Mucin 2 (MUC2) is the major intestinal mucin. O-glycans are attached to MUC2 in a potentially diverse arrangement, which is crucial for their interaction with endogenous and exogenous lectins. In the present report, a PTTTPITTTTK peptide corresponding to a portion of the MUC2 tandem repeat domain was synthesized, and incubated with UDP-N-acetyl-D-galactosamine (UDP-GalNAc) and detergent-soluble microsomes, prepared from the human colon carcinoma cell line LS174T. The products were fractionated by reverse-phase HPLC and characterized by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. Oligopeptides with GalNAc residues derived from PTTTPITTTTK, containing consecutive threonine residues, were found to be glycosylated with 1-7 GalNAc residues per single peptide. The sequences of all glycopeptides were determined. The results indicated that the predominant sites of the first through to the sixth GalNAc incorporation were Thr-3, Thr-6, Thr-5, Thr-2, Thr-4 and Thr-1, respectively. An exception was the presence of a glycopeptide with three GalNAc residues at Thr-1, Thr-4 and Thr-5. When the putative biosynthetic intermediates, PTTT*PITTTTK and PT*TTPITTTTK, were used as acceptors, all the products detected and analyzed were the same as those obtained when the unsubstituted peptide and microsome fractions of LS174T cells were incubated. Thus, the preferential order and maximum number of GalNAc incorporation into threonine residues of MUC2 core peptides are strictly regulated, when the microsome fraction of LS174T cells is used as a source of N-acetyl-D-galactosaminyltransferases.
