Glycobiology Advance Access originally published online on December 10, 2008
Glycobiology 2009 19(3):321-328; 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 Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106
3 Department of Biochemistry, Emory University, Atlanta, GA 30322
4 Department of Chemistry, Biochemistry and Pediatrics, Case Western Reserve University, Cleveland, OH 44106, USA
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; revised on December 8, 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, the peptide sequence and neighboring glycosylation effects have been implicated. To systematically address the role of the peptide sequence on the specificity of T-synthase, we used the oriented random glycopeptide: GAGAXXXX(T-O-GalNAc)XXXXAGAG (where X = G, A, P, V, I, F, Y, S, N, D, E, H, R, and 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 with 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 that there are indeed subtle specific and nonspecific 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 galactosyltransferase
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  C. L. Perrine, A. Ganguli, P. Wu, C. R. Bertozzi, T. A. Fritz, J. Raman, L. A. Tabak, and T. A. Gerken Glycopeptide-preferring Polypeptide GalNAc Transferase 10 (ppGalNAc T10), Involved in Mucin-type O-Glycosylation, Has a Unique GalNAc-O-Ser/Thr-binding Site in Its Catalytic Domain Not Found in ppGalNAc T1 or T2 J. Biol. Chem., July 24, 2009; 284(30): 20387 - 20397. [Abstract] [Full Text] [PDF]
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