Properties of catalytic, linker and chitin-binding domains of insect chitinase.
Manduca sexta (tobacco hornworm) chitinase is a glycoprotein that consists of an N-terminal catalytic domain, a Ser/Thr-rich linker region, and a C-terminal chitin-binding domain. To delineate the properties of these domains, we have generated truncated forms of chitinase, which were expressed in insect cells using baculovirus vectors. Three additional recombinant proteins composed of the catalytic domain fused with one or two insect or plant chitin-binding domains (CBDs) were also generated and characterized. The catalytic and chitin-binding activities are independent of each other because each activity is functional separately. When attached to the catalytic domain, the CBD enhanced activity toward the insoluble polymer but not the soluble chitin oligosaccharide primarily through an effect on the Km for the former substrate. The linker region, which connects the two domains, facilitates secretion from the cell and helps to stabilize the enzyme in the presence of gut proteolytic enzymes. The linker region is extensively modified by O-glycosylation and the catalytic domain is moderately N-glycosylated. Immunological studies indicated that the linker region, along with elements of the CBD, is a major immunogenic epitope. The results support the hypothesis that the domain structure of insect chitinase evolved for efficient degradation of the insoluble polysaccharide to soluble oligosaccharides during the molting process.
Arakane
Yasuyuki
Y
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Zhu
Qingsong
Q
Matsumiya
Masahiro
M
Muthukrishnan
Subbaratnam
S
Kramer
Karl J
KJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002241:Carbohydrates / Q000032:analysis; D002384:Catalysis; D020134:Catalytic Domain; D002460:Cell Line; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004064:Digestive System / Q000201:enzymology; D010450:Endopeptidases / Q000378:metabolism; D004795:Enzyme Stability; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors; D006031:Glycosylation; D015151:Immunoblotting; D019476:Insect Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D018402:Manduca / Q000201:enzymology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D002241:Carbohydrates; D019476:Insect Proteins; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases; D010450:Endopeptidases