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J Biol Chem, Vol. 275, Issue 15, 11071-11074, April 14, 2000

Mutation of Arg²⁷³ to Leu Alters the Specificity of the Yeast N-Glycan Processing Class I 1,2-Mannosidase^*

Pedro A. Romero, François Vallée^§, P. Lynne Howell^§¶, and Annette Herscovics

From the  McGill Cancer Centre, McGill University, Montréal, Québec H3G 1Y6, ^§ Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, and the ^¶ Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Class I 1,2-mannosidases (glycosyl hydrolase family 47) involved in the processing of N-glycans during glycoprotein maturation have different specificities. Enzymes in the endoplasmic reticulum of yeast and mammalian cells remove a single mannose from Man₉GlcNAc₂ to form Man₈GlcNAc₂ isomer B (lacking the 1, 2-mannose residue of the middle 1, 3-arm), whereas other 1,2-mannosidases, including Golgi 1,2-mannosidases IA and IB, can convert Man₉GlcNAc₂ to Man₅GlcNAc₂. In the present work, it is demonstrated that with a single mutation in its catalytic domain (Arg²⁷³  Leu) the yeast endoplasmic reticulum 1,2-mannosidase acquires the ability to transform Man₉GlcNAc to Man₅GlcNAc. High resolution proton nuclear magnetic resonance analysis of the products shows that the order of removal of mannose from Man₉GlcNAc is different from that of other 1,2-mannosidases that remove four mannose from Man₉GlcNAc. These results demonstrate that Arg²⁷³ is in part responsible for the specificity of the endoplasmic reticulum 1,2-mannosidase and that small differences in non-conserved amino acids interacting with the oligosaccharide substrate in the active site of class I 1,2-mannosidases are responsible for the different specificities of these enzymes.

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^* This work was supported by National Institutes of Health Grant GM31265.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: McGill Cancer Centre, McGill University, 3655 Promenade Sir-William-Osler, Montréal, Québec H3G 1Y6, Canada. E-mail: annette@med.mcgill.ca.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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