The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9

A human E membrane protein that inhibits lysis by the purified human C5b-9 proteins was isolated and characterized. After final purification, the protein migrated as an 18- to 20-kDa band by SDS-PAGE. Elution from gel slices and functional assay after SDS-PAGE (nonreduced) confirmed that all C5b-9 i...

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Veröffentlicht in:The Journal of immunology (1950) 1990-05, Vol.144 (9), p.3478-3483
Hauptverfasser: Rollins, SA, Sims, PJ
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Sims, PJ
description A human E membrane protein that inhibits lysis by the purified human C5b-9 proteins was isolated and characterized. After final purification, the protein migrated as an 18- to 20-kDa band by SDS-PAGE. Elution from gel slices and functional assay after SDS-PAGE (nonreduced) confirmed that all C5b-9 inhibitory activity of the purified protein resided in the 18- to 20-kDa band. Phosphatidylinositol-specific phospholipase C digestion of the purified protein abolished 50% of its C5b-9 inhibitory activity, and removed approximately 15% of the protein from human E. Western blots of normal and paroxysmal nocturnal hemoglobinuria E revealed an absence of the 18- to 20-kDa protein in the paroxysmal nocturnal hemoglobinuria E cells. The identity of this E protein with leukocyte Ag CD59 (P18, HRF20) was confirmed immunochemically and by N-terminal amino acid sequence analysis. A blocking antibody raised against the purified protein reacted with a single 18- to 20-kDa band on Western blots of human erythrocyte membranes. Prior incubation of human E with the F(ab) of this antibody increased subsequent lysis by the purified human C5b-9 proteins. Potentiation of C5b-9-mediated lysis was observed when erythrocytes were preincubated with this blocking antibody before C5b-9 assembly was initiated, or, when this antibody was added after 30 min, 0 degrees C incubation of C5b-8-treated E with C9. Chicken E incubated with purified CD59 were used to further characterize the mechanism of its C-inhibitory activity. Preincorporation of CD59 into these cells inhibited lysis by C5b-9, regardless of whether CD59 was added before or after assembly of the C5b-8 complex. When incorporated into the membrane, CD59 inhibited binding of 125I-C9 to membrane C5b-8 and reduced the extent of formation of SDS-resistant C9 polymer. The inhibitory effect of CD59 on 125I-C9 incorporation was most pronounced at near-saturating input of C9 (to C5b-8). By contrast, CD59 did not inhibit either C5b67 deposition onto the cell surface, or, binding of 125I-C8 to preassembled membrane C5b67. Taken together, these data suggest that CD59 exerts its C-inhibitory activity by limiting incorporation of multiple C9 into the membrane C5b-9 complex.
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Prior incubation of human E with the F(ab) of this antibody increased subsequent lysis by the purified human C5b-9 proteins. Potentiation of C5b-9-mediated lysis was observed when erythrocytes were preincubated with this blocking antibody before C5b-9 assembly was initiated, or, when this antibody was added after 30 min, 0 degrees C incubation of C5b-8-treated E with C9. Chicken E incubated with purified CD59 were used to further characterize the mechanism of its C-inhibitory activity. Preincorporation of CD59 into these cells inhibited lysis by C5b-9, regardless of whether CD59 was added before or after assembly of the C5b-8 complex. When incorporated into the membrane, CD59 inhibited binding of 125I-C9 to membrane C5b-8 and reduced the extent of formation of SDS-resistant C9 polymer. The inhibitory effect of CD59 on 125I-C9 incorporation was most pronounced at near-saturating input of C9 (to C5b-8). 