Gianni Cappugi

Purification, Characterization, and Amino Acid Sequence of Cerato-platanin, a New Phytotoxic Protein from Ceratocystis fimbriata f. sp. platani

A new phytotoxic protein (cerato-platanin) of about 12.4 kDa has been identified in culture filtrates of the Ascomycete Ceratocystis fimbriata f. sp.platani, the causal agent of canker stain disease. The toxicity of the pure protein was bioassayed by detecting the inducing necrosis in tobacco leaves. The pure protein also elicited host synthesis of fluorescent substances in tobacco and plane (Platanus acerifolia) leaves. We purified the protein from culture medium to homogeneity. Its complete amino acid sequence was determined; this protein consists of 120 amino acid residues, contains 4 cysteines (S—S-bridged), and has a high percentage of hydrophobic residues. The molecular weight calculated from the amino acid sequence agrees with that determined by mass spectrometry, suggesting that no post-transnational modification occurs. Searches performed by the BLAST program in data banks (Swiss-Prot, EBI, and GenBank™) revealed that this protein is highly homologous with two proteins produced by other Ascomycete fungi. One, produced during infection of wheat leaves, is codified by the snodprot1 gene of Phaeosphaeria nodorum (the causal agent of glume blotch of wheat), whereas the other is the rAsp f13 allergen from Aspergillus fumigatus.Furthermore, the N terminus of cerato-platanin is homologous with that of cerato-ulmin, a phytotoxic protein belonging to the hydrophobin family and produced by Ophiostoma (Ceratocystis) ulmi, a fungus responsible for Dutch elm disease.

Response to cadmium in carrot in vitro plants and cell suspension cultures

Abstract In vitro grown plants and cell suspension cultures of carrot ( Daucus carota L.) were treated with various Cd concentrations. Stress ethylene production in carrot plants was highly stimulated by 1 mM Cd. A pre-treatment with buthionine sulfoximine (BSO) did not further increase ethylene production. After being treated with Cd, both plants and cell suspensions produced phytochelatins, and no lipid peroxidation was detected. In cell cultures, the in vitro activity of phytochelatin synthase was assayed in the presence of Cd and glutathione: the first product (PC 2 ) was detected in less than 30 min. Absence of ethylene (after treatment with aminoethoxyvinylglycine (AVG), an inhibitor of ethylene-biosynthesis, or use of ethylene traps) caused both a decrease in the phytochelatin synthase activity of cell suspensions and a strong lowering in the Cd-induced SH groups in plants. However 1-aminocyclopropane-1-carboxylic acid (ACC) supply did not increase either phytochelatin synthase activity or total SH level.

The Structure of the Elicitor Cerato-platanin (CP), the First Member of the CP Fungal Protein Family, Reveals a Double ψβ-Barrel Fold and Carbohydrate Binding

Cerato-platanin (CP) is a secretion protein produced by the fungal pathogen Ceratocystis platani, the causal agent of the plane canker disease and the first member of the CP family. CP is considered a pathogen-associated molecular pattern because it induces various defense responses in the host, including production of phytoalexins and cell death. Although much is known about the properties of CP and related proteins as elicitors of plant defense mechanisms, its biochemical activity and host target(s) remain elusive. Here, we present the three-dimensional structure of CP. The protein, which exhibits a remarkable pH and thermal stability, has a double ψβ-barrel fold quite similar to those found in expansins, endoglucanases, and the plant defense protein barwin. Interestingly, although CP lacks lytic activity against a variety of carbohydrates, it binds oligosaccharides. We identified the CP region responsible for binding as a shallow surface located at one side of the β-barrel. Chemical shift perturbation of the protein amide protons, induced by oligo-N-acetylglucosamines of various size, showed that all the residues involved in oligosaccharide binding are conserved among the members of the CP family. Overall, the results suggest that CP might be involved in polysaccharide recognition and that the double ψβ-barrel fold is widespread in distantly related organisms, where it is often involved in host-microbe interactions.

A new synthesis of benzoyl phosphate: a substrate for acyl phosphatase assay.

A new method for the synthesis of benzoyl phosphate was reported. The advantages are: 1. more rapid procedure; 2. lower cost; 3. higher yield.

Rat liver low M(r) phosphotyrosine protein phosphatase isoenzymes: purification and amino acid sequences.

Two lowMr phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as lowMr acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH2-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40–73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.

