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Volume 271, Number 44, Issue of November 1, 1996 pp. 27615-27622
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Role of the Cysteine Residues in the 1,2-Mannosidase Involved in N-Glycan Biosynthesis in Saccharomyces cerevisiae
THE CONSERVED Cys³⁴⁰ AND Cys³⁸⁵ RESIDUES FORM AN ESSENTIAL DISULFIDE BOND

(Received for publication, June 11, 1996, and in revised form, July 31, 1996)

From the McGill Cancer Centre, McGill University, Montréal, Québec, Canada H3G 1Y6

The Saccharomyces cerevisiae 1,2-mannosidase, which removes one specific mannose residue from Man₉GlcNAc₂ to form Man₈GlcNAc₂, is a member of a family of 1,2-mannosidases with similar amino acid sequences. The yeast 1,2-mannosidase contains five cysteine residues, three of which are conserved. Recombinant yeast 1,2-mannosidase, produced as the soluble catalytic domain, was shown to contain two disulfide bonds and one free thiol group using 2-nitro-5-thiosulfobenzoate and 5,5-dithiobis(2-nitrobenzoate), respectively. Cys⁴⁸⁵ contains the free thiol group, as demonstrated by sequencing of labeled peptides following modification with [³H]ICH₂COOH and by high performance liquid chromatography/mass spectrometry tryptic peptide mapping. A Cys³⁴⁰-Cys³⁸⁵ disulfide was demonstrated by sequencing a purified peptide containing this disulfide and by tryptic peptide mapping. Cys⁴⁶⁸ and Cys⁴⁷¹ were not labeled with [³H]ICH₂COOH and a peptide containing these two residues was identified in the tryptic peptide map, showing that Cys⁴⁶⁸ and Cys⁴⁷¹ form the second disulfide bond. The 1,2-mannosidase loses its activity in the presence of dithiothreitol with first order kinetics, suggesting that at least one disulfide bond is essential for activity. Mutagenesis of each cysteine residue to serine showed that Cys³⁴⁰ and Cys³⁸⁵ are essential for production of recombinant enzyme, whereas Cys⁴⁶⁸, Cys⁴⁷¹, and Cys⁴⁸⁵ are not required for production and enzyme activity. These results indicate that the sensitivity to dithiothreitol is due to reduction of the Cys³⁴⁰-Cys³⁸⁵ disulfide. Since Cys³⁴⁰ and Cys³⁸⁵ are conserved residues, it is likely that this disulfide bond is important to maintain the correct structure in the other members of the 1,2-mannosidase family.

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