Site-specific core 1 O-glycosylation pattern of the porcine submaxillary gland mucin tandem repeat. Evidence for the modulation of glycan length by peptide sequence.
The sequence-specific O-linked core 1 ([R1, R2]-beta-Gal(1-3)-alpha-GalNAc-O-Ser/Thr) glycosylation pattern has been quantitatively determined for 30 of the 31 Ser/Thr residues in the 81-residue porcine submaxillary gland mucin tandem repeat. This was achieved by Edman amino acid sequencing of the isolated tandem repeat after selective removal of non-C3-substituted, peptide-linked GalNAc residues by periodate oxidation and subsequent trimming of the remaining oligosaccharides to peptide-linked GalNAc residues by mild trifluoromethanesulfonic acid/anisole treatment. The sequencing reveals 61% (range, 12-95%) of the peptide alpha-N-acetylgalactosamine (GalNAc) residues to be substituted by core 1 chains, a value in agreement with the carbon-13 NMR analysis of the native mucin. Residues with the lowest C3 substitution were typically clustered in regions of sequence with the highest densities of (glycosylated) serine or threonine. This suggests that the porcine beta3-Gal, core 1, transferase is sensitive to peptide sequence and/or neighboring core GalNAc glycosylation in vivo, in keeping with earlier in vitro enzymatic glycosylation studies (Granovsky, M., Blielfeldt, T., Peters, S., Paulsen, H., Meldal, M., Brockhausen, J., and Brockhausen, I. (1994) Eur. J. Biochem. 221, 1039-1046). These results demonstrate that the O-glycan structures in mucin domains are not necessarily uniformly distributed along the polypeptide core and that their lengths can be modulated by peptide sequence. The data further suggest that hydroxyamino acid spacing may contribute to the regulation of glycan length, thereby, providing a mechanism for maintaining an optimally expanded, protease resistant, mucin conformation.
W. A. Bernbaum Center for Cystic Fibrosis Research, Case Western Reserve University, Cleveland, Ohio 44106-4948, USA. tag@glycocon.cwru.edu
T A
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Gerken
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Owens
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Pasumarthy
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D006031:Glycosylation; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000737:chemistry*; D013363:Submandibular Gland / Q000378:metabolism*; D013552:Swine; D020080:Tandem Repeat Sequences
D009077:Mucins; D011134:Polysaccharides