Sze-Kwan Lam

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 protein with antiproliferative, antifungal and HIV-1 reverse transcriptase inhibitory activities from caper (Capparis spinosa) seeds

A protein exhibiting an N-terminal amino acid sequence with some similarity to imidazoleglycerol phosphate synthase was purified from fresh Capparis spinosa melon seeds. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, cation exchange chromatography on SP-Sepharose, and finally gel filtration by fast protein liquid chromatography on Superdex 75. The protein was adsorbed using 20 mM Tris-HCl buffer (pH 7.4) and desorbed using 1 M NaCl in the starting buffer from the DEAE-cellulose column and SP-Sepharose column. The protein demonstrated a molecular mass of 38 kDa in gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it was monomeric. The protein inhibited proliferation of hepatoma HepG2 cells, colon cancer HT29 cells and breast cancer MCF-7 cells with an IC(50) of about 1, 40 and 60 microM, respectively. It inhibited HIV-1 reverse transcriptase with IC(50) of 0.23 microM. It inhibited mycelial growth in the fungus, Valsa mali. It did not exhibit hemagglutinating, ribonuclease, mitogenic or protease inhibitory activities.

Passiflin, a novel dimeric antifungal protein from seeds of the passion fruit.

The intent was to isolate an antifungal protein from seeds of the passion fruit (Passiflora edulis) and to compare its characteristics with other antifungal proteins and bovine beta-lactoglobulin in view of its N-terminal amino acid sequence similarity to beta-lactoglobulin. The isolation procedure entailed ion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on Phenyl-Sepharose, ion-exchange chromatography on DEAE-cellulose, and FPLC-gel filtration on Superdex 75. The isolated 67-kDa protein, designated as passiflin, exhibited an N-terminal amino acid sequence closely resembling that of bovine beta-lactoglobulin. It is the first antifungal protein found to have a beta-lactoglobulin-like N-terminal sequence. Its dimeric nature is rarely found in antifungal proteins. It impeded mycelial growth in Rhizotonia solani with an IC(50) of 16 microM and potently inhibited proliferation of MCF-7 breast cancer cells with an IC(50) of 15 microM. There was no cross-reactivity of passiflin with anti-beta-lactoglobulin antiserum. Intact beta-lactoglobulin lacks antifungal and antiproliferative activities and is much smaller in molecular size than passiflin. However, it has been reported that hydrolyzed beta-lactoglobulin shows antifungal activity. The data suggest that passiflin is distinct from beta-lactoglobulin.

A dimeric high-molecular-weight chymotrypsin inhibitor with antitumor and HIV-1 reverse transcriptase inhibitory activities from seeds of Acacia confusa.

A dimeric 70-kDa chymotrypsin inhibitor with substantial N-terminal sequence homology to serine protease inhibitors was isolated from Acacia confusa seeds. The chymotrypsin inhibitor was purified using a protocol that entailed ion exchange chromatography on Q-Sepharose, SP-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. The chymotrypsin inhibitor was unadsorbed on both Q-Sepharose and SP-Sepharose. Its chymotrypsin inhibitory activity was stable from pH 3 to 10 and from 0 to 50 degrees C. It exerted antiproliferative activity toward breast cancer MCF-7 cells with an IC(50) of 10.7+/-4.2 microM. It inhibited HIV-1 reverse transcriptase with an IC(50) of 8+/-1.5 microM. It was devoid of antifungal activity toward a variety of fungal species. The distinctive features of the chymotrypsin inhibitor included dimeric nature, a high molecular mass, lack of trypsin inhibitory activity, highly potent HIV-1 reverse transcriptase inhibitory activity, specific antitumor activity and relatively high pH-stability.

Differential abilities of the mushroom ribosome-inactivating proteins hypsin and velutin to perturb normal development of cultured mouse embryos.

The teratogenicity of two fungal ribosome-inactivating proteins, hypsin from Hypsizigus mamoreus and velutin from Flammulina velutipes, was examined in this investigation using microinjection and postimplantation whole-embryo culture. The results demonstrated that hypsin induced abnormal embryonic development at 2.5 microM during the organogenesis period from E8.5 to E9.5. As its dosage increased, there was an increase in the total number of abnormal embryos, a drop in the final somite number, and a rise of abnormal structures. Structural abnormalities were detected: open cranial neural tube, abnormal branchial arches, absence of forelimb buds and twisted body axis. The otic and optic placodes were, however, less affected. Histological study of the abnormal embryos revealed a correlation of increased cell death with abnormal structures, suggesting that induction of cell death by hypsin may account for its teratogenicity. In contrast, velutin did not exert any adverse influence on mouse development.

Antifungal Protein from Passion Fruit ( Passiflora edulis ) Seeds

Publisher Summary This chapter outlines the usage of passion fruit seeds and its contribution to health and nutrition. Passion fruit has an abundance of vitamins A and C, folic acid, niacin, calcium, iron, potassium, and other nutrients. Two steroidal saponins with three and four glucose moieties, respectively, have been found to exert fungicidal effects on Aspergillus and Fusarium spp. An antifungal protein has been isolated from seeds of the passion fruit (Passiflora edulis). Passiflin specifically inhibits the fungus Rhizoctonia solani, with an IC50 of 16 mM, but no effect is seen on two other fungi, Fusarium oxysporum and Mycosphaerella arachidicola. It suppresses proliferation of breast cancer cells with an IC50 of 15 μM, but there is no effect on hepatoma HepG2 cells. It is devoid of ribonuclease, hemagglutinating, and HIV-1 reverse transcriptase inhibitory activities that may be present in some antifungal proteins. In addition to the aforementioned antifungal proteins and peptides, passion fruit has other therapeutic effects. Dietary fiber from passion-fruit rind may be promising for treatment of ailments such as diabetes, colon cancer, and other diverticular diseases. 2S albumin-like antifungal proteins and peptides distinct from passiflin in species specificity of antifungal activity have also been isolated from passion fruit.