Differential role of four cysteines on the activity of a low M r phosphotyrosine protein phosphatase

In this paper we describe the construction or five mutants of a bovine liver low M r phosphotyrosine protein phosphatase (PTPase) expressed as a fusion protein with the maltose binding protein in E. coli. Almost no changes in the kinetic parameters were observed in the fusion protein with respect to the native PTPase. Using oligonucleotide‐directed mutagenesis Cys‐17, Cys‐62 and Cys‐145 were converted to Ser while Cys‐12 was converted to both Ser and Ala. The kinetic properties of the mutants, using p‐nitrophenyl phosphate as substrate, were compared with those of the normal protein fused with the maltose binding protein or E. coli; both of the Cys‐12 mutants showed a complete loss of enzymatic activity while the specific activity of the Cys‐17 mutant was greatly decreased (200‐fold). The Cys‐62 mutant showed a 2.5‐fold decrease in specific activity, while the Cys‐145 mutant remained almost unchanged. These data confirm the involvement of Cys‐12 and Cys‐17 in the catalytic site and suggest that Cys‐62 and Cys‐145 mutations may destabilise the structure of the enzyme.

Cerato-platanin, the first member of a new fungal protein family: cloning, expression, and characterization.

The ascomcete Ceratocystis fimbriata, the causal agent of “canker stain disease,” secretes a protein of 12.4 kDa that elicits phytoalexin synthesis and plant cell death. This protein, named cerato-platanin (CP), is also located in the cell walls of ascospores, hyphae, and conidia; it contains four cysteines (S-S bridged) and is moderately hydrophobic. The cp gene consists of a single exon and has 42 bp codifying for a signal peptide of 14 residues. The recombinant protein was obtained by cloning the cp gene of the mature protein in Escherichia coli (BL21), and a refolding step was needed to achieve the native active form. In the European Molecular Biology data bank, CP is reported as the first member of the CP family; this is the first example of an set of secreted fungal proteins whose primary structure is very similar. Nonetheless, the data also revealed some structural and functional features that make CP simlar to proteins of the hydrophobin family.

New proteins orthologous to cerato-platanin in various Ceratocystis species and the purification and characterization of cerato-populin from Ceratocystis populicola

Natural variants of cerato-platanin (CP), a pathogen associated molecular pattern (PAMP) protein produced by Ceratocystis platani (the causal agent of the plane canker stain), have been found to be produced by other four species of the genus Ceratocystis, including five clones of Ceratocystis fimbriata isolated from different hosts. All these fungal strains were known to be pathogenic to plants with considerable importance in agriculture, forestry, and as ornamental plants. The putative premature proteins were deduced on the basis of the nucleotide sequence of genes orthologous to the cp gene of C. platani; the deduced premature proteins of Ceratocystis populicola and Ceratocystis variospora reduced the total identity of all the others from 87.3% to 60.3%. Cerato-populin (Pop1), the CP-orthologous protein produced by C. populicola, was purified and characterized. Pop1 was a well-structured α/β protein with a different percentage of the α-helix than CP, and it self-assembled in vitro in ordered aggregates. Moreover, Pop1 behaved as PAMP, since it stimulated poplar leaf tissues to activate defence responses able to reduce consistently the C. populicola growth.

Affinity chromatographic purification of horse muscle acylphosphatase: Evidence of the existence of multiple molecular forms

Acylphosphatase was purified from horse muscle by a new procedure involving an affinity chromatography step and subsequent ion-exchange chromatography. This procedure was considerably milder than the preceding one, gave an overall yield of about 60% of activity and permitted isolation of three molecular forms with acylphosphatase activity. All these enzymatic forms are tightly bound to Sepharose 4B-linked anti-horse muscle acylphosphatase antibodies. Two of these forms (Ho1 and Ho3) are present in larger amounts: Ho1 corresponds to the enzyme purified according to the older procedure; this enzyme is a mixed disulfide between a main chain of 98 amino acid residues and glutathione. Ho2 differs from Ho1 only in the chemical nature of the molecule(s) S-S bound to the sole cysteine present at position 21 of the main chain. Ho3 is an S-S dimer of the main polypeptide chain. Ho1, Ho2, and Ho3 elicit very similar kinetic parameters in the presence of benzoylphosphate as a substrate.

Rabbit skeletal muscle acylphosphatase: The amino acid sequence of form Ra1☆☆☆

Acylphosphatase was purified from rabbit skeletal muscle by a procedure involving an affinity chromatography step on immunoadsorbent and subsequent ion-exchange chromatography. Three molecular forms with acylphosphatase activity, named Ra1, Ra2, and Ra3, were purified and characterized with respect to molecular weight, amino acid composition, and main kinetic parameters. The amino acid sequence of Ra1 is given in the present paper. The Ra1 form consists of a single polypeptide chain of 98 amino acid residues and contains only one cysteine residue at position 21 that is S-S bound to glutathione. The polypeptide chain has an acetyl group blocking the NH2 terminus. Ra1, Ra2, and Ra3 are compared with the corresponding molecular forms isolated from skeletal muscle of horse and turkey.