Gaucher disease is the most prevalent lysosomal lipid storage disease caused by deficient glucocerebrosidase activity. It is transmitted as an autosomal recessive trait. Three clinical forms of Gaucher disease have been described: type 1, non-neuronopathic; type 2, acute neuronopathic; and type 3, subacute neuronopathic. It has been known that in most cases, the deficient glucocerebrosidase activity is due to mutations in the glucocerebrosidase gene. However, some mutant alleles remain unidentified. In this study, we performed DNA sequence analysis of 12 mutant alleles from 6 unrelated type 1 and type 2 Gaucher patients. Two novel mutations (649T and 1366G) from one type 1 and one type 2 Gaucher patient, and two rare mutations (48IT and 1604A) from two type 1 Gaucher patients were identified. To demonstrate that these mutations are deleterious and not neutral mutations, we inserted the full-length normal and mutant glucocerebrosidase cDNA into the genome of baculovirus AcUW1.lacZ and expressed the recombinant enzyme in Spodoptera frugiperda cells (Sf9). The levels of glucocerebrosidase, activities from crude extracts of transfected Sf9 cells with the Gaucher 649T, 1366G, 48IT, and 1604A alleles are 2.8%, 2.9%, 17.3% and 6.9% of that expressed by the normal allele [normal = 352.0 nmol/hr/mg protein, using a fluorogenic substrate 4-methylumbeffifery 1-β-D-glucopyranoside (4MUGP)]. The results demonstrated that the two novel mutations (1604A and 1366G) and the two rare mutations (481T and 1604A) are deleterious, resulting in profoundly deficient glucocerebrosidase activity and subsequent Gaucher disease.
To explore the feasibility of the heterologous expression of the recombinant glucocerebrosidase in the yeast Pichia pastoris, we cloned the glucocerebrosidase cDNA into transformation vectors pPIC9K and pPlCαZ downstream of the AOX1 promoter, and integrated into yeast hosts KM71 and SMD 1168 of Pichia pastoris. The recombinant glucocerebrosidase was expressed and secreted into the induced culture medium when the native targeting signal of glucocerebrosidase cDNA was replaced by an α-factor secretion signal of Saccharomyces cerevisiae. The maximum expression level under flask culture conditions reached the specific activity of 494 nmol/hr/mg protein on a natural substrate (N-palmitoyl dihydroglucocerebroside). The secreted form of recombinant glucocerebrosidase was determined to have a molecular weight of 66 KDa. After deglycosylation, the peptide backbone has a molecular weight of 58 kDa. The recombinant enzyme exhibits similar kinetic properties to that of native glucocerebrosidase. A successive two-step chromatography procedure was developed to purify the recombinant enzyme to apparent homogeneity. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/8685 |
| Date | 18 October 2017 |
| Creators | Wei, Chao |
| Contributors | Choy, Francis Y. M. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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