Molecular Identification and Characterization of Hevein Antimicrobial Peptide Genes in Two-Row and Six-Row Cultivars of Barley (Hordeum vulgare L.).

Heveins are one of the most important groups of plant antimicrobial peptides. So far, various roles in plant growth and development and in response to biotic and abiotic stresses have reported for heveins. The present study aimed to identify and characterize the hevein genes in two-row and six-row cultivars of barley. In total, thirteen hevein genes were identified in the genome of two-row and six-row cultivars of barley. The identified heveins were identical in two-row and six-row cultivars of barley and showed a high similarity with heveins from other plant species. The hevein coding sequences produced open reading frames (ORFs) ranged from 342 to 1002 bp. Most of the identified hevein genes were intronless, and the others had only one intron. The hevein ORFs produced proteins ranged from 113 to 333 amino acids. Search for conserved functional domains showed CBD and LYZ domains in barley heveins. All barley heveins comprised extracellular signal peptides ranged from 19 to 35 amino acids. The phylogenetic analysis divided barley heveins into two groups. The promoter analysis showed regulatory elements with different frequencies between two-row and six-row cultivars. These cis-acting elements included elements related to growth and development, hormone response, and environmental stresses. The expression analysis showed high expression level of heveins in root and reproductive organs of both two-row and six-row cultivars. The expression analysis also showed that barley heveins is induced by both biotic and abiotic stresses. The results of antimicrobial activity prediction showed the highest antimicrobial activity in CBD domain of barley heveins. The findings of the current study can improve our knowledge about the role of hevein genes in plant and can be used for future studies.

Ginsentide-like Coffeetides Isolated from Coffee Waste Are Cell-Penetrating and Metal-Binding Microproteins.

Coffee processing generates a huge amount of waste that contains many natural products. Here, we report the discovery of a panel of novel cell-penetrating and metal ion-binding microproteins designated coffeetide cC1a-c and cL1-6 from the husk of two popular coffee plants, Coffea canephora and Coffea liberica, respectively. Combining sequence determination and a database search, we show that the prototypic coffeetide cC1a is a 37-residue, eight-cysteine microprotein with a hevein-like cysteine motif, but without a chitin-binding domain. NMR determination of cC1a reveals a compact structure that confers its resistance to heat and proteolytic degradation. Disulfide mapping together with chemical synthesis reveals that cC1a has a ginsentide-like, and not a hevein-like, disulfide connectivity. In addition, transcriptomic analysis showed that the 98-residue micrcoproten-like coffeetide precursor contains a three-domain arrangement, like ginsentide precursors. Molecular modeling, together with experimental validation, revealed a Mg2+ and Fe3+ binding pocket at the N-terminus formed by three glutamic acids. Importantly, cC1a is amphipathic with a continuous stretch of 19 apolar amino acids, which enables its cell penetration to target intracellular proteins, despite being highly negatively charged. Our findings suggest that coffee by-products could provide a source of ginsentide-like bioactive peptides that have the potential to target intracellular proteins.

Genome-Wide Mining of Selaginella moellendorffii for Hevein-like Lectins and Their Potential Molecular Mimicry with SARS-CoV-2 Spike Glycoprotein.

