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Glycobiology, 2000, Vol. 10, No. 6 595-599
© 2000 Oxford University Press

Minimal catalytic domain of N-acetylglucosaminyltransferase V

Bozena Korczak1, Thuyanh Le, Sabine Elowe, Alessandro Datti and James W. Dennis2

GlycoDesign Inc., 480 University Avenue, Suite 900, Toronto, Ontario, Canada

UDP-GlcNAc: Man{alpha}1–6Manß-R ß1–6 N-acetylglucosaminyltransferase V (EC 2.4.1.155, GlcNAc-TV) is a Golgi enzyme that substitutes the trimannosyl core in the biosynthetic pathway for complex-type N-linked glycans. GlcNAc-TV activity is regulated by oncogenes frequently activated in cancer cells (ras, src, and her2/neu) and by activators of T lymphocytes. Overexpression of GlcNAc-TV in epithelial cells results in morphological transformation, while tumor cell mutants selected for loss of GlcNAc-TV products show diminished malignant potential in mice. In this report, we have expressed and characterized a series of N- and C-terminal deletions of GlcNAc-TV. Portions of GlcNAc-TV sequence were fused at the N-terminal domain to IgG-binding domains of staphylococcal Protein A and expressed in CHOP cells. The secreted fusion proteins were purified by IgG Sepharose affinity chromatography and assayed for enzyme activities. The peptide sequence S213–740 of GlcNAc-TV was determined to be essential for the catalytic activity, the remaining amino acids comprising a 183 amino acid stem region, a 17 amino acid transmembrane domain and a 12 amino acid cytosolic moiety. Further deletion of 5 amino acids to produce peptide R218–740 reduced enzyme activity by 20-fold. Similar Km and Vmax values for donor and acceptor were observed for peptide S213–740, the minimal catalytic domain, and peptide Q39–740, which also included the stem region. Truncation of five amino acids from the C-terminus also resulted in a 20-fold loss of catalytic activity. Secondary structure predictions suggest a high frequency of turns in the stem region, and more contiguous stretches of {alpha}-helix found in the catalytic domain.

1 To whom correspondence should be addressed

2 Present addresses: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada, and Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada


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