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Glycobiology Advance Access originally published online on August 6, 2007
Glycobiology 2007 17(10):1084-1093; doi:10.1093/glycob/cwm083
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© The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Elina Kuokkanen3,4,, Wesley Smith3, Marika Mäkinen3,4,, Heidi Tuominen4, Maija Puhka3, Eija Jokitalo3, Sandrine Duvet5, Thomas Berg2,6 and Pirkko Heikinheimo1,3

3 Institute of Biotechnology, University of Helsinki, FIN-00014, Finland
4 Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014, Finland
5 Unité de Glycobiologie Structurale et Fonctionnelle, UMR CNRS 8576, IFR 147, GDR CNRS 2590, Université des Sciences et Technologies de Lille, Villeneuve d’Ascq, France
6 Department of Medical Biochemistry, University of Tromsø, N-9037 Tromsø, Norway

1 To whom correspondence should be addressed: Tel: +358-9-191-58957; Fax: +358-9-191-59940; e-mail: pirkko.heikinheimo{at}helsinki.fi

Received on April 10, 2007; revised on July 27, 2007; accepted on July 31, 2007

A glycosyl hydrolase family 38 enzyme, neutral {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-mannosidase pool is somehow complexed. The purified recombinant enzyme is a tetramer and has a neutral pH optimum for activity. It hydrolyzed Man9GlcNAc to Man5GlcNAc in the presence of Fe2+, Co2+, and Mn2+, and uniquely to neutral {alpha}-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 {alpha}-mannosidase. Our findings from enzyme-substrate characterizations and subcellular localization studies support the suggested role for neutral {alpha}-mannosidase in hydrolysis of soluble cytosolic oligomannosides.

Key words: cytosolic enzymes / free oligosaccharides / glycoside hydrolase family 38 / M2C1 / N-glycosylation

2 Present address: Department of Pathology, University Hospital of Northern Norway, 9038, Tromsø, Norway


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