Abstract
O-glycosylated proteins are ubiquitous in eukaryotes and are responsible for a variety of biological functions. O-glycosylation is initiated by the addition of N-acetylgalactosamine to serine or threonine residues, though it is not clear how specific residues are selected for modification. We have compared serine and threonine glycosylation using peptide substrates based on sequences from erythropoietin (EPO) and von Willebrand factor (HVF) that are glycosylated in vivo. UDP-GaINAc :polypeptide N-acetylgalactosaminyltransferase was derived from rat parotid, submandibular, and sublingual glands, liver and kidney as well as from human colostrum. The threonine-containing substrates were glycosylated to a much greater extent than those containing serine for all the enzyme sources. Changes in reaction pH, donor concentration, or divalent cation were unable to increase glycosylation of serine. When the incubation time was extended, serine in the EPObased peptide was found to incorporate GalNAc at a low level, in contrast to the serine-containing HVF peptide, which did not glycosylate at all. By circular dichroism, the non-glycosylating peptide was the only one of the series that did not exhibit random coil structure. Our data suggest that although the structural and sequence requirements for O-glycosylation of serine and threonine residues are similar, serine sites are glycosylated less effectively than are threonine sites in vitro.
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