Alejandro Blanco-Labra

Digestive amylase from the larger grain borer, Prostephanus truncatus Horn

A combination of ion-exchange chromatography, preparative electrophoresis and gel filtration chromatography allowed a 1209-fold purification of one of the two major digestive alpha-amylases from larvae of the larger grain borer, Prostephanus truncatus Horn. The purified enzyme showed a molecular mass of 60.2 kDa, an isoelectric point of 4.7 and an optimal pH for activity of 6.0. The enzyme was heat labile and it was recognized by proteinaceous inhibitors from amaranth seeds (Amaranthus hypochondriacus), whereas extracts from maize (Zea mays) and tepary bean (Phaseolus acutifolius) produced very low inhibition. When the enzyme was measured at different stages of development, maximal activity was found in the second instar larvae. Activity drastically decreased to a very low level during the pupae stage and increased again at the adult stage. A zymogram of the different developmental stages showed two main bands of alpha-amylase activity, which almost disappeared at the pupae stage to increase again during the adult stage, revealing a new, smaller band. This new band may be required for a better adaptation of the adult insect to its new environment.

Cloning, sequencing, purification, and crystal structure of Grenache (Vitis vinifera) polyphenol oxidase.

The full-length cDNA sequence (P93622_VITVI) of polyphenol oxidase (PPO) cDNA from grape Vitis vinifera L., cv Grenache, was found to encode a translated protein of 607 amino acids with an expected molecular weight of ca. 67 kDa and a predicted pI of 6.83. The translated amino acid sequence was 99%, identical to that of a white grape berry PPO (1) (5 out of 607 amino acid potential sequence differences). The protein was purified from Grenache grape berries by using traditional methods, and it was crystallized with ammonium acetate by the hanging-drop vapor diffusion method. The crystals were orthorhombic, space group C222(1). The structure was obtained at 2.2 A resolution using synchrotron radiation using the 39 kDa isozyme of sweet potato PPO (PDB code: 1BT1 ) as a phase donor. The basic symmetry of the cell parameters (a, b, and c and alpha, beta, and gamma) as well as in the number of asymmetric units in the unit cell of the crystals of PPO, differed between the two proteins. The structures of the two enzymes are quite similar in overall fold, the location of the helix bundles at the core, and the active site in which three histidines bind each of the two catalytic copper ions, and one of the histidines is engaged in a thioether linkage with a cysteine residue. The possibility that the formation of the Cys-His thioether linkage constitutes the activation step is proposed. No evidence of phosphorylation or glycoslyation was found in the electron density map. The mass of the crystallized protein appears to be only 38.4 kDa, and the processing that occurs in the grape berry that leads to this smaller size is discussed.

The complete amino acid sequence of the major kunitz trypsin inhibitor from the seeds of Prosopsis juliflora

The major inhibitor of trypsin in seeds of Prosopsis juliflora was purified by precipitation with ammonium sulphate, ion-exchange column chromatography on DEAE- and CM-Sepharose and preparative reverse phase HPLC on a Vydac C-18 column. The protein inhibited trypsin in the stoichiometric ratio of 1:1, but had only weak activity against chymotrypsin and did not inhibit human salivary or porcine pancreatic alpha-amylases. SDS-PAGE indicated that the inhibitor has a Mr of ca 20,000, and IEF-PAGE showed that the pI is 8.8. The complete amino acid sequence was determined by automatic degradation, and by DABITC/PITC microsequence analysis of peptides obtained from enzyme digestions of the reduced and S-carboxymethylated protein with trypsin, chymotrypsin, elastase, the Glu-specific protease from S. aureus and the Lys-specific protease from Lysobacter enzymogenes. The inhibitor consisted of two polypeptide chains, of 137 residues (alpha chain) and 38 residues (beta chain) linked together by a single disulphide bond. The amino acid sequence of the protein exhibited homology with a number of Kunitz proteinase inhibitors from other legume seeds, the bifunctional subtilisin/alpha-amylase inhibitors from cereals and the taste-modifying protein miraculin.

Purification, Characterization, and Complete Amino Acid Sequence of a Trypsin Inhibitor from Amaranth (Amaranthus hypochondriacus) Seeds

A protein proteinase inhibitor was purified from a seed extract of amaranth (Amaranthus hypochondriacus) by precipitation with (NH4)2SO4, gel-filtration chromatography, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography. It is a 69-amino acid protein with a high content of valine, arginine, and glutamic acid, but lacking in methionine. The inhibitor has a relative molecular weight of 7400 and an isoelectric point of 7.5. It is a serine proteinase inhibitor that recognizes chymotrypsin, trypsin, and trypsin-like proteinase activities extracted from larvae of the insect Prostephanus truncatus. This inhibitor belongs to the potato-I inhibitor family, showing the closest homology (59.5%) with the Lycopersicum peruvianum trypsin inhibitor, and (51%) with the proteinase inhibitor 5 extracted from the seeds of Cucurbita maxima. The position of the lysineaspartic acid residues present in the active site of the amaranth inhibitor are found in almost the same relative position as in the inhibitor from C. maxima.

Exclusive rewards in mutualisms: ant proteases and plant protease inhibitors create a lock–key system to protect Acacia food bodies from exploitation

Myrmecophytic Acacia species produce food bodies (FBs) to nourish ants of the Pseudomyrmex ferrugineus group, with which they live in an obligate mutualism. We investigated how the FBs are protected from exploiting nonmutualists. Two‐dimensional gel electrophoresis of the FB proteomes and consecutive protein sequencing indicated the presence of several Kunitz‐type protease inhibitors (PIs). PIs extracted from Acacia FBs were biologically active, as they effectively reduced the trypsin‐like and elastase‐like proteolytic activity in the guts of seed‐feeding beetles (Prostephanus truncatus and Zabrotes subfasciatus), which were used as nonadapted herbivores representing potential exploiters. By contrast, the legitimate mutualistic consumers maintained high proteolytic activity dominated by chymotrypsin 1, which was insensitive to the FB PIs. Larvae of an exploiter ant (Pseudomyrmex gracilis) taken from Acacia hosts exhibited lower overall proteolytic activity than the mutualists. The proteases of this exploiter exhibited mainly elastase‐like and to a lower degree chymotrypsin 1‐like activity. We conclude that the mutualist ants possess specifically those proteases that are least sensitive to the PIs in their specific food source, whereas the congeneric exploiter ant appears partly, but not completely, adapted to consume Acacia FBs. By contrast, any consumption of the FBs by nonadapted exploiters would effectively inhibit their digestive capacities. We suggest that the term ‘exclusive rewards’ can be used to describe situations similar to the one that has evolved in myrmecophytic Acacia species, which reward mutualists with FBs but safeguard the reward from exploitation by generalists by making the FBs difficult for the nonadapted consumer to use.

Recombinant amaranth cystatin (AhCPI) inhibits the growth of phytopathogenic fungi.

Phytocystatins are cysteine proteinase inhibitors from plants implicated in defense mechanisms against insects and plant pathogens. We have previously characterized an amaranth cystatin cDNA and analyzed its response to different kinds of abiotic stress [37]. In order to characterize amaranth cystatin, the coding sequence was expressed in Escherichia coli using the pQE-2 vector. Recombinant cystatin was predominantly found in the soluble fraction of the cell extract. Large amounts (266 mgL(-1)) of pure recombinant protein were obtained by affinity chromatography in a single step of purification. The amaranth cystatin with a pI 6.8 and an apparent 28 kDa molecular mass inhibited papain (E.C.3.4.22.2) (Ki 115 nM), ficin (E.C.3.4.22.3) (Ki 325 nM) and cathepsin L (E.C.3.4.22.15) (Ki 12.7 nM) but not stem bromelain (E.C.3.4.22.32), and cathepsin B (E.C.3.4.22.1) activities, in colorimetric assays. Furthermore, it was able to arrest the fungal growth of Fusarium oxysporum, Sclerotium cepivorum and Rhyzoctonia solani. It was further demonstrated that recombinant AhCPI is a weak inhibitor of the endogenous cysteine proteinase activities in the fungal mycelium. These findings contribute to a better understanding of the amaranth cystatin activity and encourage further studies of this protein.

New amylase inhibitor present in corn seeds active in vitro against amylase from Fusarium verticillioides

A screening for specific amylase inhibitor levels against amylase from Fusarium verticillioides (Fusarium moniliforme), the most relevant mycotoxigenic fungus in corn, was conducted on 37 corn hybrids. The amylase inhibitor levels in these hybrids ranged from 5.5 to 16.0 amylase inhibitor units per gram of corn (AIU/g) in the MASTER and AG5011 hybrids, respectively. The hybrid with the maximum content of inhibitor was used as the source of this new protein. The inhibitor was partially purified using fractional precipitation, gel filtration on Sephadex G-75 column, high performance liquid chromatography (HPLC) Superose HR 10/30 column, and HPLC anion exchange chromatography, obtaining a 20.7-fold purification. Electrophoresis after denaturing and heating under reductive conditions showed an apparent 23.8 kDa molecular mass and an acidic isoelectric point of 5.4, which differs from previous molecular masses reported for other inhibitors present in corn seeds (14 and 22 kDa). This inhibitor showed activity against amylases from human saliva and pancreas, from the fungi F. verticillioides and Aspergillus flavus, and from the insects Acanthoscelides obtectus, Zabrotes subfasciatus, Tribolium castaneum, and Sitotroga cerealella. The mycoflora found in the corn grain indicated Fusarium sp. as the most prevalent fungi (81.1% of the samples), with a count ranging from 1.5 × 102 to 2.4 × 106 CFU/g of corn. The presence of fumonisins was detected in 21 out of the 37 hybrids studied, ranging from 0.05 to 2.67 μg of FB per gram of corn. No correlation could be established between this amylase inhibitor level in the corn seeds and the presence of Fusarium sp. or with the fumonisin content under the experimental conditions of the test.

Characterization of a Hydrophobic Amylase Inhibitor from Corn (Zea mays) Seeds with Activity Against Amylase from Fusarium verticillioides

ABSTRACT A hydrophobic 19.7-kDa amylase inhibitor (AI) was purified from corn kernels by 95% ethanol extraction and anionic exchange chromatography. The AI has an isoelectric point of 3.6 and was very stable at different pH values and high temperatures, maintaining 47.6% activity after heating to 94 degrees C for 60 min. Amino acid analysis indicated high valine, leucine, glycine, alanine, and glutamic acid/glutamine content, and especially high valine content (41.2 mol%). This inhibitor is not a glycoprotein. It required 30-min preincubation to maximize complex enzyme-inhibitor formation when the amylase from Fusarium verticillioides was tested. The optimal pH of interaction was 6.5. It showed broad-spectrum activity including the following amylases: human saliva, porcine pancreas, F. verticillioides, as well as those from some insects of agricultural importance (Acanthoscelides obtectus, Zabrotes subfasciatus, Sitophilus zeamais, and Prostephanus truncatus). This novel hydrophobic protein not only inhibited the amylase from F. verticillioides but also decreased the conidia germination. Thus, this protein represents an approach to decrease the production of fumonisin in corn, either by using it as a molecular marker to detect fungal resistance or through genetic engineering.

Effects of Tepary Bean (Phaseolus acutifolius) Protease Inhibitor and Semipure Lectin Fractions on Cancer Cells

Some natural and synthetic protease inhibitors (PI), such as the Bowman-Birk PI from soybean, have anticancer effects. We previously purified and characterized a Bowman-Birk-type PI from Tepary bean (Phaseolus acutifolius) seeds (TBPI). A semipure protein fraction containing this inhibitor, when tested its in vitro effect on transformed cells, showed a differential cytotoxic effect, as well as an increase in cell attachment to culture dishes. In this article we report that lectins were responsible for the cytotoxic effect previously observed, exhibiting a differential, antiproliferative effect on nontransformed cells and on different lineages of cancer cells. Although the purified TBPI lacked cytotoxicity, it was found to be responsible for the increase in cell adhesion, decreasing culture dishes’ extracellular matrix degradation, leading to a decrease of the in vitro cell invasion capacity. This effect coincided with the suppression of Matrix Metalloproteinase-9 activity. These results indicate that Tepary bean seeds contain at least 2 different groups of bioactive proteins with distinct effects on cancer cells.

Characterization of a highly stable trypsin-like proteinase inhibitor from the seeds of Opuntia streptacantha (O. streptacantha Lemaire)

A trypsin inhibitor from Opuntia streptacantha Lemaire (Prickly pear) seeds was purified and characterized. Of several proteases tested, this inhibitor showed specificity to trypsin-like enzymes. The major inhibitor present in these seeds showed distinctive characteristics, most notably a low molecular weight of 4.19 kDa, as determined by MALDI TOF, and an unusually high thermal stability, retaining most of the activity after heating the sample 1h to 120 degrees C with a pressure of 1 kg/cm(2). Its complete amino acid sequence was obtained through mass spectrometry, this establishing presence a blocked N-terminal region. When comparing its sequence in the MEROPS database for peptidases and peptidase inhibitors, it showed 34.48% identity with a serine-proteinase inhibitor from the I15 family.