Preliminary Characterization of Drosophila Beta-glucuronidases

Chowdhury, Priyanka (2015) Preliminary Characterization of Drosophila Beta-glucuronidases. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS dissertation of Priyanka Chowdhury (10MS40)) MS_Thesis_Priyanka_Chowdhury_10MS40.pdf - Submitted Version Restricted to Repository staff only Download (1MB)

Abstract

Beta glucuronidase is an enzyme belonging to the Glycosidase family which helps in the breakdown of glycosaminoglycans in lysosomes. Mutation in beta glucuronidase can cause accumulation of undegraded/partially degraded glycosaminoglycans in the organelle leading to Mucopolysachharidosis VII, a lysosomal storage disorder. Analysis of Drosophila melanogaster genome suggests that it encodes for two putative beta glucuronidases annotated as CG2135 and CG15117 in the FlyBase. Sequence alignment of CG2135 and CG15117 with human beta glucuronidase shows 60% and 40% similarity respectively. The three main active-site catalytic residues Glu⁴⁵¹ (proton donor), Glu⁵⁴⁰ (stabilizes intermediate) and Tyr⁵⁰⁴ (stabilizes leaving nucleophile) are conserved in both the Drosophila beta-glucuronidase isoforms. Homology modeling predicted CG2135 and CG15117 to be homotetramers as ascertained by RMSD mapping with the human counterpart. Interestingly, analysis of genomes of organisms of various phyla in Animal Kingdom revealed Drosophila to be the only genus possessing two isoforms of beta glucuronidase. Whereas all other organisms studied by us, including human, possess only one beta-glucuronidase. Of the two isoforms of beta-glucuronidase in Drosophila, CG2135 is predicted to contain the ER signal sequence, which is a primary signature for all lysosomal enzymes. However, based on bioinformatic study, CG15117 isoform appears to be a transmembrane protein. These interesting observations gave us the impetus to biochemically characterize Drosophila beta-glucuronidase isoforms with the ultimate goal of understanding their function in fly physiology. For this, we first tried to express CG2135 and CG15117 in bacterial expression system. We transformed E.coli BL21DE3 cells with CG2135 or CG15117 cDNA cloned in PET28a vectors. Upon induction with 0.5 mM IPTG, the expected ~80 KDa proteins were overexpressed in both the cases. CG15117 protein showed higher expression than CG2135 in the induced cells. Whole cell lysate of bacterial cells transformed with the above constructs showed no detectable beta-glucuronidase activity and >95% of both the induced proteins were in the insoluble fraction. Following this, we attempted the purification of Histidine tagged recombinant CG15117 protein from the soluble fraction but failed in purifying the protein. Hence to purify active proteins, we switched to a eukaryotic expression system, Drosophila Schneider 2 cell line. CG2135 and CG15117 genes cloned with Myc tag in Drosophila expression vector pMT-puro were used to transiently transfect S2 cells. Both CG2135 and CG15117 proteins were expressed in the S2 cells as determined by beta-glucuronidase assay and Western blot using anti-Myc primary antibody. Both proteins show higher activity in culture media as compared to the cell lysate indicating that a significant proportion of the expressed protein is secreted. Cloning of CG2135 and CG15117 with Histidine tag in pMT-puro is underway which will be used for large scale protein purification purposes. We thus prove that S2 cell is an appropriate expression system for purifying Drosophila beta-glucuronidase isoforms.

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