Localization of O-glycosylation sites on glycopeptide fragments from lactation-associated MUC1. All putative sites within the tandem repeat are glycosylation targets in vivo

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Item Type:	Article
Title:	Localization of O-glycosylation sites on glycopeptide fragments from lactation-associated MUC1. All putative sites within the tandem repeat are glycosylation targets in vivo
Creators Name:	Mueller, S., Goletz, S., Gooley, A., Lawson, A.M. and Hanisch, F.G.
Abstract:	Since there is no consensus sequence directing the initial GalNAc incorporation into mucin peptides, O-glycosylation sites are not reliably predictable. We have developed a mass spectrometric sequencing strategy that allows the identification of in vivo O-glycosylation sites on mucin-derived glycopeptides. Lactation-associated MUC1 was isolated from human milk and partially deglycosylated by trifluoromethanesulfonic acid to the level of core GalNAc residues. The product was fragmented by the Arg-C-specific endopeptidase clostripain to yield tandem repeat icosapeptides starting with the PAP motif. PAP20 glycopeptides were subjected to sequencing by post-source decay matrix-assisted laser desorption ionization mass spectrometry or by solid phase Edman degradation to localize the glycosylation sites. The masses of C- or N-terminal fragments registered for the mono- to pentasubstituted PAP20 indicated that GalNAc was linked to the peptide at Ser5,Thr6 (GSTA) and Thr14 (VTSA) but contrary to previous in vitro glycosylation studies also at Thr19 and Ser15 located within the PDTR or VTSA motifs, respectively. Quantitative data from solid phase Edman sequencing revealed no preferential glycosylation of the threonines. These discrepancies between in vivo and in vitro glycosylation patterns may be explained by assuming that O-glycosylation of adjacent peptide positions is a dynamically regulated process that depends on changes of the substrate qualities induced by glycosylation at vicinal sites.
Keywords:	Acetylgalactosamine, Amino Acid Sequence, Gel Chromatography, Glycopeptides, Glycosylation, High Pressure Liquid Chromatography, Human Milk, Lactation, Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry, Mesylates, Molecular Sequence Data, Mucin-1
Source:	Journal of Biological Chemistry
ISSN:	0021-9258
Publisher:	American Society for Biochemistry and Molecular Biology
Volume:	272
Number:	40
Page Range:	24780-24793
Date:	3 October 1997
Official Publication:	http://www.jbc.org/content/272/40/24780.abstract
PubMed:	View item in PubMed

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