Naturally Occurring Anti-band 3 Antibodies Bind to Protein Rather Than to Carbohydrate on Band 3 (1)
Selective recognition and opsonization of senescent and oxidatively stressed red cells appears to involve increased anti-band 3 binding and C3b-deposition via the alternative complement pathway. An endogenous proteolytic modification of band 3 protein and the occurrence of a COOH-terminal 60-kDa polypeptide serve as a senescent cell antigen with binding sites involving amino acids 539-554 and 812-830. Oligomerization of band 3 protein within the plane of the membrane enhances anti-band 3 binding by favoring bivalent binding. This binding site is localized within the 55-kDa NH2-terminal fragment of band 3 protein which can be generated by treating intact red cells with chymotrypsin. Thus, it may include the domain formed by amino acids 539-554, but not the COOH-terminal domain.
Anti-band 3 antibodies bound to purified band 3 protein, its oligomers, and among red cell membrane proteins primarily to band 3 protein. Despite antibody preparations bound exclusively to the 55-kDa NH2-terminal. chymotryptic fragment of band 3 protein and not to the carbohydrate-rich 38-kDa fragment which also remained membrane-associated and was present among the electrophoresed polypeptides. The lack of reactivity with carbohydrate was further evident from the inability of lactofemn to inhibit anti-band 3 antibody binding to band 3 protein and its 55-kDa fragment on blots.
Endoglycosidase-H removed sialic acid from glycophorin and thereby altered the electrophoretic mobility of the 14C-labeled glycophorin band. The binding of their anti band 3 antibodies to oxidatively stressed red cells was apparently abolished by endo-p-galactosidase or neuraminidase. Neither endo-p-galactosidase nor neuraminidase destroyed the binding site for anti-band 3 antibodies on band 3 protein.
The predominant binding of anti-band 3 antibodies to the 55-kDa N-terminal chymotryptic fragment of band 3 protein and the results obtained with glycosidases call for peptide rather than carbohydrate structures as potential binding sites. The findings are not only incompatible with reactivity to carbohydrates, but also with the existence of an extra binding site in the region of amino acids 812-830. On the other hand, anti-band 3 binding to amino acids 812-830 is unlikely, because the published structures of band 3 protein contains these amino acids in a cytoplasmic loop.