Isolation and characterization of a membrane protein from rat erythrocytes which inhibits lysis by the membrane attack complex of rat complement.
The membrane attack complex (MAC) of complement in humans is regulated by several membrane-bound proteins; however, no such proteins have so far been described in other species. Here we report the isolation and characterization of a rat erythrocyte membrane glycoprotein of molecular mass 21 kDa which inserts into cell membranes and is a potent inhibitor of the rat MAC. This protein, here called rat inhibitory protein (RIP), was first partially purified by column chromatography from a butanol extract of rat erythrocyte membranes. Monoclonal antibodies (Mabs) were raised against RIP and used for its affinity purification. Affinity-purified RIP was shown to inhibit in a dose-dependent manner the cobra venom factor (CVF)-mediated 'reactive' lysis of guinea pig erythrocytes by rat complement. Conversely, the anti-RIP MAbs 6D1 and TH9 were shown to markedly enhance the CVF-mediated lysis of rat erythrocytes by rat complement. RIP acted late in the assembly of the MAC (at or after the C5b-8 stage) and was releasable from the membranes of rat erythrocytes by phosphatidylinositol-specific phospholipase C. These features, together with its size, deglycosylation pattern and N-terminal amino acid sequence, lead us to conclude that RIP is the rat homologue of the human MAC-inhibitory protein CD59 antigen.
Hughes
T R
TR
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, U.K.
Piddlesden
S J
SJ
Williams
J D
JD
Harrison
R A
RA
Morgan
B P
BP
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000502:physiology; D018957:CD59 Antigens; D002845:Chromatography; D015938:Complement Membrane Attack Complex / Q000037:antagonists & inhibitors*; D004910:Erythrocyte Membrane / Q000737:chemistry* / Q000502:physiology; D006031:Glycosylation; D006168:Guinea Pigs; D006801:Humans; D008562:Membrane Glycoproteins / Q000097:blood* / Q000302:isolation & purification / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D051381:Rats; D012756:Sheep
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018957:CD59 Antigens; D015938:Complement Membrane Attack Complex; D008562:Membrane Glycoproteins; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase