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Chitinase 7 (TcCHT7) is required for cuticle lamina organization and chitin deposition in Tribolium
Insect chitinases (CHTs), which belong to family 18 glycosylhydrolases (GH-18), have been detected in molting fluid and gut tissues and are predicted to mediate the digestion of chitin present in the exoskeleton and peritrophic matrix (PM) in the gut. Based on amino acid sequence similarity and phylogenetic analysis, insect CHT family proteins have been classified into eight groups (group I to VIII). The CHTs belonging to different groups have distinctly different developmental patterns of expression and tissue specificity, suggestive of distinct biological functions.
CHT7s belong to Group III chitinase contain two catalytic domains and one chitin binding domain (CBD). The catalytic domain 1 of this group of chitinases exhibits greater sequence similarity to one another than to the catalytic domain 2 in the same protein(s), suggesting distinct functions and/or evolutionary origins for each of these two catalytic domains. This group of chitinases, unlike most insect CHTs, possesses a predicted transmembrane segment at the N-terminal region. The recombinant T. castaneum CHT7 that was expressed in Hi-5 insect cells was bound to the cell membrane. Apparently, the catalytic domains of this CHT face the extracellular space as revealed by its ability to hydrolyze an artificial chitin substrate added to the medium.
DsRNA-based functional studies (RNAi) for several CHT genes in Tribolium castaneum indicated that CHTs belong to groups I (TcCHT5) and II (TcCHT10) are critical for molting and turnover of chitin in the old cuticle. In other hand, RNAi for TcCHT7 did not affect any types of molting such as larval-larval, larval-pupal and pupal-adult. The resulting pupae or adults, however, failed to wing-expansion and abdominal contraction. Immunohistochemical analysis revealed that TcCHT7 protein is localized in newly synthesized procuticle, suggesting that TcCHT7 could be released form the plasma membrane of epidermal cells by proteolysis. Chitin seems to accumulate within the assembly zone of the elytral and body wall cuticle in dsTcCHT7-treated animals. Transmission electron microscopy revealed that down-regulation of TcCHT7 transcripts resulted in disorganization of chitin laminar and vertical canals in the procuticle. These results suggest that TcCHT7 may have critical roles in the laminar assembly and synthesis and/or deposition of cuticular chitin.
This work was supported by NRF (NRF-2012R1A2A1A01006467).
