Differential enzymatic deglycosylation reveals attachment of red cell B antigen onto the carbohydrate moiety of glycophorin A and glycophorin B.

Background and Objectives: Early studies indicate that red cell A and B antigens are attached primarily onto band 3 and GLUT1 on the erythrocyte membrane and little onto glycophorin A (GPA) and glycophorin B (GPB). But as GPA and band 3 form stable protein complexes and GPA is much more heavily glycosylated than band 3, this study re‐examined the association between ABO antigens and GPA/GPB. Materials and Methods: Band 3/GPA‐associated protein complexes were first immunoprecipitated, followed by differential enzymatic deglycosylation that removed sialic acids, N‐glycans and O‐glycans. Serological anti‐A (BIRMA 1) and anti‐B IgM (GAMA 110) could be used for western blot (WB); however, only the anti‐B IgM showed significant reactivity for the immunoprecipitates isolated by anti‐band 3. The expression of the B antigen in un‐deglycosylated and differentially deglycosylated band 3 immunoprecipitates was thus compared. Results: Besides attachment to band 3, red cell B antigen expressed substantially on GPA monomer and homodimer, GPA*GPB heterodimer, and GPB monomer and dimer via attachments through the N‐ and O‐glycans. Conclusion: Immunoprecipitation (IP), as a means of protein separation and concentration, was used in combination with a WB to differentiate glycosylation on different proteins and oligomers. This study implemented differential enzymatic deglycosylation during IP of the band 3 complexes. This combined approach allowed separate identification of the B antigen on GPA/GPB monomer and dimer and GPA*GPB heterodimer, and band 3 on the WB and verified non‐trivial expression of the B antigen on GPA and GPB on the erythrocyte surface. [ABSTRACT FROM AUTHOR]