|Zhu, Qingsong - KANSAS STATE UNIV|
|Matsumiya, Masahiro - NIHON UNIV, JAPAN|
|Muthukrishnan, Subbaratnam - KANSAS STATE UNIV|
Submitted to: Journal of Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 14, 2003
Publication Date: May 23, 2003
Citation: ARAKANE,Y. ., ZHU,Q., MATSUMIYA,M., MUTHUKRISHNAN,S., KRAMER,K.J., PROPERTIES OF CATALYTIC, LINKER AND CHITIN-BINDING DOMAINS OF INSECT CHITINASE, JOURNAL OF INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 33: 631-648. 2003. Interpretive Summary: In this collaborative study conducted with Japanese and Kansas State University scientists, we investigated some of the properties of the insect molting enzyme, chitinase, in order to better understand how different parts of this molecule work. Our results show that this molecule is made of two parts that are linked together. One part of the chitinase molecule recognizes and binds to insect or mold structures that are made from chitin. The other part of this enzyme attacks the chitin and cuts it into smaller pieces. Our data show that these two parts of the molecule act independently from each other and are responsible for its insecticidal and anti-mold activity.
Technical Abstract: 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) also were generated and characterized. The catalytic and chitin-binding activities are independent of each other because each is functional separately. When attached to the catalytic domain, the CBD enhanced activity toward the insoluble polymer but not the soluble chitin oligosaccharide. The linker region not only connects the two domains but also facilitates their secretion from the cell and helps to stabilize the enzyme in the presence of gut proteolytic enzymes. The linker region of the enzyme is heavily 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 ß (1®4)-linked oligosaccharides of GlcNAc during the molting process.