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

Isao Nishimori, Fulvio Perini, Kimberly P. Moun, Sam Sanderson, Nicole Johnson, Michael A Hollingsworth

Research output: Contribution to journalArticle

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Abstract

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 MUCI 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 MUCI 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.

Original languageEnglish (US)
Pages (from-to)3738-3744
Number of pages7
JournalCancer Research
Volume54
Issue number14
StatePublished - Jul 15 1994

Fingerprint

Acetylgalactosamine
Cell Extracts
Glycosylation
Tandem Repeat Sequences
Peptides
Amino Acid Sequence
Neoplasms
Glycopeptides
Threonine
Tumor Cell Line
Uridine Diphosphate
Mucins
Amino Acid Substitution
Substrate Specificity
Population Groups
Radioactivity
Serine
MUC1 tandem repeat peptide
Mass Spectrometry
Gases

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Nishimori, I., Perini, F., Moun, K. P., Sanderson, S., Johnson, N., & Hollingsworth, M. A. (1994). N-Acetylgalactosamine Glycosylation of MUC1 Tandem Repeat Peptides by Pancreatic Tumor Cell Extracts. Cancer Research, 54(14), 3738-3744.

N-Acetylgalactosamine Glycosylation of MUC1 Tandem Repeat Peptides by Pancreatic Tumor Cell Extracts. / Nishimori, Isao; Perini, Fulvio; Moun, Kimberly P.; Sanderson, Sam; Johnson, Nicole; Hollingsworth, Michael A.

In: Cancer Research, Vol. 54, No. 14, 15.07.1994, p. 3738-3744.

Research output: Contribution to journalArticle

Nishimori, I, Perini, F, Moun, KP, Sanderson, S, Johnson, N & Hollingsworth, MA 1994, 'N-Acetylgalactosamine Glycosylation of MUC1 Tandem Repeat Peptides by Pancreatic Tumor Cell Extracts', Cancer Research, vol. 54, no. 14, pp. 3738-3744.
Nishimori I, Perini F, Moun KP, Sanderson S, Johnson N, Hollingsworth MA. N-Acetylgalactosamine Glycosylation of MUC1 Tandem Repeat Peptides by Pancreatic Tumor Cell Extracts. Cancer Research. 1994 Jul 15;54(14):3738-3744.
Nishimori, Isao ; Perini, Fulvio ; Moun, Kimberly P. ; Sanderson, Sam ; Johnson, Nicole ; Hollingsworth, Michael A. / N-Acetylgalactosamine Glycosylation of MUC1 Tandem Repeat Peptides by Pancreatic Tumor Cell Extracts. In: Cancer Research. 1994 ; Vol. 54, No. 14. pp. 3738-3744.
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abstract = "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 MUCI 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 MUCI 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.",
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