N-glycosylation site mapping of recombinant tissue plasminogen activator by micellar electrokinetic capillary chromatography.
This report describes the N-glycosylation mapping of recombinant tissue plasminogen activator (rt-PA) using micellar electrokinetic capillary chromatography. The carbohydrate structures were tentatively assigned 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 oligosaccharides, allowing a quantitative determination of the proportion of neutral and mono- to tetrasialylated structures. Significant differences in the oligosaccharide distribution of the two variants of rt-PA, which differ by the presence (type I) or the absence (type II) of oligosaccharides at the Asn-184 site, were observed. The distribution of the oligosaccharides at each of the rt-PA glycosylation sites was then determined. Glycopeptides were prepared by tryptic digestion of rt-PA and isolated using two consecutive chromatographic procedures. The glycopeptides were finally treated with N-glycanase, and the resulting oligosaccharides were analysed by capillary electrophoresis. Oligosaccharide mapping revealed that the Asn-448 and Asn-184 sites carry the same population of complex-type oligosaccharides but that the relative amounts of each oligosaccharide vary markedly. High-pH anion-exchange chromatography performed on the desialylated oligosaccharides at each glycosylation 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 concluded that the Asn-448 site contains a greater proportion of heavily sialylated structures and has a higher degree of microheterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)
Taverna
M
M
Faculté de Pharmacie, Laboratoire de Chimie Analytique III, Chatenay-Malabry, France.
Baillet
A
A
Schlüter
M
M
Baylocq-Ferrier
D
D
D002853:Chromatography, Liquid / Q000379:methods*; D006031:Glycosylation; D008823:Micelles; D009844:Oligosaccharides / Q000737:chemistry; D010960:Plasminogen Activators / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
D008823:Micelles; D009844:Oligosaccharides; D011994:Recombinant Proteins; D010960:Plasminogen Activators