Systematic determination of the peptide acceptor preferences for the human UDP-Gal:glycoprotein-alpha-GalNAc beta 3 galactosyltransferase (T-synthase).
Mucin-type protein O-glycosylation is initiated by the addition of alpha-GalNAc to Ser/Thr residues of a polypeptide chain. The addition of beta-Gal to GalNAc by the UDP-Gal:glycoprotein-alpha-GalNAc beta 3 galactosyltransferase (T-synthase), forming the Core 1 structure (beta-Gal(1-3)-alpha-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.
Perrine
Cynthia
C
Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA.
Ju
Tongzhong
T
Cummings
Richard D
RD
Gerken
Thomas A
TA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D005700:Galactosyltransferases / Q000378:metabolism*; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D014530:Uridine Diphosphate / Q000378:metabolism*
D006020:Glycopeptides; D010455:Peptides; D014530:Uridine Diphosphate; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C076385:glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase