Molecular and biochemical characterisation of a dual proteolytic system in vine weevil larvae (Otiorhynchus sulcatus Coleoptera: Curculionidae).
The ability of phytophagous insects to utilise the relatively low nitrogen content of plant tissues is typically the limiting factor in their nutritional uptake. In the larval stage, the vine weevil feeds predominantly on root tissues of plants. The root tissue as a whole has low levels of free amino acids, and thus effective hydrolysis of dietary proteins is essential for survival. In contrast to previous reports the present study demonstrates through both molecular and biochemical studies the presence of proteolytic enzymes from two mechanistic classes, cysteine and serine proteases, in the gut of larval vine weevil; with the latter being the predominant form. cDNA clones encoding cathepsin B-like and serine-like sequences were isolated from a gut specific cDNA library; the cathepsin B-like clone has the Cys-His-Asn catalytic triad. However, the sequence showed the replacement of the conserved His-His sequence in the "occluding loop" region of the enzyme with Asp-His. This may result in a change to the substrate specificity. Two trypsin precursors contained evidence of a signal peptide, activation peptide, and conserved N-termini (IVGG). Other structural features included typical His, Asp, and Ser residues of the catalytic amino acid triad indicative of serine proteases, characteristic residues in the substrate-binding pocket, and four pairs of cysteine residues for disulfide bridges. The apparent abundance of the trypsin-like cDNA clones compared to the cathepsin B clones suggests that serine proteases are the predominant form, thus supporting data from the biochemical studies.
Edwards
M G
MG
Newcastle University, School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, Newcastle upon Tyne NE1 7RU, UK. m.g.edwards@newcastle.ac.uk
Gatehouse
John A
JA
Gatehouse
A M R
AM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D057056:Cysteine Proteases / Q000235:genetics / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000201:enzymology; D015723:Gene Library; D007814:Larva / Q000201:enzymology; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D057057:Serine Proteases / Q000235:genetics / Q000378:metabolism*; D048889:Weevils / Q000201:enzymology* / Q000235:genetics
D015345:Oligonucleotide Probes; D057056:Cysteine Proteases; D057057:Serine Proteases