RESUMEN
Cyclotides are plant-derived disulfide-rich cyclic peptides that have a natural function in plant defense and potential for use as agricultural pesticides. Because of their highly constrained topology, they are highly resistant to thermal, chemical, or enzymatic degradation. However, the stability of cyclotides at alkaline pH for incubation times of longer than a few days is poorly studied but important since these conditions could be encountered in the environment, during storage or field application as insecticides. In this study, kalata B1 (kB1), the prototypical cyclotide, was engineered to improve its long-term stability and retain its insecticidal activity via point mutations. We found that substituting either Asn29 or Gly1 to lysine or leucine increased the stability of kB1 by twofold when incubated in an alkaline buffer (pH = 9.0) for 7 days, while retaining its insecticidal activity. In addition, when Gly1 was replaced with lysine or leucine, the mutants could be cyclized using an asparaginyl endopeptidase, in vitro with a yield of â¼90% within 5 min. These results demonstrate the potential to manufacture kB1 mutants with increased stability and insecticidal activity recombinantly or in planta. Overall, the discovery of mutants of kB1 that have enhanced stability could be useful in leading to longer term activity in the field as bioinsecticides.
Asunto(s)
Ciclotidas , Insecticidas , Oldenlandia , Ciclotidas/genética , Ciclotidas/farmacología , Ciclotidas/química , Insecticidas/química , Insecticidas/farmacología , Leucina , Lisina/genética , Mutagénesis , Proteínas de Plantas/metabolismo , Oldenlandia/química , Estabilidad Proteica , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacosRESUMEN
The stinging hairs of plants from the family Urticaceae inject compounds that inflict pain to deter herbivores. The sting of the New Zealand tree nettle (Urtica ferox) is among the most painful of these and can cause systemic symptoms that can even be life-threatening; however, the molecular species effecting this response have not been elucidated. Here we reveal that two classes of peptide toxin are responsible for the symptoms of U. ferox stings: Δ-Uf1a is a cytotoxic thionin that causes pain via disruption of cell membranes, while ß/δ-Uf2a defines a new class of neurotoxin that causes pain and systemic symptoms via modulation of voltage-gated sodium (NaV) channels. We demonstrate using whole-cell patch-clamp electrophysiology experiments that ß/δ-Uf2a is a potent modulator of human NaV1.5 (EC50: 55 nM), NaV1.6 (EC50: 0.86 nM), and NaV1.7 (EC50: 208 nM), where it shifts the activation threshold to more negative potentials and slows fast inactivation. We further found that both toxin classes are widespread among members of the Urticeae tribe within Urticaceae, suggesting that they are likely to be pain-causing agents underlying the stings of other Urtica species. Comparative analysis of nettles of Urtica, and the recently described pain-causing peptides from nettles of another genus, Dendrocnide, indicates that members of tribe Urticeae have developed a diverse arsenal of pain-causing peptides.
Asunto(s)
Neurotoxinas , Péptidos , Toxinas Biológicas , Urticaceae , Humanos , Neurotoxinas/química , Dolor , Técnicas de Placa-Clamp , Péptidos/química , Péptidos/toxicidad , Toxinas Biológicas/química , Urticaceae/química , Canales de Sodio Activados por Voltaje/efectos de los fármacosRESUMEN
Multiple sclerosis (MS) is a debilitating disease that requires prolonged treatment with often severe side effects. One experimental MS therapeutic currently under development is a single amino acid mutant of a plant peptide termed kalata B1, of the cyclotide family. Like all cyclotides, the therapeutic candidate [T20K]kB1 is highly stable as it contains a cyclic backbone that is cross-linked by three disulfide bonds in a knot-like structure. This stability is much sought after for peptide drugs, which despite exquisite selectivity for their targets, are prone to rapid degradation in human serum. In preliminary investigations, it was found that [T20K]kB1 retains oral activity in experimental autoimmune encephalomyelitis, a model of MS in mice, thus opening up opportunities for oral dosing of the peptide. Although [T20K]kB1 can be synthetically produced, a recombinant production system provides advantages, specifically for reduced scale-up costs and reductions in chemical waste. In this study, we demonstrate the capacity of the Australian native Nicotiana benthamiana plant to produce a structurally identical [T20K]kB1 to that of the synthetic peptide. By optimizing the co-expressed cyclizing enzyme, precursor peptide arrangements, and transgene regulatory regions, we demonstrate a [T20K]kB1 yield in crude peptide extracts of ~ 0.3 mg/g dry mass) in whole plants and close to 1.0 mg/g dry mass in isolated infiltrated leaves. With large-scale plant production facilities coming on-line across the world, the sustainable and cost-effective production of cyclotide-based therapeutics is now within reach.
Asunto(s)
Ciclotidas , Esclerosis Múltiple , Ratones , Humanos , Animales , Ciclotidas/genética , Ciclotidas/química , Ciclotidas/metabolismo , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/genética , Australia , Nicotiana/genética , Nicotiana/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
Cyclotides are a unique family of stable and cyclic mini-proteins found in plants that have nematicidal and anthelmintic activities. They are distributed across the Rubiaceae, Violaceae, Fabaceae, Cucurbitaceae, and Solanaceae plant families, where they are posited to act as protective agents against pests. In this study, we tested the nematicidal properties of extracts from four major cyclotide-producing plants, Oldenlandia affinis, Clitoria ternatea, Viola odorata, and Hybanthus enneaspermus, against the free-living model nematode Caenorhabditis elegans. We evaluated the nematicidal activity of the cyclotides kalata B1, cycloviolacin O2, and hyen D present in these extracts and found them to be active against the larvae of C. elegans. Both the plant extracts and isolated cyclotides exerted dose-dependent toxicity on the first-stage larvae of C. elegans. Isolated cyclotides caused death or damage upon interacting with the worms' mouth, pharynx, and midgut or membrane. Cycloviolacin O2 and hyen D produced bubble-like structures around the C. elegans membrane, termed blebs, implicating membrane disruption causing toxicity and death. All tested cyclotides lost their toxicity when the hydrophobic patches present on them were disrupted via a single-point mutation. The present results provide a facile assay design to measure and explore the nematicidal activities of plant extracts and purified cyclotides on C. elegans.
Asunto(s)
Ciclotidas , Fabaceae , Nematodos , Violaceae , Animales , Antinematodos/farmacología , Caenorhabditis elegans , Ciclotidas/farmacología , Ciclotidas/química , Fabaceae/química , Extractos Vegetales/química , Proteínas de Plantas/químicaRESUMEN
Cyclotides are plant defense peptides that have been extensively investigated for pharmaceutical and agricultural applications, but key details of their posttranslational biosynthesis have remained elusive. Asparaginyl endopeptidases are crucial in the final stage of the head-to-tail cyclization reaction, but the enzyme(s) involved in the prerequisite steps of N-terminal proteolytic release were unknown until now. Here we use activity-guided fractionation to identify specific members of papain-like cysteine proteases involved in the N-terminal cleavage of cyclotide precursors. Through both characterization of recombinantly produced enzymes and in planta peptide cyclization assays, we define the molecular basis of the substrate requirements of these enzymes, including the prototypic member, here termed kalatase A. The findings reported here will pave the way for improving the efficiency of plant biofactory approaches for heterologous production of cyclotide analogs of therapeutic or agricultural value.
Asunto(s)
Ciclotidas , Proteasas de Cisteína , Papaína , Proteínas de Plantas , Ciclotidas/química , Ciclotidas/metabolismo , Proteasas de Cisteína/química , Proteasas de Cisteína/metabolismo , Defensinas/química , Defensinas/metabolismo , Modelos Moleculares , Papaína/química , Papaína/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismoRESUMEN
Knottins are topologically complex peptides that are stabilised by a cystine knot and have exceptionally diverse functions, including protease inhibition. However, approaches for tuning their activity in situ are limited. Here, we demonstrate separate approaches for tuning the activity of knottin protease inhibitors using light or streptavidin. We show that the inhibitory activity and selectivity of an engineered knottin can be controlled with light by activating a second mode of action that switches the inhibitor ON against new targets. Guided by a knottin library screen, we also identify a position in the inhibitor's binding loop that permits insertion of a biotin tag without impairing activity. Using streptavidin, biotinylated knottins with nanomolar affinity can be switched OFF in activity assays, and the anticoagulant activity of a factor XIIa inhibitor can be rapidly switched OFF in human plasma. Our findings expand the scope of engineered knottins for precisely controlling protein function.
Asunto(s)
Miniproteínas Nodales de Cistina , Cistina , Miniproteínas Nodales de Cistina/metabolismo , Humanos , Péptidos/metabolismo , Péptidos/farmacología , Proteínas , EstreptavidinaRESUMEN
Chemoenzymatic protein and peptide modification is a powerful means of generating defined, homogeneous conjugates for a range of applications. However, the use of transpeptidases is limited by the need to prepare synthetic peptide conjugates to be ligated, bulky recognition tags remaining in the product, and inefficient substrate turnover. Here, we report a peptide/protein labeling strategy that utilizes a promiscuous, engineered transpeptidase to irreversibly incorporate diverse, commercially available amines at a C-terminal asparagine. To demonstrate the utility of this approach, we prepare a protein-drug conjugate, generate a genetically inaccessible C-to-C protein fusion, and site specifically label both termini of a single protein in sequential steps.
Asunto(s)
Aminas/química , Peptidil Transferasas/química , Ingeniería de Proteínas , Aminas/metabolismo , Modelos Moleculares , Peptidil Transferasas/metabolismoRESUMEN
Enzyme-catalysed site-specific protein modifications enable the precision manufacture of conjugates for the study of protein function and/or for therapeutic or diagnostic applications. Asparaginyl ligases are a class of highly efficient transpeptidases with the capacity to modify proteins bearing only a tripeptide recognition motif. Herein, we review the types of protein modification that are accessible using these enzymes, including N- and C-terminal protein labelling, head-to-tail cyclisation, and protein-protein conjugation. We describe the progress that has been made to engineer highly efficient ligases as well as efforts to chemically manipulate the enzyme reaction to favour product formation. These enzymes are powerful additions to the protein engineer's toolbox.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Ingeniería de Proteínas , Procesamiento Proteico-PostraduccionalRESUMEN
Double-knotted peptides identified in venoms and synthetic bivalent peptide constructs targeting ion channels are emerging tools for the study of ion channel pharmacology and physiology. These highly complex and disulfide-rich peptides contain two individual cystine knots, each comprising six cysteines and three disulfide bonds. Until now, native double-knotted peptides, such as Hi1a and DkTx, have only been isolated from venom or produced recombinantly, whereas engineered double-knotted peptides have successfully been produced through enzymatic ligation using sortase A to form a seamless amide bond at the ligation site between two knotted toxins, and by alkyne/azide click chemistry, joining two peptide knots via a triazole linkage. To further pursue these double-knotted peptides as pharmacological tools or probes for therapeutically relevant ion channels, we sought to identify a robust methodology resulting in a high yield product that lends itself to rapid production and facile mutational studies. In this study, we evaluated the ligation efficiency of enzymatic (sortase A5°, butelase 1, wild-type OaAEP 1, C247A-OaAEP 1, and peptiligase) and mild chemical approaches (α-ketoacid-hydroxylamine, KAHA) for forming a native amide bond linking the toxins while maintaining the native disulfide connectivity of each pre-folded peptide. We used two NaV1.7 inhibitors: PaurTx3, a spider-derived gating modifier peptide, and KIIIA, a small cone snail-derived pore blocker peptide, which have previously been shown to increase affinity and inhibitory potency on hNaV1.7 when ligated together. Correctly folded peptides were successfully ligated in varying yields, without disulfide bond shuffling or reduction, with sortase A5° being the most efficient, resulting in 60% ligation conversion within 15 min. In addition, electrophysiology studies demonstrated that for these two peptides, the amino acid composition of the linker did not affect the activity of the double-knotted peptides. This study demonstrates the powerful application of enzymes in efficiently ligating complex disulfide-rich peptides, paving the way for facile production of double-knotted peptides.
Asunto(s)
DisulfurosRESUMEN
Mixed-linkage (1,3;1,4)-ß-glucan (MLG), an abundant cell wall polysaccharide in the Poaceae, has been detected in ascomycetes, algae, and seedless vascular plants, but not in eudicots. Although MLG has not been reported in bryophytes, a predicted glycosyltransferase from the moss Physcomitrella patens (Pp3c12_24670) is similar to a bona fide ascomycete MLG synthase. We tested whether Pp3c12_24670 encodes an MLG synthase by expressing it in wild tobacco (Nicotiana benthamiana) and testing for release of diagnostic oligosaccharides from the cell walls by either lichenase or (1,4)-ß-glucan endohydrolase. Lichenase, an MLG-specific endohydrolase, showed no activity against cell walls from transformed N. benthamiana, but (1,4)-ß-glucan endohydrolase released oligosaccharides that were distinct from oligosaccharides released from MLG by this enzyme. Further analysis revealed that these oligosaccharides were derived from a novel unbranched, unsubstituted arabinoglucan (AGlc) polysaccharide. We identified sequences similar to the P. patens AGlc synthase from algae, bryophytes, lycophytes, and monilophytes, raising the possibility that other early divergent plants synthesize AGlc. Similarity of P. patens AGlc synthase to MLG synthases from ascomycetes, but not those from Poaceae, suggests that AGlc and MLG have a common evolutionary history that includes loss in seed plants, followed by a more recent independent origin of MLG within the monocots.
Asunto(s)
Bryopsida/metabolismo , Pared Celular/metabolismo , Glucanos/metabolismo , Glicosiltransferasas/metabolismoRESUMEN
The use of enzymes for the site-specific modification of proteins/peptides has become a highly accessible, widespread approach to study protein/peptide functions or to generate therapeutic conjugates. Asparaginyl endopeptidases (AEPs) that preferentially catalyze transpeptidation reactions (AEP ligases) have emerged as enticing alternatives to established approaches, such as bacterial sortases, due to their catalytic efficiency and short tripeptide recognition motifs. However, under standard conditions, a substantial excess of the nucleophile to be conjugated is needed to reach desirable yields. Herein we report a versatile approach to shift the AEP-catalyzed transpeptidation equilibrium toward product formation via selectively quenching the nucleophilicity of the competing leaving-group peptide. Our metal-complexation-based strategy enables efficient peptide/protein labeling at the N- or C-terminus with near-equimolar concentrations of nucleophile label. Furthermore, we show that this approach can enhance protein-protein ligation and facilitate the formation of transpeptidation products that are otherwise unattainable.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Péptidos/metabolismo , Secuencias de Aminoácidos , Biocatálisis , Cobre/química , Cobre/metabolismo , Humanos , Níquel/química , Níquel/metabolismo , Péptidos/química , Unión Proteica , Ingeniería de Proteínas , Albúmina Sérica/química , Albúmina Sérica/metabolismoRESUMEN
MAIN CONCLUSION: We demonstrate the production of a structurally correct cyclotide in rice suspension cells with co-expression of a ligase-type AEP, which unlocks monocotyledons as production platforms to produce cyclotides. Cyclotides are a class of backbone-cyclic plant peptides that harbor a cystine knot composed of three disulfide bonds. These structural features make cyclotides particularly stable, and thus they have attracted significant attention for their use in biotechnological applications such as drug design. Currently, chemical synthesis is the predominant strategy to produce cyclotides for research purposes. However, synthetic production becomes costly both economically and environmentally at large scale. Plants offer an attractive alternative to chemical synthesis because of their lower cost and environmental footprint. In this study, rice suspension cells were engineered to produce the prototypical cyclotide, kalata B1 (kB1), a cyclotide with insecticidal properties from the African plant Oldenlandia affinis. Engineered rice cells produced structurally validated kB1 at yields of 64.21 µg/g (DW), which was dependent on the co-expression of a peptide ligase-competent asparaginyl endopeptidase OaAEP1b from O. affinis. Without co-expression, kB1 was predominantly produced as linear peptide. Through HPLC-MS co-elution, reduction, alkylation, enzymatic digestion, and proton NMR analysis, kB1 produced in rice was shown to be structurally identical to native kB1. This study reports the first example of an engineered plant suspension cell culture with the required molecular machinery for efficient production and cyclisation of a heterologous cyclotide.
