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 NaPIsusceptible 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.chymotrypsin mutants | inhibitor-resistant proteinase | Lepidoptera S erine peptidases from the chymotrypsin family are a large group of enzymes and, although they have a highly conserved tertiary structural fold, they have developed a range of substrate specificities critical to many biological functions, including blood coagulation and immune responses (1, 2).Chymotrypsin and trypsin are the major digestive serine proteases of insects from the order Lepidoptera, which comprises some of the most important agricultural pests. When ingested, protease inhibitors (PI) block protease activity and increase insect mortality by restricting the availability of essential amino acids. Mechanisms of insect resistance to PIs include the upregulation of enzymes that degrade the PIs (3, 4), induction of enzymes that resist inactivation by PIs (5, 6), and overproduction of enzymes to maintain normal levels of gut proteolysis (7,8).As part of a strategy to develop novel insecticidal agents, we investigated the properties of a series of 6-kDa chymotrypsin and trypsin inhibitors (C1, C2, T1-T4) from Nicotiana alata (NaPI), which are members of the potato type II family of inhibitors (pin II). In our companion paper (9), we report that larvae from the major agricultural insect pest Helicoverpa punctigera that survive consumption of NaPI have high levels of an NaPI-resistant chymotrypsin. We discovered that a potato type I inhibitor (StPin1A) abolished the NaPI-resistant chymotrypsin activity and that the combination of these two PIs in artificial diets substantially stunted the growth of another agronomically important pest, H. armigera (9). Furthermore, field-grown transgenic cotton expressing both NaPI and StPin1A showed greater insect protection over the growing season than plants expressing a single inhibitor. In the current stud...

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Fc gamma receptors bind IgG to initiate cellular responses against pathogens and soluble antigens. We have determined the three-dimensional structure of the extracellular portion of human Fc gammaRIIa to 2.0 A resolution providing a structural basis for the unique functions of the leukocyte FcR family. The receptor is composed of two immunoglobulin domains and arranged to expose the ligand-binding site at one end of domain 2. Using alanine mutants we find that the binding sites for IgG1 and 2 are similar but the relative importance of specific regions on the receptor varies. In crystals, Fc gammaRIIa molecules associate to resemble V(L)V(H) dimers, suggesting that two Fc gammaRIIa molecules could cooperate to bind IgG in an asymmetric manner.

A gene encoding a 40.3-kDa serine proteinase inhibitor (PI) precursor is expressed at high levels in the stigma of the ornamental tobacco, Nicotiana alata. The precursor is processed proteolytically in vivo to release five homologous proteinase inhibitors of approximately 6 kDa, as well as two flanking peptides. The five PIs have been purified from stigmas and identified by N-terminal sequencing, electrospray mass spectrometry and inhibition activity against chymotrypsin or trypsin. One of the PIs inhibits chymotrypsin and the other four are most active on trypsin. Cleavage occurs in a linker region (EEKKND) that is repeated six times in the precursor molecule. In the plant, the initial cleavage probably occurs between asparagine and the aspartate residues and ragged ends are formed by subsequent trimming. In vitro, the protease-sensitive linker region is selectively cleaved by the endoproteinases Asp-N, Glu-C and Lys-C to release fully active approximately 6-kDa PIs that are resistant to further proteolytic digestion. The precursor, produced by a recombinant baculovirus, inhibits chymotrypsin more effectively than trypsin. The stoichiometry of 2.6 trypsin molecules/1 precursor molecule indicates that processing is required to activate or expose all of the four trypsin inhibitory sites.

A gene encoding a 40.3‐kDa serine proteinase inhibitor (PI) precursor is expressed at high levels in the stigma of the ornamental tobacco, Nicotiana alata. The precursor is processed proteolytically in vivo to release five homologous proteinase inhibitors of approximately 6 kDa, as well as two flanking peptides. The five PIs have been purified from stigmas and identified by N‐terminal sequencing, electrospray mass spectrometry and inhibition activity against chymotrypsin or trypsin. One of the PIs inhibits chymotrypsin and the other four are most active on trypsin. Cleavage occurs in a linker region (EEKKND) that is repeated six times in the precursor molecule. In the plant, the initial cleavage probably occurs between asparagine and the aspartate residues and ragged ends are formed by subsequent trimming. In vitro, the protease‐sensitive linker region is selectively cleaved by the endoproteinases Asp‐N, Glu‐C and Lys‐C to release fully active approximately 6‐kDa PIs that are resistant to further proteolytic digestion. The precursor, produced by a recombinant baculovirus, inhibits chymotrypsin more effectively than trypsin. The stoichiometry of 2.6 trypsin molecules/1 precursor molecule indicates that processing is required to activate or expose all of the four trypsin inhibitory sites.