A novel developmental stage-specific lectin of the basidiomycete Pleurotus cornucopiae

Abstract

A novel lectin was isolated from mycelia of the basidiomycete Pleurotus cornucopiae grown on solid medium. The lectin was purified to homogeneity by mucin-Sepharose affinity chromatography. The molecular mass of the lectin was 40 kDa under reducing conditions, but the subunits were polymerized through disulfide bridges under physiological conditions. Hemagglutinating activity of this lectin was completely inhibited by 2-mercaptoethanol, indicating that the multimer is active. The activity was also inhibited by EDTA, and restored by CaCl 2 . N-Acetyl-D-galactosamine was the most potent hapten inhibitor. N-terminal amino acid sequence analysis revealed that the mycelial lectin was different from the fruit body lectin of this organism. The mycelial lectin appeared prior to fruit body formation and disappeared during the formation of fruit bodies. The lectin was localized on the surface of solid-medium-grown mycelia, and only dikaryotic, and not monokaryotic, mycelia produced the lectin. These results suggest that the appearance of this lectin is associated with fruit body formation.Lectins are carbohydrate-binding proteins that are found in various organisms (21). Although there have been several reports of fungal lectins found in fruit bodies (8-10, 13, 18, 23, 24), there are few reports of mycelial lectins (17), and their physiological functions in nature have not been completely explained yet. We have found a new lectin in the fruit body of Pleurotus cornucopiae and characterized it (8, 24). This lectin was not contained in vegetatively growing mycelia in liquid medium, but it appeared after the onset of fruit body formation on solid medium (8). In the course of studies on the function of this lectin, we found and established two strains of P. cornucopiae, named KC-1 and KC-2, with respect to the lectin content in their fruit bodies (14,15).In this study, we found that mycelia of P. cornucopiae grown on solid medium exhibit hemagglutinating activity and that the active substance is different from the lectin found in the fruit body. We describe here the purification and properties as well as localization and developmental stage-specific expression of this novel mycelial lectin, suggesting that the lectin participates in the process of fruit body formation in this organism. The results presented in this paper together with those of our other studies (8, 24) demonstrate that two lectins are produced in P. cornucopiae in a developmental stage-specific manner. MATERIALS AND METHODSOrganisms. Two strains of P. cornucopiae, KC-1 and KC-2, were studied. The former contains the fruit body lectin (PCL-F), but the latter does not (15,24). Dikaryotic mycelia of these strains were maintained on agar slants of yeast extract-malt extract-glucose medium (8). Monokaryotic mycelia were obtained by single-spore isolation.Fruit body formation. Dikaryotic mycelia on the slant were first grown in liquid yeast extract-malt extract-glucose medium at 28ЊC for 7 days with shaking (stage 0). The liquid-medium-grown mycelia were inoculate...

New lectins from aspergilli and their carbohydrate specificity

“…Pleurotus cornucopiae has been reported to synthesize a mycelial lectin in a developmental stage specific manner. This lectin was found localized on the surface of the growing mycelia on solidified medium, whereas mycelia from the broth cultures did not show any lectin activity (Oguri et al 1996). Fungal cultures in broth did not produce as many spores as the mycelia grown on solidified medium, indicating that the lectin activity was not spore specific.…”

Properties of Mycelial Aggregate-specific Lectin ofPleurotus cornucopiaeProduced inPichia pastoris

“…As shown in Table 2, the hemagglutinating activity of the expression product was inhibited most strongly by N-acetyl-D-galactosamine, and it was also inhibited by D-galactose, D-fucose, and lactose but not by N-acetyl-Dglucosamine, as was the native PCL-M. 6) These results indicate that although glycosylation occurred more than in the case of the native PCL-M, the expression product in P. pastoris is functional. Further studies using this product on the role of PCL-M in the process of fruitbody formation are in progress.…”

“…[1][2][3] We have reported that in Pleurotus cornucopiae two kinds of lectins were synthesized in a developmental stage-specific manner, one in fruitbodies and the other in mycelial aggregates. [4][5][6][7] The lectin from the mycelial aggregates (PCL-M) is active when the 40-kDa subunit forms a multimer through a disulfide bridge in the presence of Ca 2þ . 6) We cloned the cDNA of the PCL-M subunit, and sequenced (Sumisa, F., et al, unpublished results).…”

“…[4][5][6][7] The lectin from the mycelial aggregates (PCL-M) is active when the 40-kDa subunit forms a multimer through a disulfide bridge in the presence of Ca 2þ . 6) We cloned the cDNA of the PCL-M subunit, and sequenced (Sumisa, F., et al, unpublished results). PCL-M is composed of 373 amino acids, thirty-three of which constitute a signal sequence.…”

“…One of the transformants was cultured in a 3-liter table-top glass fermenter (Model TS-M; Takasugi Seisakusho, Tokyo) using basal salts as the medium with the addition of methanol as a carbon source and an inducer for gene expression as reported. 8) Since PCL-M is known to have an affinity toward porcine stomach mucin (PSM), 6) it was purified by a batch-wise affinity chromatography using PSM as a ligand. PSM was coupled with N-hydroxysuccinimideactivated Sepharose (Amersham Biosciences).…”