Chymotrypsin-like peptidases from Tribolium castaneum: A role in molting revealed by RNA interference
Introduction
Serine peptidases of insects serve numerous physiological functions that are essential for growth and development. In addition to their well-established function in digestion, they function in molting and metamorphosis, and act as highly specific regulators of innate immune responses, including melanization and the expression of antimicrobial peptides (Kanost et al., 2004, Law et al., 1977, Neurath, 1984, Srinivasan et al., 2006). The latter functions are tightly controlled by serpins, specific serine proteinase inhibitors that irreversibly inhibit their target enzymes by a suicide mechanism involving major conformational changes (Gettins, 2002).
Many serine peptidases in insects belong to the S1 family (chymotrypsin family) of PA Clan serine peptidases (EC 3.4.21), which includes the two phylogenetically distinct but structurally related S1A and S1B subfamilies (Rawlings et al., 2008). While S1B peptidases are found in all living organisms and function in intracellular protein turnover, S1A peptidases act mostly in the extracellular space where they serve different functions. The S1A subfamily includes enzymes like chymotrypsin, trypsin, elastase, granzyme and different matrix peptidases. The structures of S1A peptidases have been determined for various mammalian serine peptidases and are characterized by the prototypic chymotrypsin-fold of two β-barrels with the active site residues in the cleft between (Kraut, 1977, Page and Di Cera, 2008). The fold is stabilized by disulfide bonds between highly conserved cysteine residues. The active site of S1A peptidases is formed by the canonical catalytic triad residues Ser, His and Asp, which are stabilized by an extensive network of hydrogen bonds (Hedstrom, 2002, Rawlings and Barrett, 1994). The determination of the atomic structure of a chymotrypsin from the fire ant Solenopsis invicta demonstrated that the overall S1A fold applies also to insect serine peptidases (Botos et al., 2000). S1A family peptidases exhibit different cleavage specificities. Whereas chymotrypsin-like enzymes cleave peptidyl bonds preferentially after larger hydrophobic residues, trypsin-like enzymes prefer positively charged residues and elastase-like enzymes small hydrophobic residues at the P1 position. Which type of specificity actually is exhibited by S1A peptidases depends on the properties of the S1 substrate-binding pocket, which lies adjacent to the catalytic site (Perona and Craik, 1995). Several conserved amino acid residues in the S1 pocket have been recognized as important determinants of substrate specificity, but these may be not the sole decisive factors for specifying the cleavage site (Hedstrom, 2002). Most S1A peptidases are secreted into the extracellular space as inactive, zymogenic precursor proteins, which become activated by proteolytic cleavage at conserved sites (Bode et al., 1978, Neurath and Dixon, 1957, Page and Di Cera, 2008). Zymogen activation reflects a powerful regulatory mechanism that is frequently embedded in a cascade of successive proteolytic steps that together initiate physiological processes in due time at the right place.
Although S1 peptidases are one of the best characterized enzyme families, their precise functions in insects are still poorly understood. Recent genome annotations have allowed the first comprehensive studies of this peptidase family. The Drosophila genome contains more than 200 genes encoding serine peptidases and homologous enzymes, of which 37 contain regulatory CLIP domains at the N-terminus (Ross et al., 2003). The vast majority of these genes encode S1 peptidases with a predicted trypsin-like specificity (trypsin-like peptidases, TLP). In contrast, only 29 of these genes were predicted to encode S1A peptidases with a predicted chymotrypsin-like specificity (chymotrypsin-like peptidase, CTLP). Also in other available insect genomes the total number of genes encoding CTLPs is rather low compared to the number of genes that encode TLPs. In Apis mellifera, 57 genes encoding serine peptidases and homologous enzymes were reported, of which only six had a predicted chymotrypsin-like specificity (Zou et al., 2006). Similar results were obtained when we screened the Anopheles gambiae genome for serine peptidases. From more than 300 genes encoding S1 peptidases, less than a tenth encode CTLPs. Genomic Southern blots performed with hybridization probes specific for chymotrypsin-like peptidases (CTLPs) from Manduca sexta indicated also a low number (≤8) of genes encoding CTLPs in the Manduca genome (Broehan et al., 2008). Thus, it appears that genes encoding serine peptidases with a chymotrypsin-like specificity constitute a small subset within the large gene family encoding S1 peptidases.
