Biological and biochemical properties of two Xenopus laevis N-acetylgalactosaminyltransferases with contrasting roles in embryogenesis.
Bottom Line: Until now, the mammalian forms of these enzymes have been the best characterized.In terms of enzymatic activity, both enzymes were found to be active towards the EA2 peptide, but display differential activity towards a peptide based on the sequence of ActR-IIB, a receptor relevant to TGF-β/BMP signaling.In summary, these data demonstrate that these two enzymes from different branches of the N-acetylgalactosaminyltransferase do not only display differential substrate specificities, but also specific and distinct expression pattern and biological activities in vivo.
Affiliation: Department für Chemie, Universität für Bodenkultur, Wien, Austria; Manchester Interdisciplinary Biocentre, University of Manchester, UK.Show MeSH
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Mentions: The products of assays performed with a modified form of the EA2 peptide (PTTDSTTPAPTTR) were analysed by MS/MS in order to define the sites of glycosylation. As judged by the series of probable y-fragments (Fig. 3), xGalNAc-T6 modified first Thr7 of this peptide, probably followed by Thr12 for the diglycosylated species. On the other hand, xGalNAc-T16 appeared to solely glycosylate Thr7 under the assay conditions employed. Previous data on other N-acetylgalactosaminyltransferases would suggest that Thr7 is indeed a frequently favoured glycosylation site for those enzymes which can accept non-glycosylated peptides as substrates; in the crystal structure of human GalNAc-T2, Thr7 of the EA2 peptide is well located to be the glycosylated residue (Fritz et al., 2006). Also, human GalNAc-T1 and T11 as well as Drosophila GalNAc-T1 glycosylate either Thr6 and/or Thr7 (ten Hagen et al., 2001; Schwientek et al., 2002). The preference for xGalNAc-T6 then glycosylating the C-terminal threonine is in line with recent data on its bias towards singly glycosylated peptides where the initial glycosylation has taken place at a more N-terminal site (Gerken et al., 2013).
Affiliation: Department für Chemie, Universität für Bodenkultur, Wien, Austria; Manchester Interdisciplinary Biocentre, University of Manchester, UK.