Asunto(s)
Biotecnología , Ciclotidas , Oldenlandia , Oryza , Biotecnología/métodos , Ciclotidas/biosíntesis , Ciclotidas/genética , Oldenlandia/genética , Oryza/enzimología , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismoRESUMEN
Ruthenium-catalysed azide-alkyne cycloaddition (RuAAC) provides access to 1,5-disubstituted 1,2,3-triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor-1. NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5â Å) of the triazole linkages compared to the parent disulfide molecules. The triazole-bridged peptides also displayed superior half-lives in liver S9 stability assays compared to disulfide-bridged peptides. This work establishes a foundation for the application of 1,5-disubstituted 1,2,3-triazoles as disulfide mimetics.
Asunto(s)
Disulfuros/química , Imitación Molecular , Péptidos Cíclicos/química , Triazoles/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Ciclización , Resonancia Magnética Nuclear Biomolecular , Rutenio/químicaRESUMEN
Protein ligases of defined substrate specificity are versatile tools for protein engineering. Upon completion of the reaction, the products of currently reported protein ligases contain the amino acid sequence that is recognized by that same ligase, resulting in repeated cycles of ligation and hydrolysis as competing reactions. Thus, previous efforts to sequentially label proteins at distinct positions required ligases of orthogonal specificity. A recombinant Oldenlandia affinis asparaginyl endopeptidase, OaAEP1, is promiscuous for incoming nucleophiles. This promiscuity enabled us to define a nucleophile composed of natural amino acids that is ligated efficiently to the substrate yet yields a product that is poorly recognized by OaAEP1. Proteins modified with an efficient recognition module could be readily modified to yield a defined product bearing a cleavage-resistant motif, whereas proteins containing this inferior recognition motif remained essentially unmodified. We demonstrate the versatility of the N- or C-terminal protein modifications obtainable with this approach and modify the N- and C-termini of a single substrate protein in a sequential, site-specific manner in excellent yield.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Ingeniería de Proteínas/métodos , Proteínas/química , Secuencias de Aminoácidos , Catálisis , Cisteína Endopeptidasas/genética , Electroforesis en Gel de Poliacrilamida , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Oldenlandia/enzimología , Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticuerpos de Dominio Único/químicaRESUMEN
Chickpea (Cicer arietinum L.) is an important nutritionally rich legume crop that is consumed worldwide. Prior to cooking, desi chickpea seeds are most often dehulled and cleaved to release the split cotyledons, referred to as dhal. Compositional variation between desi genotypes has a significant impact on nutritional quality and downstream processing, and this has been investigated mainly in terms of starch and protein content. Studies in pulses such as bean and lupin have also implicated cell wall polysaccharides in cooking time variation, but the underlying relationship between desi chickpea cotyledon composition and cooking performance remains unclear. Here, we utilized a variety of chemical and immunohistological assays to examine details of polysaccharide composition, structure, abundance, and location within the desi chickpea cotyledon. Pectic polysaccharides were the most abundant cell wall components, and differences in monosaccharide and glycosidic linkage content suggest both environmental and genetic factors contribute to cotyledon composition. Genotype-specific differences were identified in arabinan structure, pectin methylesterification, and calcium-mediated pectin dimerization. These differences were replicated in distinct field sites and suggest a potentially important role for cell wall polysaccharides and their underlying regulatory machinery in the control of cooking time in chickpea.
Asunto(s)
Pared Celular/química , Cicer/citología , Cicer/genética , Harina/análisis , Pared Celular/genética , Celulosa/análisis , Culinaria , Cotiledón/química , Genotipo , Monosacáridos/análisis , Pectinas/análisis , Polisacáridos/análisis , Polisacáridos/química , Factores de TiempoRESUMEN
Hull-less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however, recalcitrance of hull-less cultivars to transformation has limited the use of these varieties. To overcome this limitation, we sought to develop an effective transformation system for hull-less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over-express genes encoding synthases for the important dietary fiber component, (1,3;1,4)-ß-glucan (mixed-linkage glucan), primarily present in starchy endosperm cell walls. Over-expression of the HvCslF6 gene, driven by an endosperm-specific promoter, produced lines where mixed-linkage glucan content increased on average by 45%, peaking at 70% in some lines, with smaller increases in transgenic HvCslH1 grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker color, were more easily crushed than wild type and were smaller. This was associated with an enlarged cavity in the central endosperm and changes in cell morphology, including aleurone and sub-aleurone cells. This work provides proof-of-concept evidence that mixed-linkage glucan content in hull-less barley grain can be increased by over-expression of the HvCslF6 gene, but also indicates that hull-less cultivars may be more sensitive to attempts to modify cell wall composition.
Asunto(s)
Ligamiento Genético , Hordeum/genética , Semillas/genética , Transformación Genética , beta-Glucanos/metabolismo , Hordeum/embriología , Fenotipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Regeneración , Plantones/metabolismo , Almidón/metabolismoRESUMEN
Four members of the UDP-Ara mutase (UAM) gene family from barley have been isolated and characterized, and their map positions on chromosomes 2H, 3H, and 4H have been defined. When the genes are expressed in Escherichia coli, the corresponding HvUAM1, HvUAM2, and HvUAM3 proteins exhibit UAM activity, and the kinetic properties of the enzymes have been determined, including Km, Kcat, and catalytic efficiencies. However, the expressed HvUAM4 protein shows no mutase activity against UDP-Ara or against a broad range of other nucleotide sugars and related molecules. The enzymic data indicate therefore that the HvUAM4 protein may not be a mutase. However, the HvUAM4 gene is transcribed at high levels in all the barley tissues examined, and its transcript abundance is correlated with transcript levels for other genes involved in cell wall biosynthesis. The UDP-l-Arap â UDP-l-Araf reaction, which is essential for the generation of the UDP-Araf substrate for arabinoxylan, arabinogalactan protein, and pectic polysaccharide biosynthesis, is thermodynamically unfavorable and has an equilibrium constant of 0.02. Nevertheless, the incorporation of Araf residues into nascent polysaccharides clearly occurs at biologically appropriate rates. The characterization of the HvUAM genes opens the way for the manipulation of both the amounts and fine structures of heteroxylans in cereals, grasses, and other crop plants, with a view toward enhancing their value in human health and nutrition, and in renewable biofuel production.
Asunto(s)
Hordeum/enzimología , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Azúcares de Uridina Difosfato/metabolismo , Regulación de la Expresión Génica de las Plantas , Transferasas Intramoleculares/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
The legume albumin-1 gene family, arising after nodulation, encodes linear a- and b-chain peptides for nutrient storage and defense. Intriguingly, in one prominent legume, Clitoria ternatea, the b-chains are replaced by domains producing ultra-stable cyclic peptides called cyclotides. The mechanism of this gene hijacking is until now unknown. Cyclotides require recruitment of ligase-type asparaginyl endopeptidases (AEPs) for maturation (cyclization), necessitating co-evolution of two gene families. Here we compare a chromosome-level C. ternatea genome with grain legumes to reveal an 8 to 40-fold expansion of the albumin-1 gene family, enabling the additional loci to undergo diversification. Iterative rounds of albumin-1 duplication and diversification create four albumin-1 enriched genomic islands encoding cyclotides, where they are physically grouped by similar pI and net charge values. We identify an ancestral hydrolytic AEP that exhibits neofunctionalization and multiple duplication events to yield two ligase-type AEPs. We propose cyclotides arise by convergence in C. ternatea where their presence enhances defense from biotic attack, thus increasing fitness compared to lineages with linear b-chains and ultimately driving the replacement of b-chains with cyclotides.
