, M. (1994) J. Biol. Chem. 269, 3563-3567), prevented Fc receptor for IgG (Fc gamma R)-dependent phagocytosis of the human monocytic cell line U937 or guinea pig neutrophils. Cross-linking of Fc gamma R on the surface of U937 cells increased PI 3-kinase activity that was immunoprecipitated with antibody against phosphotyrosine or antibody against the 85-kDa regulatory subunit of PI 3-kinase. Specific cross-linking of Fc gamma R subclass Fc gamma RI or Fc gamma RII, using monoclonal antibodies against each receptor subclass and the F(ab')2 fragment of goat antibody against mouse IgG, increased anti-phosphotyrosine-precipitable PI 3-kinase activity. Treatment of cells with anti-Fc gamma RIII antibody plus the same F(ab')2 did not affect the activity, reflecting the lack of Fc gamma RIII in U937 cells. Fcy gamma R stimulation triggered prominent tyrosine phosphorylation of several proteins, among which the 115-kDa peptide showed strong association with PI 3-kinase. Thus, Fc gamma R appears to be coupled functionally, via a tyrosine kinase, to PI 3-kinase, which may regulate the phagocytotic activity of the cells.
Androstadienes↗
Expression of a hybrid complement regulatory protein, membrane cofactor protein decay accelerating factor on Chinese hamster ovary. Comparison of its regulatory effect with those of decay accelerating factor and membrane cofactor protein.
C activation on the cell surface is supposedly regulated by membrane cofactor protein (MCP) and decay accelerating factor (DAF). These are complementary in function: MCP acts as a cofactor in factor I-mediated C3b and C4b inactivation, thus preventing the assembly of C3 convertases, whereas DAF accelerates spontaneous decay of the assembled C3 convertase. In this report, a hybrid MCP-DAF was expressed on Chinese hamster ovary cells by transfecting cDNA, and its regulatory activity was compared with those of MCP and DAF transfectants and with a transfectant expressing both MCP and DAF (MCP + DAF). The C3 deposition on sensitized CHO cells through activation of the classical pathway was blocked to a different degree with these transfectants, the order being MCP + DAF > DAF > hybrid MCP-DAF > MCP. Likewise, the C3 deposition via the alternative pathway was blocked efficiently in the order hybrid > MCP + DAF > MCP. The C-mediated cytolysis of CHO cells virtually reflected the degree of C3 fragment deposition. The MCP-DAF transfectant acquired additive protective activity against alternative pathway-mediated C3 deposition and cytolysis but was less potent in circumventing classical pathway attack than cells that expressed DAF alone or DAF + MCP. Hybrid MCP-DAF may be useful for alleviating C-mediated cell damage, especially via the alternative pathway.
Animals↗
Measles virus infects mouse fibroblast cell lines, but its multiplication is severely restricted in the absence of CD46.
Mouse cell lines (L, NIH3T3, and RMA cells) infected with the Edmonston strain of measles virus (MV) did not exhibit cytopathic effects (CPE), consistent with the finding that mice are not susceptible to MV. Northern blot analysis, however, revealed that MV genes were transcribed in infected L and NIH3T3 cells, although expression levels were much lower than those in lytically infected Vero cells. Expression of MV genes was not detected in infected RMA cells. L and NIH3T3 cells were found to synthesize viral proteins and produce infectious virions after infection. These cell lines did not express on the surface the molecule detectable by antibodies directed against human CD46, the recently identified MV receptor. L cell transfectants expressing human CD46 exhibited CPE after MV infection, and produced 50 times more viral transcripts and 20 times more infectious virions than the parental L cells. The lowest titer of MV that induced viral multiplication in L cells as detected by cocultivation with Vero cells was larger than that in CD46+ L cells by two orders of magnitude. Our results indicate that MV can infect some mouse cells in the absence of CD46, yet the presence of CD46 facilitates multiplication and cytopathogenicity of MV in mouse cells.
3T3 Cells↗
Involvement of the pertussis toxin-sensitive GTP-binding protein in regulation of expression and function of granulocyte complement receptor type 1 and type 3.
Human polymorphonuclear leukocytes (PMN) express receptors for complement (C) C3b and C3bi termed CR1 and CR3, respectively. The addition of PMA or fMLP to PMN enhances the capacity of these receptors to promote binding of C3b- and C3bi-coated erythrocytes. fMLP-dependent increase of the binding of these ligand-coated erythrocytes was completely abolished by prior exposure of the PMN to pertussis toxin (IAP). GTP-binding protein (Gi alpha) was ADP-ribosylated and dysfunctional by this treatment. On the other hand, PMA-dependent binding of these ligands, as well as control binding, was inhibited only slightly, if at all, by the IAP treatment. The levels of C receptor expression on cell surface were determined by flow cytometry using monoclonal antibody against CR1 and those against the alpha and beta chains of CR3 (CR3 is composed of alpha and beta chain). Upon exposure of PMN to the chemotactic factor or PMA, or upon incubation of the cells at 37 degrees C, the surface expression of CR1 and CR3 alpha was increased. IAP also blocked an fMLP-induced increase of CR1 and CR3 alpha, but did not block the temperature- or PMA-dependent increase of these receptors. Opsonized zymosan (SOZ), another ligand for CR3, also led to an increase of both CR1 and CR3 alpha. Neither PMA nor SOZ brought about an increase of the surface expression of CR3 beta, but fMLP caused a slight increase of CR3 beta in an IAP-sensitive manner. Based on the IAP-sensitivity of the receptor expression, therefore, it appears that at least two separate mechanisms are operative in the control of C receptors. In addition, the alpha and beta chains of CR3 are regulated independently. The present data offer evidence suggesting that C receptor functions are in part regulated through a GTP-binding protein via modulation of their surface expression.
Adenosine Diphosphate Ribose↗
Development of an ELISA assay for soluble CD35 (C3b/C4b receptor): high levels of soluble CD35 in LE-positive patients with hematological malignancies.
Malignant cells usually lack CD35 (complement receptor type 1, C3b/C4b receptor), a differentiation surface antigen. We measured soluble forms of CD35 in the plasma of normal subjects and patients with various malignant diseases. A microassay for the determination of CD35 was established based on a sandwich enzyme immunoassay using 2 monoclonal antibodies that recognize different epitopes. Soluble CD35 was not detected in any plasma samples from normal subjects or from patients with a variety of solid cancers: i.e., levels were below 20 ng/ml. On the other hand, 3 of 70 patients with hematological malignancies showed high levels of plasma CD35. The molecular mass of the soluble form was about 200 kDa, which is similar in size to membrane forms of CD35. Although the clinical conditions differed in these patients, they had high transaminase titers and detectable autoantibody. Complement titers (CH50) and the levels of membrane complement regulatory proteins were within the normal range in these patients. Although the mechanism by which it is produced remains unknown, soluble CD35 is present in significant amounts in association with immunological disorders secondary to hematological malignancies.
Enzyme-Linked Immunosorbent Assay↗
Distribution of C3-step regulatory proteins of the complement system, CD35 (CR1), CD46 (MCP), and CD55 (DAF), in hematological malignancies.
The distribution and levels of three membrane proteins, CD35, CD46, and CD55, which serve as complement regulators, were examined in normal peripheral blood and hematologically malignant cells. CD35 was negative in most leukemia cells regardless of the type of leukemia, although granulocytes, monocytes, and some populations of lymphocytes were CD35+. CD46 was present in all blood cells except erythrocytes, and levels were 2-8 times higher in most leukemia cells than in their mature counterparts, particularly in CML and CLL cells, except for those of B cell lineage. CD55, a widely-distributed phosphatidyl inositol-anchored protein, was more frequently lost in NHL cells than in other types of hematological malignancies. In this review, we discuss the roles, mechanisms, and clinical applications of cell-associated complement regulatory proteins in hematological malignancies.
Antigens, CD↗
Possibility of prevention of hyperacute rejection by DAF and CD59 in xenotransplantation.
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Animals↗
Membrane cofactor protein (CD46) protects cells predominantly from alternative complement pathway-mediated C3-fragment deposition and cytolysis.
Membrane cofactor protein (MCP) cDNA was transfected into Chinese hamster ovarian tumor (CHO) cells and the functional properties of the expressed protein were studied. Cells adherent to flasks were essential for continuous expression of MCP on CHO cells. If the cells were maintained in noncoated flasks, MCP expression was markedly reduced but upon being transferred to coated flasks reexpressed the protein. MCP expressed on CHO cells had the expected m.w., approximately 50 kDa, and possessed factor I-cofactor activity. By a propidium iodide incorporation assay and by 51Cr release assay, antibody-sensitized CHO cells expressing MCP were protected from C-mediated cytotoxicity. The inhibition of lytic activity correlated with a decrease in C3 deposition. This host cell protective activity was exerted efficiently for the alternative pathway. The classical pathway was not blocked by MCP unless the cells were presensitized with low concentrations of antibody. These results imply that MCP primarily protects host cells from alternative pathway-mediated C3 targeting. In a pathologic state such as autoimmune diseases, the binding of an autoantibody to a target may overcome this protective effect of MCP via the classical pathway.
Animals↗
Membrane cofactor protein (MCP, CD46) in seminal plasma and on spermatozoa in normal and "sterile" subjects.
A sperm protein of molecular mass 43 kDa (the spermatozoa membrane cofactor protein, smMCP) and a seminal plasma protein of 60 kDa (ssMCP) were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with four monoclonal antibodies (mAb) against membrane cofactor protein (MCP, CD46). These proteins served as factor I cofactors for the cleavage of methylamine-treated C3 (C3ma), the activity of which was blocked by M75, an MCP cofactor-activity-blocking mAb. Thus, these semen proteins are antigenic and functional homologous of MCP. On SDS-PAGE analysis these MCP migrated as single-band proteins which differed from the two-band forms of MCP expressed on other cells. smMCP was N-glycosylated but not O-glycosylated, while ssMCP was O-glycosylated: after deglycosylation of these proteins bands were detected at 38-40 kDa and 43 kDa on SDS-PAGE, respectively. These semen MCP are therefore, structurally different from the conventional MCP. ssMCP in both normal and "sterile" subject groups was determined by sandwich enzyme-linked immunosorbent assay. Seminal plasma in the two groups contained 250-700 ng/ml ssMCP. The difference between the two groups was marginal, although samples from normal subjects tended to show higher concentrations of ssMCP than samples from "sterile" subjects. No molecular difference was observed with ssMCP and smMCP in the two groups by SDS-PAGE/immunoblotting analysis. Immunohistochemical analysis suggested that MCP was positive in glandular epithelial cells and the lumen of the prostate, and in most intra-lumen cells of the testis. Using antibody M177, solubilized prostate and testis were analyzed by immunoblotting and compared with other cell MCP. The major band of MCP in the testis, but not in the prostate, was of 60 kDa, which aligned with ssMCP. No band of testis or prostate MCP, however, aligned with smMCP. ssMCP may be produced in the testis, while the origin of smMCP remains unknown. We hypothesize that ssMCP is important in the survival of spermatozoa, protecting them against local secretion of immunoglobulin and complement in the female genital tract, and that smMCP, which is expressed on acrosome-reacted spermatozoa, plays an essential role in the interaction of spermatozoa with oocytes.
Antigens, CD↗
Homologous C3 deposition and homotypic cell adhesion in a human myeloid cell line, P39.
It has been accepted that decay-accelerating factor (DAF) and membrane cofactor protein (MCP) on human cells block C3 deposition, thereby preventing homologous complement attack. In this study, we discovered that a human myeloid cell line, P39, was a target for human C3 even though it expressed normal DAF and MCP. This homologous C3 deposition was induced by serum containing Mg2+ and EGTA (Mg(2+)-EGTA serum) selectively on a P39 subline [P39(+)cells] having the capacity to form cell aggregates. Another P39 subline [P39(-)cells] growing as a separated form did not induce homologous C3 deposition. Multiple C3 fragments, C3b and C3bi, were fixed on P39(+) cells and a significant amount of C5a was released. Several distinct C3 fragment-membrane acceptor molecule complexes were immunoprecipitated with anti-C3c antibody from surface-labeled P39(+) cells treated with Mg(2+)-EGTA serum and from unlabeled cells incubated with 125I-labeled C3 and Mg(2+)-EGTA serum. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed 90, 60 and < or = 40 kDa C3 acceptors on P39(+) cells. On these cells, some of the C3 bound to these acceptors remained in the form of C3b, which can form C3/C5 convertases. P39(+) cells differed phenotypically from P39(-) cells in that P39(+) cells expressed intercellular adhesion molecule-1 (ICAM-1), whereas P39(-) cells did not. Both cells were lymphocyte function-associated antigen 1 (LFA-1)+/CR3- counter-receptors for ICAM-1. However, homotypic cell adhesion was not completely inhibited by antibodies against ICAM-1 and LFA-1, suggesting that the homotypic cell aggregation of P39(+) cells is due only in part to ICAM-1 and LFA-1. In addition, C3 deposition, the expression of ICAM-1, and cell aggregation were enhanced by both tumor necrosis factor-alpha and interferon-gamma. Although the principal causative mechanisms remain obscure, C3 deposition and cell adhesion appear in parallel in this cell line and may be involved in the modulation of cell-mediated immune reactions.
Cell Adhesion↗
Implication of membrane factors other than DAF and CD59 in complement-mediated lysis of paroxysmal nocturnal hemoglobinuria erythrocytes.
Erythrocytes from two patients (F.K. and A.M.) with paroxysmal nocturnal hemoglobinuria, which were almost completely deficient in decay-accelerating factor and CD59, were found to differ in their susceptibility to homologous complement. Whereas 50-70% of F.K. erythrocytes were lysed, almost 100% of the erythrocytes from A.M. were lysed. These observations were seen under both acidified and nonacidified conditions, and regardless of whether or not the normal human serum was adsorbed with normal erythrocytes to remove natural antibodies. Erythrocytes from two other patients, J.S. and Y.K., about 85% of which did not express CD59, showed lytic profiles similar to those of patient F.K. The differences between patients were not related to levels of natural antibody or other membrane regulatory proteins such as complement receptor type I or membrane cofactor protein. Erythrocytes from F.K. and A.M. differed in their reconstitution with decay accelerating factor and CD59. While erythrocytes from F.K. were reconstituted with CD59 in a unimodal pattern, erythrocytes from A.M. showed a bimodal pattern of reconstitution assessed by flow cytometry. After reconstitution with CD59, erythrocytes from F.K. and A.M. differed in their protection against homologous complement. It is concluded that erythrocyte membrane constituents other than the known inhibitors differ in these patients.
Antigens, CD↗
Acute promyelocytic leukemia with CD59 deficiency.
CD59 is a phosphatidyl inositol-anchored protein (which is lost in paroxysmal nocturnal hemoglobinuria (PNH) cells) with the capacity to block the formation of membrane attack complex, and protects host cells from autologous complement-mediated cytolysis. We found a patient with acute promyelocytic leukemia (APL) accompanied by disseminated intravascular coagulation (DIC), the cells of which were CD59-negative. Although the CD59 deficiency in the malignant cells was not related to PNH, we offered the possibility that DIC was induced by APL lysis secondary to the deficiency of CD59.
Aged↗
Functional properties of the allotypes of mouse complement regulatory protein, factor H: difference of compatibility of each allotype with human factor I.
