Protein glycosylation is initiated in the endoplasmic reticulum (ER) by transfer of an oligosaccharide (Glc3Man9GlcNAc2) to nascent polypeptide chains. Processing of the oligosaccharide by glucosidases I and II yields unglucosylated oligosaccharides that are reglucosylated by the UDP-Glc:glycoprotein glucosyltransferase (GT) only if the protein moieties of glycoproteins are not properly folded, as GT behaves as a sensor of glycoprotein conformations. Interaction of monoglucosylated glycoproteins (created by partialdeglucosylation of the transferred compound or by GT-mediated reglucosylation) with ER lectins (calnexin and calreticulin) facilitates acquisition of the proper tertiary structures and prevents secretion of not yet properly folded species. Folding facilitation mediated by the interaction of monoglucosylated glycoproteins and ER lectins is necessary for production of infective viral particles (vesicular stomatitis virus, HIV, hepatitis B virus) and also for the viability of yeasts when under severe ER stress conditions. The long term objective of the proposal is to gain thorough information of the structural features leading to the monoglucosylated glycoprotein-lectin interaction as this knowledge will undoubtedly provide useful information for understanding and/or preventing production of the etiological agents of several diseases. Within this long term objective, the Specific Aims of the proposed research are: a) to define the structural elements exclusively exposed in misfolded glycoproteins whose recognition is required for GT-mediated glucosylation; b) to define GT domains responsible for substrate donor (UDP-Glc), and substrate acceptor (N-oligosaccharide) recognition and for the exclusive glucosylation of misfolded species and c) to study the possibility that folding facilitation mediated by the interaction of cruzipain, a cysteine proteinase from the protozoan parasite Trypanosoma cruzi and ER lectins could be absolutely necessary for parasite infectivity. T. cruzi is the causative agent of a disease endemic in Latin America (Chagas's disease) that affects about 16 million people. Cruzipain has been identified as one of the T. cruzi virulence factors and has been found to be the only glycoprotein interacting with ER lectins in the protozoon.
