Abstract
The Drosophila melanogaster glucuronyltransferases dGlcAT-S and dGlcAT-P were reported to be expressed ubiquitously and results of in vitro activity assays indicate a functional redundancy. We analyzed both transferases in vivo and in vitro and could show significant differences in their activity towards N-and O-glycoproteins in vivo. While GlcAT-P is able to use N-linked N-acetyllactosamine chains and the O-linked T-antigen as a substrate to form non-sulfated HNK1- (GlcAβ1-3Galβ1-4GlcNAcβ1-) and glucuronyl-T-antigens in vivo, GlcAT-S adds glucuronic acid only to N-linked chains, thereby synthesizing only the non-sulfated HNK1-antigen.
Highlights
Despite the fact that Drosophila melanogaster has been used as a model organism for genetics since several decades, its application as a model organism for glycomic studies is still in its infancy
MUC1-VH was only recognised by mAb 114-2G11-A, confirming that mAb M6749 is specific for the non-sulfated HNK1-epitope while mAb 114-2G11-A generally detects terminal β1-3-linked glucuronic acid modifications
In comparison to Schneider 2 (S2) wt-cells, overexpression glucuronyltransferases, only minor quantitative differences in glycan glucuronylation were observed of dGlcAT-P led to a higher expression of the glucuronyl-T antigen as observed by an increase in the by MALDI-MS of the permethylated O-glycan chains
Summary
Despite the fact that Drosophila melanogaster has been used as a model organism for genetics since several decades, its application as a model organism for glycomic studies is still in its infancy. Previous studies on the glycome of the fruit fly Drosophila melanogaster have revealed restricted patterns of oligomannose N-linked sugar chains and simple mucin-type O-glycans dominated by the T-antigen (Galβ1-3GalNAcα-). About 20%–30% of the O-glycans in Drosophila embryos are modified with glucuronic acid [1] Despite this high percentage, a function of this modification has not been elucidated so far. To unravel the functional context of glucuronic acid in Drosophila, the elucidation of HNK-1 (GlcAβ1-3Galβ1-4GlcNAcβ-R) and glucuronyl-T (GlcAβ1-3Galβ1-3GalNAcα-R) biosynthesis are of utmost importance. GlcAT-P and GlcAT-S, on the other hand, have rigid substrate specificities towards the Galβ1-4GlcNAc epitope, synthesizing the non-sulfated HNK1-epitope (GlcAβ1-3Galβ1-4GlcNAcβ1-) on glycoproteins and glycolipids. The enzymes exhibit broad substrate specificity with formation of N-linked HNK-1 and O-linked glucuronyl-T antigens [7].
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