Biopolym. Cell. 2014; 30(6):454-461.
http://dx.doi.org/10.7124/bc.0008C0
Molecular and Cell Biotechnologies
Characterization of novel peptide-specific antibodies against the translation elongation factor eEF1A2 and their application for cancer research
1Shalak V. F., 1Vislovukh A. A., 1Novosylna O. V., 1Khoruzhenko A. I., 1Kovalenko M. I., 2Kolesanova E. F., 2Egorova E. A., 2, 3Mishin A. A., 4Krotevych M. S., 4Skoroda L. V., 1Negrutskii B. S.
  1. State Key Laboratory of Molecular and Cellular Biology
    Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  2. Institute of Biomedical Chemistry
    10/8, Pogodinskaya Str, Moscow, Russian Federation, 119121
  3. G. N. Gabrichevsky Research Institute for Epidemiology and Microbiology
    10, Admiral Makarov Str., Moscow, Russian Federation, 125212
  4. National Cancer Institute
    33/43, Lomonosova Str., Kyiv, Ukraine, 03022

Abstract

Aim. We intend to characterize the new peptide-specific antibodies against the isoform 2 of translation elongation factor 1A (eEF1A2) and determine its presence in the postoperative samples of human breast, lung and stomach tumor tissues. Methods. The analysis of antibody specificity was performed by enzyme-linked immunosorbent assay, immunoblotting and immunohistochemistry. Immunoblotting and immunohistochemistry were used for the determination of the eEF1A2 in the human tumor samples, as well as in the samples of normal tissues surrounding tumors. Results. The antibodies obtained against the eEF1A2 specifically recognized this protein in the cell extracts and histological sections and did not cross-react with the elongation factor 1A isoform 1. eEF1A2 was revealed in the postoperative samples of breast, lung and stomach tumors as well as in the putative normal tissues surrounding tumors. Conclusions. The antibodies obtained against eEF1A2 are highly specific for the antigen and can be used for the immunological studies of tumors.
Keywords: eEF1A2, anti-peptide antibodies, immunoblotting, immunohistochemistry.
Full text: (PDF, in English)

