MANNOSIDASES IN GLYCOPROTEIN BIOSYNTHESIS AND CATABOLISM

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The long term goals of this proposal are to examine the factors which influence the regulation, structure, and function of enzymes involved in the biosynthesis and catabolism of mammalian N-linked glycans. These enzymes determine the fate of the oligosaccharides on newly synthesized glycoproteins by determining the extent of processing from high mannose- type to complex-type structures. Little is known about the structure or regulation of the enzymes in this pathway. To address these problems the cDNAs encoding several of the processing and catabolic alpha-mannosidases have been isolated and have been organized into two multigene classes. This collection of alpha-mannosidase cDNAs offer several unique experimental systems for the study of the structure, function, and regulation of these enzymes as models for the regulation of glycoprotein biosynthesis and catabolism. Four specific aims are addressed in this proposal. The first specific aim is to complete the isolation of cDNA and genomic clones encoding the mammalian alpha-mannosidases. The second specific aim is to examine the structure, regulation, and function of the mammalian alpha-mannosidase family members. Heterologous expression, purification, determination of substrate specificity, and determination of in vivo tissue and cell- specific expression patterns will be compared among the enzymes in order to define the respective functions of each of members of the various enzyme classes. Enzymes from mammalian and non-mammalian sources will also be isolated for structure, function, and expression pattern studies. An additional focus of the cloning and sequencing studies on the mannosidases will be the characterization of the molecular basis of human genetic diseases characterized by a deficiency in either Golgi Man II or the lysosomal alpha-mannosidase. This is the third specific aim of the grant. The fourth specific aim will be the generation and characterization of murine models for deficiencies in the various processing and catabolic alpha-mannosidases by murine gene ablation techniques. These mouse knockout models will provide a direct demonstration of the in vivo roles of each of the multi-gene family members and generate an animal model for the eventual testing of therapeutic strategies for the lysosomal storage disease, alpha- mannosidosis.