Proteins with antifungal properties and other medicinal applications from plants and mushrooms.

Living organisms produce a myriad of molecules to protect themselves from fungal pathogens. This review focuses on antifungal proteins from plants and mushrooms, many of which are components of the human diet or have medicinal value. Plant antifungal proteins can be classified into different groups comprising chitinases and chitinase-like proteins, chitin-binding proteins, cyclophilin-like proteins, defensins and defensin-like proteins, deoxyribonucleases, embryo-abundant protein-like proteins, glucanases, lectins, lipid transfer proteins, peroxidases, protease inhibitors, ribonucleases, ribosome-inactivating proteins, storage 2S albumins, and thaumatin-like proteins. Some of the aforementioned antifungal proteins also exhibit mitogenic activity towards spleen cells, nitric oxide inducing activity toward macrophages, antiproliferative activity toward tumor cells, antibacterial activity, and inhibitory activity toward HIV-1 reverse transcriptase. In contrast to the large diversity of plant antifungal proteins, only a small number of mushroom antifungal proteins have been reported. Mushroom antifungal proteins are distinct from their plant counterparts in N-terminal sequence. Nevertheless, some of the mushroom antifungal proteins have been shown to inhibit HIV-1 reverse transcriptase activity and tumor cell proliferation.

A lectin with antifungal and mitogenic activities from red cluster pepper (Capsicum frutescens) seeds.

A monomeric mannose/glucose-binding lectin, with a molecular mass of 29.5 kDa and an N-terminal sequence GQRELKL showing resemblance to that of the lectin-like oxidized low-density lipoprotein receptor from the rabbit, has been isolated from the seeds of red cluster pepper Capsium frutescens L. var. fasciculatum. The protocol involved anion exchange chromatography on diethylamino ethanol-cellulose and Q-Sepharose and fast protein liquid chromatography on Mono Q. Its hemagglutinating activity toward rabbit erythrocytes was inhibited by D: -mannose and glucose, specifically. The activity was stable from 0 to 40 degrees C, reached a maximum at pH 7 and 8, and was potentiated by Ca2+ and Mn2+ ions. The lectin showed strong mitogenic activity toward spleen cells isolated from BALB/c mice. The mitogenic activity, which reached a peak at a lectin concentration of 0.27 microM, was inhibited specifically by D(+)-mannose. The lectin was capable of inhibiting the germination of Aspergillus flavus and Fusarium moniliforme spores and hyphal growth in the two fungi.

A mannose-specific tetrameric lectin with mitogenic and antibacterial activities from the ovary of a teleost, the cobia (Rachycentron canadum).

A tetrameric lectin, with hemagglutinating activity toward rabbit erythrocytes and with specificity toward D-mannosamine and D(+)-mannose, was isolated from the ovaries of a teleost, the cobia Rachycentron canadum. The isolation protocol comprised ion exchange chromatography on CM-cellulose and Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q, and finally gel filtration by FPLC on Superose 12. The lectin was adsorbed on all ion exchangers used. It exhibited a molecular mass of 180 kDa in gel filtration on Superose 12 and a single 45-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is a tetrameric protein. The hemagglutinating activity of the lectin was stable up to 40 degrees C and between pH 4 and pH 10. All hemagglutinating activity disappeared at 60 degrees C and at pH 1 and pH 13. The hemagglutinating activity was doubled in the presence of 0.1 microM FeCl3. The lectin exerted antibacterial activity against Escherichia coli with 50% inhibition at 250 microg. There was no antifungal activity toward Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, and Rhizoctonia solani at a dose of 300 microg. The lectin exhibited maximal mitogenic response from mouse splenocytes at a concentration of 14 microM.

An agglutinin with mitogenic and antiproliferative activities from the mushroom Flammulina velutipes.

A hemagglutinin with a molecular mass of 12 kDa was isolated from the fruiting bodies of the mushroom Flammulina velutipes. Its molecular mass is similar to that of the fungal immunomodulatory protein isolated from F. velutipes (FIP-fve) with ice-cold 5% acetic acid and 50 mM 2-mercaptoethanol as extraction medium and to that of the larger 12 kDa subunit of F. velutipes lectin isolated with phosphate buffer as extraction medium. Its hemagglutinating activity cannot be inhibited by a variety of carbohydrates tested. The activity is stable between pH 4 and pH 11. Loss in activity occurred when the temperature is raised to 60 C and 70 C. Activity is indiscernible at and above 80 C. Its N-terminal sequence shows differences from that of FIP-fve. F. velutipes hemagglutinin stimulates [3H-methyl] thymidine uptake by mouse splenocytes. It inhibits proliferation of leukemia L1210 cells with an IC50 of 13 microM.

A hemolysin from the mushroom Pleurotus eryngii.

A monomeric 17-kDa hemolysin designated as eryngeolysin was isolated from fresh fruiting bodies of the mushroom Pleurotus eryngii, using a protocol that involved gel filtration on Superdex 75, ion exchange chromatography on Mono Q and gel filtration on Superdex 75. Its N-terminal sequence demonstrated striking homology to that of its counterparts ostreolysin from the oyster mushroom Pleurotus ostreatus and aegerolysin from the mushroom Agrocybe cylindracea. Its hemolytic activity was unaffected over the pH range 4.0-12.0, but no activity was observed at pH 13 and at and below pH 2. The hemolysin was stable between 0 and 30 degrees C. At 40 degrees C, only residual activity was detectable. At and above 50 degrees C, activity was indiscernible. Eryngeolysin exhibited cytotoxicity toward leukemia (L1210) cells but not toward fungi. The hemolysin was inactivated by treatment with trypsin. It exhibited antibacterial activity against Bacillus sp. but not against other species. It inhibited basal as well as ConA-stimulated mitogenic response of murine splenocytes. N-Glycolyneuraminic acid was the only sugar capable of inhibiting the hemolytic activity. Eryngeolysin-induced hemolysis was osmotically protected by polyethylene glycol (PEG) 10000 with a mean hydrated diameter dose to 9.3 nm. However, no protection was offered by PEG 10000 to the anti-mitogenic and antiproliferative activities of eryngeolysin. The susceptibility of erythrocytes from different classes of vertebrates to eryngeolysin was mammalian > avian > reptilian > piscine.

Phaseococcin, an antifungal protein with antiproliferative and anti-HIV-1 reverse transcriptase activities from small scarlet runner beans.

From the seeds of small scarlet runner beans (Phaseolus coccineus 'Minor'), an antifungal protein with an N-terminal sequence homologous to those of defensins was isolated. The antifungal protein bound to Affi-gel blue gel and Mono S but it did not bind to DEAE-cellulose. It was further purified by gel filtration on a Superdex peptide column. It exhibited a molecular mass of 5422 Da as determined by mass spectrometry. The protein, designated as phaseococcin, suppressed mycelial growth in a number of fungi including Botrytis cinerea, Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, Physalospora piricola, and Rhizoctonia solani. It also inhibited proliferation in several Bacillus species and the leukemia cell lines HL60 and L1210 and curtailed the activity of HIV-1 reverse transcriptase. It did not affect proliferation of mouse splenocytes and neither did it inhibit protein synthesis in a cell-free rabbit reticulocyte lysate system.

Agrocybin, an antifungal peptide from the edible mushroom Agrocybe cylindracea.

An antifungal peptide with a molecular mass of 9 kDa was isolated from fresh fruiting bodies of the mushroom Agrocybe cylindracea. The isolation procedure comprised ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and FPLC-gel filtration on a Superdex 75 column. The antifungal peptide, designated as agrocybin, was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and Mono S. Agrocybin exerted antifungal activity against several fungal species but lacked inhibitory activity against bacteria when tested up to 300 microM. The activity of HIV-1 reverse transcriptase was attenuated in the presence of agrocybin. It exhibited weaker mitogenic activity than Con A on isolated murine splenocytes, but was devoid of antiproliferative activity on Hep G2 (hepatoma) cells when tested at 110 microM.

Coccinin, an antifungal peptide with antiproliferative and HIV-1 reverse transcriptase inhibitory activities from large scarlet runner beans.

An antifungal peptide, designated coccinin, with a molecular mass of 7kDa and an N-terminal sequence resembling those of defensins, was purified from the seeds of large scarlet runner beans (Phaseolus coccineus cv. 'Major'). The peptide isolated was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and Mono S. The peptide excerted antifungal activity on a number of fungal species including Botrytis cinerea, Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, Physalospora piricola, and Rhizoctonia solani. It also inhibited proliferation in the leukemia cell lines HL60 and L1210, and reduced the activity of HIV-1 reverse transcriptase. However, it did not affect proliferation of mouse splenocytes.

A ribonuclease with antimicrobial, antimitogenic and antiproliferative activities from the edible mushroom Pleurotus sajor-caju.

A 12 kDa ribonuclease preferential for poly U and with much lower activity toward poly A, poly G and poly C was isolated from fresh fruiting bodies of the mushroom Pleurotus sajor-caju. A purification procedure involving ion exchange chromatography on CM-cellulose, affinity chromatography on Red-Sepharose and Heparin-Sepharose, and fast protein liquid chromatography-gel filtration on Superdex 75 was used. The ribonuclease was adsorbed on all of the first three types of chromatographic media. It exhibited some activity toward herring sperm DNA and calf thymus DNA. The ribonuclease activity was unaffected in the presence of KCl (10 and 100 mM) and NaCl (100 mM and 1 M), but was strongly inhibited by CuSO4 (0.01 and 0.1 mM) and less potently inhibited by other divalent salts including MgCl2, CaCl2, ZnCl2, ZnSO4 and FeSO4. The optimal pH was 5.5 and the ribonuclease was stable up to 60 degrees C for 1 h. The ribonuclease inhibited mycelial growth in the fungi Fusarium oxysporum and Mycosphaerella arachidicola with an IC50 value of 95 and 72 microM, respectively. Out of the 12 species of bacteria tested, only Pseudomonas aeruginosa and Staphylococcus aureus were inhibited in growth by the ribonuclease. Viability of the tumor cells HepG2 (hepatoma) and L1210 (leukemia) was reduced with an IC50 of 0.22 and 0.1 microM, respectively in the presence of the ribonuclease. The ribonuclease inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC50 of 158 nM and 3H-methyl-thymidine uptake by murine splenocytes with an IC50 of 65 nM.

The trypsin-inhibitory, immunostimulatory and antiproliferative activities of a napin-like polypeptide from Chinese cabbage seeds.

A heterodimeric 13.8 kDa napin-like polypeptide has previously been isolated from Chinese cabbage (Brassica parachinensis) seeds with a procedure involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, FPLC-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75. In the present study the N-terminal sequence of the 8.8 kDa subunit of the polypeptide (PQGPQQRPPKLLQQQTNEEHE) was found to have pronounced homology to napins, albumins and trypsin inhibitors, but demonstrated little similarity to the 5 kDa subunit. The polypeptide stimulated nitrite production by mouse peritoneal macrophages and reduced the viability of leukaemia (L1210) cells. It inhibited trypsin with a higher potency than it inhibited chymotrypsin, but was devoid of ribonuclease and antifungal activities.

A mushroom (Ganoderma capense) lectin with spectacular thermostability, potent mitogenic activity on splenocytes, and antiproliferative activity toward tumor cells.

An 18-kDa lectin, with an N-terminal sequence displaying slight similarity to some lectins and fungal immunomodulatory proteins, was isolated from the mushroom Ganoderma capense (Lloyd) Teng. It exhibited more potent mitogenic activity than that of concanavalin A toward mouse splenocytes, and antiproliferative activity toward leukemia (L1210 and M1) cells and hepatoma (HepG2) cells. The isolation procedure entailed ion exchange chromatography on Q-Sepharose, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 75. D(+)-galactose and D(+)-galactosamine specifically inhibited the hemagglutinating activity of the lectin. The hemagglutinating activity of the lectin was not affected over the temperature range 0-100 degrees C and after exposure to 100 degrees C for 60min. The activity was stable in the pH range of 4-11, and after incubation with solutions of various chlorides (from 3.125 to 50mM) including NaCl, KCl, CaCl(2), MgCl(2), ZnCl(2), MnCl(2), and AlCl(3). However, it was potentiated by 12.5-50mM FeCl(3). The lectin was devoid of HIV-1 reverse transcriptase inhibitory and antifungal activities.

Purification of glysojanin, an antifungal protein, from the black soybean Glycine soja.

A monomeric protein, with a molecular mass of 25 kDa and an N-terminal sequence resembling a segment of chitin synthase, was isolated from the seeds of the black soybean Glycine soja. The protein, designated glysojanin, demonstrated potent antifungal activity against the fungi Fusarium oxysporum and Mycosphaerella arachidicola. It inhibited HIV-1 reverse transcriptase with an IC50 of 47 micromol/L, [methyl-3H]thymidine incorporation by mouse spleen cells with an IC50 of 175 micromol/L, and translation in the rabbit reticulocyte lysate with an IC50 of 20 micromol/L. Glysojanin was purified using a procedure that involved ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography by fast protein liquid chromatography on Mono S, and gel filtration by fast protein liquid chromatography on Superdex 75.

Lentin, a novel and potent antifungal protein from shitake mushroom with inhibitory effects on activity of human immunodeficiency virus-1 reverse transcriptase and proliferation of leukemia cells.

From the fruiting bodies of the edible mushroom Lentinus edodes, a novel protein designated lentin with potent antifungal activity was isolated. Lentin was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and Mono S. The N-terminal sequence of lentin manifested similarity to endoglucanase. Lentin, which had a molecular mass of 27.5 kDa, inhibited mycelial growth in a variety of fungal species including Physalospora piricola, Botrytis cinerea and Mycosphaerella arachidicola. Lentin also exerted an inhibitory activity on HIV-1 reverse transcriptase and proliferation of leukemia cells.

Isolation of a napin-like polypeptide with potent translation-inhibitory activity from Chinese cabbage (Brassica parachinensis cv green-stalked) seeds.

A heterodimeric napin-like polypeptide was isolated from Brassica parachinensis seeds with a procedure involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, FPLC-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75. The N-terminal sequence of the 5 kDa subunit of the polypeptide (PAGPFRIPKKRKKEE) showed high homology with other 2S storage proteins like napins and albumins. The polypeptide potently inhibited translation in a cell free system with an IC50 of 6.2 nM. The translation-inhibiting activity of the polypeptide was relatively stable in the pH range 6-11 and in the temperature range 10-50 degrees C.

Purification and characterization of a ubiquitin-like peptide with macrophage stimulating, antiproliferative and ribonuclease activities from the mushroom Agrocybe cylindracea.

A peptide, with a molecular mass of 9.5kDa and demonstrating an N-terminal sequence similar to ubiquitin, was isolated from fruiting bodies of the mushroom Agrocybe cylindracea. The peptide was isolated with a purification protocol involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, FPLC-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75. The peptide was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and Mono S. It showed antiproliferative activity on leukemia cell line (M1) and hepatoma cell line (HepG2), and enhanced nitric oxide production in murine peritoneal macrophages with a potency comparable to that of lipopolysaccharide. A pH of 6.0 was required for optimal RNase activity. Its RNase activity was stable over the temperature range of 0-60 degrees C. It exerted ribonucleolytic activity preferentially on polyC, much lower activity on polyU, and negligible activity on polyA and polyG.