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Structural Distinctive 26SK, a Ribosome-Inactivating Protein from Jatropha curcas and Its Biological Activities

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Abstract

Ribosome-inactivating proteins (RIPs) are a group of proteins exhibiting N-glycosidase activity leading to an inactivation of protein synthesis. Thirteen predicted Jatropha curcas RIP sequences could be grouped into RIP types 1 or 2. The expression of the RIP genes was detected in seed kernels, seed coats, and leaves. The full-length cDNA of two RIP genes (26SK and 34.7(A)SK) were cloned and studied. The 34.7(A)SK protein was successfully expressed in the host cells while it was difficult to produce even only a small amount of the 26SK protein. Therefore, the crude proteins were used from E. coli expressing 26SK and 34.7(A)SK constructs and they showed RIP activity. Only the cell lysate from 26SK could inhibit the growth of E. coli. In addition, the crude protein extracted from 26SK expressing cells displayed the effect on the growth of MDA-MB-231, a human breast cancer cell line. Based on in silico analysis, all 13 J. curcas RIPs contained RNA and ribosomal P2 stalk protein binding sites; however, the C-terminal region of the P2 stalk binding site was lacking in the 26SK structure. In addition, an amphipathic distribution between positive and negative potential was observed only in the 26SK protein, similar to that found in the anti-microbial peptide. These findings suggested that this 26SK protein structure might have contributed to its toxicity, suggesting potential uses against pathogenic bacteria in the future.

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Abbreviations

RIP:

Ribosome-inactivating protein

rRNA:

Ribosomal RNA

RTA:

Ricin A chain

PMSF:

Phenylmethylsulfonyl fluoride

PBS:

Phosphate buffer saline

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Acknowledgements

The authors would like to thank Associate Professor Kittisak Yokthongwattana, Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand, for providing the plasmid, pETDuet-CPN60B1.

Funding

This work was supported in part by funding (v-t(d)45.54) to CY from the Kasetsart University Research and Development Institute (KURDI), Bangkok, Thailand.

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Authors and Affiliations

  1. Interdisciplinary Program in Genetic Engineering, Graduate School, Kasetsart University, 50 Ngamwongwan Rd., Bangkok, 10900, Thailand

    Danulada Pathanraj

  2. Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Ngamwongwan Rd., Bangkok, 10900, Thailand

    Kiattawee Choowongkomon & Chotika Yokthongwattana

  3. Functional Ingredients and Food Innovation Research Group, National Center for Genetic Engineering and Biotechnology, Pathum Thani, 12120, Thailand

    Sittiruk Roytrakul

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  1. Danulada Pathanraj
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  2. Kiattawee Choowongkomon
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  4. Chotika Yokthongwattana
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Contributions

D.P. and C.Y. conceived and designed the experiments. D.P. performed the experiments. D.P., K.C., S.R., and C.Y. analyzed the data. D.P. and C.Y. wrote the manuscript with contributions from all authors. All authors read and commented on the manuscript before submission.

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Correspondence to Chotika Yokthongwattana.

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Supplementary Fig. 1

The Growth rate of E. coli host cells expressing (a) 26SK and (b) 34.7(A)SK under inducing and non-inducing conditions. EV: E. coli carrying pET28a(+) or pETDuet-1 empty vector in (a) and (b) respectively; 26SK: E. coli containing pET28(a)+-26SK; 34.7(A)SK: E. coli harboring pETDuet-34.7(A)SK: CPN60B1: E. coli harboring pETDuet-CPN60B1; N: non-inducing condition; I: inducing condition (PNG 188 kb)

High resolution image (TIF 238 kb)

Supplementary Fig. 2

Recombinant RIPs production in E. coli cells. (a) SDS-PAGE (b) Western blot of pET28a(+) empty vector, CPN60B1, 26SK and 34.7(A)SK crude proteins under inducing and non-inducing conditions. M: Prestained SDS-PAGE standard broad range protein marker; EV: pET28a(+) empty vector; CPN60B1: E. coli harboring pETDuetTM-CPN60B1; 26SK: E. coli carrying pET28(a)+-26SK; 34.7(A)SK: E. coli containing pETDuetTM-34.7(A)SK; N: non- inducing condition; I: inducing condition. (Exposure time = 120 seconds) (PNG 1669 kb)

High resolution image (TIF 36659 kb)

Supplementary Fig. 3

Electrostatic potential map of transmembrane helices located in 26SK, 33curcin, 34.7(A)SK and luffin P1. Predicted conformation of 26SK, 33curcin, and 34.7(A)SK represented by green ribbons and luffin P1 transmembrane helices and transmembrane helices of 26SK, 33curcin and 34.7(A)SK display by black ribbons. Histogram presents the charge of electrostatic potential from -1.8 (red) to 1.8 (blue). (PNG 1175 kb)

High resolution image (TIF 47415 kb)

Supplementary Table 1

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Supplementary Table 2

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Supplementary Table 6

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Supplementary Table 7

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Pathanraj, D., Choowongkomon, K., Roytrakul, S. et al. Structural Distinctive 26SK, a Ribosome-Inactivating Protein from Jatropha curcas and Its Biological Activities. Appl Biochem Biotechnol 193, 3877–3897 (2021). https://doi.org/10.1007/s12010-021-03714-6

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