Characterization, Recombinant Production and Structure-Function Analysis of NvCI, A Picomolar Metallocarboxypeptidase Inhibitor from the Marine Snail Nerita versicolor.

A very powerful proteinaceous inhibitor of metallocarboxypeptidases has been isolated from the marine snail Nerita versicolor and characterized in depth. The most abundant of four, very similar isoforms, NvCla, was taken as reference and N-terminally sequenced to obtain a 372-nucleotide band coding for the protein cDNA. The mature protein contains 53 residues and three disulphide bonds. NvCIa and the other isoforms show an exceptionally high inhibitory capacity of around 1.8 pM for human Carboxypeptidase A1 (hCPA1) and for other A-like members of the M14 CPA subfamily, whereas a twofold decrease in inhibitory potency is observed for carboxypeptidase B-like members as hCPB and hTAFIa. A recombinant form, rNvCI, was produced in high yield and HPLC, mass spectrometry and spectroscopic analyses by CD and NMR indicated its homogeneous, compact and thermally resistant nature. Using antibodies raised with rNvCI and histochemical analyses, a preferential distribution of the inhibitor in the surface regions of the animal body was observed, particularly nearby the open entrance of the shell and gut, suggesting its involvement in biological defense mechanisms. The properties of this strong, small and stable inhibitor of metallocarboxypeptidases envisage potentialities for its direct applicability, as well as leading or minimized forms, in biotechnological/biomedical uses.

Identification of cross-reactive B-cell epitopes between Bos d 9.0101(Bos Taurus) and Gly m 5.0101 (Glycine max) by epitope mapping MALDI-TOF MS.

Exposure to cow's milk constitutes one of the most common causes of food allergy. In addition, exposure to soy proteins has become relevant in a restricted proportion of milk allergic pediatric patients treated with soy formulae as a dairy substitute, because of the cross-allergenicity described between soy and milk proteins. We have previously identified several cross-reactive allergens between milk and soy that may explain this intolerance. The purpose of the present work was to identify epitopes in the purified αS1-casein and the recombinant soy allergen Gly m 5.0101 (Gly m 5) using an α-casein-specific monoclonal antibody (1D5 mAb) through two different approaches for epitope mapping, to understand cross-reactivity between milk and soy. The 1D5 mAb was immobilized onto magnetic beads, incubated with the peptide mixture previously obtained by enzymatic digestion of the allergens, and the captured peptides were identified by MALDI-TOF MS analysis. On a second approach, the peptide mixture was resolved by RP-HPLC and immunodominant peptides were identified by dot blot with the mAb. Finally, recognized peptides were sequenced by MALDI-TOF MS. This novel MS based approach led us to identify and characterize four peptides on α-casein and three peptides on Gly m 5 with a common core motif. Information obtained from these cross-reactive epitopes allows us to gain valuable insight into the molecular mechanisms of cross-reactivity, to further develop new and more effective vaccines for food allergy.

Biochemical characterization of VQ-VII, a cysteine peptidase with broad specificity, isolated from Vasconcellea quercifolia latex.

The latex from Vasconcellea quercifolia ("oak leaved papaya"), a member of the Caricaceae family, contains at least seven cysteine endopeptidases with high proteolytic activity, which helps to protect these plants against injury. In this study, we isolated and characterized the most basic of these cysteine endopeptidases, named VQ-VII. This new purified enzyme was homogeneous by bidimensional electrophoresis and MALDI-TOF mass spectrometry, and exhibited a molecular mass of 23,984 Da and an isoelectric point >11. The enzymatic activity of VQ-VII was completely inhibited by E-64 and iodoacetic acid, confirming that it belongs to the catalytic group of cysteine endopeptidases. By investigating the cleavage of the oxidized insulin B-chain to establish the hydrolytic specificity of VQ-VII, we found 13 cleavage sites on the substrate, revealing that it is a broad-specificity peptidase. The pH profiles toward p-Glu-Phe-Leu-p-nitroanilide (PFLNA) and casein showed that the optimum pH is about 6.8 for both substrates, and that in casein, it is active over a wide pH range (activity higher than 80 % between pH 6 and 9.5). Kinetic enzymatic assays were performed with the thiol peptidase substrate PFLNA (K m = 0.454 ± 0.046 mM, k cat = 1.57 ± 0.07 s(-1), k cat/K m = 3.46 × 10(3) ± 14 s(-1) M(-1)). The N-terminal sequence (21 amino acids) of VQ-VII showed an identity >70 % with 11 plant cysteine peptidases and the presence of highly conserved residues and motifs shared with the "papain-like" family of peptidases. VQ-VII proved to be a new latex enzyme of broad specificity, which can degrade extensively proteins of different nature in a wide pH range.

Characterization of the proteolytic system present in Vasconcellea quercifolia latex.

Vasconcellea quercifolia (Caricaceae) latex contains several cysteine endopeptidases with high proteolytic activity. Cysteine endopeptidases are the main active compounds used by the plant as a defense mechanism. A proteolytic preparation from V. quercifolia ("oak leaved papaya") latex was purified by cation exchange chromatography. From SDS-PAGE and blotting of the selected fractions, the N-terminal amino acid sequences of polypeptides were determined by Edman's degradation. The analysis by peptide mass fingerprinting (PMF) of the enzymes allowed their characterization and confirmed the presence of seven different cysteine proteinases in the latex of V. quercifolia. Moreover, the comparison between the tryptic maps with those deposited in databases using the MASCOT tool showed that none of the isolated proteases matched with another plant protease. Notably, a propeptidase was detected in the plant latex, which is being the first report in this sense. Furthermore, the cDNA of one of the cysteine proteases that is expressed in the latex of V. quercifolia was cloned and sequenced. The consensus sequence was aligned using the ClustalX web server, which allowed detecting a high degree of identity with cysteine proteases of the Caricaceae family and establishing the evolutionary relationship between them. We also observed a high conservation degree for those amino acid residues which are essential for the catalytic activity and tridimensional structure of the plant proteases belonging to the subfamily C1A. The PMF analysis strongly suggests that the sequence obtained corresponds to the VQ-III peptidase.

High-level expression of Falcipain-2 in Escherichia coli by codon optimization and auto-induction.

Falcipain-2, the major cysteine hemoglobinase from the human malaria parasite Plasmodium falciparum, is critical for parasite development and is considered a promising chemotherapeutic target. In order to facilitate the high-throughput screening of Falcipain-2 inhibitors from natural sources, we developed an economic and highly-productive overexpression system in Escherichia coli using a codon-optimized proFalcipain-2 construct. Very high expression levels (35-55% of total host proteins) were observed when proFalcaipain-2 expression was induced with 1mM isopropyl-1-thio-ß-D-galactopyranoside (IPTG) in several E. coli strains, with the highest level observed for BL21(DE3). A lower expression (~40% of total host proteins) was observed when BL21(DE3) was grown in ZYM-5052 auto-induction medium, containing 0.2% lactose as inducer. However, the culture grew to notably higher cellular density, increasing ~1.5 times the overall yield of the system when compared with conventional IPTG-induction. Although several conditions were modified to achieve the expression of soluble and active Falcipain-2, the enzyme was mainly obtained in the form of insoluble aggregates. After purification and refolding, ~50 mg of active enzyme were obtained per liter of culture at low cost using a regular incubator shaker, and recombinant Falcipain-2 exhibited structural and functional characteristics very similar to the natural counterpart. Due to its versatility and simplicity, this strategy can be straightforwardly adapted to other proteins from Plasmodium species or any other organism with an AT-rich genome.

Simulated gastrointestinal conditions increase adhesion ability of Lactobacillus paracasei strains isolated from kefir to Caco-2 cells and mucin.

Gastrointestinal conditions along the digestive tract are the main stress to which probiotics administrated orally are exposed because they must survive these adverse conditions and arrive alive to the intestine. Adhesion to epithelium has been considered one of the key criteria for the characterization of probiotics because it extends their residence time in the intestine and as a consequence, can influence the health of the host by modifying the local microbiota or modulating the immune response. Nevertheless, there are very few reports on the adhesion properties to epithelium and mucus of microorganisms after passing through the gastrointestinal tract. In the present work, we evaluate the adhesion ability in vitro of L. paracasei strains isolated from kefir grains after acid and bile stress and we observed that they survive simulated gastrointestinal passage in different levels depending on the strain. L. paracasei CIDCA 8339, 83120 and 83123 were more resistant than L. paracasei CIDCA 83121 and 83124, with a higher susceptibility to simulated gastric conditions. Proteomic analysis of L. paracasei subjected to acid and bile stress revealed that most of the proteins that were positively regulated correspond to the glycolytic pathway enzymes, with an overall effect of stress on the activation of the energy source. Moreover, it is worth to remark that after gastrointestinal passage, L. paracasei strains have increased their ability to adhere to mucin and epithelial cells in vitro being this factor of relevance for maintenance of the strain in the gut environment to exert its probiotic action.

ACE-inhibitory peptides from bovine caseins released with peptidases from Maclura pomifera latex.

In work reported here, a proteolytic extract prepared from Maclura pomifera latex was employed to hydrolyze bovine caseins. Densitograms of Tricine-sodium-dodecyl-sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) indicated that the caseins were considerably degraded after a 10-min reaction. The degree of hydrolysis determined by the 2,4,6-trinitrobenzenesulfonic-acid method was 17.1±0.7% after 180min of digestion. The concentration of small peptides increased with hydrolysis time, and analysis by reverse-phase high-performance liquid chromatography (RP HPLC) and mass spectrometry, revealed a virtually unchanged peptide profile. These results suggested that those proteases were highly specific, as only certain peptide bonds were cleaved. The hydrolysate of 180min displayed the highest inhibition of angiotensin-converting enzyme (ACE) showing an IC50 of 1.72±0.25mg/mL, and the analysis of the peptide fractionation in this hydrolysate by RP HPLC exhibited two peaks responsible for that activity. Fragmentation analysis through the use of iterated matrix-assisted-laser-desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF MS/MS) with the aid of bioinformatics tools enabled us to deduce two peptide sequences-one, YQEPVLGPVRGPFPIIV, having been previously reported as an ACE-inhibitor; the other, RFFVAPFPE, as yet undescribed. The presence of bioactive peptides in these casein hydrolysates argues for their potential use in the development of functional foods.

Profiling and identification of new proteins involved in brain ischemia using MALDI-imaging-mass-spectrometry.

The identification of proteins involved in brain ischemia might allow the discovery of putative biomarkers or therapeutic targets for ischemic stroke. Our aim is to study the distribution of proteins within mouse brain after an ischemic insult using MALDI imaging-mass-spectrometry and to identify relevant proteins involved in brain damage. We occluded the middle cerebral artery of C57BL/6J mice. Brain slices were analyzed by MALDI-TOF and infarct (IC) and contralateral (CL) regions were compared using ClinProTools. The ion distribution maps of relevant m/z values were obtained by FlexImagin3.0. Protein identification was conducted through a bottom-up approach consisting on complementary sample fractionation methods. Some identifications were confirmed by immunohistochemistry and western blot. We identified 102 m/z values with different abundances between IC and CL (p<0.05), from which 21 m/z peaks were selected as more relevant. Thirteen of them were found increased in the infarct region and 4 m/z values showed AUC>90% between IC and CL. Identification analyses confirmed altered expressions of ATP5i, COX6C and UMP-CMP kinase in IC compared to CL. BIOLOGICAL SIGNIFICANCE: Using MALDI-IMS we identified for the first time new proteins that might be involved in brain ischemia representing potential diagnostic biomarkers or target molecules for neurological recovery.

Cloning, sequencing, and identification using proteomic tools of a protease from Bromelia hieronymi Mez.

Fruits of Bromelia hieronymi, a tropical South American plant, possess a high content of peptidases with potential biotechnological uses. Total RNA was extracted from unripe fruits and peptidase cDNA was obtained by 3'RACE-PCR. The consensus sequence of the cysteine peptidase cDNA contained 875 bp, the 690 first ones codifying for a hypothetical polypeptide chain of the mature peptidase, named Bh-CP1 (molecular mass 24.773 kDa, pI 8.6, extinction molar coefficient 58,705 M(-1) cm(-1)). Bh-CP1 sequence shows a high percentage of identity with those of other cysteine plant proteases. The presence of highly preserved residues is observed, like those forming the catalytic site (Gln19, Cys25, His159, and Asn175, papain numbering), as well as other six Cys residues, involved in the formation of disulfide bounds. Molecular modeling results suggest the enzyme belongs to the α + ß class of proteins, with two disulfide bridges (Cys23-Cys63 and Cys57-Cys96) in the α domain, while the ß domain is stabilized by another disulfide bridge (Cys153-Cys203). Additionally, peptide mass fingerprints (PMFs) of the three peptidases previously isolated from B. hieronymi fruits (namely hieronymain I, II, and III) were performed and compared with the theoretical fingerprint of PMF of Bh-CP1, showing a partial matching between the in silico-translated protein and hieronymain II.