Structural basis of the resistance of an insect carboxypeptidase to plant protease inhibitors

Abstract

Corn earworm (Helicoverpa zea), also called tomato fruitworm, is a common pest of many Solanaceous plants. This insect is known to adapt to the ingestion of plant serine protease inhibitors by using digestive proteases that are insensitive to inhibition. We have now identified a B-type carboxypeptidase of H. zea (CPBHz) insensitive to potato carboxypeptidase inhibitor (PCI) in corn earworm. To elucidate the structural features leading to the adaptation of the insect enzyme, the crystal structure of the recombinant CPBHz protein was determined by x-ray diffraction. CPBHz is a member of the A͞B subfamily of metallocarboxypeptidases, which displays the characteristic metallocarboxypeptidase ␣͞␤-hydrolase fold, and does not differ essentially from the previously described Helicoverpa armigera CPA, which is very sensitive to PCI. The data provide structural insight into several functional properties of CPBHz. The high selectivity shown by CPBHz for C-terminal lysine residues is due to residue changes in the S1 substrate specificity pocket that render it unable to accommodate the side chain of an arginine. The insensitivity of CPBHz to plant inhibitors is explained by the exceptional positioning of two of the main regions that stabilize other carboxypeptidase-PCI complexes, the ␤8-␣9 loop, and ␣7 together with the ␣7-␣8 loop. The rearrangement of these two regions leads to a displacement of the active-site entrance that impairs the proper interaction with PCI. This report explains a crystal structure of an insect protease and its adaptation to defensive plant protease inhibitors.crystal structure ͉ inhibitor resistance ͉ Helicoverpa zea ͉ metallocarboxypeptidase T he larvae of plant-eating insects have a high need for free amino acids and nitrogen during their development. Normally, they meet these requirements by degrading dietary proteins. The digestive enzymes present in the larvae of the lepidopteran insects such as Helicoverpa zea are closely related to those secreted from the mammalian pancreas, comprising serine endopeptidases like trypsins and chymotrypsins and exopeptidases such as metallocarboxypeptidases and aminopeptidases (1, 2).Plants have evolved a defense that interferes with the insects' digestive system by expressing a number of different protease inhibitors (PIs), which are either constitutively present or strongly up-regulated in response to wound damage (3). Most PIs are small proteins that have been found in all plant species investigated thus far and occur in both reproductive and vegetative tissues. In herbivorous insects, they act by inhibiting protein digestive enzymes in the guts of insect larvae or adults, resulting in amino acid deficiencies that lead to serious developmental delay, mortality, or reduced fecundity.A number of polyphagous insects, among them H. zea, a common pest of many Solanaceous plants such as the potato (4), have adapted to the protease inhibitors of their various host plants. Their survival and larval development is not affected by the presence of such molecules in th...

Cloning and characterization of cDNAS encoding carboxypeptidase-like proteins from the gut of Hessian fly larvae [Mayetiola destructor (Say)]☆

“…Mammalian carboxypeptidase, an exopeptidase, has been well characterized (Titani et al, 1975;Rees et al, 1983;Coll et al, 1991;Guasch et al, 1992;Aloy et al, 1998), but the studies of insect carboxypeptidases are limited to a few species including black fly Simulium vittatum (Ramos et al, 1993), corn earworm Helicoverpa armingera (Bown et al, 1998;Bown and Gatehouse, 2004), mosquitoes Anopheles gambiae and Aedes aegypti (Edwards et al, 1997(Edwards et al, , 2000, tsetse fly Glossina morsitans (Yan et al, 2002), bertha armyworm Mamestra configurata (Hegedus et al, 2003), cabbage looper Trichoplusia ni (Wang et al, 2004), and corn earworm Helicoverpa zea (Bayes et al, 2005). In the current research, we report the isolation and characterization of nine unique cDNAs that encode carboxypeptidases from the Hessian fly, Mayetiola destructor (Say), one of the most destructive pests of wheat (Triticum aestivum L.) (Hatchett et al, 1987;Buntin, 1999).…”

Serine Protease Inhibitors Specifically Defend Solanum nigrum against Generalist Herbivores but Do Not Influence Plant Growth and Development

“…There are examples of herbivore-inducible PIs with no defensive function or even the opposite effect due to the counteradaptations of insects to the ingestion of PIs (Zhu-Salzman et al, 2008). Several studies demonstrated that some insects respond with constitutive or induced production of PI-insensitive proteases (Jongsma et al, 1995;Bown et al, 1997;Bayé s et al, 2005Bayé s et al, , 2006 or by proteolytically inactivating the ingested PIs to prevent binding to sensitive proteases (Girard et al, 1998;Giri et al, 1998;Zhu-Salzman et al, 2003). For many insects, the ingestion of PI-containing tissues elicits behavioral and physiological counterresponses that increase the amount of damage they inflict on plants: sublethal PI levels stimulate feeding and induce a general overproduction of proteolytic enzymes.…”

Role of cysteine proteinase inhibitors in preference of Japanese beetles (Popillia japonica) for soybean (Glycine max) leaves of different ages and grown under elevated CO2

“…4). Herbivores can respond to high PI levels in the diet by (a) overproducing or down-regulating PI-sensitive proteinase (Broadway and DuVey 1986;Zavala et al 2008b), (b) overproducing PI-insensitive proteases (Bayes et al 2005;Jongsma et al 1995), (c) degrading the inhibitors (Giri et al 1998), or (d) changing their feeding positions on the plant . Selective feeding on older, more poorly defended soybean leaves may allow JBs to optimize their growth under a variety of atmospheric conditions.…”