Two homologues encoding human UDP-glucose : glycoprotein glucosyltransferase differ in mRNA expression and enzymatic activity

UDP-glucose:glycoprotein glucosyltransferase (UGT) is a soluble protein of the endoplasmic reticulum (ER) that operates as a gatekeeper for quality co ntrol by preventing transport of improperly folded glycoproteins out of the ER. We report the isolation of two cDNAs encoding human UDP-glucose: glyco protein glucosyltransferase homologues. HUGT1 encodes a 1555 amino acid pol ypeptide that, upon cleavage of an N-terminal signal peptide, is predicted to produce a soluble 173 kDa protein with the ER retrieval signal REEL. HUG T2 encodes a 1516 amino acid polypeptide that also contains a signal peptid e and the ER retrieval signal HDEL. HUGT1 shares 55% identity with HUGT2 an d 31-45% identity with Drosophila, Caemorhabditis elegans, and Schizosaccha romyces pombe homologues, with most extensive conservation of residues in t he carboxy-terminal fifth of the protein, the proposed catalytic domain. HU GT1 is expressed as multiple mRNA species that are induced to similar exten ts upon disruption of protein folding in the ER. In contrast, HUGT2 is tran scribed as a single mRNA species that is not induced under similar conditio ns. HUGT1 and HUGT2 mRNAs are broadly expressed in multiple tissues and dif fer slightly in their tissue distribution. The HUGT1 and HUGT2 cDNAs were e xpressed by transient transfection in COS-I monkey cells to obtain similar levels of protein localized to the ER. Extracts from HUGT1-transfected cell s displayed a 27-fold increase in the transfer of [C-14]glucose from UDP-[C -14]glucose to denatured substrates. Despite its high degree of sequence id entity with HUGT1, the expressed recombinant HUGT2 protein was not function al under the conditions optimized for HUGT1. Site-directed alanine mutagene sis within a highly conserved region of HUGT1 identified four residues that are essential for catalytic function.