A rapid method for the isolation of analogues of human CD59 by preparative SDS-PAGE: application to pig CD59.
A method for the rapid isolation of functionally active analogues of human CD59 from erythrocytes (E) is described. The method, here applied to pig E, is based on the fractionation of a butanol extract of E ghosts by preparative SDS-PAGE followed by gel filtration on Superose 12. Purification was monitored using a functional complement inhibition assay. SDS-PAGE analysis of the product of this procedure indicated a single protein band with apparent M(r) of 20 kDa under reducing and non-reducing conditions. The preparation could be incorporated into guinea pig E to inhibit both CVF-reactive lysis and lysis through C8 and C9 using preformed C5b-7 sites, demonstrating that it contained a CD59-like activity. PIPLC treatment of the isolated protein abolished the inhibition. In contrast to SDS-PAGE analysis, amino-terminal sequence analysis of the preparation showed that it comprised two components. One was identified from databank searches as a fragment of pig glycophorin. These two components could not be separated by either standard or affinity chromatographic techniques. The second component was novel and had high sequence homology with human CD59, identifying it as the pig analogue. Further functional studies showed that the pig analogue of human CD59 was effective in the protection of guinea pig E against lysis by serum from a variety of species, including human.
van den Berg
C W
CW
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Harrison
R A
RA
Morgan
B P
BP
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000302:isolation & purification*; D018957:CD59 Antigens; D004546:Elapid Venoms / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004910:Erythrocyte Membrane / Q000737:chemistry; D017261:Glycosylphosphatidylinositols; D006801:Humans; D008562:Membrane Glycoproteins / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine
D015703:Antigens, CD; D018957:CD59 Antigens; D004546:Elapid Venoms; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; C016310:cobra venom factor; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase