Cowpea bruchid midgut transcriptome response to a soybean cystatin--costs and benefits of counter-defence.

The insect digestive system is the first line of defence protecting cells and tissues of the body from a broad spectrum of toxins and antinutritional factors in its food. To gain insight into the nature and breadth of genes involved in adaptation to dietary challenge, a collection of 20 352 cDNAs was prepared from the midgut tissue of cowpea bruchid larvae (Callosobruchus maculatus) fed on regular diet and diets containing antinutritional compounds. Transcript responses of the larvae to dietary soybean cystatin (scN) were analysed using cDNA microarrays, followed by quantitative real-time PCR (RT-PCR) confirmation with selected genes. The midgut transcript profile of insects fed a sustained sublethal scN dose over the larval life was compared with that of insects treated with an acute high dose of scN for 24 h. A total of 1756 scN-responsive cDNAs was sequenced; these clustered into 967 contigs, of which 653 were singletons. Many contigs (451) did not show homology with known genes, or had homology only with genes of unknown function in a Blast search. The identified differentially regulated sequences encoded proteins presumptively involved in metabolism, structure, development, signalling, defence and stress response. Expression patterns of some scN-responsive genes were consistent in each larval stage, whereas others exhibited developmental stage-specificity. Acute (24 h), high level exposure to dietary scN caused altered expression of a set of genes partially overlapping with the transcript profile seen under chronic lower level exposure. Protein and carbohydrate hydrolases were generally up-regulated by scN whereas structural, defence and stress-related genes were largely down-regulated. These results show that insects actively mobilize genomic resources in the alimentary tract to mitigate the impact of a digestive protease inhibitor. The enhanced or restored digestibility that may result is possibly crucial for insect survival, yet may be bought at the cost of weakened response to other stresses.

Functional expression of an insect cathepsin B-like counter-defence protein.

Insects are capable of readjusting their digestive regimes in response to dietary challenge. Cowpea bruchids (Callosobruchus maculatus) strongly induce C. maculatus cathepsin B-like cysteine protease 1 (CmCatB1) transcripts when fed diet containing a soybean cysteine protease inhibitor soyacystatin N (scN). CmCatB1 shares significant sequence similarity with cathepsin B-like cysteine proteases. In this study, we isolated another cDNA, namely CmCatB2 that encodes a protein sequence otherwise identical to CmCatB1, but lacking a 70-amino-acid internal section. CmCatB1 and CmCatB2 probably resulted from alternate splicing events. Only the CmCatB1 transcript, however, exhibited differential expression in response to dietary scN. Further, this expression was only detectable in larvae, which is the developmental stage associated with food ingestion. The scN-activated and developmentally regulated CmCatB1 expression pattern suggests it may have a unique function in insect counter-defence against antinutritional factors. Heterologously expressed recombinant CmCatB1 protein exhibited enzymatic activity in a pH-dependent manner. Activity of the protein was inhibited by both the cysteine protease inhibitor E-64 and the cathepsin B-specific inhibitor CA-074, verifying its cathepsin B-like cysteine protease nature. Interestingly, the enzymatic activity was unaffected by the presence of scN. Together, we have provided functional evidence suggesting that CmCatB1 confers inhibitor-insensitive enzymatic activity to cowpea bruchids, which is crucial for insect survival when challenged by dietary protease inhibitors.

Cowpea bruchid Callosobruchus maculatus counteracts dietary protease inhibitors by modulating propeptides of major digestive enzymes.

