N-GLYCOSYLATION SITE MAPPING OF RECOMBINANT TISSUE-PLASMINOGEN ACTIVATOR BY MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY

This report describes the N-glycosylation mapping of recombinant tissu e plasminogen activator (rt-PA) using micellar electrokinetic capillar y chromatography. The carbohydrate structures were tentatively assigne d by comparison with the anion-exchange fractionated oligosaccharides and by a comparison with previously reported data, The separation was shown to rely mainly on the degree of sialylation of the oligosacchari des, allowing a quantitative determination of the proportion of neutra l and mono- to tetrasialylated structures, Significant differences in the oligosaccharide distribution of the two variants of rt-PA, which d iffer by the presence (type I) or the absence (type II) of oligosaccha rides at the Asn-184 site, were observed, The distribution of the olig osaccharides al each of the rt-PA glycosylation sites was then determi ned, Glycopeptides were prepared by tryptic digestion of rt-PA and iso lated using two consecutive chromatographic procedures, The glycopepti des were finally treated with N-glycanase, and the resulting oligosacc harides were analysed by capillary electrophoresis, Oligosaccharide ma pping revealed that the Asn-448 and Asn-184 sites carry the same popul ation of complex-type oligosaccharides but that the relative amounts o f each oligosaccharide vary markedly, High-pH anion-exchange chromatog raphy performed on the desialylated oligosaccharides at each glycosyla tion site showed that the degree of microheterogeneity was related not only to the degree of sialylation but also to structural differences in the oligosaccharide sequences. From the results as a whole, we conc luded that the Asn-448 site contains a greater proportion of heavily s ialylated structures and has a higher degree of microheterogeneity, Th e Asn-117 site was demonstrated to contain a significant number of mon osialylated structures (18.4%) in addition to the high-mannose-type ol igosaccharides.