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Biochemical and structural properties of three recombinant (R), highly homologous, plant bifunctional nucleases from tomato (R-TBN1), hop (R-HBN1) and Arabis brassica (R-ABN1) were determined. These nucleases cleave single- and double-stranded substrates, as well as both RNA and DNA with nearly the same efficiency. In addition, they are able to cleave several artificial substrates and highly stable viroid RNA. They also possess 3'-nucleotidase activity; therefore, they can be classified as nuclease I family members. Interestingly, poly(G) is resistant to cleavage and moreover it inhibits dsDNase, ssDNase and RNase activity of the studied nucleases. All three nucleases exhibit zinc-dependence and a strong stimulatory effect of Zn²+ for dsDNA cleavage. 3-D models, predicted on the basis of experimental structure of P1 nuclease, show nine amino acid residues responsible for interactions with zinc atoms, located in the same positions as in P1 nuclease. It was also shown that R-TBN1, R-HBN1, and R-ABN1 are all N-glycosylated. Oligosaccharidic chains constitute about 16% of their MW. In addition, an anticancer potential of the R-ABN1 is compared in this work with previously tested R-TBN1, and R-HBN1. R-ABN1 injected intravenously showed 70% inhibitory effect on growth of human prostate carcinoma in athymic mice., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Nuclease from tomato (TBN1) was produced by in planta biotechnology purified and tested for its anticarcinogenic properties. The nuclease was cytostatic after its intratumoral administration to nude mice bearing human melanoma or prostate carcinoma or after tumor targeting by TBN1 administration intravenously as conjugate with polyethylene glycol (PEG). Inhibitory effects of TBN1 on tumor growth were comparable to effects of bovine seminal RNase (BS-RNase), but the inhibition was reached at about ten times lower protein concentration. Simultaneously, TBN1 exhibited a lower degree of embryotoxicity compared to BS-RNAse and other nucleases. TBN1 showed significant stability in vivo, because it was readily detected after its administration intratumorally or intravenously by the fluorescence methods. Intravenous administration of TBN1-PEG caused significant inhibition of tumor proliferation without obvious degenerative changes, while direct administration of TBN1 into melanoma tumors led to rapid tumor tissue degeneration. The fact can be essential for the mode of TBN1 biological action that mature nuclease is a small (36 kDa) thermostable glycoprotein that has ability to destroy human 28S, 18S, 7S and 5.8S RNA, circular RNAs, double-stranded RNA in vitro and shows DNase and 3'nucleotidase activities.

Recombinant plant nucleases R-TBN1 and R-HBN1 were isolated to homogeneity and examined for their antitumor effects and cytotoxicity. Although antiproliferative effects of both recombinant nucleases were not significant on the ML-2 cell culture in vitro, the nucleases were strongly cytostatic in vivo after their administration intravenously as stabilized conjugates with polyethylene glycol (PEG). Recombinant nucleases were as effective against melanoma tumors as previously studied pine pollen (PN) and mung bean nucleases and their effects were reached at about 10 times lower concentrations compared to the use of bovine seminal RNase (BS-RNase). Because the recombinant nucleases R-HBN1 and R-TBN1 share only 67.4% amino acid identity and showed only partial immunochemical cross-reactivity, their similar anticancerogenic effects can be mainly explained by their catalytical similarity. Both recombinant nucleases showed lower degree of aspermatogenesis compared to BS-RNAse and PN nuclease. Unlike BS-RNase, aspermatogenesis induced by both recombinant nucleases could not be prevented by the homologous antibody complexes. Owing to relatively low cytotoxicity on the one hand, and high efficiency at low protein levels on the other, recombinant plant nucleases R-HBN1 and R-TBN1 appear to be stable biochemical agents that can be targeted as potential antitumor cytostatics.