Heterologous expression and characterization of two chitinase 5 enzymes from the migratory locustLocusta migratoria

Abstract

Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix of midgut. In Locusta migratoria, two duplicated Cht5s (LmCht5-1 and LmCht5-2) have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate (4MU-(GlcNAc)3 ), and higher Km value for the longer substrate (CM-Chitin-RBV) compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain (CBD) tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5-1 and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-(GlcNAc)3 , whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBV. These findings are helpful for further research to clarify their different roles in insect growth and development.

Identification and RNAi‐based function analysis of chitinase family genes in diamondback moth, Plutella xylostella

“…Initially, the DBM genome database (GCF_000330985.1) was used to search chitinase genes by using the amino acid sequences of those from Drosophila melanogaster , Tribolium castaneum , Anopheles gambiae , Locusta migratoria , M. sexta and Bombyx mori published previously (Table S2). As a result, 16 chitinase‐like genes were obtained.…”

“…The first full‐length cDNA of insect chitinase was cloned from Manduca sexta by Kramer et al . Since then, hundreds of chitinase genes have discovered in many insect species belonging to different orders, including Diptera, Lepidoptera, Coleoptera, Hemiptera and Orthoptera . Most recently, based on amino acid sequence similarity and phylogenetic analyses of the catalytic domains, insect chitinases were divided into 11 groups (groups I to X, and Lepidoptera‐specific group h) .…”

Composition and emission dynamics of migratory locust volatiles in response to changes in developmental stages and population density

“…The achievements in locust genome (Wang et al ., ), transcriptomes (Wang & Kang, ) and RNAi techniques (Ma et al ., ; Guo et al ., ; Wang & Kang, ) provide powerful tools to determine the molecular mechanisms of locust pheromone productions. Moreover, their functions were established by manipulating the expression levels of related genes in the biosynthetic pathways (Li et al ., ; Song et al ., ). Related discoveries will be useful in developing environment‐friendly behavioral regulators for locust management.…”

Contributions of dsRNases to differential RNAi efficiencies between the injection and oral delivery of dsRNA in Locusta migratoria

“…The procedure for protein extraction and purification was adapted from that described by Li et al 22 In brief, the desired enzymes were highly expressed when the Sf9 cells were transfected with P2 virus. After confirmation of expression, large amounts of Sf9 cells were infected for protein production and extraction.…”