N-acetylgalactosamine incorporation into a peptide containing consecutive threonine residues by UDP-N-acetyl-D-galactosaminide:polypeptide N-acetylgalactosaminyltransferases

A limited number of glycosylation products were generated in a cell-free system from a portion of the MUC2 tandem repeat, PTTTPITTTTK, when microsome fractions of human colon carcinoma LS174T cells were used as the source of UDP-N-acetyl-D-galactosaminide:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-T) in our previous work. The structures of all products suggested that there were only two biosynthetic pathways in the GalNAc incorporation into this peptide. In the present report, the putative biosynthetic intermediates, PTTT*PITTTTK (asterisk designates a GalNAc residue), PT*TTPITTTTK, PTT*T*PITT*T*TK, and PT*TTPIT*T*T*TK, of these two hypothetical pathways were used as acceptors to prove that these two pathways do exist. The incubation products of these glycopeptides, microsome fractions of LS174T cells, and UDP-GalNAc were fractionated by reverse-phase HPLC and their structures were determined using MALDI-TOF MS and peptide sequencing. The products from PTTT*PITTTTK were PTTT*PITTT*TK, PTTT*PITT*T*TK, PTT*T*PI-TT*T*TK, PTT*T*PIT*T*T*TK, PT*T*T*PIT*T*T*TK, and PT*T*T*PIT*T*T*T*K. The products from PTT*-T*PITT*T*TK exactly corresponded to the products with five to seven GalNAc residues from PTTT*PITTTTK. The products from PT*TTPITTTTK were PT*TTPITT*TTK, PT*TTPIT*T*TTK, and PT*TTPIT*T*T*TK. PT*TTP-IT*T*T*TK was not converted further under the applied condition. All the products detected and analyzed were the same as those obtained when the unsubstituted peptide and microsome fractions of LS174T cells were incubated. Immunocytochemical analysis indicated that LS174T cells contain at least four pp-GalNAc-Ts (-T1, -T2, -T3, and -T4), suggesting that control of the order and the maximum number of GalNAc incorporation into this peptide is regulated through the coordinated actions of these and possibly other pp-GalNAc-Ts.