Abstract
CD59 antigen is a membrane glycoprotein that inhibits the activity of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The inhibitory function of CD59 derives from its capacity to interact with both the C8 and C9 components of MAC, preventing assembly of membrane-inserted C9 polymer. MAC-inhibitory activity of CD59 is species-selective and is most effective when both C8 and C9 derive from human or other primate plasma. Rabbit C8 and C9, which can substitute for human C8 and C9 in MAC, mediate virtually unrestricted lysis of human cells expressing CD59. In order to identify the segment of human C8 that is recognized by CD59, recombinant peptides containing human or rabbit C8 sequence were expressed in Escherichia coli and purified. CD59 was found to specifically bind to a peptide corresponding to residues 334-385 of the human C8 alpha-subunit, and to require a disulfide bond between Cys345 and Cys369. No specific binding was observed to the corresponding sequence from rabbit C8 alpha (residues 334-386). To obtain functional evidence that this segment of human C8 alpha is selectively recognized by CD59, recombinant C8 proteins were prepared by co-transfecting COS-7 cells with human/rabbit chimeras of the C8 alpha cDNA, and cDNAs encoding the C8 beta and C8 gamma chains. Hemolytic activity of MAC formed with chimeric C8 was analyzed using target cells reconstituted with CD59. These experiments confirmed that CD59 recognizes a conformationally sensitive epitope that is within a segment of human C8 alpha internal to residues 320-415. Our data also suggest that optimal interaction of CD59 with this segment of human C8 alpha is influenced by N-terminal flanking sequence in C8 alpha and by human C8 beta, but is unaffected by C8 gamma.
Highlights
From the Wlood Research Institute, Blood Center ofSoutheastern Wisconsin, Milwaukee, Wisconsin 53233 and the §Department of Chemistry and Biochemistry, School ofMedicine, University ofSouth Carolina, Columbia, South Carolina 29208
Interacts with both the C8 and C9 components of membrane attack complex (MAC) during its assembly at the cell surface, thereby inhibiting formation of the membrane-inserted C9 homopolymer responsible for MAC cytolytic activity [2, 3], This serves to protect hu blood and vascular cells from injury arising through activation of complement in plasma, CD59's inhibitory activity is dependent upon the species of origin of C8 and C9, with greatest inhibitory activity observed when C8 and C9 are from hu or other primates
Analysis ofthe physical association of CD59 with components of MAC suggested that separate binding sites for CD59 are contained within the a-chain ofhu C8 and within hu C9.(9) Consistent with the evidence for a CD 59 binding site in hu C8a, C8 hybrids formed by combining rb C8a-y with hu C8,B displayed unrestricted hemolytic activity toward hu erythrocytes [10]
Summary
Blood Center of Southeastern Wisconsin, P, O. Expression and Purification of Recombinant CBa Peptides-Fusion proteins containing the maltose-binding protein (MBP) and a defined segment ofhu or rb C8a were expressed in E. coli, purified, and cleaved with factor Xa to release recombinant C8a peptides. Construction of CB and Chimeric CBa Expression Vectors-To express hu or rb C8 in COS-7 cells, cDNAs encoding full-length C8a, C8f3, and C8y subunits were cloned into the expression vector pcDNA3 (Invitrogen) as described elsewhere.f The hulrb chimeras of C8a were prepared either in pBluescript II (Stratagene) or by modifying fulllength pcDNA3 constructs. ChE were washed extensively and suspended in GVBS, and the membrane C5b67 complex assembled by mixing cells (1.4 x 109/ml final concentration) with hu C5b6 (13 ILg/ml final concentration) followed by addition ofhu C7 (1 ug/ml). Hemolysis was determined after 30 min at 37 DC, with correction for nonspecific lysis (always
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