Beta-mannosidase, a lysosomal enzyme which acts exclusively at the last step of oligosaccharide catabolism in glycoprotein degradation, functions to cleave the single beta-linked mannose sugar found in all N-linked oligosaccharides of glycoproteins. Deficiency of this enzyme results in beta-mannosidosis, a lysosomal storage disease characterized by the cellular accumulation of small oligosaccharides. This autosomal recessive genetic disease is the most recently identified human acid glycosyl hydrolase deficiency. In the human beta-mannosidosis cases described to date the clinical presentation is relatively mild for a lysosomal storage disease and very variable. Nothing is known about the molecular basis for human beta-mannosidosis. The reasons for the variability are unknown and will be addressed in this proposal. Bovine beta-mannosidase cDNA has been cloned and sequenced. A full length cDNA clone has been prepared and verified by its in vitro expression. Using this information, it is now possible to isolate and characterize the normal human beta-mannosidase cDNA and gene, to identify the mutations that cause beta-mannosidosis, and to explore the molecular pathology of this disease.
The specific aims of this proposal ar: 1. Clone and characterize cDNA for human beta-mannosidase. Human beta-mannosidase cDNA will be cloned using information obtained from the bovine cDNA. A full length human cDNA will be constructed for expression in transfected cells. 2. Clone and characterize the human genomic beta-mannosidase sequence. The genomic structure of the beta- mannosidase gene from any species is not yet known. We plan to clone the human beta-mannosidase gene, determine its transcriptional start site(s) and intron/exon placement in the gene. Achievement of specific aims 1 and 2 will characterized and will provide the reagents required to peruse mutation analysis and structure-function analysis. 3. Determine the molecular pathology to human beta-mannosidosis. The levels of beta-mannosidase protein and mRNA will be evaluated in fibroblast cell cultures derived from beta-mannosidase patients. The mutations responsible for each case of beta-mannosidosis will be identified and correlated with the severity and symptoms in the presentation of the disease. Experiments in Specific aim 3 will delineate the mutations which cause the human disease to provide insight into the nature of the extreme variability of the human disease. Information of the molecular pathology of beta-mannosidosis will allow prediction about the clinical spectrum of the disease and suggest where to look for additional cases of this potentially underreported disease. Structure-function studies will lead to further understanding of the enzyme providing information useful for potential treatment of diseases involving glycosyl hydrolases.
