Aleuria aurantia lectin exhibits antifungal activity against Mucor racemosus.

Aleuria aurantia lectin (AAL) is an L-fucose-specific lectin produced in the mycelia and fruit-bodies of the widespread ascomycete fungus Aleuria aurantia. It is extensively used in the detection of fucose, but its physiological role remains unknown. To investigate this, we analyzed the interaction between AAL and, a zygomycete fungus Mucor racemosus, which is assumed to contain fucose in its cell wall.

Molecular and enzymatic characterization of a subfamily I.4 lipase from an edible oil-degrader Bacillus sp. HH-01.

An edible-oil degrading bacterial strain HH-01 was isolated from oil plant gummy matter and was classified as a member of the genus Bacillus on the basis of the nucleotide sequence of the 16S rRNA gene. A putative lipase gene and its flanking regions were cloned from the strain based on its similarity to lipase genes from other Bacillus spp. The deduced product was composed of 214 amino acids and the putative mature protein, consisting of 182 amino acids, exhibited 82% amino acid sequence identity with the subfamily I.

Molecular characterization and antifungal activity of a family 46 chitosanase from Amycolatopsis sp. CsO-2.

An actinomycete strain, Amycolatopsis sp. CsO-2, produces a 27-kDa chitosanase. To reveal the molecular characteristics of the enzyme, its corresponding gene ctoA was cloned by a reverse genetic technique, based on the N-terminal amino acid sequence of the protein.

Molecular structure and properties of lectin from tomato fruit.

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro(4) motifs inserted between the first and second chitin-binding domains.

Molecular characterization of a novel family-46 chitosanase from Pseudomonas sp. A-01.

Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique.

The dasABC gene cluster, adjacent to dasR, encodes a novel ABC transporter for the uptake of N,N'-diacetylchitobiose in Streptomyces coelicolor A3(2).

N,N'-Diacetylchitobiose [(GlcNAc)(2)] induces the transcription of chitinase (chi) genes in Streptomyces coelicolor A3(2). Physiological studies showed that (GlcNAc)(2) addition triggered chi expression and increased the rate of (GlcNAc)(2) concentration decline in culture supernatants of mycelia already cultivated with (GlcNAc)(2), suggesting that (GlcNAc)(2) induced the synthesis of its own uptake system. Four open reading frames (SCO0531, SCO0914, SCO2946, and SCO5232) encoding putative sugar-binding proteins of ABC transporters were found in the genome by probing the 12-bp repeat sequence required for regulation of chi transcription.

An enzyme-linked immunosorbent assay (ELISA) to determine the specificity of the sugar-binding protein NgcE, a component of the ABC transporter for N-acetylglucosamine in Streptomyces olivaceoviridis.

The ATP-binding cassette (ABC) transporter Ngc for N-acetylglucosamine (GlcNAc) of the chitin-degrader Streptomyces olivaceoviridis comprises the solute-binding protein NgcE, which has highest affinity for GlcNAc and N,N'-diacetylchitobiose {(GlcNAc)2} and reduced affinity for longer chitooligomers. NgcE was used to develop a generally applicable enzyme-linked immunosorbent assay (ELISA) system. As a prerequisite, the reducing end of (GlcNAc)2 was coupled with the ethylamino group of 2-(4-aminophenyl)ethylamine.

Characterization, occurrence, and molecular cloning of a lectin from Grifola frondosa: jacalin-related lectin of fungal origin.

A lectin named GFL was isolated from the fruiting body of the basidiomycete mushroom Grifola frondosa, which belongs to Aphyllophorales. The lectin had a molecular mass of 24 kDa on SDS-PAGE. The hemagglutinating activity of GFL was not inhibited by any monosaccharide, and inhibited only by porcine stomach mucin so far as tested.

Bacillus circulans MH-K1 chitosanase: amino acid residues responsible for substrate binding.

To identify the amino acids responsible for the substrate binding of chitosanase from Bacillus circulans MH-K1 (MH-K1 chitosanase), Tyr148 and Lys218 of the chitosanase were mutated to serine and proline, respectively, and the mutated chitosanases were characterized. The enzymatic activities of Y148S and K218P were found to be 12.5% and 0.

Molecular properties of mycelial aggregate-specific lectin of Pleurotus cornucopiae.

By cloning and sequencing cDNA, the primary structure of a mycelial aggregate-specific lectin of Pleurotus cornucopiae was determined. The amino acid sequence was novel and elucidated unique properties of this lectin: It was composed of 373 amino acids, 33 of which constitute a signal sequence. The sequence of the mature lectin consisted of two homologous regions having five glycosylation recognition signals and six cysteine residues.

Production of fruiting-body lectins of Pleurotus cornucopiae in methylotrophic yeast Pichia pastoris.

Fruiting-body lectin genes obtained from Pleurotus cornucopiae were expressed in Pichia pastoris Because of glycosylation of the products, their molecular mass was larger than that of the corresponding native lectins. They showed binding activity to porcine stomach mucin in the enzyme-linked lectin assay system, but did not agglutinate red blood cells.

Properties of mycelial aggregate-specific lectin of Pleurotus cornucopiae produced in Pichia pastoris.

cDNA of a mycelial aggregate-specific lectin of Pleurotus cornucopiae was expressed in Pichia pastoris, and the expression product was purified and characterized. The product was functional, and the hemagglutinating activity was inhibited most strongly by the addition of N-acetyl-D-galactosamine as was the native lectin. The native lectin is a glycoprotein having five glycosylation recognition signals, and the expression product showed slightly larger molecular mass than that of the native one due to further glycosylation.

Involvement of tyrosines at fucose-binding sites of Aleuria aurantia lectin: non-equal response to site-directed mutagenesis among five sites.

Since the involvement of Tyr residues in the fucose-binding of Aleuria aurantia lectin (AAL) was proved by chemical modification using the Tyr-specific reagent tetranitromethane, site-directed mutagenesis was attempted. Since the tertiary structure of AAL was determined recently to be a six-bladed beta-propeller fold, and five fucose-binding sites per subunit were found, based on positions of Tyr residues in the tertiary structure, three classes of mutants were constructed: 1) Tyr on the 2nd beta-strand of each blade (beta-2 mutants), 2) Tyr or Trp on the 3rd beta-strand (beta-3 mutants), and 3) Tyr outside of binding sites (other-Y mutants). The mutagenized cDNA was expressed in Escherichia coli as His-tag-AAL, and the hemagglutinating activity was assayed.

Production of functional lectin in Pichia pastoris directed by cloned cDNA from Aleuria aurantia.

A plasmid bearing a nucleotide sequence of fucose-specific lectin of Aleuria aurantia was constructed and expressed in a methylotrophic yeast, Pichia pastoris. The product showed almost the same hemagglutinating activity as the lectin produced in Escherichia coli, the properties of which were quite similar to the native one. Because of glycosylation of the product, the molecular mass was larger than that of the native one, and it acquired higher thermostability.

Crystal structure of fucose-specific lectin from Aleuria aurantia binding ligands at three of its five sugar recognition sites.

Aleuria aurantia possesses a fucose-specific lectin (AAL) that is widely used as a specific probe for fucose. Fucosylated sugars often play pivotal roles in many cellular processes. We have determined the crystal structure of AAL at 2.

Characterization and sequence of tomato 2S seed albumin: a storage protein with sequence similarities to the fruit lectin.

We found a 2S storage albumin from the seed of tomato ( Lycopersicon esculentum L. cv. Cherry) that cross-reacted with antiserum to the fruit lectin, and named it Lec2SA.

X-ray crystallographic characterization and phasing of a fucose-specific lectin from Aleuria aurantia.

A fucose-specific lectin from Aleuria aurantia was crystallized in its native form and was also cocrystallized with HgCl(2). Crystallization was performed using the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant. Both the Hg-free native crystals and the Hg cocrystals belong to the hexagonal space group P6(5)22.

Two genes encoding fruit body lectins of Pleurotus cornucopiae: sequence similarity with the lectin of a nematode-trapping fungus.

Our previous studies on the fruit body lectin of Pleurotus cornucopiae revealed the existence of three isolectins, composed of two homodimers and one heterodimer of 16- and 15-kDa subunits. In this study, two genes encoding the lectins were cloned and characterized. Both genes encoded 144 amino acids and only 5 amino acids were different within the coding region, but the nucleotide sequences of the 5'-upstream and 3'-downstream regions differed extensively.

Purification and properties of a lectin from ascomycete mushroom, Ciborinia camelliae.

A lectin was isolated from an ascomycete mushroom, Ciborinia camelliae which was specific to N-acetyl-D-galactosamine. On SDS-polyacrylamide gel electrophoresis; this lectin gave a single band of approximately 17-kDa in the presence of 2-mercaptoethanol, but formed dimers, trimers and tetramers in its absence. Amino acid analysis revealed the lectin contained two cysteines and no methionine.