A Study of the Effect of Human Prothrombin’s Signal Peptide on the Secretion Efficiency of Recombinant Human FIX in the HEK293T Cell Line

Authors
M. Behmanesh 1
S. Khorshidi 1
A. Zomorodipour 2
M. Behmanesh 1
1 Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
2 Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
Abstract
Objective: Eukaryotic proteins generally have signal peptides which are not only crucial for their secretion efficiencies but are important for their expression levels. Coagulation factor IX (FIX) is a glycoprotein that plays a fundamental role in the blood coagulation pathway. Reduced levels or dysfunctional FIX are associated with hemophilia B. This study investigates the function of the human prothrombin signal peptide in an attempt to improve the human FIX (hFIX) secretion efficiency in a heterologous expression system. With this aim, we have used the SignalP and PrediSi programs for in silico evaluation of the signal peptide efficiency prior to conducting this experiment.
Methods: We used molecular techniques to amplify and join the coding region of the human prothrombin signal peptide to the cDNA of mature hFIX. The chimeric fragment was examined for transient expression in a mammalian cell line (HEK293T) in comparison with the native hFIX, under a CMVp regulation. Using the neural network-based prediction programs, we evaluated the scores for cleavage position and secretion efficiency of the human prothrombin and hFIX signal peptides. The expression efficiencies of hFIX expressed by the recombinant cells were analyzed by RT-PCR and ELISA.
Results: In silico analysis more efficiently predicted the human prothrombin signal peptide with a high score compared to the native hFIX signal peptide. This data was confirmed by the RT-PCR and ELISA results obtained from expression analyses at the RNA and protein levels, respectively.
Conclusion: The present study showed that the signal peptide derived from the human prothrombin has the potential for efficient secretion of hFIX as evidenced by the results taken from a transient expression system. The results were consistent with in silico analysis. This replacement could be evaluated in a stable state condition.

Keywords

[1]     Mannucci PM, Tuddenham EG. The hemophilias--from royal genes to gene therapy. N Engl J Med 2001; 344(23): 1773-9.
[2]     Vatandoost J, Zomorodipour A, Sadeghizadeh M, Aliyari R, Bos MH, Ataei F. Expression of biologically active human clotting factor IX in Drosophila S2 cells: γ-carboxylation of a human vitamin K-dependent protein by the insect enzyme. Biotechnol Prog 2012; 28(1): 45-51.
[3]     Jackson CM, Nemerson Y. Blood coagulation. Annu Rev Biochem 1980; 49: 765-811.
[4]     Bandyopadhyay PK. Vitamin K-dependent gamma-glutamylcarboxylation: an ancient posttranslational modification. Vitam Horm 2008; 78: 157-84.
[5]     Nielsen H, Engelbrecht J, Brunak S, von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 1997; 10(1): 1-6.
[6]     Martoglio B, Dobberstein B. Signal sequences: more than just greasy peptides. Trends Cell Biol 1998; 8(10): 410-5.
[7]     Zhang L, Leng Q, Mixson AJ. Alteration in the IL-2 signal peptide affects secretion of proteins in vitro and in vivo. J Gene Med 2005; 7(3): 354-65.
[8]     Menne KM, Hermjakob H, Apweiler R. A comparison of signal sequence prediction methods using a test set of signal peptides. Bioinformatics 2000; 16(8): 741-2.
[9]     Jorgensen MJ, Cantor AB, Furie BC, Brown CL, Shoemaker CB, Furie B. Recognition site directing vitamin K-dependent gamma-carboxylation resides on the propeptide of factor IX. Cell 1987; 48(2): 185-91.
[10] Bristol JA, Ratcliffe JV, Roth DA, Jacobs MA, Furie BC, Furie B. Biosynthesis of prothrombin: intracellular localization of the vitamin K-dependent carboxylase and the sites of gamma-carboxylation. Blood 1996; 88(7): 2585-93.
[11] Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 2004; 340(4): 783-95.
[12] Hiller K, Grote A, Scheer M, Münch R, Jahn D. PrediSi: prediction of signal peptides and their cleavage positions. Nucleic Acids Res 2004; 32(Web Server issue): W375-9.
[13] Eisenberg D, Weiss RM, Terwilliger TC, Wilcox W. Hydrophobic moments and protein structure. Faraday Symp Chem Soc 1982; 17: 109-20.
[14] Zanen G, Houben EN, Meima R, Tjalsma H, Jongbloed JD, Westers H, Oudega B, Luirink J, van Dijl JM, Quax WJ. Signal peptide hydrophobicity is critical for early stages in protein export by Bacillus subtilis. FEBS J 2005; 272(18): 4617-30.
[15] Kim YK, Shin HS, Tomiya N, Lee YC, Betenbaugh MJ, Cha HJ. Production and N-glycan analysis of secreted human erythropoietin glycoprotein in stably transfected Drosophila S2 cells. Biotechnol Bioeng 2005; 92(4): 452-61.
[16] Sam MR, Zomorodipour A, Shokrgozar MA, Ataei F, Haddad-Mashadrizeh A, Amanzadeh A. Enhancement of the human factor IX expression, mediated by an intron derived fragment from the rat aldolase B gene in cultured hepatoma cells. Biotechnol Lett 2010; 32(10): 1385-92.
[17] Soejima Y, Lee JM, Nagata Y, Mon H, Iiyama K, Kitano H, Matsuyama M, Kusakabe T. Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system. Cent Eur J Biol 2013; 8(1): 1-7.
[18] Stern B, Olsen LC, Tröße C, Ravneberg H, Pryme IF. Improving mammalian cell factories: The selection of signal peptide has a major impact on recombinant protein synthesis and secretion in mammalian cells. Trends Cell Mol Biol 2007; 2: 1-17.
[19] Serruto D, Galeotti CL. The signal peptide sequence of a lytic transglycosylase of Neisseria meningitidis is involved in regulation of gene expression. Microbiology 2004; 150(Pt 5): 1427-37.
[20] Hatsuzawa K, Tagaya M, Mizushima S. The hydrophobic region of signal peptides is a determinant for SRP recognition and protein translocation across the ER membrane. J Biochem 1997; 121(2): 270-7.
[21] Bird P, Gething MJ, Sambrook J. The functional efficiency of a mammalian signal peptide is directly related to its hydrophobicity. J Biol Chem 1990; 265(15): 8420-5.
[22] Knappskog S, Ravneberg H, Gjerdrum C, Trösse C, Stern B, Pryme IF. The level of synthesis and secretion of Gaussia princeps luciferase in transfected CHO cells is heavily dependent on the choice of signal peptide. J Biotechnol 2007; 128(4): 705-15.
Pathobiology Reserach
Volume 18, Issue 2
June 2015
Pages 27-39