Characterization and subcellular localization of human neutral class IIα-mannosidase cytosolic enzymes/free oligosaccharides/ glycosidehydrolase family 38/M2C1/N-glycosylation

Abstract

A glycosyl hydrolase family 38 enzyme, neutral α-mannosidase, has been proposed to be involved in hydrolysis of cytosolic free oligosaccharides originating either from ER-misfolded glycoproteins or the N-glycosylation process. Although this enzyme has been isolated from the cytosol, it has also been linked to the ER by subcellular fractionations. We have studied the subcellular localization of neutral α-mannosidase by immunofluorescence microscopy and characterized the human recombinant enzyme with natural substrates to elucidate the biological function of this enzyme. Immunofluorescence microscopy showed neutral α-mannosidase to be absent from the ER, lysosomes, and autophagosomes, and being granularly distributed in the cytosol. In experiments with fluorescent recovery after photo bleaching, neutral α-mannosidase had slower than expected two-phased diffusion in the cytosol. This result together with the granular appearance in immunostaining suggests that portion of the neutral α-mannosidase pool is somehow complexed. The purified recombinant enzyme is a tetramer and has a neutral pH optimum for activity. It hydrolyzed Man9 GlcNAc to Man5GlcNAc in the presence of Fe2+, Co2+, and Mn2+, and uniquely to neutral α-mannosidases from other organisms, the human enzyme was more activated by Fe2+ than Co2+. Without activating cations the main reaction product was Man8GlcNAc, and Cu2+ completely inhibited neutral α-mannosidase. Our findings from enzyme-substrate characterizations and subcellular localization studies support the suggested role for neutral α-mannosidase in hydrolysis of soluble cytosolic oligomannosides. © The Author 2007. Published by Oxford University Press. All rights reserved.