Chymotrypsin-like peptidases from Tribolium castaneum: A role in molting revealed by RNA interference

Broehan, Gunnar; Arakane, Yasuyuki; Beeman, Richard W.; Kramer, Karl J.; Muthukrishnan, Subbaratnam; Merzendorfer, Hans

doi:10.1016/j.ibmb.2009.10.009

Cited by 49 publications

(36 citation statements)

References 51 publications

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“…Its depletion might be attributable to digestion by molting fluid proteases. In that regard, it is interesting to point out that RNAi of transcripts for a chymotrypsin-like protein that is present in cast cuticles results in molting defects (14). Immunolocalization of the proteolytic enzyme(s) involved in cuticular protein turnover will lend further support to our conclusion that the envelope alone cannot account for the stability of the newly synthesized cuticle in the presence of molting enzymes.…”

Section: Susceptibility Of Old Cuticle To Digestion During Molting Comentioning

confidence: 69%

Knickkopf protein protects and organizes chitin in the newly synthesized insect exoskeleton

Chaudhari

Arakane

Specht

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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108

127

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During each molting cycle of insect development, synthesis of new cuticle occurs concurrently with the partial degradation of the overlying old exoskeleton. Protection of the newly synthesized cuticle from molting fluid enzymes has long been attributed to the presence of an impermeable envelope layer that was thought to serve as a physical barrier, preventing molting fluid enzymes from accessing the new cuticle and thereby ensuring selective degradation of only the old one. In this study, using the red flour beetle, Tribolium castaneum, as a model insect species, we show that an entirely different and unexpected mechanism accounts for the selective action of chitinases and possibly other molting enzymes. The molting fluid enzyme chitinase, which degrades the matrix polysaccharide chitin, is not excluded from the newly synthesized cuticle as previously assumed. Instead, the new cuticle is protected from chitinase action by the T. castaneum Knickkopf (TcKnk) protein. TcKnk colocalizes with chitin in the new cuticle and organizes it into laminae. Down-regulation of TcKnk results in chitinase-dependent loss of chitin, severe molting defects, and lethality at all developmental stages. The conservation of Knickkopf across insect, crustacean, and nematode taxa suggests that its critical roles in the laminar ordering and protection of exoskeletal chitin may be common to all chitinous invertebrates.RNAi | nikkomycin | phylogenetic tree | transmission electron microscopy | chitin synthase D uring development, insects must undergo periodic molting to accommodate growth and to overcome the rigid constraints imposed by portions of their chitinous exoskeletons (1, 2). This process entails the complete replacement of the entire outer shell of the insect, including digestion; resorption and recycling of the inner, more pliable layers; and shedding of the outer, more highly sclerotized and waterproofed layers, which are either discarded or, in some cases, ingested for further recycling (1-4). The molting process is hormonally initiated by 20-hydroxyecdysone and begins with the epidermis secreting what will become the outer layers of the new cuticle that separate the epidermal layer from the overlying old cuticle. An "apolytic space" then forms, separating new (inner) from old (outer) cuticles (5). With the delicate epidermis now protected by the first layers of new cuticle, the molting fluid in the apolytic space can digest the inner layers of the outer (old) cuticle. According to long-held dogma, protection of the new cuticle from degradation by molting fluid enzymes is conferred by a thin, nonchitinous envelope (previously termed the "cuticulin" layer or the "outer epicuticle") deposited by the epidermal cells just before the secretion of new cuticular chitin underneath (3). This envelope was believed to form a protective barrier against proteolytic and chitinolytic enzymes of the molting fluid, thereby confining their actions to the proximal layers of the old (outer) exoskeleton while preventing digestion of the newly deposit...

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Section: Susceptibility Of Old Cuticle To Digestion During Molting Comentioning

confidence: 69%

Knickkopf protein protects and organizes chitin in the newly synthesized insect exoskeleton

Chaudhari

Arakane

Specht

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

108

127

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“…Prior to RNAi analysis, the spatiotemporal pattern of gene expression is determined via RT-PCR to ensure RNAi is performed at appropriate time points. Chitin/cuticle pathway genes, including those required for chitin synthesis (Arakane et al, 2005b;, molting, survival and fecundity (Arakane et al, 2010b;Arakane et al, 2009;Broehan et al, 2010;Hogenkamp et al, 2007;Zhu et al, 2008) and tanning of the epidermal cuticle (Arakane et al, 2010a;Arakane et al, 2009;Arakane et al, 2005a) have been functionally characterized, revealing a wealth of potential biotargets for arthropod-specific pest control. While the candidate gene approach has been of enormous value in T. castaneum, it imposes limitations due to reliance on sequence conservation.…”

Section: Red Flour Beetlementioning

confidence: 99%

RNAi in Agriculturally-Important Arthropods

Aronstein¹,

Oppert²,

Lorenzen³

2011

RNA Processing

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“…Chymotrypsin B plays an important role in intracellular protein turnover, while chymotrypsin A is prevalent in the extracellular space and performs different functions (Broehan et al , 2010). The chymotrypsin A sub-family contains a variety of enzymes, such as chymotrypsin, trypsin, elastase, granzyme and different matrix peptidases, with different cleavage specificities.…”

Section: Introductionmentioning

confidence: 99%

“…In the lepidopteran, Spodoptera exigua , chymotrypsin was found likely to mediate the proteolytic remodeling in the gut during larval-pupal transition (Herrero et al , 2005). The injection of dsRNA for chymotrypsin 5C/6C in the red flour beetle, Tribolium castaneum , resulted in severe molting defects, which indicate that chymotrypsin plays an important role in molting process (Broehan et al , 2010). In addition, chymotrypsin was associated with immune defense reactions against bacteria in D. melanogaster (de Morais et al , 2005).…”

Section: Introductionmentioning

confidence: 99%

Characterization and expression analysis of chymotrypsin after bacterial challenge in the mud crab, Scylla paramamosain

Gong

Xie

et al. 2014

Genet. Mol. Biol.

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Chymotrypsin is one of the serine proteases families that have various biological functions. A chymotrypsin gene was isolated from hepatopancreas of the mud crab, Scylla paramamosain (designated SpCHY) in this study. The full-length cDNA of SpCHY contained 942 nucleotides with a polyadenylation sequence and encoded a peptide of 270 amino acids with a signal peptide of 17 amino acids. The SpCHY gene contains seven exons, six introns, a TATA box and several transcription factor binding sites that were found in 5’-promoter region which is 1221 bp in length. Real-time quantitative PCR analysis indicated that the expression level of SpCHY mRNA in hepatopancreas was significantly higher than that in other tissues. Immunocytochemistry and in situ hybridization exhibited the CHY-like reactivity presented in resorptive cells of the hepatopancreas. After bacterial challenge with Vibrio alginolyticus, the expression level of SpCHY mRNA was extremely up-regulated at 3 h in hepatopancreas. Our results suggest that SpCHY might play an important role in the mud crab’s immune response.

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Chymotrypsin-like peptidases from Tribolium castaneum: A role in molting revealed by RNA interference

Cited by 49 publications

References 51 publications

Knickkopf protein protects and organizes chitin in the newly synthesized insect exoskeleton

Knickkopf protein protects and organizes chitin in the newly synthesized insect exoskeleton

RNAi in Agriculturally-Important Arthropods

Characterization and expression analysis of chymotrypsin after bacterial challenge in the mud crab, Scylla paramamosain

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