Endoglycosidase H digestion of Yuk b(Pen a) alloantigen

Abstract

We characterized a platelet specific alloantigen (Yuk b). In immunoblotting, anti-Yuk b antibody was found to react with both 110 kDa and 96 kDa bands under nonreducing condition. Immunoblotting followed by separation by two-dimensional electrophoresis (isoelectric focusing/SDS-PAGE) showed that the 96 kDa band had a pI of 5.1–5.8, while the 110 kDa band had a pI of 5.2–5.7. The 96 kDa band was identified as glycoprotein (GP) IIIa on the basis of periodic acid Schiff staining, but the 110 kDa band was not characterized. These results implied that it is difficult to determine differences in antigenic epitopes between Yuk b and P1 A1 antigens by the electrophoresis. Yuk b antigen, unlike P1 A1 antigen, was partially destroyed by chymotrypsin treatment. Furthermore, endoglycosidase H digestion resulted in loss of the Yuk b epitope, while the P1A1 determinant was retained on the three bands with lower molecular weights after endoglycosidase H digestion. The transfer of Yuk b antigens recognized by anti-Yuk b antibody into the supernatant of platelets treated with endoglycosidase H was also found using mixed passive hemagglutination test. The results indicated that the Yuk b epitope might be located to the endoglycosidase H digested N-linked high mannose carbohydrate chains of GP IIIa or hybrid type chains of GP IIIa, which is different from the location of P1 A1 epitope.