The biosynthesis of N-glycans involves the stepwise removal of glucose and mannose residues in the endoplasmic reticulum and the Golgi complex. The mannose trimming events are initiated by members of a multigene family of a1,2-mannosidases that are temporally and spatially expressed in distinctive cell types in mammalian tissues. We have cloned six members of the a1,2-mannosidase multigene family and are presently carrying out detailed structural studies on one of the family members. The cDNA encoding murine Golgi a-mannosidase IA was subcloned into a vector for the inducible expression of the cDNA in the methylotropic yeast, Pichia pastoris. Enzyme expression was induced by culturing the cells in a methanol-containing medium, and the enzyme has been isolated and purified in milligram quantities from the culture medium. The homogeneous enzyme preparation has been subjected to initial screening of crystallization conditions, and initial microcrystals have been obtained. We are presently isolating additional enzyme for large scale crystallization studies and structure determined by X-ray diffraction. These studies would represent the first determination of the structure of a mammalian Golgi-processing enzyme and could lead to greater insight into the structural basis for the differences in substrate specificities among the Class I a-mannosidases.
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