Multidisciplinary research efforts on potential COVID-19 vaccine and therapeutic candidates have increased since the pandemic outbreak of SARS-CoV-2 in 2019. This search has become imperative due to the increasing emergences and limited widely available medicines. The presence of bioactive anti-SARS-CoV-2 molecules was examined from various plant sources. Among them is a group of proteins called lectins that can bind carbohydrate moieties. In this article, we present ten novel, chitin-specific Hevein-like lectins that were derived from Selaginella moellendorffii v1.0's genome. The capacity of these lectin homologs to bind with the spike protein of SARS-CoV-2 was examined. Using the HDOCK server, 3D-modeled Hevein-domains were docked to the spike protein's receptor binding domain (RBD). The Smo446851, Smo125663, and Smo99732 interacted with Asn343-located complex N-glycan and RBD residues, respectively, with binding free energies of -17.5, -13.0, and -26.5 Kcal/mol. The molecular dynamics simulation using Desmond and the normal-state analyses via torsional coordinate association for the Smo99732-RBD complex using iMODS is characterized by overall higher stability and minimum deformity than the other lectin complexes. The three lectins interacting with carbohydrates were docked against five individual mutations that frequently occur in major SARS-CoV-2 variants. These were in the spike protein's receptor-binding motif (RBM), while Smo125663 and Smo99732 only interacted with the spike glycoprotein in a protein-protein manner. The precursors for the Hevein-like homologs underwent additional characterization, and their expressional profile in different tissues was studied. These in silico findings offered potential lectin candidates targeting key N-glycan sites crucial to the virus's virulence and infection.

Hololectin Interdomain Linker Determines Asparaginyl Endopeptidase-Mediated Maturation of Antifungal Hevein-Like Peptides in Oats.

Heveins and hevein-containing (hev-) lectins play important roles in stress and pathogenic responses in plants but cause health concerns in humans. Hev-hololectins contain multiple modular hev-peptide domains and are abundantly present in cereals and pseudocereals. However, it is unclear why some cereal hev-hololectins are presented as different forms of proteolytically processed proteoforms. Here we show the precursor architectures of hev-hololectins lead to different processing mechanisms to give either hololectins or hevein-like peptides. We used mass spectrometry and datamining to screen hev-peptides from common cereals, and identified from the oat plant Avena sativa nine novel hevein-like peptides, avenatide aV1-aV9. Bioinformatic analysis revealed that asparaginyl endopeptidase (AEP) can be responsible for the maturation of the highly homologous avenatides from five oat hev-hololectin precursors, each containing four tandemly repeating, hev-like avenatide domains connected by AEP-susceptible linkers with 13-16 residues in length. Further analysis of cereal hev-hololectins showed that the linker lengths provide a distinguishing feature between their cleavable and non-cleavable precursors, with the cleavables having considerably longer linkers (>13 amino acids) than the non-cleavables (<6 amino acids). A detailed study of avenatide aV1 revealed that it contains eight cysteine residues which form a structurally compact, metabolic-resistant cystine-knotted framework with a well-defined chitin-binding site. Antimicrobial assays showed that avenatide aV1 is anti-fungal and inhibits the growth of phyto-pathogenic fungi. Together, our findings of cleavable and non-cleavable hololectins found in cereals expand our knowledge to their biosynthesis and provide insights for hololectin-related health concerns in human.

Anti-Fungal Hevein-like Peptides Biosynthesized from Quinoa Cleavable Hololectins.

Chitin-binding hevein-like peptides (CB-HLPs) belong to a family of cysteine-rich peptides that play important roles in plant stress and defense mechanisms. CB-HLPs are ribosomally synthesized peptides that are known to be bioprocessed from the following two types of three-domain CB-HLP precursor architectures: cargo-carrying and non-cargo-carrying. Here, we report the identification and characterization of chenotides biosynthesized from the third type of precursors, which are cleavable hololectins of the quinoa (Chenopodium quinoa) family. Chenotides are 6-Cys-CB-HLPs of 29-31 amino acids, which have a third type of precursor architecture that encompasses a canonical chitin-binding domain that is involved in chitin binding and anti-fungal activities. Microbroth dilution assays and microscopic analyses showed that chenotides are effective against phyto-pathogenic fungi in the micromolar range. Structure determination revealed that chenotides are cystine knotted and highly compact, which could confer resistance against heat and proteolytic degradation. Importantly, chenotides are connected by a novel 18-residue Gly/Ala-rich linker that is a target for bioprocessing by cathepsin-like endopeptidases. Taken together, our findings reveal that chenotides are a new family of CB-HLPs from quinoa that are synthesized as a single multi-modular unit and bioprocessed to yield individual mature CB-HLPs. Importantly, such precursors constitute a new family of cleavable hololectins. This unusual feature could increase the biosynthetic efficiency of anti-fungal CB-HLPs, to provide an evolutionary advantage for plant survival and reproduction.