References

[1] Negrutskii BS, El'skaya AV. Eukaryotic translation elongation factor 1 alpha: structure, expression, functions, and possible role in aminoacyl-tRNA channeling. Prog Nucleic Acid Res Mol Biol. 1998;60:47-78.
[2] El'skaya AV, Negrutskii BS, Shalak VF, Vislovukh AA, Vlasenko DO, Novosylna AV, Lukash TO, Veremieva MV. Specific features of protein biosynthesis in higher eukaryotes. Biopolym Cell. 2013; 29(3):177-87.
[3] Mateyak MK, Kinzy TG. eEF1A: thinking outside the ribosome. J Biol Chem. 2010;285(28):21209-13.
[4] Li D, Wei T, Abbott CM, Harrich D. The unexpected roles of eukaryotic translation elongation factors in RNA virus replication and pathogenesis. Microbiol Mol Biol Rev. 2013;77(2):253-66.
[5] Lukash TO, Turkivska HV, Negrutskii BS, El'skaya AV. Chaperone-like activity of mammalian elongation factor eEF1A: renaturation of aminoacyl-tRNA synthetases. Int J Biochem Cell Biol. 2004;36(7):1341-7.
[6] Kahns S, Lund A, Kristensen P, Knudsen CR, Clark BF, Cavallius J, Merrick WC. The elongation factor 1 A-2 isoform from rabbit: cloning of the cDNA and characterization of the protein. Nucleic Acids Res. 1998;26(8):1884-90.
[7] Anand N, Murthy S, Amann G, Wernick M, Porter LA, Cukier IH, Collins C, Gray JW, Diebold J, Demetrick DJ, Lee JM. Protein elongation factor EEF1A2 is a putative oncogene in ovarian cancer. Nat Genet. 2002;31(3):301-5.
[8] Tomlinson VA, Newbery HJ, Wray NR, Jackson J, Larionov A, Miller WR, Dixon JM, Abbott CM. Translation elongation factor eEF1A2 is a potential oncoprotein that is overexpressed in two-thirds of breast tumours. BMC Cancer. 2005;5:113.
[9] Li R, Wang H, Bekele BN, Yin Z, Caraway NP, Katz RL, Stass SA, Jiang F. Identification of putative oncogenes in lung adenocarcinoma by a comprehensive functional genomic approach. Oncogene. 2006;25(18):2628-35.
[10] Vislovukh A, Kratassiouk G, Porto E, Gralievska N, Beldiman C, Pinna G, El'skaya A, Harel-Bellan A, Negrutskii B, Groisman I. Proto-oncogenic isoform A2 of eukaryotic translation elongation factor eEF1 is a target of miR-663 and miR-744. Br J Cancer. 2013;108(11):2304-11.
[11] Novosylna AV, Timchenko AA, Tiktopulo EI, Serdyuk IN, Negrutskii BS, El'skaya AV. Characterization of physical properties of two isoforms of translation elongation factor 1A. Biopolym Cell. 2007; 23(5):386-90.
[12] Timchenko AA, Novosylna OV, Prituzhalov EA, Kihara H, El'skaya AV, Negrutskii BS, Serdyuk IN. Different oligomeric properties and stability of highly homologous A1 and proto-oncogenic A2 variants of mammalian translation elongation factor eEF1. Biochemistry. 2013;52(32):5345-53.
[13] Kanibolotsky DS, Novosyl'na OV, Abbott CM, Negrutskii BS, El'skaya AV. Multiple molecular dynamics simulation of the isoforms of human translation elongation factor 1A reveals reversible fluctuations between "open" and "closed" conformations and suggests specific for eEF1A1 affinity for Ca2+-calmodulin. BMC Struct Biol. 2008;8:4.
[14] Ruest LB, Marcotte R, Wang E. Peptide elongation factor eEF1A-2/S1 expression in cultured differentiated myotubes and its protective effect against caspase-3-mediated apoptosis. J Biol Chem. 2002;277(7):5418-25.
[15] Chang R, Wang E. Mouse translation elongation factor eEF1A-2 interacts with Prdx-I to protect cells against apoptotic death induced by oxidative stress. J Cell Biochem. 2007;100(2):267-78.
[16] Whitlock NA, Lindsey K, Agarwal N, Crosson CE, Ma JX. Heat shock protein 27 delays Ca2+-induced cell death in a caspase-dependent and -independent manner in rat retinal ganglion cells. Invest Ophthalmol Vis Sci. 2005;46(3):1085-91.
[17] Talapatra S, Wagner JD, Thompson CB. Elongation factor-1 alpha is a selective regulator of growth factor withdrawal and ER stress-induced apoptosis. Cell Death Differ. 2002;9(8):856-61.
[18] Pecorari L, Marin O, Silvestri C, Candini O, Rossi E, Guerzoni C, Cattelani S, Mariani SA, Corradini F, Ferrari-Amorotti G, Cortesi L, Bussolari R, Raschell? G, Federico MR, Calabretta B. Elongation Factor 1 alpha interacts with phospho-Akt in breast cancer cells and regulates their proliferation, survival and motility. Mol Cancer. 2009;8:58.
[19] Cao H, Zhu Q, Huang J, Li B, Zhang S, Yao W, Zhang Y. Regulation and functional role of eEF1A2 in pancreatic carcinoma. Biochem Biophys Res Commun. 2009;380(1):11-6.
[20] Tomlinson VA, Newbery HJ, Bergmann JH, Boyd J, Scott D, Wray NR, Sellar GC, Gabra H, Graham A, Williams AR, Abbott CM. Expression of eEF1A2 is associated with clear cell histology in ovarian carcinomas: overexpression of the gene is not dependent on modifications at the EEF1A2 locus. Br J Cancer. 2007;96(10):1613-20.
[21] Pinke DE, Kalloger SE, Francetic T, Huntsman DG, Lee JM. The prognostic significance of elongation factor eEF1A2 in ovarian cancer. Gynecol Oncol. 2008;108(3):561-8.
[22] Sun Y, Wong N, Guan Y, Salamanca CM, Cheng JC, Lee JM, Gray JW, Auersperg N. The eukaryotic translation elongation factor eEF1A2 induces neoplastic properties and mediates tumorigenic effects of ZNF217 in precursor cells of human ovarian carcinomas. Int J Cancer. 2008;123(8):1761-9.
[23] Zhu H, Lam DC, Han KC, Tin VP, Suen WS, Wang E, Lam WK, Cai WW, Chung LP, Wong MP. High resolution analysis of genomic aberrations by metaphase and array comparative genomic hybridization identifies candidate tumour genes in lung cancer cell lines. Cancer Lett. 2007;245(1-2):303-14.
[24] Schlaeger C, Longerich T, Schiller C, Bewerunge P, Mehrabi A, Toedt G, Kleeff J, Ehemann V, Eils R, Lichter P, Schirmacher P, Radlwimmer B. Etiology-dependent molecular mechanisms in human hepatocarcinogenesis. Hepatology. 2008;47(2):511-20.
[25] Yamashita S, Tsujino Y, Moriguchi K, Tatematsu M, Ushijima T. Chemical genomic screening for methylation-silenced genes in gastric cancer cell lines using 5-aza-2'-deoxycytidine treatment and oligonucleotide microarray. Cancer Sci. 2006;97(1):64-71.
[26] Kolesanova EF, Farafonova TE, Aleshina EIu, Pyndyk NV, Verem'eva MV, Novosil'naia AV, Kovalenko MI, Shalak VF, Negrutski? BS. [Preparation of monospecific antibodies against isoform 2 of translation elongation factor 1A (eEF1A2)]. Biomed Khim. 2014;60(1):51-62.
[27] Shalak VF, Budkevich TV, Negrutski? BS, El'skaia AV. A fast and effective method for purification of elongation factor 1 alpha from rabbit liver. Ukr Biokhim Zh. 1997;69(2):104-9.
[28] Veremieva M, Khoruzhenko A, Zaicev S, Negrutskii B, El'skaya A. Unbalanced expression of the translation complex eEF1 subunits in human cardioesophageal carcinoma. Eur J Clin Invest. 2011;41(3):269-76.
[29] Paland N, Kamer I, Kogan-Sakin I, Madar S, Goldfinger N, Rotter V. Differential influence of normal and cancer-associated fibroblasts on the growth of human epithelial cells in an in vitro cocultivation model of prostate cancer. Mol Cancer Res. 2009;7(8):1212-23.
[30] Gao MQ, Kim BG, Kang S, Choi YP, Park H, Kang KS, Cho NH. Stromal fibroblasts from the interface zone of human breast carcinomas induce an epithelial-mesenchymal transition-like state in breast cancer cells in vitro. J Cell Sci. 2010;123(Pt 20):3507-14.