Proteinase inhibitors from the molting fluid of the pharate adult tobacco hornworm,Manduca sexta

Abstract

The molting fluid of pharate adult Manduca sexta was found to contain at least two types of proteinase inhibitor activities. One inhibited the native cuticle degrading trypsin‐like proteinase, MFP1, while the other was found to be highly specific for subtilisin‐like enzymes. The developmental profiles of both these inhibitor activities were investigated. MFP‐1 inhibitor activity was found to be present in the molting fluid of all stages of pre‐ecdysial development, except stage 7, which possessed the highest levels of MFP‐1 activity. The inhibitor was estimated to have a relative molecular mass of 14.5 k and was found to be heat stable. A role in regulation of cuticle degradation is suggested. Subtilisin inhibitor activity was found in molting fluid from all eight stages of pre‐ecdysial development, although there was some variation observed between the stages when inhibitor activities were visualized using PAGE zymograms. A subtilisin inhibitor was purified using Sep‐Pak cartridges and Reverse Phase HPLC. The inhibitor was found to be of low relative molecular mass (11 k), heat stable, and highly specific for fungal enzymes such as PR1 from the entomopathogen Metarhizium anisopliae. Therefore, a role in insect defense is suggested. Arch Insect Biochem. Physiol. 43:33–43, 2000. © 2000 Wiley‐Liss, Inc.

A Kunitz type protease inhibitor related protein is synthesized in Drosophila prepupal salivary glands and released into the moulting fluid during pupation

“…The moulting fluid of the silkworm Cecropia is characterized by high levels of chitinase and protease activities (Passonneau and Williams, 1953). In the moulting fluid of Manduca sexta high levels of the trypsin-like MFP-1 proteinase have been identified and related to cuticle degrading activities (Samuels and Reynolds, 2000). In our present D. virilis study, we show that prior to pupation the moulting fluid mainly comprises serine proteases, which suddenly disappear.…”

“…We assume it improbable that protein turnover abolishes these proteolytic activities because proteases in extracellular fluids like pupation fluids should be very stable and should require specific developmental control mechanisms at the post-transcriptional level. This assumption is substantiated by the observation that in the moulting fluid of the pharate adult hornworm Manduca sexta, the levels of the trypsin-like protease MFP-1 are inversely correlated with MFP-1-inhibitor activity during development (Samuels and Reynolds, 2000).…”

“…The validity of their hypothesis that homologs of these inhibitors should be present in all insect cocoons is substantiated by our finding that in samples obtained from the D. virilis moulting fluid at pupation increased levels of inhibitor activities directed against the serine proteases and the bacterial metalloprotease thermolysin are detectable. Both, Kunitz-type inhibitors (Ramesh et al, 1988;Samuels and Reynolds, 2000) and the insect metalloprotease inhibitor (Wedde et al, 1998) have been implicated in contributing to humoral defence of lepidopterans. Among the immune-related proteins that are induced and secreted into the hemolymph of G. mellonella during humoral immune response a Kunitz-type inhibitor has been identified that is active against the major serine protease produced by the fungal insect pathogen Metarhizium anisopliae (Fröbius et al, 2000).…”

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

“…In case of the chymotrypsin-like peptidase SeCT34 from Spodoptera exigua it has been suggested that the zymogen is activated by chymotrypsin rather than trypsin (Herrero et al, 2005). TcCTLP-5C and TcCTLP-6C may be also targets for regulatory serine peptidase inhibitors that are present in the molting fluid as well (Samuels and Reynolds, 2000). In any case, chymotrypsin-like peptidases apparently act in concert with other molting peptidases to degrade proteins in the old cuticle, which may involve also some of those chymotrypsin-like peptidases that we did not examine so far.…”

Seasonal and spatial influences on gene expression in Antarctic krill Euphausia superba

“…Trypsin may also be the activator of enzymes involved in the degradation of cuticle, such as β-Nacetylglucosaminidase, present in the moulting fluid of insects (Samuels et al 1993) and crustaceans (Warner & Matheson 1998). Klein et al (1996) found that both trypsin mRNA and trypsin enzyme activity are at their highest levels during premoult (stage D1) of the shrimp Penaeus vannamei.…”