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Molecular cloning, expression and characterization of a novel vacuolar protein sorting 4 gene in silkworm, Bombyx
Hengchuan Xia1, Chunxia Zhang, Fan Feng
1Institute of Life Sciences, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu 212013, People's Republic of China.
Abstract:
The vacuolar protein sorting 4 (Vps4) protein is essential for the multivesicular body (MVB) pathway, virus budding process and cytokinesis. Vps4 has been identified and characterized from many species, but not from silkworm Bombyx mori. In this study, we firstly identified and cloned the silkworm homologous gene for VPS4, expressed it in Escherichia coli, purified and characterized the protein designated as BmVps4. The BmVps4 cDNA contains an open reading frame of 1,314 bp, and encodes a protein of 438 amino acid residues. BmVps4 is of high sequence-similarity to Vps4 proteins from other species. The recombinant BmVps4 shows ATPase activity, which can be stimulated by Mg(2+) and inhibited by dominant mutations. Together, our data suggest BmVps4 is the genuine silkworm homologue of Vps4. To our knowledge, this is the first-time characterization of any silkworm MVB proteins. This study will facilitate further investigation of silkworm MVB pathway and its possible roles in the infection and budding of B. mori nuclear polyhedrosis virus (BmNPV), which is one of the most common and severe pathogens for silkworms. The cloned BmVps4 sequence is deposited in GenBank (Accession number GQ995504).
Insights
Researchers identified and characterized silkworm vacuolar protein sorting 4 (Vps4), crucial for cell processes. This discovery aids understanding of silkworm viruses and the multivesicular body pathway.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Vacuolar protein sorting 4 (Vps4) is vital for the multivesicular body (MVB) pathway, virus budding, and cytokinesis in various organisms.
- Vps4 characterization is lacking in the silkworm, Bombyx mori, a significant insect in sericulture.
Purpose of the Study:
- To identify, clone, and characterize the silkworm Bombyx mori Vps4 homolog (BmVps4).
- To establish a foundation for studying the MVB pathway and its role in silkworm viral infections, particularly BmNPV.
Main Methods:
- Gene identification and cloning of the silkworm VPS4 homolog.
- Recombinant protein expression in Escherichia coli and subsequent purification.
- Biochemical characterization of the purified BmVps4 protein, including ATPase activity assays.
Main Results:
- The silkworm VPS4 gene (BmVps4) was successfully identified and cloned, encoding a 438-amino acid protein.
- BmVps4 exhibits high sequence similarity to Vps4 proteins from other species.
- Recombinant BmVps4 demonstrated Mg(2+)-stimulated ATPase activity, sensitive to dominant mutations.
Conclusions:
- BmVps4 is confirmed as the genuine silkworm homolog of Vps4.
- This study provides the first characterization of an MVB pathway protein in silkworms.
- The findings facilitate future research on the silkworm MVB pathway and its implications for BmNPV pathogenesis.
