Glycoprotein Biogenesis: Optimization of N- Acetylglucosaminidase, Lysosomal Neuraminidase, and Protective Protein Cathepsin A Expression
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Humans depend on lysosomal enzymes for proper macromolecular degradation. In their absence, lysosomal catabolism is impaired, leading to Lysosomal Storage Diseases (LSDs) (Meikle, 2003). LSDs are characterized by accumulations of metabolic intermediates (Meikle, 2003; Meikle et al., 1999). Accumulation of these biomolecules over time can lead to irreversible arterial blockage, nervous tissue damage, and progressive neurodegeneration (Meikle, 2003; Naganawa et al., 2000). Most LSDs are incurable (Neufeld et al., 1975). Determining the structure of functional lysosomal enzymes is the first step toward understanding LSDs. X-ray crystallography is one technique used to understand protein structure. To perform crystallography, researchers must generate enough protein for crystallization. Using a small polyclonal lepidopteran stable cell line, I have generated two enzymes - Protective Protein Cathepsin A (PPCA), and Lysosomal Neuraminidase 1 (NEU1). Stable cell line generation entails introduction of recombinant protein coding genes into lepidopteran cells, selecting the best protein producers through antibiotic resistance and protein immunodot blot assays. I also employed a baculovirus expression system to generate NEU1 (O’Reilly et al., 1994). Further work would include protein purification and crystallization of proteins produced. This 1 2 work has long-range applications in the rational design of small molecules that stabilize native state protein conformations (Fan and Ishii, 2007a).