Functional analysis of TcCTLP-5C2, a chymotrypsin-like serine protease needed for molting in Tribolium castaneum.

In a previous study, we have characterized a gene family encoding chymotrypsin-like proteases from the red flour beetle, Tribolium castaneum (TcCTLPs). We identified 14 TcCTLP genes that were predominantly expressed in the midgut, where they presumably function in digestion. Two genes (TcCTLP-6C and TcCTLP-5C2), however, additionally showed considerable expression in the carcass, and RNAi studies demonstrated that they are required for molting (Broehan et al., 2010; Insect Biochem. Mol. Biol 40, 274-83). Thus, the enzyme has distinct functions in different physiological environments. To study molecular adaptations that facilitate enzyme function in different environments, we performed an in-depth analysis of the molecular and enzymatic properties of TcCTLP-5C2. We expressed different mutated versions of TcCTLP-5C2 in form of factor Xa activatable pro-enzymes in insect cells using a baculoviral expression system, and purified the recombinant proteins by affinity chromatography. By measuring and comparing the enzyme activities, we obtained information about the significance of single amino acid residues in motifs that determine substrate specificity and pH tolerance. Further, we showed that TcCTLP-5C2 is modified by N-glycosylation at amino acid position N137, which lies opposite to the catalytic cleft. Comparison of the enzymatic properties of non-glycosylated and glycosylated TcCTLP-5C2 versions showed that N-glycosylation decreases Vmax (maximum velocity) and kcat (turnover) while leaving the Km (specificity) unchanged. Thus, we provide evidence that N-glycosylation alters catalytic behavior by allosteric effects presumably due to altered structural dynamics as observed for chemically glycosylated enzymes.

Functional analysis of the ATP-binding cassette (ABC) transporter gene family of Tribolium castaneum.

BACKGROUND: The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. Most are integral membrane proteins that transport a broad spectrum of substrates across lipid membranes. In insects, ABC transporters are of special interest because of their role in insecticide resistance. RESULTS: We have identified 73 ABC transporter genes in the genome of T. castaneum, which group into eight subfamilies (ABCA-H). This coleopteran ABC family is significantly larger than those reported for insects in other taxonomic groups. Phylogenetic analysis revealed that this increase is due to gene expansion within a single clade of subfamily ABCC. We performed an RNA interference (RNAi) screen to study the function of ABC transporters during development. In ten cases, injection of double-stranded RNA (dsRNA) into larvae caused developmental phenotypes, which included growth arrest and localized melanization, eye pigmentation defects, abnormal cuticle formation, egg-laying and egg-hatching defects, and mortality due to abortive molting and desiccation. Some of the ABC transporters we studied in closer detail to examine their role in lipid, ecdysteroid and eye pigment transport. CONCLUSIONS: The results from our study provide new insights into the physiological function of ABC transporters in T. castaneum, and may help to establish new target sites for insect control.

Ammonia uptake in Manduca sexta midgut is mediated by an amiloride sensitive cation/proton exchanger: Transport studies and mRNA expression analysis of NHE7, 9, NHE8, and V-ATPase (subunit D).

In this study participation of cation/proton exchangers (NHE) in ammonia uptake in the medial midgut of Manduca sexta larvae was investigated employing a modified Ussing chamber. There was a mean inward net ammonia (NH3 + NH4+) flux of 194 ± 17 nmol cm⁻² h⁻¹ across the isolated epithelium under conditions of 0.1 mmol L⁻¹ ammonia on both sides of the tissue and a 100-fold inwardly directed P(NH3)-gradient (pH 8.5 luminal side, pH 6.5 hemolymphal side). Employing a 100-fold NH4+ gradient amiloride applied to the luminal side inhibited the influx in a dose-dependent manner, with a maximal inhibition of 75% at 20 mmol L⁻¹ and an estimated IC50 = 2 mmol L⁻¹. Amiloride also caused a dose-dependent but smaller decrease in the short-circuit current (I(sc)). No inhibition by apical or basal applied amiloride was noticed on cellular metabolic ammonia release, of which ca. 1/3 and 2/3 was secreted towards the apical and basal side, respectively. Using molecular methods full and partial sequence information of two putative cation/proton exchangers (MsNHE8, MsNHE7, 9) were obtained, both containing the characteristic amiloride binding motif. An mRNA expression analysis revealed ubiquitous expression patterns for both proteins, with similar expression levels for NHE8 in all tissues investigated and lower mRNA abundances for MsNHE7, 9 in the midgut sections of the caterpillar. In contrast, in this tissue high expression levels of the V-ATPase (D subunit) were detected, likely the sole pump responsible for energizing goblet cell K+ excretion, but also involved in columnar cell ammonia uptake.