High Resistance of Potato to Early Blight Is Achieved by Expression of the Pro-SmAMP1 Gene for Hevein-Like Antimicrobial Peptides from Common Chickweed (Stellaria media).

In the common chickweed Stellaria media, two antimicrobial peptides (AMPs), SmAMP1.1a and SmAMP1.2a, have been shown to be proteolytically released as products of the expression of a single gene, proSmAMP1. In this study, the gene proSmAMP1 was introduced into two potato varieties, Zhukovsky ranny and Udacha. These early-maturing varieties were shown to be susceptible to early blight caused by Alternaria spp. Most transgenic lines of either variety having strong expression of the target gene demonstrated high levels of resistance to Alternaria spp. during three years of cultivation, but did not otherwise differ from the initial varieties. Disease severity index (DSI) was introduced as a complex measure of plant susceptibility to early blight, taking into account the diameter of lesions caused by the Alternaria spp., the fungus sporulation intensity and its incubation period duration. Across all transgenic lines, the DSI inversely correlated both with the target gene expression and the copy number in the plant genome. Our results are promising for improving the resistance of potato and other crops to early blight by expression of AMPs from wild plants.

Plant antimicrobial peptides: structures, functions, and applications.

Antimicrobial peptides (AMPs) are a class of short, usually positively charged polypeptides that exist in humans, animals, and plants. Considering the increasing number of drug-resistant pathogens, the antimicrobial activity of AMPs has attracted much attention. AMPs with broad-spectrum antimicrobial activity against many gram-positive bacteria, gram-negative bacteria, and fungi are an important defensive barrier against pathogens for many organisms. With continuing research, many other physiological functions of plant AMPs have been found in addition to their antimicrobial roles, such as regulating plant growth and development and treating many diseases with high efficacy. The potential applicability of plant AMPs in agricultural production, as food additives and disease treatments, has garnered much interest. This review focuses on the types of plant AMPs, their mechanisms of action, the parameters affecting the antimicrobial activities of AMPs, and their potential applications in agricultural production, the food industry, breeding industry, and medical field.

Fragments of a Wheat Hevein-Like Antimicrobial Peptide Augment the Inhibitory Effect of a Triazole Fungicide on Spore Germination of Fusarium oxysporum and Alternaria solani.

There are increasing environmental risks associated with extensive use of fungicides for crop protection. Hence, the use of new approaches using natural plant defense mechanisms, including application of plant antimicrobial peptides (AMPs), is of great interest. Recently, we studied the structural-function relationships between antifungal activity and five hevein-like AMPs from the WAMP (wheat AMP) family of Triticum kiharae Dorof. et Migush. We first discovered that short peptides derived from the central, N-, and C-terminal regions of one of the WAMPs (WAMP-2) were able to augment the inhibitory effect of Folicur® EC 250, a triazole fungicide, on spore germination of the wheat pathogenic fungi, including Fusarium spp. and Alternaria alternata. In this research, we explored the ability of chemically synthesized WAMP-2-derived peptides for enhancing the sensitivity of two other Fusarium and Alternaria species, F. oxysporum and A. solani, causing wilt and early blight of tomato, respectively, to Folicur®. The synthesized WAMP-2-derived peptides synergistically interacted with the fungicide and significantly increased its efficacy, inhibiting conidial germination at much lower Folicur® concentrations than required for the same efficiency using the fungicide alone. The experiments on co-applications of some of WAMP-2-fragments and the fungicide on tomato leaves and seedlings, which confirmed the results obtained in vitro, are described.

Plant Antimicrobial Peptides: State of the Art, In Silico Prediction and Perspectives in the Omics Era.

