| Summary: | Digestion of proteins in the midgut of lepidopteran larvae relies on different types
of peptidases, among the trypsins and chymotrypsins. In this work four chymotrypsinlike
peptidases (MsCTLP1–4) were identified from the larval midgut of M. sexta, which
are distantly related to another chymotrypsin (MsCT), a previously described peptidase
present in the larval midgut of M. sexta. MsCTLP1–4 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. Examination of MsCTLP expression in different
tissues showed that most of the peptidases were predominantly expressed in the anterior
and median midgut, while some were found in the Malpighian tubules. Expression
analysis of MsCTLPs at different physiological states revealed that the mRNA amounts
did not differ considerably in feeding and starving larvae except for MsCTLP2, whose
mRNA dropped significantly upon starvation. During molting, however, the mRNA
amounts of all MsCTLPs dropped significantly. Immunological determination of
MsCTLP1 amounts showed that the mature peptidase was only detectable in the gut
lumen of feeding and re-fed larvae, but not in that of starving or molting larvae,
suggesting that MsCTLP1 secretion is suspended during starvation or molt. Differential
regulation of transcript levels as well as their partial expression in Malpighian tubules
might point to a role, which is distinct from digestion for at least some MsCTLPs. In
line with this assumption, MsCTLP1 was shown to interact with the chitin synthase 2
(MsCHS2), necessary for chitin synthesis in the course of peritrophic matrix formation
in the midgut of M. sexta. The occurrence of this interaction in vivo is supported by colocalization
and co-immunoprecipitation. The data suggest that chitin synthesis is
controlled by an intestinal proteolytic signaling cascade linking chitin synthase activity
to the nutritional state of the larvae. As MsCTLP1 appears to be involved in such
signaling cascades, other midgut peptidases could have other targets and may therefore
regulate different activities.
To gain more insight into the functions of CTLPs, the gene family encoding these
peptidases in the genome of the red flour beetle, T. castaneum, was analyzed. Using an
extended search pattern, 14 TcCTLP genes were identified that encode peptidases with
S1 specificity pocket residues typically found in chymotrypsin-like enzymes. Analysis
of the expression patterns of seven TcCTLP genes at various developmental stages
revealed that 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-5C and TcCTLP-6C 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, western blots were
performed using protein extracts from larval exuviae. The extracts reacted with
antibodies to TcCTLP-5C and TcCTLP-6C suggesting that the corresponding
peptidases are secreted into the molting fluid. Finally, systemic RNAi experiments were
performed. While injections of dsRNAs to TcCTLP-5A/B and TcCTLP-6A/D/E into
penultimate larvae did not affect growth or development, injection of dsRNA for TcCTLP-5C and TcCTLP-6C resulted in severe molting defects. Recombinant
expressed TcCTLP-5C2 was moreover activated by trypsin and was able to hydrolyze
AAPF, hence making TcCTLP-5C the first described chymotrypsin-like peptidase ever
to be involved in molting.
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