Among the inherited metabolic diseases, Gaucher disease (GD) is of particular importance since it is the most prevalent lysosomal storage disease. GD has marked clinical heterogeneity within and among the three Types: These distinct Types result from the deficient activity (5-20%) of acid beta-glucosidase (beta-Glc). The overall objective of the proposed research is to elucidate the molecular bases of the various enzymatic defects which underlie this remarkable phenotypic heterogeneity in Gaucher disease (GD). Already the rich genetic diversity in the GD Types and variants has been indicated by the identification of seven missense mutations and additional mutant alleles in the GD population. This heterogeneity will be determined by sequencing amplified beta-Glc cDNAs from patients with unidentified alleles and/or having residual enzymes with unique biochemical properties (e.g., increased stability). Ten cDNA libraries already have been made in this laboratory. The frequency of mutations and their correlation with phenotypic severity will be determined from population studies (>300 GD patients) using amplified genomic DNA and allele specific probes. To account for the large phenotypic variation among genotypically identical (beta-Glc locus) patients, our GLBA (SAP-2) cDNA will be evaluated as a candidate modifier gene by determining the correlation of RFLPs at this locus with GD severity in selected affected siblings with differing degrees of involvement. The functional perturbations of beta-Glc caused by authentic GD mutations will be determined by systematic kinetic, processing and/or stability studies of the enzyme expressed from mutant CDNAs using our eukaryotic (Baculovirus, CHO or human) systems. Additional residues critical to beta-Glc catalytic activity or negatively-charged phospholipid (NCP) interactions will be assigned using affinity ligands (e.g., photoactivable substrate or NCPs analogues and/or binding site specific antibodies) to identify beta-Glc peptides for amino acid sequence analysis. Our demonstration that beta-Glc expressed in an unglycosylated form was inactive implicates N-glycosylation site occupancy in the modulation of catalytic activity. To elucidate this role, our beta-Glc cDNA having each of the five potential N-glycosylation sites individually destroyed (Asn->Gln) or those with various combinations of mutated sites will be expressed and the catalytic properties systematically evaluated. Similarly the localization (by cell fractionation and immunoelectronmicroscopy) of truncated beta-Glcs expressed from deletion mutants transfected into CRIMneg fibroblasts will define the mannose-6-phosphate independent signals for lysomal targeting of this membrane associated enzyme. The results of these studies should provide a basis for the design of biologicals with enhanced activities for future therapeutic interventions in GD. In addition, phenotype/genotype correlations by molecular analyses will facilitate the presymptomatic prediction of GD severity as a necessary prelude to the critical evaluation of the efficacy of genic therapeutic strategies in this prototype inborn error or metabolism.
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