Investigating the Subcellular Localisation of β-Glucuronidase enzymes of Drosophila melanogaster

Sabar, Aniket (2025) Investigating the Subcellular Localisation of β-Glucuronidase enzymes of Drosophila melanogaster. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Aniket Sabar (20MS033)) 20MS033_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (3MB)

Abstract

Beta-glucuronidase (β-GUS) is a lysosomal hydrolase responsible for the degradation of glycosaminoglycans (GAGs), with functional impairment linked to the lysosomal storage disorder Mucopolysaccharidosis type VII (MPS VII) in humans. Uniquely, Drosophila melanogaster possesses two β-GUS paralogs, CG2135 and CG15117, providing a valuable model to study subcellular targeting and the evolutionary consequences of gene duplication. This report investigates the subcellular localisation of CG2135 and CG15117 through a combination of bioinformatic analyses, immunofluorescence microscopy, and differential centrifugation-based subcellular fractionation. While sequence and structural comparisons confirmed functional conservation of both paralogs, key differences were observed: CG2135 contains an N-terminal signal peptide, suggesting lysosomal targeting, whereas CG15117 lacks this motif, indicating a potentially non-canonical localisation. The differential fractionation experiments yielded clearer insights. CG2135 enzyme activity was enriched in microsomal/lysosomal fractions, while CG15117 activity was predominantly found in cytosolic fractions. These findings strongly suggest that the two enzymes exhibit distinct subcellular distributions. However, it is important to note that this study does not precisely determine the exact intracellular locations of either paralog. Instead, the data provide strong indications—rather than definitive confirmation—of contrasting localisation patterns between CG2135 and CG15117. Despite this limitation, the consistency of results across knockout genotypes supports a robust conclusion: CG2135 and CG15117 occupy different subcellular compartments, following gene duplication. This work contributes to our understanding of lysosomal enzyme targeting, the adaptability of duplicated genes, and intracellular compartmentalisation. It lays the groundwork for future studies to investigate the precise localisation mechanisms and functional implications of β-GUS paralogs in Drosophila melanogaster.

Actions (login required)

View Item