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Biological and biochemical properties of two Xenopus laevis N-acetylgalactosaminyltransferases with contrasting roles in embryogenesis.

Voglmeir J, Laurent N, Flitsch SL, Oelgeschläger M, Wilson IB - Comp. Biochem. Physiol. B, Biochem. Mol. Biol. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department für Chemie, Universität für Bodenkultur, Wien, Austria; Manchester Interdisciplinary Biocentre, University of Manchester, UK.

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Tandem mass spectrometric analysis of xGalNAcT products. A modified form of the EA2 peptide was employed in a MALDI-ToF-based assay and the fragmentation patterns of the substrate (A; m/z 1345), the mono- and di-glycosylated products of xGalNAc-T6 (B and C; m/z 1548 and 1751) and the sole product of xGalNAc-T16 (m/z 1548) were compared after overnight incubation. Series of putative y-fragments are annotated with amino acids (one-letter code) commencing with the C-terminal arginine residue (y1 ion of 175); a mass difference of 304 corresponds to a glycosylated threonine, whereas mass differences of − 17 may result from loss of ammonia from y-ions. The sequences of the glycosylated peptides are shown with the putatively-modified threonines indicated with asterisks.
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f0015: Tandem mass spectrometric analysis of xGalNAcT products. A modified form of the EA2 peptide was employed in a MALDI-ToF-based assay and the fragmentation patterns of the substrate (A; m/z 1345), the mono- and di-glycosylated products of xGalNAc-T6 (B and C; m/z 1548 and 1751) and the sole product of xGalNAc-T16 (m/z 1548) were compared after overnight incubation. Series of putative y-fragments are annotated with amino acids (one-letter code) commencing with the C-terminal arginine residue (y1 ion of 175); a mass difference of 304 corresponds to a glycosylated threonine, whereas mass differences of − 17 may result from loss of ammonia from y-ions. The sequences of the glycosylated peptides are shown with the putatively-modified threonines indicated with asterisks.

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).


Biological and biochemical properties of two Xenopus laevis N-acetylgalactosaminyltransferases with contrasting roles in embryogenesis.

Voglmeir J, Laurent N, Flitsch SL, Oelgeschläger M, Wilson IB - Comp. Biochem. Physiol. B, Biochem. Mol. Biol. (2014)

Tandem mass spectrometric analysis of xGalNAcT products. A modified form of the EA2 peptide was employed in a MALDI-ToF-based assay and the fragmentation patterns of the substrate (A; m/z 1345), the mono- and di-glycosylated products of xGalNAc-T6 (B and C; m/z 1548 and 1751) and the sole product of xGalNAc-T16 (m/z 1548) were compared after overnight incubation. Series of putative y-fragments are annotated with amino acids (one-letter code) commencing with the C-terminal arginine residue (y1 ion of 175); a mass difference of 304 corresponds to a glycosylated threonine, whereas mass differences of − 17 may result from loss of ammonia from y-ions. The sequences of the glycosylated peptides are shown with the putatively-modified threonines indicated with asterisks.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4291152&req=5

f0015: Tandem mass spectrometric analysis of xGalNAcT products. A modified form of the EA2 peptide was employed in a MALDI-ToF-based assay and the fragmentation patterns of the substrate (A; m/z 1345), the mono- and di-glycosylated products of xGalNAc-T6 (B and C; m/z 1548 and 1751) and the sole product of xGalNAc-T16 (m/z 1548) were compared after overnight incubation. Series of putative y-fragments are annotated with amino acids (one-letter code) commencing with the C-terminal arginine residue (y1 ion of 175); a mass difference of 304 corresponds to a glycosylated threonine, whereas mass differences of − 17 may result from loss of ammonia from y-ions. The sequences of the glycosylated peptides are shown with the putatively-modified threonines indicated with asterisks.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Department für Chemie, Universität für Bodenkultur, Wien, Austria; Manchester Interdisciplinary Biocentre, University of Manchester, UK.

Show MeSH
Related in: MedlinePlus