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 activity against most mammalian erythrocytes, although the amino acid sequences of both proteins show 77% identity. In an attempt to define the region of human C9 responsible for conferring its lytic activity, or conversely, the region of equine C9 responsible for its restriction, recombinant 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 purified native proteins. Exchange of a fragment extending from residues 145 to 290 in horse C9 with the corresponding one from human C9 created a fully hemolytic protein. This region contains the putative hinge region but not the membrane-interacting domain. Nonlytic chimeric C9 proteins inhibited hemolysis and binding of human C9 to EAC1-8 cells, indicating that they bind to their receptor, but subsequent unfolding or insertion into the membrane is impaired. These results suggest that restriction factors, such as glycophorin, CD59, or homologous restriction factor, on erythrocytes may limit the activity of horse C9 by interacting 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 horse 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 be achieved by insertion of the human C9 hinge region into horse C9.
Tomlinson
S
S
Department of Comparative and Experimental Pathology, University of Florida Health Science Center, Gainesville 32610, USA.
Wang
Y
Y
Ueda
E
E
Esser
A F
AF
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001483:Base Sequence; D018957:CD59 Antigens; D002478:Cells, Cultured; D003186:Complement C9 / Q000737:chemistry* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006461:Hemolysis / Q000187:drug effects / Q000502:physiology*; D006736:Horses; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D015703:Antigens, CD; D018957:CD59 Antigens; D003186:Complement C9; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins