Frederico Bruno Mendes Batista Moreno

New insights into the complex mixture of latex cysteine peptidases in Calotropis procera

The latex of Calotropis procera is a rich source of proteolytic activity. This latex is known to contain two distinct cysteine peptidases: procerain and procerain B. In this study, new cysteine peptidases were purified from C. procera latex. The enzymes were purified by two sequential ion-exchange chromatography steps (CM-Sepharose plus Resource S(®)) at pH 5.0 and 6.0. The purified enzymes had molecular mass spectra corresponding to CpCP-1=26,213, CpCP-2=26,133 and CpCP-3=25,086 Da. These enzymes exhibited discrete differences in terms of enzymatic activity at a broad range of pH and temperature conditions and contained identical N-terminal amino acid sequences. In these respects, these three new proteins are distinct from those previously studied (procerain and procerain B). Circular dichroism analysis revealed that the new peptidases contain extensive secondary structures, α(15-20%) and β(26-30%), that were stabilized by disulfide bonds. The purified enzymes exhibited plasma-clotting activity mediated by a thrombin-like mechanism. The set of results suggest the three isolated polypeptides correspond to different post-translationally processed forms of the same protein.

cDNA cloning and 1.75 Å crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP‐HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 ± 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N‐acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed β(1–4) glycosidic bonds linking 2‐acetoamido‐2‐deoxy‐β‐d‐glucopyranose units in chitin. The full‐length amino acid sequence of Parkia platycephala lectin 2, determined by N‐terminal sequencing and cDNA cloning, and its three‐dimensional structure, established by X‐ray crystallography at 1.75 Å resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (βα)8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182.

Crystallization and preliminary X-ray diffraction analysis of the lectin from Canavalia gladiata seeds

The seed lectin from Canavalia gladiata was purified and crystallized. Orthorhombic crystals belonging to space group C222(1) grew within three weeks at 293 K using the hanging-drop vapour-diffusion method. Using synchrotron X-ray radiation, a complete structural data set was collected at 2.3 A resolution. The preliminary crystal structure of the lectin, determined by molecular replacement, had a correlation coefficient of 0.569 and an R factor of 0.412.

Purification, partial characterization and preliminary X-ray diffraction analysis of a mannose-specific lectin from Cymbosema roseum seeds

A lectin from Cymbosema roseum seeds (CRL) was purified, characterized and crystallized. The best crystals grew in a month and were obtained by the vapour-diffusion method using a precipitant solution consisting of 0.1 M Tris-HCl pH 7.8, 8%(w/v) PEG 3350 and 0.2 M proline at a constant temperature of 293 K. A data set was collected to 1.77 A resolution at a synchrotron-radiation source. CRL crystals are orthorhombic, belonging to space group P2(1)2(1)2(1). Crystallographic refinement and full amino-acid sequence determination are in progress.

Crystallization and preliminary X-ray diffraction analysis of a new chitin-binding protein from Parkia platycephala seeds.

A chitin-binding protein named PPL-2 was purified from Parkia platycephala seeds and crystallized. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 55.19, b = 59.95, c = 76.60 A, and grew over several days at 293 K using the hanging-drop method. Using synchrotron radiation, a complete structural data set was collected to 1.73 A resolution. The preliminary crystal structure of PPL-2, determined by molecular replacement, presents a correlation coefficient of 0.558 and an R factor of 0.439. Crystallographic refinement is in progress.

Crystallization and preliminary X-ray diffraction analysis of a lectin from Canavalia maritima seeds

A lectin from Canavalia maritima seeds (ConM) was purified and submitted to crystallization experiments. The best crystals were obtained using the vapour-diffusion method at a constant temperature of 293 K and grew in 7 d. A complete structural data set was collected to 2.1 A resolution using a synchrotron-radiation source. The ConM crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 67.15, b = 70.90, c = 97.37 A. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%. Crystallographic refinement is under way.

New crystal forms of Diocleinae lectins in the presence of different dimannosides.

Studying the interactions between lectins and sugars is important in order to explain the differences observed in the biological activities presented by the highly similar proteins of the Diocleinae subtribe. Here, the crystallization and preliminary X-ray data of Canavalia gladiata lectin (CGL) and C. maritima lectin (CML) complexed with Man(alpha1-2)Man(alpha1)OMe, Man(alpha1-3)Man(alpha1)OMe and Man(alpha1-4)Man(alpha1)OMe in two crystal forms [the complexes with Man(alpha1-3)Man(alpha1)OMe and Man(alpha1-4)Man(alpha1)OMe crystallized in space group P3(2) and those with Man(alpha1-2)Man(alpha1)OMe crystallized in space group I222], which differed from those of the native proteins (P2(1)2(1)2 for CML and C222 for CGL), are reported. The crystal complexes of ConA-like lectins with Man(alpha1-4)Man(alpha1)OMe are reported here for the first time.

Diocleinae Lectins: Clues to Delineate Structure/Function Correlations

Lectins are a structurally heterogeneous group of carbohydrate-binding proteins of non-immune origin comprising distinct families of evolutionarily related proteins (van Damme et al. 1998) (consult also the 3D Lectin Database at http://www.cermay.cnrs.fr/databank/lectine). Sugar recognition mechanisms have evolved independently in diverse protein frameworks, and lectins are ubiquitous in animals, plants and microorganisms. Lectins play biological roles in many cellular processes, such as: cell communication, host defense, fertilisation, development, parasitic infection and tumour metastasis, by deciphering the glycocodes encoded in the structure of glycans attached to soluble and integral cell membrane glycoconjugates (Gabius and Gabius 1997).

A novel peroxidase purified from Marsdenia megalantha latex inhibits phytopathogenic fungi mediated by cell membrane permeabilization.

An antifungal class III peroxidase was purified from Marsdenia megalantha latex (named Mo-POX) using DEAE-cellulose and gel filtration chromatography on a Superose 12 HR 10/30 column. Mm-POX has an apparent molecular mass of 67.0kDa and a pI of 5.2, shares identity with other peroxidases, and follows Michaelis-Menten kinetics. It has a high affinity for guaiacol and hydrogen peroxide. The pH and temperature optima for Mm-POX were 5.0-7.0 and 60°C, respectively. The catalytic activity of Mm-POX was decreased in the presence of classic peroxidase inhibitors including azide, dithiothreitol, ethylenediamine tetraacetic acid, and sodium metabisulfite and high concentrations of Na+, Mn+, and salicylic acid. In contrast, Ca+ and Mg+, even at low concentrations, enhanced the Mm-POX enzymatic activity. This protein inhibited the germination of the conidia of the phytopathogenic fungi Fusarium oxysporum and Fusarium solani by acting through a membrane permeabilization mechanism. Mm-POX also induced oxidative stress in F. solani. Mm-POX is the first enzyme to be isolated from the M. megalantha species and it has potential use in the control of plant disease caused by important phytopathogenic fungi. This adds biotechnological value to this enzyme.

Proteome of the rete testis fluid from tropically-adapted Morada Nova rams

The rete testis has a close relationship with sperm development and may have other functions besides serving as an intercalated channel. The aim of this study was to identify and characterize the proteins of rete testis fluid (RTF) from tropically-adapted Morada Nova rams. Testicles obtained from six Morada Nova rams were dissected and the head of the epididymis was separated to access the efferent ducts. Rete testis fluid was obtained by gentle massage of the testis. The fluid was centrifuged to remove cell debris and sperm. RTF samples (containing 400μg protein) were separated by 2-D SDS-PAGE and gels, analyzed using PDQuest software (Bio Rad, USA). Proteins were identified using tandem mass spectrometry. Gene ontology and protein network were analyzed using the software tool for searching annotations of proteins (STRAP) and STRING database. Gels had, on average, 227±13.5 spots and 51% of the proteins were found above 40kDa, corresponding to 65% of the intensity of all spots detected. Based on gene ontology analysis, the most common biological processes associated with RTF proteins were regulation (24.3%) and cellular process (23.3%). Binding (27.3%) and catalytic activity (19.3%) corresponded to the most frequent molecular functions. Albumin, clusterin, serotransferrin, immunoglobulin gamma-1 chain and alpha-2-HS-glycoprotein were the most abundant proteins in the ram rete testis fluid. In conclusion, proteins identified in the ram rete testis fluid are linked to several physiological processes associated with sperm protection and spermatogenesis.