Cowpea bruchids, when challenged by consumption of the soybean cysteine protease inhibitor scN, reconfigure expression of their major CmCP digestive proteases and resume normal feeding and development. Previous evidence indicated that insects selectively induced CmCPs from subfamily B, that were more efficient in autoprocessing and possessed not only higher proteolytic, but also scN-degrading activities. In contrast, dietary scN only marginally up-regulated genes from the more predominant CmCP subfamily A that were inferior to subfamily B. To gain further molecular insight into this adaptive adjustment, we performed domain swapping between the two respective subfamily members B1 and A16, the latter unable to autoprocess or degrade scN even after intermolecular processing. Swapping the propeptides did not qualitatively alter autoprocessing in either protease isoform. Incorporation of either the N- (pAmBA) or C-terminal (pAmAB) mature B1 segment into A16, however, was sufficient to prime autoprocessing of A16 to its mature form. Further, the swap at the N-terminal mature A16 protein region (pAmBA) resulted in four amino acid changes. Replacement of these amino acid residues by the corresponding B1 residues, singly and pair-wise, revealed that autoprocessing activation in pAmBA resulted from cumulative and/or coordinated individual effects. Bacterially expressed isolated propeptides (pA16 and pB1) differed in their ability to inhibit mature B1 enzyme. Lower inhibitory activity in pB1 is likely attributable to its lack of protein stability. This instability in the cleaved propeptide is necessary, although insufficient by itself, for scN-degradation by the mature B1 enzyme. Taken together, cowpea bruchids modulate proteolysis of their digestive enzymes by controlling proCmCP cleavage and propeptide stability, which explains at least in part the plasticity cowpea bruchids demonstrate in response to protease inhibitors.

Functional roles of specific bruchid protease isoforms in adaptation to a soybean protease inhibitor.

Upon challenge by the soybean cysteine protease inhibitor soyacystatin N (scN), cowpea bruchids reconfigure their major digestive cysteine proteases (CmCPs) in adaptation to the inhibitor and resume normal feeding and development. We have previously shown that CmCPB transcripts were 116.3-fold more abundant in scN-adapted bruchid guts than in unadapted guts, while CmCPA transcripts were only 2.5-fold higher. In order to further elucidate the functional significance of this differential regulation, we expressed three CmCPA and one CmCPB isoforms (A9, A13, A16 and B1) using a bacterial expression system, and characterized their activities. In contrast to the precursors of CmCPAs (proCmCPAs), proCmCPB1 exhibited more efficient autocatalytic conversion from the latent proenzyme to its active mature protease form, and demonstrated higher intrinsic proteolytic activity. Among proCmCPAs, dependence on exogenous enzymatic processing varies: while maturation of proCmCPA13 and proCmCPA16 was impaired in the absence of external proteolytic activity, proCmCPA9 appeared to utilize a two-step autoprocessing mechanism. Although all CmCPs are scN-sensitive, scN was degraded by CmCPB1 when outnumbered by the protease, but scN remained intact in the presence of excessive CmCPA9. These results provide further evidence that differential expression of CmCPs under scN challenge brings about adaptation to the inhibitor. High induction of unique cysteine protease isoforms with superior autoprocessing and proteolytic efficacy represents a strategy cowpea bruchids use to cope with dietary scN.

Transcriptional regulation in cowpea bruchid guts during adaptation to a plant defence protease inhibitor.

Cowpea bruchid, when fed on a diet containing the soybean cysteine protease inhibitor soyacystatin N (scN), activates an array of counter-defence genes to adapt to the negative effects of the inhibitor and regain its normal rate of feeding and development. A collection of 1920 cDNAs was obtained by differential subtraction with cDNAs prepared from guts of the 4th instar larvae of scN-adapted (reared on scN-containing diet) and scN-unadapted (reared on regular scN-free diet) cowpea bruchids. Subsequent expression profiling using DNA microarray and Northern blot analyses identified ninety-four transcript species from this collection that are responsive to dietary scN. scN-adapted insects induced genes encoding protein and carbohydrate digestive enzymes, probably to help meet their carbon and nitrogen requirements. Up-regulation of antimicrobial and detoxification protein genes may represent a generalized defence response. Genes down-regulated by scN reflected physiological adjustments of the cowpea bruchids to scN challenge. A large portion of the responsive genes, presumably involved in carrying out the counter-defence response, were of unknown function. The full-length cDNA of an scN-inducible cathepsin B-like cysteine protease was obtained. Its transcriptional response to scN during larval development contrasts with the pattern of the cathepsin L family, the major digestive enzymes. These results suggest cathepsin B-like cysteine proteases may play a crucial role in cowpea bruchid adaptation to dietary scN.

