Structural study of the glycosylated and unglycosylated forms of a genetic variant of human serum albumin (63 Asp--Asn)

A genetic variant of human serum albumin (alloalbumin) exhibited atypical electrophoretic mobility and chromatographic behavior apparently because of the effect of a point substitution on the molecular conformation. Three forms of albumin were isolated by DEAE HPLC chromatography: normal albumin, and two variant forms V1 and V2. The point substitution (Asp-63 → Asn) generated a canonical tripeptide acceptor sequence for glycosylation with an N-linked oligosaccharide (Asn-Lys-Ser). Neuraminidase digestion followed by electrophoresis showed that the V2 variant form was glycosylated and the V1 form was not. Time-of-flight mass spectrometry yielded a molecular weight of about 2000 for the carbohydrate. Structural analysis of the carbohydrate was done by chromatographic comparison of the pyridylaminated derivatives with standards and was confirmed by proton NMR of the three pronase glycopeptides and of the pyridylaminated oligosaccharide. The oligosaccharide had a complex biantennary structure with two sialic acid residues. In normal albumin Asp-63 is exposed and is adjacent to the first disulfide bond, Cys-62 → Cys-53. The apparent effect on molecular conformation resulting in incomplete glycosylation and atypical electrophoretic mobility suggests that glycosylation may interfere with disulfide bond formation at this site.