Sequencing and characterization of the citrus weevil, Diaprepes abbreviatus, trypsin cDNA

Abstract

Trypsin mRNA from the citrus weevil, Diaprepes abbreviatus, was reverse transcribed and amplified by PCR. A cDNA species of 513 bp was cloned and sequenced. The 3 H and 5 H ends of the gene (262 bp and 237 bp, respectively) were amplified by rapid amplification of cDNA ends, cloned and sequenced. The deduced sequence of the trypsin cDNA (860 bp) encodes for 250 amino acids including 11 amino acids of activation and signal peptides and exhibited 16.8% identity to trypsin genes of selected Lepidoptera and Diptera. A three-dimensional model of Diaprepes trypsin contained two domains of b-barrel sheets as has been found in Drosophila and Neobellieria. The catalytic active site is composed of the canonical triad of His41, Asp92 and Ser185 and a specificity pocket occupied by Asp179 with maximal activity at pH 10.4. Southern blot analysis indicated that at least two copies of the gene are encoded by Diaprepes midgut. Northern blot analysis detected a single RNA band below 1.35 kb at different larval ages (28±100 days old). The message increased with age and was most abundant at 100 days. Trypsin activity, on the other hand, reached a peak at 50 days and fell rapidly afterwards indicating that the trypsin message is probably regulated translationally. Feeding of soybean trypsin inhibitor and Aedes aegypti trypsin modulating oostatic factor affected trypsin activity and trypsin biosynthesis, respectively. These results indicate that Diaprepes regulates trypsin biosynthesis with a trypsin modulating oostatic factor-like signal.

Biosynthesis and Control of Mosquito Gut Proteases

“…gambiae and Ae. aegypti late trypsins is only 40.5% (15) three dimensional models using ribbon drawings made by MOLSCRIPT as was done for the fleshfly Neobellieria bullata, for Drosophila melanogaster and for the citrus weevil Diaprepes abbreviatus (15,16) show that the 3 D folding of these enzymes is very similar (Fig. 3).…”

Molecular and biochemical characterisation of a dual proteolytic system in vine weevil larvae (Otiorhynchus sulcatus Coleoptera: Curculionidae)

“…The sequence similarities suggest that this sequence corresponds to a digestive protease. The 50 most similar sequences to VW5 as determined by a BLASTP similarity search are all insect or arthropod trypsins, with the most similar sequence from a coleopteran species, the citrus weevil Diaprepes abbreviatus (Yan et al, 1999). On the other hand, although the enzyme encoded by VW11 has sequence homology to trypsins, the active site residues suggest that this is not a trypsin-like protease.…”

Proteolytic gut activities in the rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae)

“…Nevertheless, aspartic and/or serine proteases have been identified in some of these species (Purcell et al, 1992;Wilhite et al, 2000). There are also some curculionids with serine protease activity and alkaline pH optima, such as the red palm weevil, Rhynchophorus ferrugineus (Alarcon et al, 2002), and the citrus weevil, Diaprepes abbreviatus (Yan et al, 1999).…”