title: O-GalNAc incorporation into a cluster acceptor site of three consecutive threonines. Distinct specificity of GalNAc-transferase isoforms.
abstract: O-Glycosylation of three consecutive Thr residues in a fluorescein-conjugated peptide PTTTPLK - which mimics a portion of mucin 2 - by four isozymes of UDP-N-acetylgalactosaminyltransferases (pp-GalNAc-T1, T2, T3, or T4) was investigated. Partially glycosylated versions of this peptide, PT*TTPLK, PTTT*PLK, PT*TT*PLK, PTT*T*PLK, PT* degrees TTPLK, and PTTT* degrees PLK (*, N-acetylgalactosamine; degrees, galactose), were also tested. The products were separated by RP-HPLC and characterized by MALDI-TOF MS and peptide sequencing. The first and the third Thr residues act as the peptide's initial glycosylation sites for pp-GalNAc-T4, which were different from the sites for pp-GalNAc-T1 and T2 (the first Thr residue) or T3 (the third Thr residue) shown in our previous report. All pp-GalNAc-T isozymes tested exhibited distinct specificities toward glycopeptides. The most notable findings were: (a) prior incorporation of an N-acetylgalactosamine residue at the third Thr greatly enhanced N-acetylgalactosamine incorporation into the other Thr residues when pp-GalNAc-T2, T3, or T4 were used; (b) the enhancing effect of the N-acetylgalactosamine residue on the third Thr was completely abrogated by galactosylation of this N-acetylgalactosamine; (c) prior incorporation of an N-acetylgalactosamine at the first Thr did not have any enhancing effect; (d) pp-GalNAc-T2 was unique as it transferred N-acetylgalactosamine into the second Thr residue only when N-acetylgalactosamine was attached to the third one.
authors.affiliation: Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Takeuchi
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kato
authors.forename: Helle
authors.initials: H
authors.lastname: Hassan
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Irimura
mesh_terms: D000116:Acetylgalactosamine / Q000737:chemistry*; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000494:pharmacology; D005700:Galactosyltransferases / Q000737:chemistry*; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000737:chemistry*
chemical_list: D003902:Detergents; D006020:Glycopeptides; D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013912:Threonine; D014536:Uridine Diphosphate N-Acetylgalactosamine; D005700:Galactosyltransferases; D000116:Acetylgalactosamine