Abstract
A gut-specific chitinase gene was cloned from the mulberry longicorn beetle, Apriona germari. The A. germari chitinase (AgChi) gene spans 2894 bp and consists of five introns and six exons coding for 390 amino acid residues. AgChi possesses the chitinase family 18 active site signature and three N-glycosylation sites. Southern blot analysis of genomic DNA suggests that AgChi is a single copy gene. The AgChi cDNA was expressed as a 46-kDa polypeptide in baculovirus-infected insect Sf9 cells and the recombinant AgChi showed activity in a chitinase enzyme assay. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-linked glycosylation, revealed that AgChi is N-glycosylated, but the carbohydrate moieties are not essential for chitinolytic activity. Northern and Western blot analyses showed that AgChi was specifically expressed in the gut; AgChi was expressed in three gut regions, indicating that the gut is the prime site for AgChi synthesis in A. germari larvae.
Highlights
Chitin is the second most abundant polysaccharide in nature and is a linear homopolymer of E-1,4-linked N-acetylglucosamine (GlcNAc) residues
On the basis of these characteristics, we propose that A. germari chitinase (AgChi) is a member of the same family as all other insect chitinases identified to date
We described the cloning and characterization of the beetle A. germari chitinase gene, which shows sequence similarity to members of family 18 of the glycosyl hydrolase superfamily
Summary
Chitin is the second most abundant polysaccharide in nature and is a linear homopolymer of E-1,4-linked N-acetylglucosamine (GlcNAc) residues. It is one of the most unique biochemical constituents found in the cell walls of fungi, the cuticle and peritrophic membrane of insects, the eggshells of nematodes, and the integument of arthropods. Chitinolytic enzymes that catalyze the hydrolysis of chitin have been found in chitin-containing organisms as well as in microorganisms that do not have chitin. The enzymes, chitinases (EC 3.2.1.14), from various organisms have various biological functions. Chitin is the major component of the cuticle and peritrophic membrane, where it functions as a protective structural polysaccharide. Chitin degradation is achieved through tissuespecific expression of chitinases (reviewed by Merzendorfer & Zimoch, 2003)
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