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 endogeneous and exogeneous
lectins. In the present report, five
oligopeptides [PTTTPITTTT(K), ITTTTTVTPT(K), TVTPTPTPTG(K), PTPTGTQTPT(K) and TQTPTTTPIT(K)] corresponding to portions of the MUC2 tandem repeat domain were 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 PTTTPITTTT(K), 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).
Oligopeptides containing alternating
threonine residues [TVTPTPTPTG(K) and PTPTGTQTPT(K)] were not fully glycosylated under the same conditions or even after prolonged incubations. Thus, the preferential order and maximum number of GalNAc incorporation into
threonine residues of MUC2 core
peptides depends on the
peptide sequence, when the microsome fraction of LS174T cells is used as a source of N-acetyl-D-galactosaminyltransferases.