Endoglycosidase H digestion of Yukb (Pena) alloantigen

We characterized a platelet specific alloantigen (Yukb). In immunoblotting, anti-Yukb 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 Yukb and PlA1 antigens by the electrophoresis. Yukb antigen, unlike PlA1 antigen, was partially destroyed by chymotrypsin treatment. Furthermore, endoglycosidase H digestion resulted in loss of the Yukb epitope, while the PlA1 determinant was retained on the three bands with lower molecular weights after endoglycosidase H digestion. The transfer of Yukb antigens recognized by anti-Yukb antibody into the supernatant of platelets treated with endoglycosidase H was also found using mixed passive hemagglutination test. The results indicated that the Yukb 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 PlA1 epitope.