ALPHA-MANNOSIDASES IN GLYCOPROTEIN PROCESSING

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The long term goals of this proposal are to examine the factors which influence the regulation, structure, and function of resident enzymes of the Golgi complex by examining the alpha-mannosidases of the N-glycan processing pathway. 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 alpha-mannosidases have been isolated. These alpha- mannosidases represent the first of the mammalian processing hydrolases to be cloned and offer several unique experimental systems for the study of their structure and regulation. Two specific aims are addressed in this proposal. The first specific aim is to complete the isolation of cDNA and genomic clones for two of the processing alpha-mannosidases, alpha-mannosidase I (Man I) and alpha-mannosidase II (Man II). These clones and their derived sequences will act as the fundamental base of information for the studies on regulation and protein structure. The primary focus of the cloning and sequencing studies on Man II will be the characterization of the molecular basis of HEMPAS disease, a human genetic disease characterized by a deficiency in Man II. These studies will utilize the rapid PCR amplification and sequencing of HEMPAS cDNAs in combination with the heterologous expression of mutant and control Man II constructs in COS cells to address the functional consequences of each mutation. Similar studies will be performed to determine the molecular basis of Man I deficiency in the ricin-resistant cell line, clone 6. The second specific aim examines the regulation and structure of the alpha- mannosidases. The tissue-specific and developmental expression of the enzymes will be examined at the transcript, polypeptide, and enzyme level and will be compared with the expression of other Golgi glycosyltransferases to examine the coordinate regulation of the enzymes in the pathway. Domain structure studies and an enzymatic comparison of related alpha-mannosidase activities will also be carried out in overexpression constructs in mammalian cells. Successful completion of the proposed studies will provide fundamental knowledge about the structure, regulation, and function of N-glycan processing in animal cells. The determination of the molecular basis of the human genetic deficiency in Man II, HEMPAS, will also be of great value in determining the sequences essential for enzyme function and their consequences in the alteration of oligosaccharide structures. Finally, a detailed examination of the expression of the alpha- mannosidases in the context of the glycosylation pathway as a whole should yield a cohesive picture of the coordinated regulation of the pathway in animal systems.