Cowpea bruchid Callosobruchus maculatus uses a three-component strategy to overcome a plant defensive cysteine protease inhibitor.

The soybean cysteine protease inhibitor, soyacystatin N (scN), negatively impacts growth and development of the cowpea bruchid, Callosobruchus maculatus[Koiwa et al. (1998) Plant J 14: 371-379]. However, the developmental delay and feeding inhibition caused by dietary scN occurred only during the early developmental stages (the 1st, 2nd and 3rd instars) of the cowpea bruchid. The 4th instar larvae reared on scN diet (adapted) exhibited rates of feeding and development which were comparable to those feeding on an scN-free diet (unadapted) prior to pupation. Total gut proteolytic capacity at this larval stage significantly increased in the scN-adapted insects. The elevated enzymatic activity was attributed to a differential expression of insect gut cysteine proteases (representing the major digestive enzymes), and of aspartic proteases. scN degradation by the gut extract was observed only in adapted bruchids, and this activity appeared to be a combined effect of scN-induced cysteine and aspartic proteases. Thirty cDNAs encoding cathepsin L-like cysteine proteases were isolated from insect guts, and they were differentially regulated by dietary scN. Our results suggest that the cowpea bruchid adapts to the challenge of scN by qualitative and quantitative remodelling of its digestive protease complement, and by activating scN-degrading protease activity.

Functional mechanics of the plant defensive Griffonia simplicifolia lectin II: resistance to proteolysis is independent of glycoconjugate binding in the insect gut.

Griffonia simplicifolia lectin II (GSII) is a plant defensive protein that significantly delays development of the cowpea bruchid Callosobruchus maculatus (F.). Previous structure/function analysis by site-directed mutagenesis indicated that carbohydrate binding and resistance to insect gut proteolysis are required for the anti-insect activity of this lectin. However, whether there is a causal link between carbohydrate binding and resistance to insect metabolism remains unknown. Two proteases principally responsible for digestive proteolysis in third and fourth instar larvae of C. maculatus were purified by activated thiol sepharose chromatography and resolved as cathepsin L-like proteases, based on N-terminal amino acid sequence analysis. Digestion of bacterially expressed recombinant GSII (rGSII) and its mutant protein variants with the purified gut proteases indicates that carbohydrate binding, presumably to a target ligand in insect gut, and proteolytic resistance are independent properties of rGSII, and that both facilitate its efficacy as a plant defensive molecule.

Phage display selection of hairpin loop soyacystatin variants that mediate high affinity inhibition of a cysteine proteinase.

Two hairpin-loop domains in cystatin family proteinase inhibitors form an interface surface region that slots into the active site cleft of papain-like cysteine proteinases, and determine binding affinity. The slot region surface architecture of the soybean cysteine proteinase inhibitor (soyacystatin N, scN) was engineered using techniques of in vitro molecular evolution to define residues that facilitate interaction with the proteinase cleft and modulate inhibitor affinity and function. Combinatorial phage display libraries of scN variants that contain mutations in the essential motifs of the first (QVVAG) and second (EW) hairpin-loop regions were constructed. Approximately 1010-1011 phages expressing recombinant scN proteins were subjected to biopanning selection based on binding affinity to immobilized papain. The QVVAG motif in the first hairpin loop was invariant in all functional scN proteins. All selected variants (30) had W79 in the second hairpin-loop motif, but there was diversity for hydrophobic and basic amino acids in residue 78. Kinetic analysis of isolated scN variants identified a novel scN isoform scN(LW) with higher papain affinity than the wild-type molecule. The variant contained an E78L substitution and had a twofold lower Ki (2.1 pM) than parental scN, due to its increased association rate constant (2.6 +/- 0.09 x 107 M-1sec-1). These results define residues in the first and second hairpin-loop regions which are essential for optimal interaction between phytocystatins and papain, a prototypical cysteine proteinase. Furthermore, the isolated variants are a biochemical platform for further integration of mutations to optimize cystatin affinity for specific biological targets.

