Cloning and expression of a fat body-specific chitinase cDNA from the spider, Araneus ventricosus.
A fat body-specific chitinase cDNA was cloned from the spider, Araneus ventricosus. The cDNA encoding A. ventricosus chitinase (AvChit1) is 1515 bp long with an open reading frame (ORF) of 431 amino acid residues. AvChit1 possesses the chitinase family 18 active site signature and one N-glycosylation site. The deduced amino acid sequence of AvChit1 cDNA showed 43% identity to both Glossina morsitans morsitans chitinase and a human chitotriosidase, and 30-40% to some insect chitinases which lack both the serine/threonine and chitin binding domains. Southern blot analysis of genomic DNA suggested the presence of AvChit1 gene as a single copy. Northern and Western blot analysis and enzyme activity assay showed the tissue-specific expression of AvChit1 in the A. ventricosus fat body. The AvChit1 cDNA was expressed as a 61 kDa polypeptide in baculovirus-infected insect Sf9 cells and the recombinant AvChit1 showed activity in the chitinase enzyme assay using 0.1% glycol chitin as a substrate. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that AvChit1 is N-glycosylated, but the carbohydrate moieties are not essential for chitinolytic activity.
Han
Ji Hee
JH
College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea.
Lee
Kwang Sik
KS
Li
Jianhong
J
Kim
Iksoo
I
Je
Yeon Ho
YH
Kim
Doh Hoon
DH
Sohn
Hung Dae
HD
Jin
Byung Rae
BR
D000595:Amino Acid Sequence; D000818:Animals; D002688:Chitinases / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005216:Fat Body / Q000201:enzymology*; D018628:Gene Dosage; D006031:Glycosylation; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013112:Spiders / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D002688:Chitinases