Three allotypes of mouse factor H, H.1, H.2, and H.3 were purified from the sera of mice with different factor H allotypes, and their functional properties were investigated. The three allotypes all bound to heparin, DNA, Con A, and methylamine-treated mouse C3 (C3(MA)mo) with similar affinities for each protein immobilized, showed identical mobilities on SDS-PAGE, and were reacted well with rabbit polyclonal antibody against H.1 and H.2. Factor I-cofactor activity of these factor H allotypes was measured using highly purified material of mouse, guinea-pig, and human origin. In a homologous system, these allotypes expressed indistinguishable mouse factor I (Imo)-cofactor activity for the cleavage of C3(MA)mo. Imo-cofactor activity was again indistinguishable in these allotypes when methylamine-treated human C3 (C3(MA)hu) or methylamine-treated guinea-pig C3 (C3(MA)gp) was substituted for the C3(MA)mo substrate. The cofactor activity of these factor H allotypes, however, was augmented 4-5 times if C3(MA)hu) was used instead of C3(MA)mo, and was barely detected if C3(MA)gp was employed. In contrast, differences in the potency of the cofactor activity for the three allotypes were revealed if human factor 1 (Ihu) was substituted for Imo: the order of the efficiency for the cleavage of C3(MA)hu was H.2 > H.1 = H.3. These results, taken together with the finding that the homologous combinations of mouse and human factors H and I expressed greater activity for the cleavage of C3(MA)hu than did the heterologous combinations of factor H and factor I, suggest that mouse factor H allotypes discriminate species of protease factor I but not those of substrate (C3(MA), and H.2 possesses the best compatibility for Ihu in C3(MA)hu inactivation.
Animals↗
A monoclonal antibody against human decay-accelerating factor (DAF, CD55), D17, which lacks reactivity with semen-DAF.
Human decay-accelerating factor (DAF, CD55) is a phosphatidyl inositol-anchored glycoprotein consisting, from the N-terminus, of 4 short consensus repeats (SCR), a Ser/Thr (ST)-rich region providing O-glycosylation sites, and the membrane-anchoring unit. A mAb, named D17, was raised against purified erythrocyte-DAF. This mAb recognized DAF on blood cells and most cell lines as determined by flow cytometry and immunoblotting. Its reactivity was similar to but weaker than that of two other well-characterized mAbs to DAF, IA10 (seeing an epitope within SCR1) and 1C6 (seeing an epitope within SCR3). The reactivity of D17 with erythrocyte DAF became increased by treatment with sialidase/O-glycanase, suggesting that its epitope is located close to the O-glycosylation sites, probably within the ST-rich region or SCR4. D17 barely blocked the decay-accelerating activity of DAF. Using the three mAbs, tissue-associated and soluble forms of DAF were identified by SDS-PAGE/immunoblotting and immunohistochemical staining. IA10 and 1C6 recognized a 50 kDa protein in spermatozoa lysate and two proteins of Mr 70 and 55 kDa, respectively, in seminal fluid. These represented membrane-associated and soluble forms of DAF, which were neither recognized by mAb against membrane cofactor protein (MCP, CD46) and C3b/C4b receptor (CR1, CD35) nor by non-immune IgG. In contrast to IA10 and 1C6, D17 did not recognize either spermatozoa-DAF or seminal plasma-DAF, or the deglycosylated or untreated forms of them. Immunohistochemical analysis showed that testis was stained with IA10 but not with D17.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Prolonging discordant xenograft survival with anticomplement reagents K76COOH and FUT175.
The guinea pig heart, when transplanted into the rat heterotopically, is rejected within 30 min via activation of the alternative complement pathway. Natural antibody does not contribute to rejection. This xenotransplantation model was used to assess the effect of anti-complement reagents on discordant xenograft survival. In vivo administration of K76COOH (K76) to rats induced only slight suppression of factors B and D and a marked decrease of C3, leading to the depression of ACH50 (reflecting the potency of the alternative pathway). On the other hand, FUT175 (FUT) reduced C3 activity by about 80% and inhibited factor B activity nearly 100% < 1 hr after the administration, but inhibited factor D activity only marginally. FUT abrogated ACH50 for > 6 hr. Of note, the xenograft beating time was prolonged approximately 3 times by FUT but not by K76, suggesting that direct inhibition of plasma serine protease factor B results in the complete suppression of ACH50 and graft survival. The administration of both K76 and FUT resulted in the longest graft survival, but the effects of these reagents were abolished by additional antigraft antibody. Anticomplement reagents that block factor B and C3 are therefore effective for prolongation of discordant xenograft survival when the graft rejection is associated with the complement alternative pathway.
Animals↗