Glycobiology Advance Access published online on December 10, 2008
Glycobiology, doi:10.1093/glycob/cwn143
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Systematic determination of the peptide acceptor preferences for the human UDP-Gal: glycoprotein-
-GalNAc β3 galactosyltranferase (T-synthase)
2 Departments of Chemistry
4 Biochemistry and Pediatrics, Case Western Reserve University, Cleveland, OH 44106
3 Department of Biochemistry Emory University, Atlanta, GA 30322
1 To whom correspondence should be addressed: Tel:+1-216-368-4556; Fax: +1-216-368-4223; e-mail: txg2{at}cwru.edu
Received on November 11, 2008; accepted on December 8, 2008
Mucin-type protein O-glycosylation is initiated by the addition of 
-GalNAc to Ser/Thr residues of a polypeptide chain. The addition of β-Gal to GalNAc by the UDP-Gal: glycoprotein-–GalNAc β3 galactosyltransferase (T-synthase), forming the Core 1 structure (β-Gal(1–3)- -GalNAc-O-Ser/Thr), is a common and biologically significant subsequent step in O-glycan biosynthesis. What dictates the sites of Core 1 glycosylation is poorly understood, however, peptide sequence and neighboring glycosylation effects have been implicated (Eur. J. Biochem. 221:1039–1046; J. Biol. Chem. 277:7736–7751; Biochemistry 43:4137–4142). To systematically address the role of peptide sequence on the specificity of T-synthase, we used the oriented random glycopeptide: GAGAXXXX(T-O-GalNAc)XXXXAGAGK, (where X = G,A,P,V,I,F,Y,S,N,D,E,H,R & K) as a substrate. The Core 1 glycosylated product was isolated on immobilized PNA (Arachis hypogaea) lectin and its composition determined by Edman amino acid sequencing for comparison to the initial substrate composition, from which transferase preferences were obtained. From these studies, elevated preferences for Gly at the +1 position with moderately high preferences for Phe and Tyr in the +3 position relative to the acceptor Thr-O-GalNAc were found. A number of smaller Pro enhancements were also observed. Basic residues, i.e., Lys, Arg and His, in any position were disfavored, suggesting electrostatic interactions as an additional important component modulating transferase specificity. This work suggests there are indeed subtle specific and non-specific protein targeting sequence motifs for this transferase.
Key words:
Core 1 Transferase
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O-glycosylation
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sequence motifs
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T-synthase
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UDP-Gal:Glycoprotein--GalNAc β3 Galactosyltranferase
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