A cDNA encoding a chitinase from the epithelial cell line of Chironomus tentans (Insecta, Diptera) and its functional expression

Abstract

A cDNA coding for chitinase was isolated from Chironomus cells, which possesses conserved regions I and II characteristic for family 18 chitinases, a C-terminus enriched in Glu and Pro without the typical "PEST-region," putative glycosylation sites, a reduced number of C-terminal cysteines, and no typical chitin binding domain. Northern blots revealed one specific signal with an apparent size of 2.3 kb. The cDNA was expressed in the baculovirus/Spodoptera system as a His-tag fusion protein, which was secreted as a functionally active enzyme into the medium and could be separated from endogenous viral and Spodoptera-specific chitinases.

Two chitinase 5 genes from Locusta migratoria: Molecular characteristics and functional differentiation

“…The first cDNA of MsCht5 belonging to Group I was isolated from Manduca sexta in 1993 (Kramer et al, 1993). Since then, the orthologous cDNAs of MsCht5 have been identified or sequenced from at least 15 different insect species including Bombyx mori, Hyphantria cunea (Kim et al, 1998), Chironomus tentans (Feix et al, 2000), Spodoptera litura (Shinoda et al, 2001), Choristoneura fumiferana (Zheng et al, 2002), Helicoverpa armigera (Ahmad et al, 2003), D. melanogaster (Zhu et al, 2004), An. gambiae, Aedes aegypti, Culex quinquefasciatus, T. castaneum (Zhu et al, 2008a;Zhang et al, 2011aZhang et al, , 2011b, Lacanobia oleracea (Fitches et al, 2004), Spodoptera exigua (Zhang et al, 2012), Mamestra brassicae (Paek et al, 2012) and Ostrinia furnacalis (Wu et al, 2013).…”

Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases

“…The basic structure consists of three domains that include (1) the catalytic region, (2) a PEST-like region, enriched in the amino acids proline, glutamate, serine and threonine, and (3) a cysteine-rich region Fig.·4B). The last two domains, however, do not seem to be necessary for chitinase activity because naturally occurring chitinases that lack these regions are still enzymatically active (Girard and Jouanin, 1999;Feix et al, 2000;Yan et al, 2002). In agreement with these observations, C-terminus-truncated versions of the recombinant Manduca chitinase still exhibit catalytic activity Zhu et al, 2001).…”

“…In some insects, the catalytic region is followed by a less conserved domain containing a putative PEST-like region that is also found near the C-terminus of the yeast chitinase (Kim et al, 1998;Kuranda and Robbins, 1991;Kramer et al, 1993;Royer et al, 2002). As already mentioned, insect chitinases without a PEST-like region have also been described in the literature (Girard and Jouanin, 1999;Feix et al, 2000;Yan et al, 2002). PEST-like regions presumably increase the susceptibility of the enzyme to proteolysis by a calcium-dependent protease or to degradation via the 26S proteasome (Rogers et al, 1986;Rechsteiner and Rogers, 1996).…”

“…While the chitinases from Manduca, Bombyx and fall webworm moth Hyphantria cunea exhibit the typical tripartite domain structure (Kramer et al, 1993;Kim et al, 1998), some other chitinases lack PEST-like or the typical chitin-binding regions (Girard and Jouanin, 1999;Feix et al, 2000;Yan et al, 2002). Other insects, in turn, may express multi-modular enzymes.…”

Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects

“…Chitinase genes from many insects have been characterized, including lepidopteran species such as Manduca sexta (Kramer et al, 1993), Bombyx mori (Kim et al, 1998), Spodoptera litura (Shinoda et al, 2001), Spodoptera frugiperda (Bolognesi et al, 2005), Hyphantria cunea (Kim et al, 1998) and Choristoneura fumiferana (Zheng et al, 2002); dipteran species such as Aedes aegypti (de la Vega et al, 1998), Anopheles gambiae (Shen and Jacobs-Lorena, 1997), Glossina morsitans (Yan et al, 2002), Lutzomyia longipalpis (Ramalho-Ortigao and Traub-Cseko, 2003) and Chironomus tentans (Feix et al, 2000); hymenopteran species, Chelonas sp. (Krishnan et al, 1994); and coleopteran species such as Phaedon cochleariae (Girard and Jouanin, 1999) and Tenebrio molitor (Royer et al, 2002;Genta et al, 2006).…”