Even before the perception or interaction with pathogens, plants rely on constitutively guardian molecules, often specific to tissue or stage, with further expression after contact with the pathogen. These guardians include small molecules as antimicrobial peptides (AMPs), generally cysteine-rich, functioning to prevent pathogen establishment. Some of these AMPs are shared among eukaryotes (eg, defensins and cyclotides), others are plant specific (eg, snakins), while some are specific to certain plant families (such as heveins). When compared with other organisms, plants tend to present a higher amount of AMP isoforms due to gene duplications or polyploidy, an occurrence possibly also associated with the sessile habit of plants, which prevents them from evading biotic and environmental stresses. Therefore, plants arise as a rich resource for new AMPs. As these molecules are difficult to retrieve from databases using simple sequence alignments, a description of their characteristics and in silico (bioinformatics) approaches used to retrieve them is provided, considering resources and databases available. The possibilities and applications based on tools versus database approaches are considerable and have been so far underestimated.

Structure of intact chitinase with hevein domain from the plant Simarouba glauca, known for its traditional anti-inflammatory efficacy.

Chitinase from the leaves of Simarouba glauca, a plant used in traditional anti-inflammatory therapy is purified and characterized. Peptide mass finger print analysis revealed the protein as an endo-chitinase which was further confirmed using chitin-agar assay. The enzyme exhibited significant anti-fungal efficacy against phyto-pathogens such as Macrophomina phaseolina, Fusarium oxysporum and Sclerotium rolfsii. Chitinolysis was also examined against insoluble chitin using SEM. Using X-ray diffraction data up to 1.66 Å, the structure was determined by Molecular Replacement using crystal structure of GH19 Chitinase-like protein from Hevea brasiliensis. During structure refinement, an extra domain could be traced and identified as hevein domain. To our knowledge, this is the first report of any chitinase with intact hevein domain. The GH19 chitinase and hevein domains though connected by a lengthy loop, are restricted to be close by disulfide bridges. These bridges connecting each domain with the loop may be important for proper chitin feeding into the active site. By considering reports on hevein and chitinase domains as well as the traditional use of the plant, this report of an intact hevein-chitinase protein and their relative orientation may add further insights for the usefulness of this protein.

Mo-HLPs: New flocculating agents identified from Moringa oleifera seeds belong to the hevein-like peptide family.

Cationic peptides found in Moringa oleifera seeds belong to different protein families and are described as the main flocculating agents of the species. In this study we report the identification and isolation of four new flocculant peptides, called Mo-HLPs 1-4, belonging to the family of hevein-like peptides, previously only known for their members' antimicrobial activity. Purification of the peptides followed two sequential membrane ultrafiltration steps and separation by reverse-phase liquid chromatography. Proteomic analyses showed that Mo-HLPs are extremely basic (pI >10) cysteine-rich molecules with molecular masses between 4.5 and 4.8 kDa and with a highly conserved chitin-binding domain. Searches in BLAST revealed high similarity of Mo-HLPs with hevein and other hevein-like peptides and 90% identity with morintides, which are members of the 8C-hevein-like subfamily found in M. oleifera leaves. Mo-HLPs microflocculation assays showed distinct coagulation/flocculation efficiencies, promoting turbidity reduction levels between 67 and 89% in synthetic turbid water. Activity variations were attributed to the substitution of some amino acids among the isoforms, which may have altered the final net charge of the molecules. The identification of Mo-HLPs represents the discovery of a new group of cationic peptides involved in the flocculation properties of M. oleifera seeds. SIGNIFICANCE: The study reveals the presence of hevein-like peptides in Moringa oleifera seeds. It is reported for the first time that members of this family have properties to act as flocculating agents of importance for water treatment processes. The identification of these peptides as well as new functional assignment broadens the horizon for speculation on new species which could act as sources of green coagulants for sustainable water treatment, and contributes to the knowledge about occurrence, distribution, molecular and active diversity of peptides belonging to the hevein-like family.

