A cellulase (endo-b-1,4-glucanase, EC 3.2.1.4) was purified from the gut of larvae of the yellow-spotted longicorn beetle Psacothea hilaris by acetone precipitation and elution from gels after native PAGE and SDS/PAGE with activity staining. The purified protein formed a single band, and the molecular mass was estimated to be 47 kDa. The purified cellulase degraded carboxymethylcellulose (CMC), insoluble cello-oligosaccharide (average degree of polymerization 34) and soluble cello-oligosaccharides longer than cellotriose, but not crystalline cellulose or cellobiose. The specific activity of the cellulase against CMC was 150 lmolAE min. TLC analysis showed that the cellulase produces cellotriose and cellobiose from insoluble cellooligosaccharides. However, a glucose assay linked with glucose oxidase detected a small amount of glucose, with a productivity of 0.072 lmolAEmin. The optimal pH of P. hilaris cellulase was 5.5, close to the pH in the midgut of P. hilaris larvae. The N-terminal amino-acid sequence of the purified P. hilaris cellulase was determined and a degenerate primer designed, which enabled a 975-bp cDNA clone containing a typical polyadenylation signal to be obtained by PCR and sequencing. The deduced aminoacid sequence of P. hilaris cellulase showed high homology to members of glycosyl hydrolase family 5 subfamily 2, and, in addition, a signature sequence for family 5 was found. Thus, this is the first report of a family 5 cellulase from arthropods.Keywords: cDNA cloning; cellulase; endoglucanase; insect; purification.Cellulase (endo-b-1,4-glucanase) is a widespread enzyme in micro-organisms such as bacteria and fungi [1,2]. Until recently it was believed that cellulose digestion in animals was mediated by microbial cellulase activity in their intestine, and that no animals possessed endogenous cellulase. This traditional view of cellulase activity in animals was challenged by two reports of endogenous animal cellulase genes from plant-parasitic nematodes and a termite [3,4]. Since these discoveries, a number of other animal cellulase genes have been reported (summarized in [5]).Glycosyl hydrolases are categorized into 90 families according to amino-acid sequence similarity and hydrophobic cluster analysis, and among them, cellulases are found in 14 families [6,7] (refer also to: http://afmb.cnrs-mrs.fr/ CAZY/index.html). Known animal cellulases belong to three glycosyl hydrolase families (GHFs): GHF 5 (plantparasitic nematodes), GHF 9 (termites, cockroaches and crayfish) and GHF 45 (mussel and beetle). These three families are structurally unrelated and their evolutionary origins are likely to be independent.Larvae of the yellow-spotted longicorn beetle, Psacothea hilaris, feed on mulberry and fig trees, tunneling inside the stems and ingesting the living wood. The major constituent is cellulose (44.6%), followed by hemicellulose (28.5%); soluble sugars constitute only 4.7% of the dry weight of the wood [8]. The habitat of P. hilaris larvae suggests that they possess the ability to digest cellulo...
Until recently, the textbook view of cellulose hydrolysis in animals was that gut-resident symbiotic organisms such as bacteria or unicellular eukaryotes are responsible for the cellulases produced. This view has been challenged by the characterization and sequencing of endogenous cellulase genes from some invertebrate animals, including plant-parasitic nematodes, arthropods and a mollusc. Most of these genes are completely unrelated in terms of sequence, and their evolutionary origins remain unclear. In the case of plant-parasitic nematodes, it has been suggested that their ancestor obtained a cellulase gene via horizontal gene transfer from a prokaryote, and similar suggestions have been made about a cellulase gene recently discovered in a sea squirt. To improve understanding about the evolution of animal cellulases, we searched for all known types of these enzymes in GenBank, and performed phylogenetic comparisons. Low phylogenetic resolution was found among most of the sequences examined, however, positional identity in the introns of cellulase genes from a termite, a sea squirt and an abalone provided compelling evidence that a similar gene was present in the last common ancestor of protostomes and deuterostomes. In a different enzyme family, cellulases from beetles and plant-parasitic nematodes were found to cluster together. This result questions the idea of lateral gene transfer into the ancestors of the latter, although statistical tests did not allow this possibility to be ruled out. Overall, our results suggest that at least one family of endogenous cellulases may be more widespread in animals than previously thought.
The privet tree, Ligustrum obtusifolium (Oleaceae), defends its leaves against insects with a strong lysine-decreasing activity that make proteins non-nutritive. This is caused by oleuropein, an iridoid glycoside. We previously found that some privet-specialist caterpillars adapt by secreting glycine in the digestive juice as a neutralizer that prevents the loss of lysine. Here, we extended the survey into 42 lepidopteran and hymenopteran species. The average concentration of glycine in digestive juice for 11 privet-feeding species (40.396 mM) was higher than that for 32 non-privet-feeding species (2.198 mM). The glycine concentrations exceeded 10 mM in 7 out of 11 privet-feeding species. In Macrophya timida (Hymenoptera), it reached 164.8 mM. Three out of the four remaining privet-feeding species had other amino acids instead. Larvae of a privet-specialist butterfly, Artopoetes pryeri (Lycaenidae), had a high concentration (60.812 mM) of GABA. In two other specialists, β-alanine was found. GABA, β-alanine, and glycine as well as alanine, amines, and ammonium ion inhibited the lysine decrease, indicating that amino residues are responsible for the inhibition. However, the three amino acids found in the specialists were far more effective (20 mM showed 80% inhibition) than the rest (>140 mM was required for 80% inhibition). Our results show a clear and rare case of the apparent convergent evolution of herbivores' molecular adaptations of feeding on a plant with a chemical defense in a manner that minimizes the cost of adaptation. The novel role of GABA in plant-herbivore interactions shown here is probably the first reported non-neuronal role of animal-derived GABA.
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