Abstract
The Saccharomyces cerevisiae alpha1,2-mannosidase, which removes one specific mannose residue from Man9GlcNAc2 to form Man8GlcNAc2, is a member of a family of alpha1,2-mannosidases with similar amino acid sequences. The yeast alpha1,2-mannosidase contains five cysteine residues, three of which are conserved. Recombinant yeast alpha1, 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 alpha1, 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 alpha1, 2-mannosidase family.
Highlights
The processing ␣1,2-mannosidase present in the endoplasmic reticulum of Saccharomyces cerevisiae removes one specific mannose residue from Man9GlcNAc2 to form Man8GlcNAc2 during the formation of N-linked oligosaccharides [1,2,3,4]
Expression of S. cerevisiae ␣1,2-Mannosidase in P. pastoris—In order to obtain sufficient protein for structural analysis, the catalytic domain of the yeast ␣1,2-mannosidase was cloned downstream of the PHO1 signal peptide and strong alcohol oxidase (AOX1) promoter in the pHIL-S1 vector
Recombinant ␣1,2-mannosidase produced from S. cerevisiae had been shown to have similar properties as the native ␣1,2-mannosidase [14], and the recombinant ␣1,2-mannosidase from P. pastoris has the same specific activity and Km as the enzyme produced in S. cerevisiae
Summary
The processing ␣1,2-mannosidase present in the endoplasmic reticulum of Saccharomyces cerevisiae removes one specific mannose residue from Man9GlcNAc2 to form Man8GlcNAc2 during the formation of N-linked oligosaccharides [1,2,3,4]. Based on sequence homology and common properties [9], these enzymes were grouped as Class 1 ␣1,2mannosidases They all contain an EF-hand Ca2ϩ binding consensus sequence and require Ca2ϩ for activity. ␣1,2-mannosidases have been cloned from Drosophila melanogaster [11], Penicilium citrinum [12], and Aspergillus saitoi [13], which have similar amino acid sequences to the yeast and mammalian enzymes. In the present work we demonstrate that the yeast ␣1,2mannosidase has two disulfide bonds and one sulfhydryl group in its catalytic domain. Their location is documented by peptide analysis, and their respective roles in enzyme activity is established by site-directed mutagenesis. One of these two disulfide bonds is essential for catalytic activity, and the free sulfhydryl residue is not required
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