White Cabbage ( Brassica chinensis ) Seeds and Their Health Promoting Activities

Publisher Summary This chapter highlights the various nutritional aspects of white cabbage seeds. The seeds of Brassica sp. and the napins they contain constitute an important resource for animal nutrition and industrial oil production. White cabbage seeds produce a heterodimeric 11-kDa napin-like polypeptide with health-promoting activities. The polypeptide displays antibacterial activity against Pseudomonas aeruginosia , Bacillus subtilis , B. megaterium , and B. cereus . A heterodimeric 11-kDa napin-like polypeptide composed of a smaller (4-kDa) subunit and a larger (10-kDa) subunit was tested for trypsin-inhibitory activity, which has been reported for other napins, and for other activities, including translation inhibitory, antibacterial, antifungal, and ribonuclease activities, which are found in other seed proteins, such as antifungal proteins, lectins, ribosome-inactivating proteins, trypsin inhibitors, and ribonucleases. The 7-kDa subunit of the polypeptide closely resembles trypsin inhibitor in sequence. The polypeptide inhibits trypsin with a higher potency than its chymotrypsin inhibitory activity. Allergic response after consumption of cabbage is rare. Swelling of the face and throat has been reported in a woman after eating cabbage. Digestive discomfort may arise due to gas originating from cabbage.

Antifungal and Antiproliferative Activity of Spotted Bean (Phaseolus vulgaris cv.)

Publisher Summary This chapter highlights the antiproliferative and antifungal activities of spotted beans. The seeds of Phaseolus vulgaris cv., “spotted bean,” produce a peptide, which exhibits a molecular mass of 7.3 kDa and an N-terminal sequence typical of leguminous defensins. It can be obtained from the beans with a procedure used for isolating other defensins, including anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and cation exchange chromatography on SP-Sepharose. It inhibits fungal growth in Mycospharella arachidicola and Fusarium oxysporum with an IC50 of 1.8 μM for the latter fungus. It also inhibits proliferation of L1210 and MBL2 cancer cells with IC50 values of 0.23 μM and 9 μM, respectively. The seeds display diuretic, hypoglycemic, and hypotensive activities. The seed flour is used in the treatment of ulcers, and cancer of the blood. When boiled with garlic, they can be used to treat intractable coughs. The toxic lectin phytohemagglutinin is present in many varieties of beans, but is highly concentrated in red kidney beans. It is devoid of deoxyribonuclease, nuclease, hemagglutinating, and protease inhibitory activities which may be present in some of the antifungal proteins.

Antifungal and Mitogenic Activities of Cluster Pepper ( Capsicum frutescens ) Seeds

Publisher Summary This chapter explores the effects of pepper seeds on fungus and exhibiting mitogenic activities. Cluster pepper seeds produce a lectin, which inhibits spore germination and hyphal growth in two fungal species: Aspergillus flavus and Fusarium moniliforme . Lectins are proteins that selectively agglutinate blood cells of a particular blood group in the ABO blood group system. However, there is no inhibitory activity on other fungi, including Botrytis cinerea , F. graminearum , F. solani , and Physalospora piricola . The lectin elicits a mitogenic response from murine splenocytes in vitro, with a reduced potency compared with that of the jackbean lectin concanavalin A. Using anion exchange chromatography on DEAE-cellulose and Q-Sepharose and fast protein liquid chromatography on MonoQ can isolate it. It is adsorbed on all three ion exchangers. It exhibits similarity to the N-terminal sequence in rabbit lectin-like oxidized low-density lipoprotein receptor-1, and the C-terminal sequences of some viral polymerases. In traditional Chinese medicine, pepper is used for the treatment of anorexia, indigestion, gastrointestinal distension, rheumatism, loin pain, frostbite, and furunculosis. The fruits and leaves of the cluster pepper contain capsaicin, and can irritate the eye, mouth, and skin. Eating excessive quantities of pepper can also produce gastrointestinal symptoms.

Therapeutic Use of Caper ( Capparis spinosa ) Seeds

Publisher Summary This chapter appraises the therapeutic properties of caper seeds. Traditionally, Capparis spinosa has been used for the treatment of flatulence, rheumatism, liver function, arteriosclerosis, and kidney infection and as an anthelmintic and tonic. Its root bark extract has been employed for anemia, arthritis, edema, and gout. In Israel, capers have also been used for treating hypoglycemia. Capers have an abundance of the powerful antioxidant, quercetin. The seeds have a high content of protein, oil, and fiber and have a potential value as food. They produce two proteins: one is a lectin with hemagglutinating activity, whereas the other is a non-lectin protein without hemagglutinating activity. Both proteins exert antifungal activity toward Valsa mali. The caper plant exerts diuretic and antihypertensive actions. Flavonoids and hydrocinammic acids with antioxidant and anti-inflammatory actions are present in the plant extract. The methanolic extract of flowering buds is able to counteract the adverse effects induced by the proinflammatory cytokine on human chondrocyte cultures. C. spinosa , abundant in the antioxidant flavonoid rutin, may have the following applications in humans: anti-inflammatory, antimicrobial, antioxidant, hepatoprotective, and photoprotective. Some lectins, ribonucleases, protease inhibitors, and antifungal proteins manifest antiproliferative activity toward tumor cells.