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Published on: July 23, 2017
Isolation of an aspartic proteinase precursor from the egg of a hard tick, Boophilus microplus
C Logullo1, I da S Vaz, M H Sorgine
1Departamento de Bioquímica Médica, ICB-CCS-UFRJ, Cidade Universitária, Rio de Janeiro, RJ, Brazil. logullo@server.bioqmed.ufrj.br
Abstract:
An aspartic proteinase precursor, herein named BYC (Boophilus Yolk pro-Cathepsin) was isolated from eggs of the hard tick, Boophilus microplus. As judged by electrophoresis on sodium dodecyl sulfate polyacrylamide slab gel (SDS-PAGE), purified BYC presented 2 bands of 54 and 49 kDa, bearing the same NH2-terminal amino acid sequence. By Western blot analysis, BYC was also found in the haemolymph, indicating an extraovarian site of synthesis. Several organs were incubated in culture medium with [35S]methionine, and only the gut and fat body showed synthesis of BYC polypeptides. Protein sequencing of both the NH2-terminal and an internal sequence obtained after cyanogen bromide (CNBr) cleavage of BYC revealed homology with several aspartic proteinase precursors. Incubation at pH 3.5 resulted in autoproteolysis of BYC, which produced the mature form of the enzyme, that displayed pepstatin-sensitive hydrolytic activity against haemoglobin. Western blot analysis using anti-BYC monoclonal antibodies showed proteolytic processing of BYC during embryogenesis and suggested activation of the enzyme during development. A role of BYC in degradation of vitellin, the major yolk protein of tick eggs, is discussed.
Insights
A novel aspartic proteinase precursor, Boophilus Yolk pro-Cathepsin (BYC), was identified in hard ticks. This enzyme is synthesized in extraovarian tissues and plays a role in degrading yolk proteins during tick embryogenesis.
Area of Science:
- Biochemistry
- Molecular Biology
- Parasitology
Background:
- Hard ticks like Boophilus microplus are significant agricultural pests.
- Understanding tick egg development and protein utilization is crucial for pest control strategies.
Purpose of the Study:
- To isolate and characterize a novel aspartic proteinase precursor from Boophilus microplus eggs.
- To investigate the synthesis, processing, and potential function of this precursor during tick development.
Main Methods:
- Isolation and purification of the proteinase precursor BYC using SDS-PAGE.
- Western blot analysis to determine BYC localization and synthesis sites.
- Protein sequencing (N-terminal and CNBr cleavage) to identify homology.
- Enzyme activity assays at acidic pH and analysis of autoproteolysis.
- Monoclonal antibody-based Western blot to track processing during embryogenesis.
Main Results:
- A 54/49 kDa aspartic proteinase precursor, BYC, was purified from Boophilus microplus eggs.
- BYC is synthesized in the gut and fat body, and found in hemolymph, indicating extraovarian synthesis.
- BYC undergoes autoproteolysis at pH 3.5 to yield a mature, pepstatin-sensitive enzyme active against hemoglobin.
- Proteolytic processing and activation of BYC occur during tick embryogenesis.
Conclusions:
- BYC is a vitellin-degrading aspartic proteinase essential for tick egg development.
- Extraovarian synthesis and developmental activation highlight BYC's critical role in tick reproduction and survival.
