CHIMERIC HORSE HUMAN RECOMBINANT C9 PROTEINS IDENTIFY THE AMINO-ACID-SEQUENCE IN HORSE C9 RESPONSIBLE FOR RESTRICTION OF HEMOLYSIS

Equine C9, in contrast to human C9, has extremely low hemolytic activi ty against must mammalian erythrocytes, although the amino acid sequen ces of both proteins show 77% identity, In an attempt to define the re gion of human C9 responsible for conferring its lytic activity, or con versely, the region oi equine C9 responsible for its restriction, reco mbinant human and equine C9 and four chimeric human/equine C9 proteins were constructed and expressed in COS-7 cells. Recombinant human and equine C9 displayed hemolytic profiles similar to those of the purifie d native proteins. Exchange of a fragment extending from residues 145 to 240 in horse C9 with the corresponding one from human C9 created a fully hemolytic protein, This region contains the putative hinge regio n but not the membrane-interacting domain. Nonlytic chimeric C9 protei ns inhibited hemolysis and binding of human C9 to EAC1-8 cells, indica ting that they bind to their receptor, but subsequent unfolding or ins ertion into the membrane is impaired. These results suggest that restr iction factors, such as glycophorin, CD59, or homologous restriction f actor, on erythrocytes may limit the activity of horse C9 by interacti ng with its hinge region, In support of this conclusion direct binding of CD59 to immobilized horse C9 was detected by ligand blotting, and it was observed that a polyclonal anti-CD59 Ab enhanced human and hors e C9-mediated hemolysis of human EAC1-7, but the increase in hemolytic activity of horse C9 by inhibition of CD59 was less than what could b e achieved by insertion of the human C9 hinge region into horse C9.