O‐GalNAc incorporation into a cluster acceptor site of three consecutive threonines

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*°TTPLK, and PTTT*°PLK (*, N ‐acetylgalactosamine; °, 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.