Use of phage display to select novel cystatins specific for Acanthoscelides obtectus cysteine proteinases

Melo, Francislete Rodrigues; Mello, Marcia O.; Franco, Octávio L.; Rigden, Daniel J.; Mello, Luciane V.; Genú, Aline Marques; Silva-Filho, Márcio C.; Gleddie, S.; Grossi-de-Sá, Maria Fátima

doi:10.1016/s1570-9639(03)00264-4

Cited by 22 publications

(17 citation statements)

References 30 publications

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“…In this context, it is worth mentioning that Arai et al (1991) found that replacement of the V residue in the active site of OC-I had no effect on the binding of papain. In phage display experiments, Koiwa et al (2001) did not find any binding variants in this region, but Melo et al (2003)-using the same technique-found two different variants of this motif, DVVSA and NTSSA, with a lower affinity for papain but a similar inhibitory effect against a cysteine proteinase from the coleopteran insect Acanthoscelides obtectus. Thus, variants of the active-site motif may be associated with different inhibitory properties against different cysteine proteinases.…”

Section: Discussionmentioning

confidence: 97%

Comparative phylogenetic analysis of cystatin gene families from arabidopsis, rice and barley

Martínez¹,

Abraham²,

Carbonero³

et al. 2005

Mol Genet Genomics

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The plant cystatins or phytocystatins comprise a family of specific inhibitors of cysteine proteinases. Such inhibitors are thought to be involved in the regulation of several endogenous processes and in defence against pests and pathogens. Extensive searches in the complete rice and Arabidopsis genomes and in barley EST collections have allowed us to predict the presence of twelve different cystatin genes in rice, seven in Arabidopsis, and at least seven in barley. Structural comparisons based on alignments of all the protein sequences using the CLUSTALW program and searches for conserved motifs using the MEME program have revealed broad conservation of the main motifs characteristic of the plant cystatins. Phylogenetic analyses based on their deduced amino acid sequences have allowed us to identify groups of orthologous cystatins, and to establish homologies and define examples of gene duplications mainly among the rice and barley cystatin genes. Moreover, the absence of a counterpart between the two monocots, as well as strong variations in the motifs that interact with the cysteine proteinases, may be related to a species-specific evolutionary process. This cystatin classification should facilitate the assignment of proteinase specificities and functions to other cystatins as new information is obtained.

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Section: Discussionmentioning

confidence: 97%

Comparative phylogenetic analysis of cystatin gene families from arabidopsis, rice and barley

Martínez¹,

Abraham²,

Carbonero³

et al. 2005

Mol Genet Genomics

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“…Qasim et al, 1997;Mason et al, 1998;Ogawa et al, 2002;Pavlova and Bjö rk, 2003) and molecular phage display procedures involving random mutagenesis in specific regions of the inhibitor sequence (e.g. Laboissiere et al, 2002;Ceci et al, 2003;Melo et al, 2003;Stoop and Craik, 2003). A structural model for the human stefin B:papain complex (Stubbs et al, 1990), in particular, has been instrumental in the identification of relevant target sites for the molecular improvement of cystatins against Cys proteases.…”

mentioning

confidence: 99%

“…In an early study, Urwin et al (1995) engineered a variant of oryzacystatin I with improved nematicidal activity by deleting the residue Asp-86 from the original sequence, after deducing the possible interfering effect of this amino acid during the inhibitory process. More recently, Melo et al (2003) produced a phage display library derived from a nematode cystatin randomly mutagenized in the first (central) inhibitory loop to select mutated cystatins with high inhibitory activity against digestive proteases of the major bean weevil Acanthoscelides obtectus.…”

mentioning

confidence: 99%

“…Several studies used protein engineering approaches to improve the effectiveness of protease inhibitors toward herbivorous insect or nematode proteases (Urwin et al, 1995;Inanaga et al, 2001;Koiwa et al, 2001;Ceci et al, 2003;Martinez et al, 2003;Melo et al, 2003), but little attention has been paid to the impact of such modifications in a multitrophic context. The ability of recombinant plant cystatins to alter digestive protease activities in insect predatory arthropods via their herbivorous prey fed the modified plant has been documented in recent years (Bouchard et al, 2003a(Bouchard et al, , 2003bFerry et al, 2003;Alvarez-Alfageme et al, 2007), as well as some unintended pleiotropic effects of these inhibitors in planta significantly altering developmental processes and stress responses in the modified plant (Gutierrez-Campos et al, 2001;Belenghi et al, 2003;Van der Vyver et al, 2003;Vaillancourt, 2005).…”

mentioning

confidence: 99%

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Tailoring the Specificity of a Plant Cystatin toward Herbivorous Insect Digestive Cysteine Proteases by Single Mutations at Positively Selected Amino Acid Sites

et al. 2008

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Plant cystatins, similar to other defense proteins, include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Using 29 single mutants of the eighth domain of tomato (Solanum lycopersicum) multicystatin, SlCYS8, we assessed here the potential of site-directed mutagenesis at positively selected amino acid sites to generate cystatin variants with improved inhibitory potency and specificity toward herbivorous insect digestive cysteine (Cys) proteases. Compared to SlCYS8, several mutants (22 out of 29) exhibited either improved or lowered potency against different model Cys proteases, strongly suggesting the potential of positively selected amino acids as target sites to modulate the inhibitory specificity of the cystatin toward Cys proteases of agronomic significance. Accordingly, mutations at positively selected sites strongly influenced the inhibitory potency of SlCYS8 against digestive Cys proteases of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). In particular, several variants exhibited improved potency against both cystatin-sensitive and cystatininsensitive digestive Cys proteases of this insect. Of these, some variants also showed weaker activity against leaf Cys proteases of the host plant (potato [Solanum tuberosum]) and against a major digestive Cys protease of the two-spotted stinkbug Perillus bioculatus, an insect predator of Colorado potato beetle showing potential for biological control. Overall, these observations suggest the usefulness of site-directed mutagenesis at positively selected amino acid sites for the engineering of recombinant cystatins with both improved inhibitory potency toward the digestive proteases of target herbivores and weaker potency against nontarget Cys proteases in the host plant or the environment.

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“…Soyacystatin N variants with higher activity against papain were also identified when variants carried mutations in the first and second hairpin loop [30]. Variants originating from screening a phage display library derived from a nematode cystatin and mutagenized in the first inhibitory hairpin loop were more potent against digestive proteases of the bean weevil, Acanthoscelides obtectus [31]. We also recently improved cystatin potency against papain when a rather weak-acting papaya cystatin was mutagenized in the Q-X aa -V-X aa -G motif to obtain a cystatin more similar to rice OC-I [19].…”

Section: Conserved Cystatin Motifsmentioning

confidence: 99%

Review: The future of cystatin engineering

et al. 2016

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Use of phage display to select novel cystatins specific for Acanthoscelides obtectus cysteine proteinases

Cited by 22 publications

References 30 publications

Comparative phylogenetic analysis of cystatin gene families from arabidopsis, rice and barley

Comparative phylogenetic analysis of cystatin gene families from arabidopsis, rice and barley

Tailoring the Specificity of a Plant Cystatin toward Herbivorous Insect Digestive Cysteine Proteases by Single Mutations at Positively Selected Amino Acid Sites

Review: The future of cystatin engineering

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