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N-Acetylgalactosamine Glycosylation of MUC1 Tandem Repeat Peptides by Pancreatic Tumor Cell Extracts¹

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805 [I. N., F. P., K. P. M., S. D. S., N. J., M. A. H.]; First Department of Internal Medicine, Kochi Medical School, Kochi 783, Japan [I. N.]; Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304 [R. L. C., M. L. G.]; and Theoretical Biology and Biophysics, T10, MS-K710, Los Alamos National Laboratory Los Alamos, New Mexico 87545 [D. F.]

Synthetic peptides corresponding to the human mucin MUC1 tandem repeat domain (20 residues) were glycosylated in vitro by using UDP-N-[³H]acetyl-D-galactosamine (GalNAc) and lysates of pancreatic tumor cell lines. 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 glycosylation along the MUC1 tandem repeat protein backbone. Purified glycopeptides were sequenced on a gas-phase sequencer, and glycosylated positions were determined by measuring the incorporated radioactivity in fractions 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 following positions: GVTSAPDTRPAPGSTAPPAH (underlined T indicates position 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 linked to each 20-residue repeat unit in the peptides. The data presented here show that acceptor substrate specificity of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase detected in lysates of pancreatic 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 activity was evaluated by using several peptides that contain single or double amino acid substitutions (relative to the native human MUC1 sequence). These included substitutions in the residues that were glycosylated and substitutions of the surrounding primary amino acid sequence. The results of these studies suggest that primary amino acid sequence, length, and relative position of the residue to be glycosylated dramatically affect the ability of peptides to serve as acceptor substrates for the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase.

¹ This work was supported in part by NIH Grants CA57362, DK47662, and CA36727; American Cancer Society Grant SIG 16; and the Nebraska Cancer and Smoking Disease Research Program. The four-sector tandem mass spectrometer was purchased with funds awarded by former National Science Foundation Regional Instrumentation Grant CHE8620177 and the University of Nebraska-Lincoln. Ongoing support for this facility was provided by National Science Foundation Biology Division Grant DIR901762. N. J. was supported by NIH Training Grant T32 CA09476.

² To whom requests for reprints should be addressed, at Eppley Institute for Research in Cancer and Applied Diseases, University of Nebraska Medical Center, 600 South 42nd Street, Box 986805, Omaha, NE 68198-6805.

Received 3/21/94. Accepted 5/13/94.

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