Inherited lysosomal beta-mannosidase (MANBA) deficiency, discovered and characterized int he goat by the investigators, is associated with profound neurological deficits, regional dysmyelinogenesis and tissue accumulation of uncatabolized oligosaccharides. Recently, the first human cases of beta- mannosidosis were identified. We intend to define the pathogenesis of caprine beta-mannosidosis and to participate in its development as an important, ethically acceptable animal model for gene therapy of inherited metabolic diseases. This investigation is designed as a necessary step to these long term goals. The main thrusts are: first, to purify and characterize the normal MANBA gene product and to produce a MANBA antibody; and second, to analyze the beta-mannosidosis metabolic perturbations. I. MANBA studies: The goat kidney MANBA enzyme will be purified to homogeneity by extraction, heat denaturation, ammonium sulfate fractionation and sequential chromatographic methods. Immunochemical, biochemical and physical properties of the purified MANBA gene product will be determined. Pure MANBA will be digested and 6-10 polypeptide fragments will be partly sequenced. Monospecific polyclonal antibody to MANBA will be produced. Oligonucleotides will be synthesized complementary to part of one or of the polypeptide fragments. II. Metabolic perturbations of beta-mannosidosis target cells and tissues: Novel complex oligosaccharides with a beta-mannose terminus (identified in preliminary studies) will be isolated from affected tissues by gel permeation and medium pressure liquid chromatography. They will be characterized by gas chromatography, mass spectrometry, and H-NMR. Carbohydrate composition analysis, methylation linkage studies and enzymatic degradations will be utilized to determine structures and anomeric configurations. The tissue localization of these oligosaccharides will be determined. III. Maintenance of the caprine beta-mannosidosis colony is required to produce affected animals for the proposed research and to preserve the beta-mannosidosis allele. These studies are intended to: a. lead to the cloning of the MANBA gene and ultimately to gene therapy of the human inherited metabolic disease; b. further define the pathogenesis and metabolic basis of beta-mannosidosis; and c. delineate heretofore unknown pathways of glycoprotein metabolism.
Showing the most recent 10 out of 36 publications