Bacterial expression, purification and biophysical characterization of wheat germ agglutinin and its four hevein-like domains.

Wheat germ agglutinin (WGA), a chitin binding lectin, has attracted increasing interest because of its unique characteristics such as conformational stability, binding specificity and transcytosis capacity. To pave the way for the study of the molecular basis of WGA's structural stability and binding capacity, as well as to facilitate its use in biomedical and biotechnological developments, we produced recombinant WGA and its 4 isolated hevein-like domains in a bacterial system. All the proteins were expressed as fusion constructs linked to a thioredoxin domain, which was enzymatically or chemically released. The structural and ligand-binding properties of recombinant WGA were similar to the wild lectin. The 4 isolated domains folded and were ligand-binding competent, indicating that each domain constitutes an independent folding unity. The biophysical characterization of the recombinant domains sheds new light on the intricate folding and binding behavior of this emblematic lectin.

A rapid and effective method for purification of a heat-resistant lectin from potato (Solanum tuberosum) tubers.

The potato lectin has been identified to consist of two chitin-binding modules, each containing twin hevein domains. Based on the thermotolerance of the hevein polypeptide, a simple, rapid, and effective protocol for the small-scale purification of the potato lectin has been developed in this study. The method involves only one anion exchange chromatographic step beyond the ammonium sulfate precipitation and the heating treatment. With this method, the potato lectin, a glycoprotein with molecular mass of approximately 60 kDa was found and purified to homogeneity with 9513.3 u/mg of specific hemagglutination (HA) activity in 76.8% yield. The homogeneity was confirmed by SDS-PAGE electrophoresis and reverse-phase HPLC analysis. The purified lectin was identified using MS-based peptide sequencing (MALDI-TOF/TOF) and showed a 100% Confidence Interval as being homologous to hevein domains in potato lectin. The periodic acid-Schiff staining and ferric-orcinol assay for pentose, as well as its HA activity inhibition by chitosan oligomers further confirmed the purified lectin as a potato chitin-binding lectin. It is noteworthy that the purified potato lectin exhibited heat resistance, by which, together with a short time precipitation by ammonium sulfate, more than 96% of the total proteins in the crude extract were removed. The lectin therefore was easily resolved from the other remining proteins on a DEAE-methyl polyacrylate column.

Investigation of Antimicrobial Peptide Genes Associated with Fungus and Insect Resistance in Maize.

Antimicrobial peptides (AMPs) are small defense proteins present in various organisms. Major groups of AMPs include beta-barrelin, hevein, knottin, lipid transfer protein (LTP), thionin, defensin, snakin, and cyclotide. Most plant AMPs involve host plant resistance to pathogens such as fungi, viruses, and bacteria, whereas a few plant AMPs from the cyclotide family carry insecticidal functions. In this research, a genome-wide investigation on antimicrobial peptide genes in maize genome was conducted. AMPs previously identified from various plant species were used as query sequences for maize genome data mining. Thirty-nine new maize AMPs were identified in addition to seven known maize AMPs. Protein sequence analysis revealed 10 distinguishable maize AMP groups. Analysis of mRNA expression of maize AMP genes by quantitative real-time polymerase chain reaction (qRT-PCR) revealed different expression patterns in a panel of 10 maize inbred lines. Five maize AMP genes were found significantly associated with insect or fungus resistance. Identification of maize antimicrobial peptide genes will facilitate the breeding of host plant resistance and improve maize production.

Vaccatides: Antifungal Glutamine-Rich Hevein-Like Peptides from Vaccaria hispanica.