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

Chymotrypsin-like peptidases (CTLPs) of insects are primarily secreted into the gut lumen where they act as digestive enzymes. We studied the gene family encoding CTLPs in the genome of the red flour beetle, Tribolium castaneum. Using an extended search pattern, we identified 14 TcCTLP genes that encode peptidases with S1 specificity pocket residues typically found in chymotrypsin-like enzymes. We further analyzed the expression patterns of seven TcCTLP genes at various developmental stages. While some TcCTLP genes were exclusively expressed in feeding larval and adult stages (TcCTLP-5A/B, TcCTLP-6A), others were also detected in non-feeding embryonic (TcCTLP-5C, TcCTLP-6D) and pupal stages (TcCTLP-5C, TcCTLP-6C/D/E). TcCTLP genes were expressed predominantly in the midgut, where they presumably function in digestion. However, TcCTLP-6C and TcCTLP-5C also showed considerable expression in the carcass. The latter two genes might therefore encode peptidases that act as molting fluid enzymes. To test this hypothesis, we performed western blots using protein extracts from larval exuviae. The extracts reacted with antibodies to TcCTLP-5C and TcCTLP-6E suggesting that the corresponding peptidases are secreted into the molting fluid. Finally, we performed systemic RNAi experiments. While injections of five TcCTLP-dsRNAs into penultimate larvae did not affect growth or development, injection of dsRNA for TcCTLP-5C and TcCTLP-6C resulted in severe molting defects.

Cloning and expression analysis of midgut chymotrypsin-like proteinases in the tobacco hornworm.

Digestion of proteins in the midgut of lepidopteran larvae relies on different trypsin and chymotrypsin isoforms. In this study we describe three chymotrypsin-like proteinases (CTLP2-4) from the larval midgut of Manduca sexta, which are closely related to CTLP1 and less closely related to another chymotrypsin (CT), two previously described proteinases present in the larval midgut of M. sexta. CTLP1-4 fit perfectly into a novel subgroup of insect CTLPs by sequence similarity and by the replacement of GP by SA in the highly conserved GDSGGP motif. When we examined CTLP expression in different tissues, most of the proteinases were predominantly expressed in the anterior and median midgut, while some were found in the Malpighian tubules. When we examined CTLP expression at different physiological states, we observed that the CTLP mRNA amounts did not differ considerably in feeding and starving larvae except for CTLP2, whose mRNA dropped significantly upon starvation. During moulting, however, the mRNA amounts of all CTLPs dropped significantly. When we immunologically examined CTLP amounts, mature proteinases were only detectable in the gut lumen of feeding and re-fed larvae, but not in that of starving or moulting larvae, suggesting that CTLP secretion is suspended during starvation or moult.

A chymotrypsin-like serine protease interacts with the chitin synthase from the midgut of the tobacco hornworm.

The chitin portion of the peritrophic matrix in the midgut of the tobacco hornworm, Manduca sexta, is produced by chitin synthase 2 (CHS2), a transmembrane family II glycosyltransferase, located at the apical tips of brush border microvilli. To look for proteins that potentially interact with CHS2, we performed yeast two-hybrid screening, identifying a novel chymotrypsin-like protease (CTLP1) that binds to the extracellular carboxyterminal domain of CHS2. The occurrence of this interaction in vivo is supported by co-localization and co-immunoprecipitation data. Based on our findings we propose that chitin synthesis is controlled by an intestinal proteolytic signalling cascade linking chitin synthase activity to the nutritional state of the larvae.