Asunto(s)
Proteínas de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Clitoria/metabolismo , Clitoria/genética , Ciclotidas/genética , Ciclotidas/química , Ciclotidas/metabolismo , Fijación del Nitrógeno/genética , Evolución Molecular , Ciclización , Filogenia , Familia de Multigenes , Duplicación de Gen , Fabaceae/genética , Fabaceae/metabolismo , Albúminas/metabolismo , Albúminas/genética , Genoma de Planta , Cisteína EndopeptidasasRESUMEN
Upon wetting, chia (Salvia hispanica L.) nutlets produce a gel-like capsule of polysaccharides called mucilage that comprises a significant part of their dietary fibre content. Seed/nutlet mucilage is often used as a texture modifying hydrocolloid and bulking dietary fibre due to its water-binding ability, though the utility of mucilage from different sources is highly structure-function dependent. The composition and structure of chia nutlet mucilage is poorly defined, and a better understanding will aid in exploiting its dietary fibre functionality, particularly if, and how, it is utilised by gut microbiota. In this study, microscopy, chromatography, mass spectrometry and glycome profiling techniques showed that chia nutlet mucilage is highly complex, layered, and contains several polymer types. The mucilage comprises a novel xyloamylose containing both ß-linked-xylose and α-linked-glucose, a near-linear xylan that may be sparsely substituted, a modified cellulose domain, and abundant alcohol-soluble oligosaccharides. To assess the dietary fibre functionality of chia nutlet mucilage, an in vitro cumulative gas production technique was used to determine the fermentability of different chia nutlet preparations. The complex nature of chia nutlet mucilage led to poor fermentation where the oligosaccharides appeared to be the only fermentable substrate present in the mucilage. Of note, ground chia nutlets were better fermented than intact whole nutlets, as judged by short chain fatty acid production. Therefore, it is suggested that the benefits of eating chia as a "superfood", could be notably enhanced if the nutlets are ground rather than being consumed whole, improving the bioaccessibility of key nutrients including dietary fibre.
Asunto(s)
Mucílago de Planta , Salvia , Salvia hispanica , Fermentación , Salvia/química , Polisacáridos/química , Semillas/química , Oligosacáridos/análisis , Fibras de la Dieta/análisis , Mucílago de Planta/químicaRESUMEN
Cyclic disulfide-rich peptides have attracted significant interest in drug development and biotechnology. Here, we describe a protocol for producing cyclic peptide precursors in Pichia pastoris that undergo in vitro enzymatic maturation into cyclic peptides using recombinant asparaginyl endopeptidases (AEPs). Peptide precursors are expressed with a C-terminal His tag and secreted into the media, enabling facile purification by immobilized metal affinity chromatography. After AEP-mediated cyclization, cyclic peptides are purified by reverse-phase high-performance liquid chromatography and characterized by mass spectrometry, peptide mass fingerprinting, NMR spectroscopy, and activity assays. We demonstrate the broad applicability of this protocol by generating cyclic peptides from three distinct classes that are either naturally occurring or synthetically backbone cyclized, and range in size from 14 amino acids with one disulfide bond, to 34 amino acids with a cystine knot comprising three disulfide bonds. The protocol requires 14 d to identify and optimize a high-expressing Pichia clone in small-scale cultures (24 well plates or 50 mL tubes), after which large-scale production in a bioreactor and peptide purification can be completed in 10 d. We use the cyclotide Momordica cochinchinensis trypsin inhibitor II as an example. We also include a protocol for recombinant AEP production in Escherichia coli as AEPs are emerging tools for orthogonal peptide and protein ligation. We focus on two AEPs that preferentially cyclize different peptide precursors, namely an engineered AEP with improved catalytic efficiency [C247A]OaAEP1b and the plant-derived MCoAEP2. Rudimentary proficiency and equipment in molecular biology, protein biochemistry and analytical chemistry are needed.