Cloning and characterization of mammalian UDP-glucose glycoprotein: glucosyltransferase and the development of a specific substrate for this enzyme

The endoplasmic reticulum enzyme UDP-glucose glycoprotein:glucosyltransfera se (UGGT) has the unique property of recognizing incompletely folded glycop roteins and, if they carry an N-linked Man(9)GlcNAc(2), oligosaccharide, of catalyzing the addition of a glucose residue from UDP-glucose. Using pepti de sequence information, we have isolated the complete cDNA of rat liver UG GT and expressed it in insect cells. The cDNA specifies an open reading fra me which codes for a protein of 1527 residues including an 18 amino acid si gnal peptide. The protein has a C-terminal tetrapeptide (HEEL) characterist ic of endoplasmic reticulum luminal proteins. The purified recombinant enzy me shows the same preference for unfolded polypeptides with N-linked Man(9) GlcNAc(2), glycans as the enzyme purified from rat liver. A genetically eng ineered Saccharomyces cerevisiae strain capable of producing glycoproteins with Man(9)GlcNAc(2) core oligosaccharides was constructed and secreted aci d phosphatase (GO-AcP) was purified. GO-AcP was used as an acceptor glycopr otein for UGGT and found to be a better substrate than the previously used soybean agglutinin and thyroglobulin, Recombinant rat UGGT has a K-m, of 44 mu M for UDP-glucose. A proteolytic fragment of UGGT was found to retain e nzymatic activity thus localizing the catalytic site of the enzyme to the C -terminal 37 kDa of the protein. Using site-directed mutagenesis and photoa ffinity labeling, we have identified residues D1334, D1336, Q1429, and N143 3 to be necessary for the catalytic activity of the enzyme.