Glycobiology, 2000, Vol. 10, No. 4 403-412
© 2000 Oxford University Press
Cloning and characterization of mammalian UDP-glucose glycoprotein: glucosyltransferase and the development of a specific substrate for this enzyme
2Genetics Group, Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec H4P 2R2, Canada, 3Department of Anatomy and Cell Biology, McGill University, Montréal, Québec H3A 2B2, Canada, 4Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA, 5Laboratoire d’Ingénierie des Macromolécules, Institut de Biologie Structurale J.-P. Ebel, CNRS, Grenoble, France, and 6Instituto de Investigaciones Bioquimicas Fundacion Campomar, Antonio Machado 151, 1405 Buenos Aires, Argentina
The endoplasmic reticulum enzyme UDP-glucose glycoprotein:glucosyltransferase (UGGT) has the unique property of recognizing incompletely folded glycoproteins and, if they carry an N-linked Man9GlcNAc2 oligosaccharide, of catalyzing the addition of a glucose residue from UDP-glucose. Using peptide sequence information, we have isolated the complete cDNA of rat liver UGGT and expressed it in insect cells. The cDNA specifies an open reading frame which codes for a protein of 1527 residues including an 18 amino acid signal peptide. The protein has a C-terminal tetrapeptide (HEEL) characteristic of endoplasmic reticulum luminal proteins. The purified recombinant enzyme shows the same preference for unfolded polypeptides with N-linked Man9GlcNAc2 glycans as the enzyme purified from rat liver. A genetically engineered Saccharomyces cerevisiae strain capable of producing glycoproteins with Man9GlcNAc2 core oligosaccharides was constructed and secreted acid phosphatase (G0-AcP) was purified. G0-AcP was used as an acceptor glycoprotein for UGGT and found to be a better substrate than the previously used soybean agglutinin and thyroglobulin. Recombinant rat UGGT has a Km of 44 µM for UDP-glucose. A proteolytic fragment of UGGT was found to retain enzymatic activity thus localizing the catalytic site of the enzyme to the C-terminal 37 kDa of the protein. Using site-directed mutagenesis and photoaffinity labeling, we have identified residues D1334, D1336, Q1429, and N1433 to be necessary for the catalytic activity of the enzyme.
1 To whom correspondence should be addressed at: Genetics Group, National Research Council of Canada, Biotechnology Research Institute, 6100 Royalmount Avenue, Montréal, Québec H4P 2R2, Canada
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