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Antiviral Cystine Knot ?-Amylase Inhibitors from Alstonia scholaris.

ABSTRACT: Cystine knot ?-amylase inhibitors are cysteine-rich, proline-rich peptides found in the Amaranthaceae and Apocynaceae plant species. They are characterized by a pseudocyclic backbone with two to four prolines and three disulfides arranged in a knotted motif. Similar to other knottins, cystine knot ?-amylase inhibitors are highly resistant to degradation by heat and protease treatments. Thus far, only the ?-amylase inhibition activity has been described for members of this family. Here, we show that cystine knot ?-amylase inhibitors named alstotides discovered from the Alstonia scholaris plant of the Apocynaceae family display antiviral activity. The alstotides (As1-As4) were characterized by both proteomic and genomic methods. All four alsotides are novel, heat-stable and enzyme-stable and contain 30 residues. NMR determination of As1 and As4 structures reveals their conserved structural fold and the presence of one or more cis-proline bonds, characteristics shared by other cystine knot ?-amylase inhibitors. Genomic analysis showed that they contain a three-domain precursor, an arrangement common to other knottins. We also showed that alstotides are antiviral and cell-permeable to inhibit the early phase of infectious bronchitis virus and Dengue infection, in addition to their ability to inhibit ?-amylase. Taken together, our results expand membership of cystine knot ?-amylase inhibitors in the Apocynaceae family and their bioactivity, functional promiscuity that could be exploited as leads in developing therapeutics.

SUBMITTER: Nguyen PQ

PROVIDER: S-EPMC4692237 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Antiviral Cystine Knot α-Amylase Inhibitors from Alstonia scholaris.

Nguyen Phuong Quoc Thuc PQ Ooi Justin Seng Geap JS Nguyen Ngan Thi Kim NT Wang Shujing S Huang Mei M Liu Ding Xiang DX Tam James P JP

The Journal of biological chemistry 20151106 52

Cystine knot α-amylase inhibitors are cysteine-rich, proline-rich peptides found in the Amaranthaceae and Apocynaceae plant species. They are characterized by a pseudocyclic backbone with two to four prolines and three disulfides arranged in a knotted motif. Similar to other knottins, cystine knot α-amylase inhibitors are highly resistant to degradation by heat and protease treatments. Thus far, only the α-amylase inhibition activity has been described for members of this family. Here, we show t ...[more]

PMID: 26546678

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Project description:The alpha(v)beta(3) integrin receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4-kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to alpha(v)beta(3) integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a six amino acid loop of AgRP with a nine amino acid loop containing the Arg-Gly-Asp integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting to identify clones with high affinity to detergent-solubilized alpha(v)beta(3) integrin receptor. Select integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various integrin receptors. These studies showed that the engineered AgRP peptides bound to cells expressing alpha(v)beta(3) integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown to bind specifically to alpha(v)beta(3) integrins and had only minimal or no binding to alpha(v)beta(5), alpha(5)beta(1), and alpha(iib)beta(3) integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally occurring ligand for alpha(v)beta(3) and other integrins. Next, to evaluate whether the other three loops of AgRP could modulate integrin specificity, we made second-generation libraries by individually randomizing these loops in one of the high-affinity integrin-binding variants. Screening of these loop-randomized libraries against alpha(v)beta(3) integrins resulted in peptides that retained high affinities for alpha(v)beta(3) and had increased specificities for alpha(v)beta(3) over alpha(iib)beta(3) integrins. Collectively, these data validate AgRP as a scaffold for protein engineering and demonstrate that modification of a single loop can lead to AgRP-based peptides with antibody-like affinities for their target.

Dataset Information

Antiviral Cystine Knot ?-Amylase Inhibitors from Alstonia scholaris.

Publications

Antiviral Cystine Knot α-Amylase Inhibitors from Alstonia scholaris.

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