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 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.
Lockert
D H
DH
Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee 53233, USA.
Kaufman
K M
KM
Chang
C P
CP
Hüsler
T
T
Sodetz
J M
JM
Sims
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D001665:Binding Sites; D018957:CD59 Antigens; D002460:Cell Line; D003185:Complement C8 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003169:Complement Inactivator Proteins / Q000378:metabolism; D000939:Epitopes; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D015703:Antigens, CD; D018957:CD59 Antigens; D003185:Complement C8; D003169:Complement Inactivator Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins