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Optimizing Human Epidermal Growth Factor for its Endurance and Specificity Via Directed Evolution: Functional Importance of Leucine at Position 8

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Abstract

Epidermal growth factor regulates cell proliferation and migration by binding and activating the extracellular region of its receptor. Many engineered EGFs with enhanced agonist activity are produced, yet the short half-life and poor environmental tolerance of EGF limits its physiological application. By using a more stable porcine EGF as the template in the staggered extension process, two mutants of human and porcine EGF, hL8P and pP8L, were obtained with improved targeting towards normal cells. The comparative studies of hEGF and mutants were conducted to evaluate their activities as agonists. The mutants showed specificity for the proliferation and migration of normal and cancer cells, i.e. both pP8L and hL8P promoted the proliferation and migration of normal cells while delayed that of cancer cells. Both mutants preserved the structure thermostability of hEGF and pP8L showed improved acidic tolerance than hEGF. For the metabolism analysis in HEK293T cells, hL8P and pP8L demonstrated the lower rate of both mutants-EGFR internalization and biodegradation, but higher recycling to the cell surface. This study not only enhanced EGF agonist activity without loss of endurance but also provided a new application foreground for trauma therapy and cancer targeting therapy.

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Fig. 1

source EGF, and p means porcine source EGF. d ELISA detects EGFR affinity of phage display libraries. The dashed line is the absorbance of the hEGF sample. Data represents the mean ± SD (*p < 0.05; **p < 0.01)

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References

  1. Abdolalizadeh J, Nouri M, Zolbanin JM, Barzegari A, Baradaran B, Barar J, Coukos G, Omidi Y (2013) Targeting cytokines: production and characterization of anti-TNF-alpha scFvs by phage display technology. Curr Pharm Des 19(15):2839–2847

  2. Bai S, Ingram P, Chen YC, Deng N, Pearson A, Niknafs YS, O'Hayer P, Wang Y, Zhang ZY, Boscolo E, Bischoff J, Yoon E, Buckanovich RJ (2016) EGFL6 regulates the asymmetric division, maintenance, and metastasis of ALDH+ ovarian cancer cells. Cancer Res 76(21):6396–6409

  3. Bergeron JJ, Di Guglielmo GM, Dahan S, Dominguez M, Posner BI (2016) Spatial and temporal regulation of receptor tyrosine kinase activation and intracellular signal transduction. Annu Rev Biochem 85:573–597

  4. Bhirde AA, Patel V, Gavard J, Zhang G, Sousa AA, Masedunskas A, Leapman RD, Weigert R, Gutkind JS, Rusling JF (2009) Targeted killing of cancer cells in vivo and in vitro with EGF-directed carbon nanotube-based drug delivery. ACS Nano 3(2):307–316

  5. Calnan DP, Fagbemi A, Berlanga-Acosta J, Marchbank T, Sizer T, Lakhoo K, Edwards AD, Playford RJ (2000) Potency and stability of C terminal truncated human epidermal growth factor. Gut 47(5):622–627

  6. Carpenter G, Cohen S (1979) Epidermal growth factor. Annu Rev Biochem 48:193–216

  7. Chen J, Li H, Chen J (2017) Human epidermal growth factor coupled to different structural classes of cell penetrating peptides: a comparative study. Int J Biol Macromol 105(Pt 1):336–345

  8. Cochran JR, Kim YS, Lippow SM, Rao B, Wittrup KD (2006) Improved mutants from directed evolution are biased to orthologous substitutions. Protein Eng Des Sel 19(6):245–253

  9. Coco WM, Encell LP, Levinson WE, Crist MJ, Loomis AK, Licato LL, Arensdorf JJ, Sica N, Pienkos PT, Monticello DJ (2002) Growth factor engineering by degenerate homoduplex gene family recombination. Nat Biotechnol 20(12):1246–1250

  10. Currin A, Swainston N, Day PJ, Kell DB (2015) Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently. Chem Soc Rev 44(5):1172–1239

  11. Di Maida F, Mari A, Scalici Gesolfo C, Cangemi A, Allegro R, Sforza S, Cocci A, Tellini R, Masieri L, Russo A, Carini M, Minervini A, Serretta V (2019) Epidermal growth factor receptor (egfr) cell expression during adjuvant treatment after transurethral resection for non-muscle-invasive bladder cancer: a new potential tool to identify patients at higher risk of disease progression. Clin Genitourin Cancer 17(4):e751–e758

  12. Engler DA, Montelione GT, Niyogi SK (1990) Human epidermal growth factor: distinct roles of tyrosine 37 and arginine 41 in receptor binding as determined by site-directed mutagenesis and nuclear magnetic resonance spectroscopy. FEBS Lett 271(1–2):47–50

  13. GraphPad Software Inc., La Jolla California USA, 2018. https://www.graphpad.com/scientific-software/prism

  14. Greenfield NJ (2006) Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1(6):2876–2890

  15. Haglund K, Sigismund S, Polo S, Szymkiewicz I, Di Fiore PP, Dikic I (2003) Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation. Nat Cell Biol 5(5):461–466

  16. Hardwicke J, Moseley R, Stephens P, Harding K, Duncan R, Thomas DW (2010) Bioresponsive dextrin-rhEGF conjugates: in vitro evaluation in models relevant to its proposed use as a treatment for chronic wounds. Mol Pharm 7(3):699–707

  17. Johannes L, Parton RG, Bassereau P, Mayor S (2015) Building endocytic pits without clathrin. Nat Rev Mol Cell Biol 16(5):311–321

  18. Kang J, Tada S, Sakuragi M, Abe H, Ito R, Ishikawa J, Kurata S, Kitajima T, Son TI, Aigaki T, Ito Y (2013) An epidermal growth factor derivative with binding affinity for hydroxyapatite and titanium surfaces. Biomaterials 34(38):9747–9753

  19. Kirchhausen T, Owen D, Harrison SC (2014) Molecular structure, function, and dynamics of clathrin-mediated membrane traffic. Cold Spring Harb Perspect Biol 6(5):a016725

  20. Köstler WJ, Yarden Y (2011) The EGFR/ErbB family in breast cancer: from signalling to therapy. Springer, Basel, pp 1–32

  21. Kovacs E, Zorn JA, Huang Y, Barros T, Kuriyan J (2015) A structural perspective on the regulation of the epidermal growth factor receptor. Annu Rev Biochem 84:739–764

  22. Lenferink AE, van Zoelen EJ, van Vugt MJ, Grothe S, van Rotterdam W, van De Poll ML, O'Connor-McCourt MD (2000) Superagonistic activation of ErbB-1 by EGF-related growth factors with enhanced association and dissociation rate constants. J Biol Chem 275(35):26748–26753

  23. Liang CC, Park AY, Guan JL (2007) In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2(2):329–333

  24. Lu C, Mi LZ, Grey MJ, Zhu J, Graef E, Yokoyama S, Springer TA (2010) Structural evidence for loose linkage between ligand binding and kinase activation in the epidermal growth factor receptor. Mol Cell Biol 30(22):5432–5443

  25. Lutz S (2010) Beyond directed evolution–semi-rational protein engineering and design. Curr Opin Biotechnol 21(6):734–743

  26. Ma Y, Yu J, Lin J, Wu S, Li S, Wang J (2016) High-efficient Expression purification, and functional characterization of native human epidermal growth factor. Biomed Res Int 2016:3758941

  27. Maheswaran S, Sequist LV, Nagrath S, Ulkus L, Brannigan B, Collura CV, Inserra E, Diederichs S, Iafrate AJ, Bell DW, Digumarthy S, Muzikansky A, Irimia D, Settleman J, Tompkins RG, Lynch TJ, Toner M, Haber DA (2008) Detection of mutations in EGFR in circulating lung-cancer cells. N Engl J Med 359(4):366–377

  28. Nelson RS, Valadon P (2017) A universal phage display system for the seamless construction of Fab libraries. J Immunol Methods 450:41–49

  29. Nicholson AG, Gonzalez D, Shah P, Pynegar MJ, Deshmukh M, Rice A, Popat S (2010) Refining the diagnosis and EGFR status of non-small cell lung carcinoma in biopsy and cytologic material, using a panel of mucin staining, TTF-1, cytokeratin 5/6, and P63, and EGFR mutation analysis. J Thorac Oncol 5(4):436–441

  30. Ogiso H, Ishitani R, Nureki O, Fukai S, Yamanaka M, Kim JH, Saito K, Sakamoto A, Inoue M, Shirouzu M, Yokoyama S (2002) Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell 110(6):775–787

  31. Panosa C, Tebar F, Ferrer-Batalle M, Fonge H, Seno M, Reilly RM, Massaguer A, Dev R (2013) Development of an epidermal growth factor derivative with EGFR blocking activity. PLoS ONE 8(7):e69325

  32. Provencher SW, Glockner J (1981) Estimation of globular protein secondary structure from circular dichroism. Biochemistry 20(1):33–37

  33. Santana H, Gonzalez Y, Campana PT, Noda J, Amarantes O, Itri R, Beldarrain A, Paez R (2013) Screening for stability and compatibility conditions of recombinant human epidermal growth factor for parenteral formulation: effect of pH, buffers, and excipients. Int J Pharm 452(1–2):52–62

  34. Schlessinger J (2000) Cell signalling by receptor tyrosine kinases. Cell 103(2):211–225

  35. Sebastian S, Settleman J, Reshkin SJ, Azzariti A, Bellizzi A, Paradiso A (2006) The complexity of targeting EGFR signalling in cancer: from expression to turnover. Biochim Biophys Acta 1766(1):120–139

  36. Senderoff RI, Wootton SC, Boctor AM, Chen TM, Giordani AB, Julian TN, Radebaugh GW (1994) Aqueous stability of human epidermal growth factor 1–48. Pharm Res 11(12):1712–1720

  37. Sigismund S, Argenzio E, Tosoni D, Cavallaro E, Polo S, Di Fiore PP (2008) Clathrin-mediated internalization is essential for sustained EGFR signalling but dispensable for degradation. Dev Cell 15(2):209–219

  38. Souriau C, Fort P, Roux P, Hartley O, Lefranc MP, Weill M (1997) A simple luciferase assay for signal transduction activity detection of epidermal growth factor displayed on phage. Nucleic Acids Res 25(8):1585–1590

  39. Sreerama N, Venyaminov SY, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis. Anal Biochem 287(2):243–251

  40. Stemmer WP (1994) DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci USA 91(22):10747–10751

  41. Tang X, Liu H, Yang S, Li Z, Zhong J, Fang R (2016) Epidermal growth factor and intestinal barrier function. Mediat Inflamm 2016:1927348

  42. Todd E, Gurba KN, Botzolakis EJ, Stanic AK, Macdonald RL (2014) GABAA receptor biogenesis is impaired by the gamma2 subunit febrile seizure-associated mutation, GABRG2(R177G). Neurobiol Dis 69:215–224

  43. Tosoni D, Puri C, Confalonieri S, Salcini AE, De Camilli P, Tacchetti C, Di Fiore PP (2005) TTP specifically regulates the internalization of the transferrin receptor. Cell 123(5):875–888

  44. Wang X, Zhang W, Tang J, Huang R, Li J, Xu D, Xie Y, Jiang R, Deng L, Zhang X, Chai Y, Qin X, Sun B (2016) LINC01225 promotes occurrence and metastasis of hepatocellular carcinoma in an epidermal growth factor receptor-dependent pathway. Cell Death Dis 7:e2130

  45. Wilson KJ, Gilmore JL, Foley J, Lemmon MA, Riese DJ 2nd (2009) Functional selectivity of EGF family peptide growth factors: implications for cancer. Pharmacol Ther 122(1):1–8

  46. Yamazaki T, Zaal K, Hailey D, Presley J, Lippincott-Schwartz J, Samelson LE (2002) Role of Grb2 in EGF-stimulated EGFR internalization. J Cell Sci 115(Pt 9):1791–1802

  47. L. Yan, W. Wu, Z. Wang, C. Li, X. Lu, H. Duan, J. Zhou, X. Wang, P. Wan, Y. Song, J. Tang, Y. Han, Comparative study of the effects of recombinant human epidermal growth factor and basic fibroblast growth factor on corneal epithelial wound healing and neovascularization in vivo and in vitro, Ophthalmic research Citations(may) (2012) include.

  48. Yang CH, Wu PC, Huang YB, Tsai YH (2004) A new approach for determining the stability of recombinant human epidermal growth factor by thermal Fourier transform infrared (FTIR) microspectroscopy. J Biomol Struct Dyn 22(1):101–110

  49. Zhao H, Zha W (2006) In vitro 'sexual' evolution through the PCR-based staggered extension process (StEP). Nat Protoc 1(4):1865–1871

  50. Zhao H, Giver L, Shao Z, Affholter JA, Arnold FH (1998) Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol 16(3):258–261

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Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 31371163, No. 31771273) and Fundamental Research Funds for the Shenzhen of China (Nos. JCYJ20170307140505192, JCYJ20180307163444601 and 20180129171138130), and grants from Guangdong Province (No. 2017A030313875).

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Correspondence to Qing Zhang.

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Yujie Sun, Nanyan Yang, Fei Li, Jinxin Ou, Xin Liu and Qing Zhang declare that they have no conflict of interest.

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Sun, Y., Yang, N., Li, F. et al. Optimizing Human Epidermal Growth Factor for its Endurance and Specificity Via Directed Evolution: Functional Importance of Leucine at Position 8. Int J Pept Res Ther (2020). https://doi.org/10.1007/s10989-020-10050-6

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Keywords

  • Epidermal growth factor
  • Staggered extension process
  • Directed evolution