A plant defensive cystatin (soyacystatin) targets cathepsin L-like digestive cysteine proteinases (DvCALs) in the larval midgut of western corn rootworm (Diabrotica virgifera virgifera).

Feeding bioassay results established that the soybean cysteine proteinase inhibitor N (soyacystatin N, scN) substantially inhibits growth and development of western corn rootworm (WCR), by attenuating digestive proteolysis [Zhao, Y. et al. (1996) Plant Physiol. 111, 1299-1306]. Recombinant scN was more inhibitory than the potent and broad specificity cysteine proteinase inhibitor E-64. WCR digestive proteolytic activity was separated by mildly denaturing SDS-PAGE into two fractions and in-gel assays confirmed that the proteinase activities of each were largely scN-sensitive. Since binding affinity to the target proteinase [Koiwa, H. et al. (1998) Plant J. 14, 371-380] governs the effectiveness of scN as a proteinase inhibitor and an insecticide, five peptides (28-33 kDa) were isolated from WCR gut extracts by scN affinity chromatographic separation. Analysis of the N-terminal sequence of these peptides revealed similarity to a cathepsin L-like cysteine proteinase (DvCAL1, Diabrotica virgifera virgifera cathepsin L) encoded by a WCR cDNA. Our results indicate that cathepsin L orthologs are pivotal digestive proteinases of WCR larvae, and are targets of plant defensive cystatins (phytocystatins), like scN.

Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II.

Griffonia simplicifolia leaf lectin II (GSII), a plant defense protein against certain insects, consists of an N-acetylglucosamine (GlcNAc)-binding large subunit with a small subunit having sequence homology to class III chitinases. Much of the insecticidal activity of GSII is attributable to the large lectin subunit, because bacterially expressed recombinant large subunit (rGSII) inhibited growth and development of the cowpea bruchid, Callosobruchus maculatus (F). Site-specific mutations were introduced into rGSII to generate proteins with altered GlcNAc binding, and the different rGSII proteins were evaluated for insecticidal activity when added to the diet of the cowpea bruchid. At pH 5.5, close to the physiological pH of the cowpea bruchid midgut lumen, rGSII recombinant proteins were categorized as having high (rGSII, rGSII-Y134F, and rGSII-N196D mutant proteins), low (rGSII-N136D), or no (rGSII-D88N, rGSII-Y134G, rGSII-Y134D, and rGSII-N136Q) GlcNAc-binding activity. Insecticidal activity of the recombinant proteins correlated with their GlcNAc-binding activity. Furthermore, insecticidal activity correlated with the resistance to proteolytic degradation by cowpea bruchid midgut extracts and with GlcNAc-specific binding to the insect digestive tract. Together, these results establish that insecticidal activity of GSII is functionally linked to carbohydrate binding, presumably to the midgut epithelium or the peritrophic matrix, and to biochemical stability of the protein to digestive proteolysis.

Phage display selection can differentiate insecticidal activity of soybean cystatins.

Plant cysteine proteinase inhibitors (phytocystatins) have been implicated as defensive molecules against Coleopteran and Hemipteran insect pests. Two soybean cystatins, soyacystatin N (scN) and soyacystatin L (scL), have 70% sequence identity but scN is a much more potent inhibitor of papain, vicilin peptidohydrolase and insect gut proteinases. When these cystatins were displayed on phage particles, papain-binding affinity and CPI activity of scN were substantially greater than those of scL, in direct correlation with their relative CPI activity as soluble recombinant proteins. Furthermore, scN substantially delayed cowpea weevil (Callosobruchus maculatus (F.)) growth and development in insect feeding bioassays, whereas scL was essentially inactive as an insecticide. Papain biopanning selection of phage-displayed soyacystatins resulted in a 200-1000-fold greater enrichment for scN relative to scL. These results establish that binding affinity of cystatins can be used in phage display biopanning procedures to select variants with greater insecticidal activity, illustrating the potential of phage display and biopanning selection for directed molecular evolution of biological activity of these plant defensive proteins.