Hevein and hevein-like peptides are disulfide-constrained chitin-binding cysteine-rich peptides. They are divided into three subfamilies, 6C-, 8C-, and 10C-hevein-like peptides, based on the number of cysteine residues. In addition, hevein-like peptides can exist in two forms, short and long. The long C-terminal form found in hevein and 10C-hevein-like peptides contain a C-terminal protein cargo. In contrast, the short form without a protein cargo is found in all three subfamilies. Here, we report the discovery and characterization of two novel glutamine-rich and protein cargo-free 8C-hevein-like peptides, vaccatides vH1 and vH2, from Vaccaria hispanica of the Caryophyllaceae family. Proteomic analyses showed that the vaccatides are 40-41 amino acids in length and contain a chitin-binding domain. NMR determination revealed that vaccatide vH2 displays a highly compact structure with a N-terminal cystine knot and an addition C-terminal disulfide bond. Stability studies showed that this compact structure renders vaccatide vH2 resistant to thermal, chemical and proteolytic degradation. The chitin-binding vH2 was shown to inhibit the mycelium growth of four phyto-pathogenic fungal strains with IC50 values in the micromolar range. Our findings show that vaccatides represent a new family of 8C-hevein-like peptides, which are protein cargo-free and glutamine-rich, characteristics that differentiate them from the prototypic hevein and the 10C-hevein-like peptides. In summary, this study enriches the existing library of hevein-like peptides and provides insight into their molecular diversity in sequence, structure and biosynthesis. Additionally, their highly disulfide-constrained structure could be used as a scaffold for developing metabolically and orally active peptidyl therapeutics.

Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study.

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations.

Morintides: cargo-free chitin-binding peptides from Moringa oleifera.

BACKGROUND: Hevein-like peptides are a family of cysteine-rich and chitin-binding peptides consisting of 29-45 amino acids. Their chitin-binding property is essential for plant defense against fungi. Based on the number of cysteine residues in their sequences, they are divided into three sub-families: 6C-, 8C- and 10C-hevein-like peptides. All three subfamilies contain a three-domain precursor comprising a signal peptide, a mature hevein-like peptide and a C-terminal domain comprising a hinge region with protein cargo in 8C- and 10C-hevein-like peptides. RESULTS: Here we report the isolation and characterization of two novel 8C-hevein-like peptides, designated morintides (mO1 and mO2), from the drumstick tree Moringa oleifera, a drought-resistant tree belonging to the Moringaceae family. Proteomic analysis revealed that morintides comprise 44 amino acid residues and are rich in cysteine, glycine and hydrophilic amino acid residues such as asparagine and glutamine. Morintides are resistant to thermal and enzymatic degradation, able to bind to chitin and inhibit the growth of phyto-pathogenic fungi. Transcriptomic analysis showed that they contain a three-domain precursor comprising an endoplasmic reticulum (ER) signal sequence, a mature peptide domain and a C-terminal domain. A striking feature distinguishing morintides from other 8C-hevein-like peptides is a short and protein-cargo-free C-terminal domain. Previously, a similar protein-cargo-free C-terminal domain has been observed only in ginkgotides, the 8C-hevein-like peptides from a gymnosperm Ginkgo biloba. Thus, morintides, with a cargo-free C-terminal domain, are a stand-alone class of 8C-hevein-like peptides from angiosperms. CONCLUSIONS: Our results expand the existing library of hevein-like peptides and shed light on molecular diversity within the hevein-like peptide family. Our work also sheds light on the anti-fungal activity and stability of 8C-hevein-like peptides.

Production in Pichia pastoris, antifungal activity and crystal structure of a class I chitinase from cowpea (Vigna unguiculata): Insights into sugar binding mode and hydrolytic action.

A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates. The highest hydrolytic activity of the cowpea chitinase toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChiI activity (approximately 92%) was retained after heating to 50 °C for 30 min, whereas treatment with 5 mM Cu2+ caused a reduction of 67% in the enzyme's chitinolytic activity. The recombinant protein had antifungal activity as revealed by its ability to inhibit the spore germination and mycelial growth of Penicillium herquei. The three-dimensional structure of VuChiI was resolved at a resolution of 1.55 Å by molecular replacement. The refined model had 245 amino acid residues and 381 water molecules, and the final R-factor and Rfree values were 14.78 and 17.22%, respectively. The catalytic domain of VuChiI adopts an α-helix-rich fold, stabilized by 3 disulfide bridges and possessing a wide catalytic cleft. Analysis of the crystallographic model and molecular docking calculations using chito-oligosaccharides provided evidences about the VuChiI residues involved in sugar binding and catalysis, and a possible mechanism of antifungal action is suggested.

The Distribution of Lectins across the Phylum Nematoda: A Genome-Wide Search.

Nematodes are a very diverse phylum that has adapted to nearly every ecosystem. They have developed specialized lifestyles, dividing the phylum into free-living, animal, and plant parasitic species. Their sheer abundance in numbers and presence in nearly every ecosystem make them the most prevalent animals on earth. In this research nematode-specific profiles were designed to retrieve predicted lectin-like domains from the sequence data of nematode genomes and transcriptomes. Lectins are carbohydrate-binding proteins that play numerous roles inside and outside the cell depending on their sugar specificity and associated protein domains. The sugar-binding properties of the retrieved lectin-like proteins were predicted in silico. Although most research has focused on C-type lectin-like, galectin-like, and calreticulin-like proteins in nematodes, we show that the lectin-like repertoire in nematodes is far more diverse. We focused on C-type lectins, which are abundantly present in all investigated nematode species, but seem to be far more abundant in free-living species. Although C-type lectin-like proteins are omnipresent in nematodes, we have shown that only a small part possesses the residues that are thought to be essential for carbohydrate binding. Curiously, hevein, a typical plant lectin domain not reported in animals before, was found in some nematode species.

Ginkgotides: Proline-Rich Hevein-Like Peptides from Gymnosperm Ginkgo biloba.

Hevein and hevein-like peptides belong to the family of chitin-binding cysteine-rich peptides. They are classified into three subfamilies, the prototypic 8C- and the 6C- and 10C-hevein-like peptides. Thus far, only five 8C-hevein-like peptides have been characterized from three angiosperms and none from gymnosperm. To determine their occurrence and distribution in the gymnosperm, Ginkgo biloba leaves were examined. Here, we report the discovery and characterization of 11 novel 8C-hevein-like peptides, namely ginkgotides gB1-gB11. Proteomic analysis showed that the ginkgotides contain 41-44 amino acids (aa), a chitin-binding domain and are Pro-rich, a distinguishing feature that differs from other hevein-like peptides. Solution NMR structure determination revealed that gB5 contains a three ß-stranded structure shaped by a cystine knot with an additional disulfide bond at the C-terminus. Transcriptomic analysis showed that the ginkgotide precursors contain a three-domain architecture, comprised of a C-terminal tail (20 aa) that is significantly shorter than those of other 8C- and 10C-hevein-like peptides, which generally contain a protein cargo such as a Barwin-like protein (126 aa) or class I chitinase (254 aa). Transcriptomic data mining found an additional 48 ginkgotide homologs in 39 different gymnosperms. Phylogenetic analysis revealed that ginkgotides and their homologs belong to a new class of 8C-hevein-like peptides. Stability studies showed that ginkgotides are highly resistant to thermal, acidic and endopeptidase degradation. Ginkgotides flanked at both the N- and C-terminal ends by Pro were resistant to exopeptidase degradation by carboxypeptidase A and aminopeptidase. Antifungal assays showed that ginkgotides inhibit the hyphal growth of phyto-pathogenic fungi. Taken together, ginkgotides represent the first suite of hevein-like peptides isolated and characterized from gymnosperms. As a group, they represent a novel class of 8C-hevein-like peptides that are Pro-rich and protein-cargo free. Our findings also suggest that the ginkgotide scaffold could be useful for engineering metabolic-stable peptide therapeutics.