Abstract
Trypsin is a serine protease that is synthesized by the gut epithelial cells of female mosquitoes; it is the enzyme that digests the blood meal. To study its molecular regulation, Culex quinquefasciatus late trypsin was purified by diethylaminoethyl (DEAE), affinity, and C18 reverse-phase high performance liquid chromatography (HPLC) steps, and the N-terminal amino acid sequence was determined for molecular cloning. Five overlapping segments of the late trypsin cDNA were amplified by PCR, cloned, and the full sequence (855 bp) was characterized. Three-dimensional models of the pro-trypsin and activated trypsin were built and compared with other trypsin models. Trypsin modulating oostatic factor (TMOF) concentrations in the hemolymph were determined by ELISA and compared with trypsin activity in the gut after the blood meal. The results showed that there was an increase in TMOF concentrations circulating in the hemolymph which has correlated to the reduction of trypsin activity in the mosquito gut. Northern blot analysis of the trypsin transcripts after the blood meal indicated that trypsin activity also followed the increase and decrease of the trypsin transcript. Injections of different amounts of TMOF (0.025 to 50 μg) decreased the amounts of trypsin in the gut. However, Northern blot analysis showed that TMOF injections did not cause a decrease in trypsin transcript abundance, indicating that TMOF probably affected trypsin translation.
Highlights
Anautogenous female mosquitoes take a blood meal to develop their eggs in the ovaries and are important vectors of human infectious diseases such as malaria, dengue, and yellow fever
We demonstrate that AeaTMOF affected the translation of the late trypsin transcript in the gut epithelial cells as it has been shown for Neobellieria in which NeoTMOF (10−9 M), a hexapeptide (NPTNLH) different than AeaTMOF, inhibited the translation of the late trypsin transcript (Borovsky et al, 1996)
To find out the relationship between trypsin activity in the gut of female C. quinquefasciatus and Trypsin modulating oostatic factor (TMOF) circulating in the hemolymph after the blood meal, the guts and hemolymph were assayed for trypsin activity and TMOF concentrations, respectively
Summary
Anautogenous female mosquitoes take a blood meal to develop their eggs in the ovaries and are important vectors of human infectious diseases such as malaria, dengue, and yellow fever. To digest the blood meal to free amino acids female mosquitoes, TMOF Controls Trypsin Translation synthesize trypsin, and chymotrypsin-like enzymes (Borovsky, 2003). Female C. quinquefasciatus that were fed on sugar before entering diapause were reported to synthesize trypsin-like enzymes in their midgut. These enzymes were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by tandem mass spectrometry (MS/MS) analyses or by molecular cloning and characterization. These studies suggested that the early trypsin might play a role in the activation of the late trypsin after the blood meal. Their role in digesting the blood meal or whether they are continued to be synthesized or downregulated after the blood remains unknown (Robitch and Denlinger, 2005; Borges-Veloso et al, 2015)
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