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After final purification, the protein migrated as an 18- to 20-kDa band by SDS-PAGE. Elution from gel slices and functional assay after SDS-PAGE (nonreduced) confirmed that all C5b-9 inhibitory activity of the purified protein resided in the 18- to 20-kDa band. Phosphatidylinositol-specific phospholipase C digestion of the purified protein abolished 50% of its C5b-9 inhibitory activity, and removed approximately 15% of the protein from human E. Western blots of normal and paroxysmal nocturnal hemoglobinuria E revealed an absence of the 18- to 20-kDa protein in the paroxysmal nocturnal hemoglobinuria E cells. The identity of this E protein with leukocyte Ag CD59 (P18, HRF20) was confirmed immunochemically and by N-terminal amino acid sequence analysis. A blocking antibody raised against the purified protein reacted with a single 18- to 20-kDa band on Western blots of human erythrocyte membranes. Prior incubation of human E with the F(ab) of this antibody increased subsequent lysis by the purified human C5b-9 proteins. Potentiation of C5b-9-mediated lysis was observed when erythrocytes were preincubated with this blocking antibody before C5b-9 assembly was initiated, or, when this antibody was added after 30 min, 0 degrees C incubation of C5b-8-treated E with C9. Chicken E incubated with purified CD59 were used to further characterize the mechanism of its C-inhibitory activity. Preincorporation of CD59 into these cells inhibited lysis by C5b-9, regardless of whether CD59 was added before or after assembly of the C5b-8 complex. When incorporated into the membrane, CD59 inhibited binding of 125I-C9 to membrane C5b-8 and reduced the extent of formation of SDS-resistant C9 polymer. The inhibitory effect of CD59 on 125I-C9 incorporation was most pronounced at near-saturating input of C9 (to C5b-8). By contrast, CD59 did not inhibit either C5b67 deposition onto the cell surface, or, binding of 125I-C8 to preassembled membrane C5b67. Taken together, these data suggest that CD59 exerts its C-inhibitory activity by limiting incorporation of multiple C9 into the membrane C5b-9 complex.</description><subject>Antigens, Differentiation - physiology</subject><subject>Biological and medical sciences</subject><subject>CD59 Antigens</subject><subject>Complement</subject><subject>Complement C9 - metabolism</subject><subject>Complement Membrane Attack Complex - metabolism</subject><subject>Complement System Proteins - metabolism</subject><subject>Erythrocyte Membrane - physiology</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Fundamental immunology</subject><subject>Glycosylphosphatidylinositols</subject><subject>Humans</subject><subject>In Vitro Techniques</subject><subject>Macromolecular Substances</subject><subject>Membrane Proteins - physiology</subject><subject>Molecular immunology</subject><subject>Phosphatidylinositols - metabolism</subject><subject>Polymers</subject><subject>Polysaccharides - metabolism</subject><subject>Protein Binding</subject><issn>0022-1767</issn><issn>1550-6606</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1990</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpNkE2PFCEQhonRrLOrv8CYcDB66pHuARqOZtTVZBMv65lANTis0LTAOJl_L-OMH6dK6v2oyoPQi56sKaHy7YOPcT-nsO4pXcv1ho7iEVr1jJGOc8IfoxUhw9D1Ix-foutSHgghnAz0Cl31XLY1WaFyv7MYUlyCjXaunZ933via8hFrqP6nr0ecHN6-ZxJnW_xkC_Yz9rVg0IuGk14TNiHB9yZAykvKuvo0_47JtmtytNFkPVu8ZaaTz9ATp0Oxzy_zBn39-OF--6m7-3L7efvurgNKeO0GrpnT3GigkpqJCE4dFWRwQg-gnXSTGM0kGFgAB8y5aZzM6IbBTdTAJDY36PW5d8npx96WqqIvYENon6R9UaMcN1RI2oybsxFyKiVbp5bso85H1RN1Qq3-oFYNtZLqhLqlXl7q9yba6V_mzLbpry66LqCDawDAl782xgSh9HT8zdm28992B5-tKlGH0Ep7dTgc_jv4C-eTmR4</recordid><startdate>19900501</startdate><enddate>19900501</enddate><creator>Rollins, SA</creator><creator>Sims, PJ</creator><general>Am Assoc Immnol</general><general>American Association of Immunologists</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>19900501</creationdate><title>The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9</title><author>Rollins, SA ; Sims, PJ</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c406t-26a5fa6bac494bd0864f4802f8a2caf9fd87bd85ceccfc5ffd7db7f22fd4bcd83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1990</creationdate><topic>Antigens, Differentiation - physiology</topic><topic>Biological and medical sciences</topic><topic>CD59 Antigens</topic><topic>Complement</topic><topic>Complement C9 - metabolism</topic><topic>Complement Membrane Attack Complex - metabolism</topic><topic>Complement System Proteins - metabolism</topic><topic>Erythrocyte Membrane - physiology</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Fundamental immunology</topic><topic>Glycosylphosphatidylinositols</topic><topic>Humans</topic><topic>In Vitro Techniques</topic><topic>Macromolecular Substances</topic><topic>Membrane Proteins - physiology</topic><topic>Molecular immunology</topic><topic>Phosphatidylinositols - metabolism</topic><topic>Polymers</topic><topic>Polysaccharides - metabolism</topic><topic>Protein Binding</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Rollins, SA</creatorcontrib><creatorcontrib>Sims, PJ</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of immunology (1950)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Rollins, SA</au><au>Sims, PJ</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9</atitle><jtitle>The Journal of immunology (1950)</jtitle><addtitle>J Immunol</addtitle><date>1990-05-01</date><risdate>1990</risdate><volume>144</volume><issue>9</issue><spage>3478</spage><epage>3483</epage><pages>3478-3483</pages><issn>0022-1767</issn><eissn>1550-6606</eissn><coden>JOIMA3</coden><abstract>A human E membrane protein that inhibits lysis by the purified human C5b-9 proteins was isolated and characterized. After final purification, the protein migrated as an 18- to 20-kDa band by SDS-PAGE. Elution from gel slices and functional assay after SDS-PAGE (nonreduced) confirmed that all C5b-9 inhibitory activity of the purified protein resided in the 18- to 20-kDa band. Phosphatidylinositol-specific phospholipase C digestion of the purified protein abolished 50% of its C5b-9 inhibitory activity, and removed approximately 15% of the protein from human E. Western blots of normal and paroxysmal nocturnal hemoglobinuria E revealed an absence of the 18- to 20-kDa protein in the paroxysmal nocturnal hemoglobinuria E cells. The identity of this E protein with leukocyte Ag CD59 (P18, HRF20) was confirmed immunochemically and by N-terminal amino acid sequence analysis. A blocking antibody raised against the purified protein reacted with a single 18- to 20-kDa band on Western blots of human erythrocyte membranes. Prior incubation of human E with the F(ab) of this antibody increased subsequent lysis by the purified human C5b-9 proteins. Potentiation of C5b-9-mediated lysis was observed when erythrocytes were preincubated with this blocking antibody before C5b-9 assembly was initiated, or, when this antibody was added after 30 min, 0 degrees C incubation of C5b-8-treated E with C9. Chicken E incubated with purified CD59 were used to further characterize the mechanism of its C-inhibitory activity. Preincorporation of CD59 into these cells inhibited lysis by C5b-9, regardless of whether CD59 was added before or after assembly of the C5b-8 complex. When incorporated into the membrane, CD59 inhibited binding of 125I-C9 to membrane C5b-8 and reduced the extent of formation of SDS-resistant C9 polymer. The inhibitory effect of CD59 on 125I-C9 incorporation was most pronounced at near-saturating input of C9 (to C5b-8). By contrast, CD59 did not inhibit either C5b67 deposition onto the cell surface, or, binding of 125I-C8 to preassembled membrane C5b67. Taken together, these data suggest that CD59 exerts its C-inhibitory activity by limiting incorporation of multiple C9 into the membrane C5b-9 complex.</abstract><cop>Bethesda, MD</cop><pub>Am Assoc Immnol</pub><pmid>1691760</pmid><doi>10.4049/jimmunol.144.9.3478</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record>
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subjects Antigens, Differentiation - physiology
Biological and medical sciences
CD59 Antigens
Complement
Complement C9 - metabolism
Complement Membrane Attack Complex - metabolism
Complement System Proteins - metabolism
Erythrocyte Membrane - physiology
Fundamental and applied biological sciences. Psychology
Fundamental immunology
Glycosylphosphatidylinositols
Humans
In Vitro Techniques
Macromolecular Substances
Membrane Proteins - physiology
Molecular immunology
Phosphatidylinositols - metabolism
Polymers
Polysaccharides - metabolism
Protein Binding
title The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9
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