MECHANISM OF CLASS I ALPHA-MANNOSIDASES INVOLVED IN N-GLYCAN PROCESSING

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Quality control in the endoplasmic reticulum (ER) determines the fate of newly synthesized glycoproteins toward either correct folding or disposal by ER-associated degradation. Initiation of the disposal process involves selective trimming of N-glycans attached to misfolded glycoproteins by ER alpha-mannosidase I and subsequent recognition by the ER-localized lectin, EDEM, both members of glycosylhydrolase family 47. The unusual inverting hydrolytic mechanism catalyzed by members of this family has been investigated by a combination of kinetic and binding analyses of wild type and mutant forms of human ER alpha-mannosidase I as well as structural analysis of a co-complex with an uncleaved thio-disaccharide substrate analog. The data reveal the roles of catalytic acid and base residues and the identification of a novel 3S1 sugar conformation for the bound substrate analog. Implications for the mechanism of action of this family of glycosidases are discussed. NMR was used in identifying the synthetic intermediates of the thio-disaccharide, as well as describing its solution conformation.