Peptides Derived From the α-Core and γ-Core Regions of a Putative Silybum marianum Flower Defensin Show Antifungal Activity Against Fusarium graminearum.

Fusarium graminearum is the etiological agent of Fusarium head blight (FHB), a disease that produces a significant decrease in wheat crop yield and it is further aggravated by the presence of mycotoxins in the affected grains that may cause health problems to humans and animals. Plant defensins and defensin-like proteins are antimicrobial peptides (AMPs); they are small basic, cysteine-rich peptides (CRPs) ubiquitously expressed in the plant kingdom and mostly involved in host defence. They present a highly variable sequence but a conserved structure. The γ-core located in the C-terminal region of plant defensins has a conserved ß-hairpin structure and is a well-known determinant of the antimicrobial activity among disulphide-containing AMPs. Another conserved motif of plant defensins is the α-core located in the N-terminal region, not conserved among the disulphide-containing AMPs, it has not been yet extensively studied. In this report, we have cloned the putative antimicrobial protein DefSm2, expressed in flowers of the wild plant Silybum marianum. The cDNA encodes a protein with two fused basic domains of an N-terminal defensin domain (DefSm2-D) and a C-terminal Arg-rich and Lys-rich domain. To further characterize the DefSm2-D domain, we built a 3D template-based model that will serve to support the design of novel antifungal peptides. We have designed four potential antifungal peptides: two from the DefSm2-D α-core region (SmAPα1-21 and SmAPα10-21) and two from the γ-core region (SmAPγ27-44 and SmAPγ29-35). We have chemically synthesized and purified the peptides and further characterized them by electrospray ionization mass spectrometry (ESI-MS) and Circular dichroism (CD) spectroscopy. SmAPα1-21, SmAPα10-21, and SmAPγ27-44 inhibited the growth of the phytopathogen F. graminearum at low micromolar concentrations. Conidia exposure to the fungicidal concentration of the peptides caused membrane permeabilization to the fluorescent probe propidium iodide (PI), suggesting that this is one of the main contributing factors in fungal cell killing. Furthermore, conidia treated for 0.5h showed cytoplasmic disorganization as observed by transmission electron microscopy (TEM). Remarkably, the peptides derived from the α-core induced morphological changes on the conidia cell wall, which is a promising target since its distinctive biochemical and structural organization is absent in plant and mammalian cells.

Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from Araujia angustifolia latex.

Araujiain aII, the protease with highest specific activity purified from latex of Araujia angustifolia (Apocynaceae), shows optimum proteolytic activity at alkaline pH, and it is completely inhibited by the irreversible inhibitor of cysteine proteases trans-epoxysucciny-L: -leucyl-amido(4-guanidino) butane. It exhibits esterolytic activity on several N-α-Cbz-amino acid p-nitrophenyl esters with a preference for Gln, Ala, and Gly derivatives. Kinetic enzymatic assays were performed with the thiol proteinase substrate p-Glu-Phe-Leu-p-nitroanilide (K (m) = 0.18 ± 0.03 mM, k (cat) = 1.078 ± 0.055 s(-1), k (cat)/K (m) = 5.99 ± 0.57 s(-1) mM(-l)). The enzyme has a pI value above 9.3 and a molecular mass of 23.528 kDa determined by mass spectrometry. cDNA of the peptidase was obtained by reverse transcription-PCR starting from total RNA isolated from latex. The deduced amino acid sequence was confirmed by peptide mass fingerprinting analysis. The N-terminus of the mature protein was determined by automated sequencing using Edman's degradation and compared with the sequence deduced from cDNA. The full araujiain aII sequence was thus obtained with a total of 213 amino acid residues. The peptidase, as well as other Apocynaceae latex peptidases, is a member of the subfamily C1A of cysteine proteases. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was suggested by molecular modeling.

Characterization of papain-like isoenzymes from latex of Asclepias curassavica by molecular biology validated by proteomic approach.

Latices from Asclepias spp are used in wound healing and the treatment of some digestive disorders. These pharmacological actions have been attributed to the presence of cysteine proteases in these milky latices. Asclepias curassavica (Asclepiadaceae), "scarlet milkweed" is a perennial subshrub native to South America. In the current paper we report a new approach directed at the selective biochemical and molecular characterization of asclepain cI (acI) and asclepain cII (acII), the enzymes responsible for the proteolytic activity of the scarlet milkweed latex. SDS-PAGE spots of both purified peptidases were digested with trypsin and Peptide Mass Fingerprints (PMFs) obtained showed no equivalent peptides. No identification was possible by MASCOT search due to the paucity of information concerning Asclepiadaceae latex cysteine proteinases available in databases. From total RNA extracted from latex samples, cDNA of both peptidases was obtained by RT-PCR using degenerate primers encoding Asclepiadaceae cysteine peptidase conserved domains. Theoretical PMFs of partial polypeptide sequences obtained by cloning (186 and 185 amino acids) were compared with empirical PMFs, confirming that the sequences of 186 and 185 amino acids correspond to acI and acII, respectively. N-terminal sequences of acI and acII, characterized by Edman sequencing, were overlapped with those coming from the cDNA to obtain the full-length sequence of both mature peptidases (212 and 211 residues respectively). Alignment and phylogenetic analysis confirmed that acI and acII belong to the subfamily C1A forming a new group of papain-like cysteine peptidases together with asclepain f from Asclepias fruticosa. We conclude that PMF could be adopted as an excellent tool to differentiate, in a fast and unequivocal way, peptidases with very similar physicochemical and functional properties, with advantages over other conventional methods (for instance enzyme kinetics) that are time consuming and afford less reliable results.

Morrenain b I, a papain-like endopeptidase from the latex of Morrenia brachystephana Griseb. (Asclepiadaceae).

A new cysteine endopeptidase (morrenain b I) has been purified and characterized from the latex of stems and petiols of Morrenia brachystephana Griseb. (Asclepiadaceae). Morrenain b I was the minor proteolytic component in the latex but showed higher specific activity than morrenain b II, which was the main active fraction. Both enzymes showed similar pH profiles and molecular masses, but kinetic parameters and N-terminal sequences were quite distinct, demonstrating that they are different enzymes instead of different forms of the same enzyme.