Secreted alkaline ribonucleases of micromycetes

V.V. Ulyanova^*, P.Yu. Vankov^**, P.V. Zelenikhin^***, R. Shah Mahmud^****, A.I. Kolpakov^*****, O.N. Ilinskaya^******

Kazan Federal University, Kazan, 420008 Russia

E-mail: ^*ulyanova.vera@gmail.com, ^**vankov93@bk.ru, ^***pasha_mic@mail.ru, ^****raihan.shah@gmail.com, ^*****ljoscha@mail.ru, ^******Ilinskaya_kfu@mail.ru

Received April 8, 2019

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DOI: 10.26907/2542-064X.2020.3.335-349

For citation: Ulyanova V.V., Vankov P.Yu., Zelenikhin P.V., Shah Mahmud R., Kolpakov A.I., Ilinskaya O.N. Secreted alkaline ribonucleases of micromycetes. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2020, vol. 162, no. 3, pp. 335–349. doi: 10.26907/2542-064X.2020.3.335-349. (In Russian)

Abstract

Many ribonucleases (RNases) have a selective cytotoxic effect on tumor cells. Of particular interest are enzymes that are phylogenetically distant from mammalian RNases, such as amphibian, fungal, and bacterial RNases, all insensitive to the action of the mammalian RNAse inhibitor. We screened 15 micromycete isolates for the presence of secreted alkaline ribonuclease. Seven isolates demonstrated the highest ribonucleolytic activity. Based on the phylogenetic analysis, the most active of them were identified as Fusarium sporotrichioides. The maximum level of activity (19000 units/mL) in the culture fluid of F. sporotrichioides was recorded on the third day of growth of the fungus. The obtained data on the thermal stability of the secreted F. sporotrichioides RNase, its molecular weight (11 kDa), and biosynthesis on a medium with a low phosphate content suggest that the enzyme belongs to the RNase T1/N1/U2 family. The culture fluid of F. sporotrichioides grown on the phosphate-deficient medium reduced the viability of A549 human lung adenocarcinoma cells by 16.3%; if the micromycete was grown on a medium with a high phosphate content, no toxicity of the culture fluid was detected. Thus, the secreted RNAse contributes to cytotoxicity. Further description and comprehensive study of the properties of the new RNase will help to establish the mechanisms of its cytotoxicity and identify the areas of application of the enzyme.

Keywords: micromycetes, Fusarium, ribonuclease, thermostability, cytotoxicity, A549 lung adenocarcinoma cells

Acknowledgements. We are grateful to Galina Yurievna Yakovleva (PhD in Biology, Associate Professor of the Department of Microbiology of Kazan Federal University) for the gift of strains of micromycetes needed for this research.

The study was supported by the Russian Foundation for Basic Research (project no. 17-00-00060 (KOMFI)), as well as performed according to the Russian Government Program of Competitive Growth of Kazan Federal University.

Figure Captions

Fig. 1. Ribonucleolytic activity exhibited by the culture fluid of seven micromycetes on the third day of cultivation; (1) – Fusarium sp. isolate 1; (2) – Penicillium sp. isolate 1; (3) – Mucor sp.; (4) – Penicillium sp. isolate 2; (5) – Penicillium sp. isolate 3; (6) – Fusarium sp. isolate 2, (7) – control well with 25 μL of Ogato’s sterile medium; (8) – Penicillium sp. isolate 4; (9) – control well with 25 μL of water. The isolate, which is most active in relation to the extracellular ribonuclease synthesis, is marked with a circle.

Fig. 2. Taxonomic positions of two micromycetes – Fusarium sp. 2 (a) and Mucor (b), which had the highest ribonucleolytic activity. Unrooted phylogenetic tree was inferred from the ITS1-ITS4 fragments by the neighbor-joining method. Numbers in the tree nodes indicate bootstrap support indices generated using 1000 replicates. Scale – substitutions per nucleotide position.

Fig. 3. Dynamics of the ribonucleolytic activity level in the culture fluid of Fusarium sporotrichioides and Mucor circinelloides in unmodified Ogato’s medium.

Fig. 4. Changes in the RNase activity of the culture fluid of Fusarium sporotrichioides grown on Ogato’s medium with modifications: Key: 1 – Ogato’s medium, 2 – Ogato’s medium with 0.1 g/L CaCO₃, 3 – Ogato’s medium with the nitrate concentration reduced by 5 times, 4 – Ogato’s medium with regular peptone replaced by the phosphate-deficient one.

Fig. 5. Changes in the viability of A549 cells depending on the concentration of the culture fluid of Fusarium sporotrichioides grown on Ogato’s phosphate-deficient medium for three days. Cell growth upon the addition of unmodified Ogato’s medium was taken as 100%. The proportion of the introduced culture fluid of Fusarium sporotrichioides is given as a percentage of the total volume of the culture medium of cells (black columns). Shaded column – the viability of cells upon the addition of 10% culture fluid of the micromycete grown on Ogato’s medium with phosphate. Mean values ± standard deviations are shown; * p < 0.05 compared with the control variant.

Fig. 6. RNase detection in the culture fluid of Fusarium sporotrichioides by the denaturing gradient gel electrophoresis (a) and zymography (b). Numbers indicate the culturing time of the micromycete on Ogato’s medium with regular (+Phosphate) and phosphate-deficient (–Phosphate) peptone: 3 – three days, 7 – seven days. Bi – preparation of binase, a secreted alkaline RNase of Bacillus pumilus. M – protein molecular weight marker (Mm).

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