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Discovery and Characterization of Pseudocyclic Cystine‐Knot Α‐Amylase Inhibitors with High Resistance to Heat and Proteolyt

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Discovery and Characterization of Pseudocyclic Cystine‐Knot Α‐Amylase Inhibitors with High Resistance to Heat and Proteolyt Discovery and characterization of pseudocyclic cystine-knot a-amylase inhibitors with high resistance to heat and proteolytic degradation Phuong Q. T. Nguyen, Shujing Wang, Akshita Kumar, Li J. Yap, Thuy T. Luu, Julien Lescar and James P. Tam School of Biological Sciences, Nanyang Technological University, Singapore Keywords Obesity and type 2 diabetes are chronic metabolic diseases, and those cis-proline; cystine knot; pseudocyclics; affected could benefit from the use of a-amylase inhibitors to manage a wrightide; -amylase inhibitors starch intake. The pseudocyclics, wrightides Wr-AI1 to Wr-AI3, isolated from an Apocynaceae plant show promise for further development as Correspondence a J. P. Tam, School of Biological Sciences, orally active -amylase inhibitors. These linear peptides retain the stability Nanyang Technological University, known for cystine-knot peptides in the presence of harsh treatment. They Singapore are resistant to heat treatment and endopeptidase and exopeptidase degra- Fax: +65 6515 1632 dation, which is characteristic of cyclic cystine-knot peptides. Our NMR Tel: +65 6316 2833 and crystallography analysis also showed that wrightides, which are cur- E-mail: [email protected] rently the smallest proteinaceous a-amylase inhibitors reported, contain the backbone-twisting cis-proline, which is preceded by a nonaromatic residue (Received 22 February 2014, revised 19 June 2014, accepted 15 July 2014) rather than a conventional aromatic residue. The modeled structure and a molecular dynamics study of Wr-AI1 in complex with yellow mealworm doi:10.1111/febs.12939 a-amylase suggested that, despite having a similar structure and cys- tine-knot fold, the knottin-type a-amylase inhibitors may bind to insect a-amylase via a different set of interactions. Finally, we showed that the precursors of pseudocyclic cystine-knot a-amylase inhibitors and their bio- synthesis in plants follow a secretory protein synthesis pathway. Together, our findings provide insights for the use of the pseudocyclic a-amylase inhibitors as useful leads for the development of orally active peptidyl bio- actives, as well as an alternative scaffold for cyclic peptides for engineering metabolically stable human a-amylase inhibitors. Database The nucleotide sequences for Wr-AI1 to Wr-AI3 have been deposited in the GenBank database under GenBank accession numbers KF679826, KF679827, and KF679828, respectively. The Wr-AI1 solution structure solved for 10 ensembles with the lowest target function is available in the Protein Data Bank under accession code 2MAU. The coordinates of Wr-AI1 crystal structure are available in the Protein Data Bank database under accession code 4BFH. Introduction Small disulfide-rich, proteinaceous bioactives are (CK) motif, with a three-disulfide knotted structure prominent in toxins, hormones, growth factors, and formed by two disulfide bonds, together with the protease inhibitors [1]. Many contain a cystine-knot connecting backbones, forming an embedded ring Abbreviations AAI, amaranth a-amylase inhibitor; CCK, cyclic cystine-knot; CK, cystine-knot; ER, endoplasmic reticulum; HSA, human salivary a-amylase; PCK, pseudocylic cystine-knot; PDB, Protein Data Bank; TMA, Tenebrio molitor a-amylase; UPLC, ultra-performance liquid chromatography. FEBS Journal 281 (2014) 4351–4366 ª 2014 FEBS 4351 Pseudocyclic cystine-knot a-amylase inhibitors P. Q. T. Nguyen et al. through which the third bond penetrates [2]. Of par- proteomic and genomic methods, we identified three ticular interest in drug development is the knottin AAI-like a-amylase inhibitors, wrightide-amylase- family of CK peptides containing 25–45 residues, and inhibitors Wr-AI1 to Wr-AI3, from the medicinal often possessing protease inhibitory functions, from plant Wrightia religiosa (Apocynaceae family). We which the name was derived [3]. Knottins form com- showed that they are resistant not only to heat treat- pact and defined structures with extensive internal ment and endopeptidase degradation, but also to exo- hydrogen bonding, endowing them with resistance to peptidase. The structure of Wr-AI1 was analyzed in proteolytic degradation by endopeptidases and dena- both solution and crystal form by NMR and X-ray turation by heat or chemicals, as shown by numerous crystallography (to 1.25-A resolution), respectively. studies, including those using sequencing experiments Modeling the Wr-AI1–TMA complex with docking to determine their primary structures. Certain CK and molecular dynamics suggests that a-amylase inhi- peptides of the knottin family have further evolved bition by knottins occurs via an overall shape-fitting as macrocycles such as cyclotides, harboring cyclic mechanism rather than through a particular set of CKs (CCKs) with no termini, a feature that has polar or ionic interactions in the TMA active site made them resistant to degradation by exopeptidases pocket. We also showed that the precursors of knot- [4]. Cyclotides, generally consisting of 28–37 residues, tin-type a-amylase inhibitors contain a three-domain are known be ultrastable to proteolytic and heat deg- structure common to CK peptides. Taken together, radation, and possess robust qualities comparable to our findings provide new insights into the sequence, those of small-molecule drug candidates. All of these structure and biosynthesis of CK a-amylase inhibitors, features bode well for the development of orally which could be used as stable scaffolds in engineering active peptidyl bioactives. human a-amylase inhibitors. In a program to identify potentially orally active peptidyl bioactives for the treatment of metabolic dis- Results eases such as diabetes, we have initiated MS profiling to identify cysteine-rich peptidyl a-amylase inhibitors Isolation of a-amylase inhibitors from in traditional medicines. Plants and microorganisms W. religiosa produce a diverse group of proteinaceous a-amylase inhibitors that function in defense pathways. These Our preliminary MS profiling of crude extracts of inhibitors vary greatly in structure and size, ranging W. religiosa leaves and flowers revealed strong posi- from small peptides (3 kDa), such as amaranth a-amy- tive signals in the mass range of 3–5 kDa, indicative lase inhibitor (AAI) [5], to large proteins, such as of cysteine-rich peptides (Fig. 1). We therefore per- a-AI1, a 23-kDa a-amylase inhibitor from kidney bean formed extraction of the putative cysteine-rich pep- (Phaseolus vulgaris) [6]. They are structurally classified tides from fresh W. religiosa leaves from Vietnam into seven groups: knottin-type, c-thionin-like, CM- and Singapore in 50% ethanol, and purified them proteins, Kunitz-type, thaumatin-like, legume-lectin- through several rounds of RP-HPLC and strong cat- like, and microbial [7]. These classes of a-amylase ion-exchange HPLC. The most abundant peptides inhibitor have attracted attention as tools in agricul- from Vietnam and Singapore leaves were named ture and for antidiabetes management. wrightide-amylase-inhibitors Wr-AI1 and Wr-AI2, The smallest proteinaceous a-amylase inhibitor respectively. Each purified wrightide was fully known to date, the 3-kDa AAI, is currently the only reduced by dithiothreitol, and then digested with member of the knottin-type group to be reported. AAI trypsin and chymotrypsin. The resulting fragments comprises 32 residues harboring a CK core. This were sequenced by tandem MS, and their sequences inhibitor specifically inhibits the yellow mealworm were deduced by analyzing b-ions and y-ions (Fig. 2). Tenebrio molitor a-amylase (TMA), but is inactive By genetic analysis, we also obtained the sequence of against human and bovine a-amylases [5]. Although wrightide Wr-AI3, which could not be detected in the detailed structural study of the inhibition mechanism MS profile. of AAI on TMA has been reported, little is known Wrightides Wr-AI1 to Wr-AI3, all 30 residues in about its knottin-type homologs or their genetic pre- length, contain six cysteines, three glycines, and two cursors. prolines. Together, these three residues account for Here, we report on the discovery and characteriza- > 35% of the sequences. Wrightides share high tion of a group of linear knottins with characteristics sequence homology with each other (93–96%), differ- and a potential for use in drug development compara- ing by one or two residues (Fig. 3), and high sequence ble to those of CCK peptides. Using a combination of homology with AAI (48%). 4352 FEBS Journal 281 (2014) 4351–4366 ª 2014 FEBS P. Q. T. Nguyen et al. Pseudocyclic cystine-knot a-amylase inhibitors Fig. 1. Tissue-specific and region-specific expression profiles of wrightides from flower and leaf of W. religiosa. A 50% ethanol extract of 1 g of each plant sample was purified with a C18 solid- phase extraction column. The eluate with 80% acetonitrile was profiled with MALDI- TOF MS to determine the occurrence of putative CK peptides in different W. religiosa plant parts from Singapore and Vietnam. Solution structure of Wr-AI1 determined by donors on the basis of the structures. Wr-AI1 contains 1H-NMR two prolines, whose conformations were identified as cis-Pro17 and trans-Pro23. The proline cis and trans With the distance, dihedral angle and hydrogen bond 1 conformations were confirmed by the observation of restraints derived from H-NMR experiments N a N NOE crosspeaks H Asp16–H Pro17 and H Gln22– (Table 1), the solution structures of Wr-AI1 showed d a H , respectively, as the H strips of these four resi- that it adopts a similar CK scaffold as AAI, with the Pro23 dues were not identified from the noise region of H O same three disulfide linkages: CysI–IV, CysII–V, and 2 around 4.7 p.p.m. CysIII–VI, where CysIII–VI is the penetrating disulfide bond (Fig. 3A,B). The structure contains three short b-strands: Tyr7–Cys8, His19–Cys20, and Gly27–Ala30; Structure of Wr-AI1 determined by X-ray His19–Ala30 forms a b-hairpin (Fig. 3C). The crystallography b-strands are connected by four b-turns, two pointing Wr-AI1 showed a high propensity to form fiber-like towards the N-terminal and C-terminal ends on one precipitates at neutral pH.
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