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An extraovarian aspartic protease accumulated in tick oocytes with vitellin-degradation activity
Paula C Pohl1, Marcos Henrique F Sorgine, Alexandre T Leal
1Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
Abstract:
An aspartic endopeptidase named THAP, from the eggs of the tick Riphicephalus (Boophilus) microplus, has been suggested to be involved in vitellin-degradation. Here we characterized this enzyme further, showing that THAP mRNA is present in the fat body, midgut and ovary of ticks, in two developmental stages (partially and fully engorged females). However, higher transcription levels were found in fully engorged vitellogenic females. The THAP protein was detected in the haemolymph, midgut and fat body and, in higher quantity, in the ovary of fully engorged females, and it was present throughout embryo development. The protein is synthesized as a higher molecular mass form and after the onset of embryogenesis THAP is converted into an active form by autocatalysis. We also produced a recombinant protein (rTHAP) in E. coli that was active in the fluorogenic peptide substrate and able to hydrolyze vitellin from 7-day-old eggs in a reaction that is heme-sensitive and inhibited by pepstatin A. However, rTHAP does not hydrolyze vitellin from 1 and 12-day-old eggs. As a result, we suggest a model for THAP synthesis, transport, storage and activation and for the role it plays in embryonic development by participating in vitellin processing.
Insights
The tick enzyme THAP (tick aspartic endopeptidase) is crucial for embryonic development, processing vitellin in Rhipicephalus (Boophilus) microplus eggs. Its synthesis, activation, and role in vitellin breakdown are detailed, highlighting its importance in tick reproduction.
Area of Science:
- Biochemistry
- Molecular Biology
- Parasitology
Background:
- Tick Rhipicephalus (Boophilus) microplus eggs contain an aspartic endopeptidase, THAP, potentially involved in vitellin degradation.
- Vitellin is a major yolk protein essential for embryonic development in many oviparous species.
Purpose of the Study:
- To further characterize the THAP enzyme from Rhipicephalus (Boophilus) microplus.
- To elucidate the synthesis, transport, activation, and role of THAP in embryonic development and vitellin processing.
Main Methods:
- Analysis of THAP mRNA expression in different tick tissues and developmental stages using quantitative methods.
- Detection of THAP protein in various tick tissues and during embryonic development using immunological techniques.
- Production and characterization of recombinant THAP (rTHAP) in E. coli, including enzymatic activity assays and substrate specificity studies.
Main Results:
- THAP mRNA is expressed in the fat body, midgut, and ovary, with higher levels in engorged females; the protein is found in hemolymph, midgut, fat body, and ovary, and throughout embryogenesis.
- THAP is synthesized as a precursor and activated by autocatalysis post-embryogenesis.
- Recombinant THAP (rTHAP) exhibits enzymatic activity on vitellin from 7-day-old eggs, a process sensitive to heme and inhibited by pepstatin A, but does not hydrolyze vitellin from 1- or 12-day-old eggs.
Conclusions:
- A model for THAP synthesis, transport, storage, and activation is proposed.
- THAP plays a specific role in vitellin processing during embryonic development in Rhipicephalus (Boophilus) microplus.
- The stage-specific activity of THAP on vitellin suggests a precise regulatory mechanism for nutrient utilization during tick embryogenesis.
