IDENTITY OF THE SEGMENT OF HUMAN-COMPLEMENT C8 RECOGNIZED BY COMPLEMENT REGULATORY PROTEIN CD59

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C5b-9 membrane attack complex (MAC), thereby protecting human cell s from lysis by human complement, The inhibitory function of CD59 deri ves from its capacity to interact with both the C8 and C9 components o f MAC, preventing assembly of membrane-inserted C9 polymer, MAC-inhibi tory activity of CD59 is species-selective and is most effective when both C8 and C9 derive from human or other primate plasma, Rabbit CS an d C9, which can substitute for human CS and C9 in MAC, mediate virtual ly unrestricted lysis of human cells expressing CD59, In order to iden tify the segment of human C8 that is recognized by CD59, recombinant p eptides containing human or rabbit C8 sequence were expressed in Esche richia coli and purified. CD59 was found to specifically bind to a pep tide corresponding to residues 334-385 of the human CS alpha-subunit, and to require a disulfide bond between Cys(345) and Cys(369). NO spec ific binding was observed to the corresponding sequence from rabbit C8 alpha (residues 334-386). To obtain functional evidence that this seg ment of human C8 alpha is selectively recognized by CD59, recombinant C8 proteins were prepared by co-transfecting COS-7 cells with human/ra bbit chimeras of the C8 alpha cDNA, and cDNAs encoding the C8 beta and C8 gamma chains. Hemolytic activity of MAC formed with chimeric C8 wa s analyzed using target cells reconstituted with CD59. These experimen ts confirmed that CD59 recognizes a conformationally sensitive epitope that is within a segment of human C8 alpha internal to residues 320-4 15, Our data also suggest that optimal interaction of CD59 with this s egment of human C8 alpha is influenced by N-terminal flanking sequence in C8 alpha and by human C8 beta, but is unaffected by CS gamma.