|Zhu, Yu cheng|
Submitted to: Journal of Insect Biochemistry and Molecular Biology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/15/2002
Publication Date: 12/30/2002
Citation: ZHU,Y. ., SPECHT,C.A., DITTMER,N.T., MUTHUKRISHNAN,S., KANOST,M.R., KRAMER,K.J., SEQUENCE OF A CDNA AND EXPRESSION OF THE GENE ENCODING A PUTATIVE EPIDERMAL CHITIN SYNTHASE OF MANDUCA SEXTA, JOURNAL OF INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 32: 1497-1506. 2002. Interpretive Summary: This paper reports the sequence of a cDNA for a very important enzyme in insect developmental biology, chitin synthase (CHS). Chitin is the structural polysaccharide found in the exoskeleton and gut, and CHS controls its synthesis. In the past, CHS has been the target of insect growth regulators, but it has been very difficult to study because of its very large size and localization in the cellular plasma membrane. Obtainin the cloned cDNA is a significant step that will allow development of reagents for use in future research on the expression, properties and regulation of CHS, as well as for the development of chitin synthesis inhibitors (CSIs). Together with scientists at Kansas State University and Boston University, we analyzed and discussed the nucleotide and amino acid sequences, and made comparisons with similar enzymes from other organisms, including nematodes and fungi. The insect enzyme is expressed in the epidermis and apparently functions to make chitin that is deposited in the exoskeleton. The results obtained provide new information about structure-activity relationships, and add to the knowledge base for biosynthetic enzymes and insect molecular science. The long-term goal of this research is to facilitate a more effective application of CSIs for the control of insect pests.
Technical Abstract: Glycosyltransferases are enzymes that synthesize oligosaccharides, polysaccharides and glycoconjugates. One type of glycosyltransferase is chitin synthase, a very important enzyme in biology, which is utilized by insects, fungi, and other invertebrates to produce chitin, a polysaccharide of B-1, 4-linked N-acetylglucosamine. Chitin is an important component of the insect's exoskeletal cuticle and gut lining. To characterize a chitin synthase gene of the tobacco hornworm, degenerate primers were designed from two highly conserved regions in fungal and nematode chitin synthase gene sequences and then used to amplify a similar region from hornworm cDNA. A full-length cDNA of 5152 nucleotides was assembled for the putative chitin synthase gene, and sequencing of genomic DNA verified the contiguity of the sequence. The gene has an ORF of 4692 nucleotides that encodes a transmembrane protein of 1564 amino acid residues with a mass of approximately 179 kDa. It is most similar to putative chitin synthases fro other insects and nematodes, with 67% identify to both the blowfly, Lucilia cuprina, and the fruit fly, Drosophila melanogaster, enzymes. The similarity with fungal chitin synthases is restricted to the putative catalytic domain, and the protein has, at equivalent positions, several amino acids that are essential for activity as revealed by mutagenesis of the fungal enzymes. A 5.3-kb transcript was identified by northern blot hybridization of RNA from larval epidermis, suggesting that the enzyme functions to make chitin deposited in the cuticle. Gene expression is regulated in the epidermis, with the amount of transcript increasing during phases of cuticle deposition.