The protein transduction domain (PTD) of the HIV-I TAT protein has been shown to be capable of crossing cellular membranes and even the blood-brain barrier while carrying cargo molecules along with it. Exploiting this property to deliver biologically active acid β-glucosidase (GBA) would be of use to improve the current treatment of Gaucher disease by enzyme replacement therapy. Genetic fusion of the TAT PTD to GBA was performed and the resulting gene was inserted into an insect expression vector, p2ZOptcxF, to allow for heterologous protein production in Sf9 cells. A TAT fusion with enhanced green fluorescent protein (EGFP) was also created to serve as a control. The insect vector encoded a cellulose-binding domain to allow for affinity purification of the heterologous proteins. The Sf9 system produced 0.9-1.2 μg/ml quantities of EGFP fusion proteins, but only low ng/ml levels of GBA fusions. The addition of the cellulose binding domain decreased protein expression, but could be used for purification when μg/ml quantities of protein were produced. This suggests that expression of soluble, unglycosylated proteins such as EGFP can be achieved in the p2ZOptcxF/Sf9 system and to a lesser extent complex, highly-glycosylated proteins such as GBA can also be produced. In order to improve the expression of GBA fusions further optimization of the vector, selection and production must be undertaken.

No description available.

Gaucher disease, an autosomal recessive disorder, is characterized by a heterogeneous set of signs and symptoms caused by a deficiency in the lysosomal enzyme glucocerebrosidase. As a single gene enzyme deficiency, Gaucher disease is a prime candidate for enzyme replacement therapy. Such therapy exists, though the exorbitant cost prevents many fiom receivii treatment. Thus, a more cost-effective method of producing glucocerebrosidase was examined. The Pichiapastoris yeast system was chosen, but resultant production levels were low. Two variants of green fluorescent protein (GFP), red-shifted GFP (RSGFP) and enhanced GFP (EGFP), were employed as molecular reporters to track enzyme production and isolation. No expression of glucocerebrosidase was evident, indicating that the P. pastoris system was not an appropriate choice for glucocerebrosidase production. Both GFP variants were successfully expressed, with EGFP levels apparently greater than RSGFP levels. To study glucocerebrosidase production and trafficking in a higher eukaryotic system, EGFP-tagged glucocerebrosidase constructs were expressed in HeLa cells. Though EGFP was readily visualized, few cells expressing glucocerebrosidase constructs were present. No co-localization with organelle markers was evident. Examination at the RNA level indicated successfid transcription, however, an apparent translational inefficiency was encountered. To shed light on the possible cause of this inefficiency, two approaches were taken: one examined expression of truncated glucocerebrosidase constructs in HeLa cells, the other included co-transfection with small interfering RNAs (siRNAs) in both HeLa and COS- 1 cells. In the first approach, greater expression was seen itom the EGFPtagged construct devoid of the proposed inhibitory binding site than itom the EGFPtagged construct containing the binding site. Expression of both truncated constructs was greater than that of EGFP-tagged glucocerebrosidase starting at either initiation codon, indicating a more complex mechanism of translational control than strictly inhibition fiom the proposed site. In the second approach, a siRNA was designed to block TCP80, which has been suggested to inhibit glucocerebrosidase translation. Co-transfection studies of siJ3NAs (control, EGFP and TCP80) and glucocerebrosidasel EGFP plasmids were performed in HeLa and COS-1 cells. In both cell types, all constructs were successfblly expressed when co-transfected with control siRNA, as indicated by RNA and protein examination. Introduction of TCP80 siRNA in both cell types did not serve to increase glucocerebrosidase expression as expected, but instead decreased such expression. EGFP expression was readily knocked down in HeLa and COS-1 cells by GF'P-targeted siRNk Knockdown was evident in the expression of glucocerebrosidase/EGFP constructs, indicating that hsion with EGFP may serve as a means to introduce a foreign gene, then knock its expression down at a desired time by introduction of a GFP-targeted siRNA.

No description available.

No description available.

Gaucher disease (GD) is caused by an inherited deficiency of the human lysosomal enzyme acid β-glucosidase (GBA, EC 3.2.1.45). Absence of functional enzyme results in lysosomal glycolipid accumulation. This disorder primarily affects organs of the reticuloendothelial system and disease severity ranges from mild hepatosplenomegaly to extreme neurological degeneration. Disease symptoms have been shown to be greatly ameliorated by enzyme replacement therapy (ERT). Limitations to therapy include the high cost of current ERT and its inability to treat neurological symptoms. In the present study I sought to produce a GBA-fusion enzyme in an economical manner that can be used to treat neurological GD. I explored the use of Pichia pastoris as an economical recombinant protein expression system for production of human GBA. In addition, I synthesized a protein transduction domain (PTD)-GBA fusion protein for its potential to be used as a neurotherapeutic. The results show that GBA-PTD4 can be expressed and purified from P. pastoris. Hydrophobic interaction chromatography and gel filtration chromatography were successful in purifying GBA-PTD4. Further optimization of expression and purification techniques is required for effective large scale production of recombinant enzyme. / Graduate

