Molecular basis for the resistance of an insect chymotrypsin to a potato type II proteinase inhibitor.
Plants produce a variety of proteinase inhibitors (PIs) that have a major function in defense against insect herbivores. In turn, insects have developed strategies to minimize the effect of dietary PIs on digestion. We have discovered that Helicoverpa larvae that survive consumption of a multidomain serine PI from Nicotiana alata (NaPI) contain high levels of a chymotrypsin that is not inhibited by NaPI. Here we describe the isolation of this NaPI-resistant chymotrypsin and an NaPI-susceptible chymotrypsin from Helicoverpa larvae, together with their corresponding cDNAs. We investigated the mechanism of resistance by mutating selected positions of the NaPI-susceptible chymotrypsin using the corresponding amino acids of the NaPI-resistant chymotrypsin. Four critical residues that conferred resistance to NaPI were identified. Molecular modeling revealed that a Phe-->Leu substitution at position 37 in the chymotrypsin results in the loss of important binding contacts with NaPI. Identification of the molecular mechanisms that contribute to PI resistance in insect digestive proteases will enable us to develop better inhibitors for the control of lepidopteran species that are major agricultural pests worldwide.
Dunse
K M
KM
Department of Biochemistry, La Trobe University, Melbourne, VIC 3086, Australia.
Kaas
Q
Q
Guarino
R F
RF
Barton
P A
PA
Craik
D J
DJ
Anderson
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D002918:Chymotrypsin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D019476:Insect Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D007814:Larva / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009036:Moths / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D010940:Plant Proteins / Q000378:metabolism* / Q000494:pharmacology; D011480:Protease Inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000378:metabolism*
D019476:Insect Proteins; C445075:PIN2 protein, Solanum americanum; D010940:Plant Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D001120:Arginine; D002918:Chymotrypsin