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Glycosylation of the N-terminal potential N-glycosylation sites in the human alpha1,3-fucosyltransferase V and -VI (hFucTV and -VI).

Abstract：

:Human alpha1,3-fucosyltransferase V and -VI (hFucTV and -VI) each contain four potential N-glycosylation sites (hFucTV: Asn60, Asn105, Asn167 and Asn198 and hFucTVI: Asn46, Asn91, Asn153 and Asn184). Glycosylation of the two N-terminal potential N-glycosylation sites (hFucTV: Asn60, Asn105 and hFucTVI: Asn46 and Asn91) have never been studied in detail. In the present study, we have analysed the glycosylation of these potential N-glycosylation sites. Initially, we compared the molecular mass of hFucTV and -VI expressed in COS-7 cells treated with tunicamycin with the mass of the proteins in untreated cells. The difference in molecular mass between the proteins in treated and untreated cells corresponded to the presence of at least three N-linked glycans. We then made a series of mutants, in which the asparagine residues in the N-terminal potential N-glycosylation sites were replaced by glutamine. Western blotting analyses demonstrated that both sites in hFucTV were glycosylated, whereas in hFucTVI only one of the sites (Asn91) was glycosylated. All the single mutants and the hFucTVI N46Q/N91Q double mutant exhibited enzyme activities that did not differ considerably from the wt activities. However, the enzyme activity of the hFucTV N60Q/N105Q double mutant was reduced to approximately 40% of the wt activity. In addition, castanospermine treatment diminished the enzyme activity and hence trimming of the N-linked glycans are required for expression of full enzyme activity of both hFucTV and -VI. The present study demonstrates that both of the N-terminal potential N-glycosylation sites in hFucTV and one of the sites in hFucTVI are glycosylated. Individually, their glycosylation does not contribute considerably to expression of enzyme activity. However, elimination of both sites in hFucTV reduces the enzyme activity.

journal_name

Glycoconj J

journal_title

Glycoconjugate journal

authors

Christensen LL,Bross P,Ørntoft TF

doi

10.1023/a:1010917229243

subject

Has Abstract

pub_date

2000-12-01 00:00:00

pages

859-65

issue

eissn

0282-0080

issn

1573-4986

journal_volume

pub_type

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Glycosylation of the N-terminal potential N-glycosylation sites in the human alpha1,3-fucosyltransferase V and -VI (hFucTV and -VI).