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Kex2 protease converts the endoplasmic reticulum 1,2-mannosidase of Candida albicans into a soluble cytosolic form

Héctor M. Mora-Montes^1,

Oliver Bader^2,

Bernhard Hube^2,

¹ Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apartado Postal 187, Guanajuato Gto. CP 36000, Mexico
² Robert Koch-Institut, FG16, Nordufer 20, D-13353 Berlin, Germany
³ Departamento de Genética y Biología Molecular, CINVESTAV del IPN, Apartado Postal 14-740, México DF 07000, Mexico
⁴ School of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK

Correspondence
Arturo Flores-Carreón
floresca{at}quijote.ugto.mx

Cytosolic -mannosidases are glycosyl hydrolases that participate in the catabolism of cytosolic free N-oligosaccharides. Two soluble -mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in Candida albicans. We demonstrate that addition of pepstatin A during the preparation of cell homogenates enriched -mannosidase E-I at the expense of E-II, indicating that the latter is generated by proteolysis during cell disruption. E-I corresponded to a polypeptide of 52 kDa that was associated with mannosidase activity and was recognized by an anti-1,2-mannosidase antibody. The N-mannan core trimming properties of the purified enzyme E-I were consistent with its classification as a family 47 1,2-mannosidase. Differential density-gradient centrifugation of homogenates revealed that 1,2-mannosidase E-I was localized to the cytosolic fraction and Golgi-derived vesicles, and that a 65 kDa membrane-bound 1,2-mannosidase was present in endoplasmic reticulum and Golgi-derived vesicles. Distribution of -mannosidase activity in a kex2 null mutant or in wild-type protoplasts treated with monensin demonstrated that the membrane-bound 1,2-mannosidase is processed by Kex2 protease into E-I, recognizing an atypical cleavage site of the precursor. Analysis of cytosolic free N-oligosaccharides revealed that cytosolic 1,2-mannosidase E-I trims free Man₈GlcNAc₂ isomer B into Man₇GlcNAc₂ isomer B. This is believed to be the first report demonstrating the presence of soluble 1,2-mannosidase from the glycosyl hydrolases family 47 in a cytosolic compartment of the cell.

Present address: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.

Present address: Intitut für medizinische Mikrobiologie, Universität Göttingen, Kreuzbergring 57, D-37075 Göttingen, Germany.

Present address: Friedrich Schiller University and Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knoell Institute, Beutenbergstrasse 11a, 07745 Jena, Germany.