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

Gaucher disease, the most common lysosomal storage disorder, results from a deficiency in the enzyme glucocerebrosidase. Inherited as an autosomal recessive disorder, Gaucher disease is clinically heterogeneous with both non-neuronopathic (Type 1) and neuronopathic (Types 2 and 3) subtypes. Although over 100 mutations in the glucocerebrosidase (GBA) gene have been identified, there still exists a poor correlation between individual genotypes and observed phenotypes, particularly for the neuronopathic subtypes. Using DNA isolated from archival tissue samples and standard molecular biology techniques, two novel and two rare mutations were identified in three individuals with neuronopathic Gaucher disease. One mutation identified only in aboriginals of Cree descent was further characterized by heterologous expression in baculovirus-infected Sf9 cells and displayed moderate levels of residual enzyme activity, despite the corresponding disease severity observed. Heterologous expression studies were extended to examine systems for high-level glucocerebrosidase expression for biochemical analysis and biotherapeutics. While the methylotrophic yeast Pichia pastoris was found to express minimal amounts of human glucocerebrosidase even when selected for high gene copy number, stable transfected Sf9 cells were found to produce functional glucocerebrosidase at a level of 1.0–1.3mg/L of cell culture. A subsequent analysis of synonymous codon usage bias in Pichia pastoris identified a significant difference in codon choice between the expression host and the GBA gene. Codon optimization studies using a 5′ fragment of the GBA gene fused to a luciferase reporter gene found that alterations in both G+C content and codon bias increased expression levels 7.5 to 10 fold. This suggests that codon optimization of the entire GBA gene could significantly improve production levels of this important enzyme in Pichia pastoris and other expression hosts. / Graduate

Gaucher’s disease (GD) is an autosomal recessive lysosomal storage disorder which is caused by a mutation in the gene encoding acid β-glucocerebrosidase (GBA, EC 3.2.1.45). Deficient activity in GBA leads to a wide variety of clinical phenotypes, including visceral symptoms such as hepatospenomegaly as well as neurological symptoms. Current enzyme replacement therapy is effective in treating visceral symptoms but cannot cross the blood-brain barrier to target neurological manifestations. Another drawback to current therapy is the high cost to patients due to present protein expression strategies. Recently, protein transduction domains, such as the synthetic PTD4 domain, have been proposed as a therapeutic strategy for drug delivery to the central nervous system. In the present study, we use an economical yeast expression system, Pichia pastoris, to produce a recombinant fusion protein GBA-PTD4, and semi-preparative hydrophobic interaction chromatography and gel filtration chromatography for purification. Results show that final preparations are near homogenous, with GBA-PTD4 accounting for approximately 76% of total protein and only one major contaminant. A cell line expressing GBA without a transduction domain was also created in anticipation of further cellular uptake studies. Future research will focus on large scale enzyme expression in fermentation systems and more direct purification methods such as immunoaffinity chromatography for better protein recovery. / Graduate

The goal of this project has been to develop therapeutic strategies for protein misfolding diseases caused by excessive degradation of misfolded proteins and loss of protein function. The focus for this work is lysosomal storage disorders (LSDs), a group of more than 50 known inherited metabolic diseases characterized by deficiency in hydrolytic enzymes and consequent buildup of lysosomal macromolecules. Gaucher’s Disease (GD) is used as a representative of the family of LSDs in this study. GD is caused by mutations in the gene encoding lysosomal glucocerebrosidase (GC) and consequent accumulation of the GC substrate, glucocerebroside. The most prevalent mutations among GD patients are single amino acid substitutions that do not directly impair GC activity, but rather destabilize its native folding. GC normally folds in the ER and trafficks through the secretory pathway to the lysosomes. GC variants containing destabilizing mutations misfold and are retrotranslocated to the cytoplasm for ER-associated degradation (ERAD). However, evidence shows that if misfolding-prone, mutated GC variants are forced to fold into their 3D native structure, they retain catalytic activity. This study describes strategies to remodel the network of cellular pathways that maintain protein homeostasis and to create a folding environment favorable to the folding of unstable, degradation-prone lysosomal enzyme variants. We demonstrated that folding and trafficking of mutated GC variants can be achieved by modulating the protein folding network in fibroblasts derived from patients with GD to i) upregulate the expression of ER luminal chaperones, ii) inhibit the ERAD pathway, and iii) enhance the pool of mutated GC in the ER amenable to folding rescue. We also demonstrated that the same cell engineering strategies that proved successful in rescuing the folding and activity of mutated GC enable rescue of mutated enzyme variants in fibroblasts derived from patients with Tay-Sachs disease, a LSD caused by deficiency of lysosomal hexosaminidase A activity. As a result, the current study provides insights for the development of therapeutic strategies for GD based on the modulation of general cellular pathways that maintain protein homeostasis that could in principle be applied to the treatment of multiple LSDs.