N-ACETYLGALACTOSAMINE GLYCOSYLATION OF MUC1 TANDEM REPEAT PEPTIDES BYPANCREATIC TUMOR-CELL EXTRACTS

Synthetic peptides corresponding to the human mucin MUC1 tandem repeat domain (20 residues) were glycosylated in vitro by using UDP-N-[H-3]a cetyl-D-galactosamine (GalNAc) and lysates of pancreatic tumor cell li nes. Results obtained with peptides of different lengths (from one to five repeats) suggest that increasing the number of tandem repeats has neither a positive nor a negative effect on the density of glycosylat ion along the MUC1 tandem repeat protein backbone. Purified glycopepti des were sequenced on a gas-phase sequencer, and glycosylated position s were determined by measuring the incorporated radioactivity in fract ions collected following each round of Edman degradation. The results showed that two of three threonine residues on the MUC1 tandem repeat peptides were glycosylated by pancreatic tumor cell lysates at the fol lowing positions: GVTSAPDTRPAPGSTAPPAH (underlined T indicates positio n of GalNAc attachment). None of the serine residues were glycosylated . Determination of the mass of the glycopeptides by mass spectrometry confirmed that a maximum of two molecules of GalNAc were covalently li nked to each 20-residue repeat unit in the peptides. The data presente d here show that acceptor substrate specificity of the UDP-GalNAc:poly peptide N-acetylgalactosaminyltransferase detected in lysates of pancr eatic and breast tumor cell lines is identical and is limited to some but not all threonines in the MUC1 tandem repeat peptide sequence. The influence of primary amino acid sequence on acceptor substrate activi ty was evaluated by using several peptides that contain single or doub le amino acid substitutions (relative to the native human MUC1 sequenc e). These included substitutions in the residues that were glycosylate d and substitutions of the surrounding primary amino acid sequence. Th e results of these studies suggest that primary amino acid sequence, l ength, and relative position of the residue to be glycosylated dramati cally affect the ability of peptides to serve as acceptor substrates f or the UDP-GalNAc:polypeptide N-acetyl-galactosaminyltransferase.