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Title: Characterization of recombinant chitinase-like proteins of Drosophila melanogaster and Tribolium castaneum

Author
item ZHU, QINGSONG - KANSAS STATE UNIV
item ARAKANE, YASUYUKI - KANSAS STATE UNIV
item Beeman, Richard
item KRAMER, KARL - 5430-05-30 RETIRED
item MUTHUKRISHNAN, SUBBARATNAM - KANSAS STATE UNIV

Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/27/2007
Publication Date: 3/1/2008
Citation: Zhu, Q., Arakane, Y., Beeman, R.W., Kramer, K., Muthukrishnan, S. 2008. Characterization of recombinant chitinase-like proteins of Drosophila melanogaster and Tribolium castaneum. Insect Biochemistry and Molecular Biology 38: 467-477.

Interpretive Summary: Chitin is the main component of insect exoskeleton, conferring both rigidity and flexibility, and protecting the insect from injury, predation, infection and desiccation. Chitin is also the major component of a membrane that coats the lining of the midgut, protecting it from abrasion and self-digestion. Until recently, very little has been known about the enzymes that are needed for digestion and reutilization of the old cuticle during the insect molting cycle. We isolated a variety of different types of chitinases from the fruit fly and the red flour beetle, and found that they had a range of different activities in the degradation of chitin. Each of these enzymes appears to have a slightly different function, but most or all are vital for insect survival. Each of these newly-discovered genes can become a target in screening assays for new biopesticides that disrupt molting and related physiological processes.

Technical Abstract: Insect chitinase (CHT) family proteins are encoded by as many as 16 genes depending upon species. We have classified these proteins in three species into five different groups based on amino acid sequence similarities (Zhu et al., 2007). The functions of most of the individual proteins of this family during growth and development are largely unknown. To help determine their enzymatic properties and physiological roles, we expressed representative members belonging to this protein family from Drosophila melanogaster (Dm) and Tribolium castaneum (Tc), and characterized their kinetic and carbohydrate-binding properties. Seven proteins, including DmCHT 4, 5, 9 and DmDS47 from Drosophila, and TcCHT5, TcIDGF2 and TcIDGF4 from Tribolium, belonging to groups I, IV or V of the chitinase-like family were expressed in a baculovirus-insect cell line expression system, purified and characterized. Their enzymatic and chitin-binding properties were compared to those of the well-characterized chitinase, MsCHT535, from Manduca sexta (Ms). All of these proteins, except those belonging to group V that are related to imaginal disc growth factors (IDGFs), exhibited chitinolytic activity against the long polymeric substrate, CM-Chitin-RBV, and/or the short oligomeric substrate, MU-(GlcNAc)3. TcCHT5, DmCHT5 and MsCHT535, which are members of group I chitinases, cleaved both polymeric and oligomeric substrates. Their enzymatic properties, including pH optima, kinetic parameters, and susceptibility to substrate inhibition by chitooligosaccharides, were similar. Two group IV chitinases, DmCHT4 and DmCHT9, also were characterized. DmCHT4 had one optimum pH of 6 towards the polymeric substrate and no detectable chitinolytic activity towards an oligosaccharide substrate. DmCHT9 had high activity from pH 4 to 8 towards the polymeric substrate and exhibited low activity towards the oligosaccharide substrate. The group V proteins, TcIDGF2 and TcIDGF4, contain all of the catalytically critical residues within conserved region II of family 18 chitinases as well as the C-terminal chitin-binding domain (CBD), but neither exhibited chitinolytic activity. Another group V protein, DmDS47, which lacks the critical glutamate residue in region II and the C-terminal CBD, also exhibited no chitinolytic activity. However, all three of the group V proteins bound to chitin tightly. A comparison of the amino acid sequences and homology model structures of group V proteins with enzymatically active members of the chitinase family indicated that the presence of additional loops of amino acids within the beta-8-barrel structure of these proteins interferes with productive substrate binding and/or catalysis.