Those CTLPs that have been identified so far in insects are secreted into the gut lumen where they act as digestive enzymes in concert with various other types of peptidases (Law et al., 1977, Srinivasan et al., 2006, Terra et al., 1996). However, CTLPs may not be restricted to the intestinal tract in insects, as chymotrypsin-like activities have been reported also in the molting fluid (Brookhart and Kramer, 1990). The molting fluid fills the exuvial space between the old and the new cuticle and exhibits chitinolytic and proteolytic activities that facilitate degradation of the inner layers of the old cuticle as part of the molting process before ecdysis. Several peptidases have been identified in insect molting fluids, including trypsin-like serine peptidases, carboxypeptidases, cysteine peptidases and metallopeptidases such as aminopeptidases (Dong et al., 2007, Liu et al., 2006, Liu et al., 2009, Ote et al., 2005, Rabossi et al., 2008, Samuels et al., 1993a, Samuels et al., 1993b, Sui et al., 2009, Wei et al., 2007).
To gain more insights into the functions of CTLPs, we have characterized the gene family encoding these peptidases in the red flour beetle, Tribolium castaneum, a well-established genetic and genomic insect model (Brown et al., 2003, Richards et al., 2008). We used an extended search pattern to identify S1A peptidases with S1 specificity pocket residues that are typically found in enzymes that exhibit chymotrypsin-like activity. In doing so, we identified 14 genes in the Tribolium genome that encode CTLPs (TcCTLPs). Analysis of the expression patterns of seven TcCTLP genes revealed that most enzymes were expressed during feeding stages in the midgut, which is consistent with a role in digestion of food material. However, two of these genes, TcCTLP-5C and TcCTLP-6C, were found to be expressed also in the carcass and were immuno-detected in protein extracts from larval exuviae. Finally, RNAi experiments demonstrated that TcCTLP-5C and TcCTLP-6C are essential enzymes in the molting process.
Section snippets
Insects
The T. castaneum strain GA-1 (Haliscak and Beeman, 1983) was used in this study. Insects were reared at 30 °C under standard conditions (Beeman and Stuart, 1990).
Cloning of cDNAs encoding TcCTLPs
The cDNAs for the seven TcCTLPs, TcCTLP-5A (embl|CBC01177.1), TcCTLP-5B (embl|CBC01166.1), TcCTLP-5C1 (embl|CBC01175.1), TcCTLP-5C2 (embl|CBC01172.1), TcCTLP-6A (embl|CBC01171.1), TcCTLP-6C (embl|CBC01169.1), TcCTLP-6D (embl|CBC01179.1) and TcCTLP-6E (embl|CBC01181.1) identified in the Tribolium genome were amplified by RT-PCR using
Identification of CTLP-encoding genes in the Tribolium genome
To identify genes encoding chymotrypsin-like peptidases, we performed a tblastn search of the Tribolium genome database at NCBI using the M. sexta CTLP1 (MsCTLP1; embl|CAL92020.1; Broehan et al., 2007) as query. From the resulting list of homologous sequences, we selected those that showed a chymotrypsin-like substrate specificity pocket as defined by Perona and Craik (1995). Accordingly, proteins containing the residues Ser/Thr190, Gly216, and Gly/Ala/Ser226 in the primary substrate-binding
Discussion
Although chymotrypsin-like activities have been reported in molting fluids from lepidopteran species (Brookhart and Kramer, 1990), so far no CTLP has been identified to be involved in molting. In this study, we provide evidence for the first time that CTLPs are required for molting in the coleopteran, T. castaneum. To identify putative CTLP genes in the Tribolium genome (Richards et al., 2008), we performed a Blast search that yielded about 160 genes encoding S1A peptidases from which we
Acknowledgments
This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 431 and GRK 612) and National Science Foundation (IOS-0615818). Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
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