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Probiotics and Antimicrobial Proteins

, Volume 11, Issue 3, pp 1034–1041 | Cite as

Secretory Expression of a Chimeric Peptide in Lactococcus lactis: Assessment of its Cytotoxic Activity and a Deep View on Its Interaction with Cell-Surface Glycosaminoglycans by Molecular Modeling

  • Abbas Tanhaeian
  • Mahmoud Reza Jaafari
  • Farajollah Shahriari Ahmadi
  • Roghayyeh Vakili‐Ghartavol
  • Mohammad Hadi SekhavatiEmail author
Article
  • 64 Downloads

Abstract

Nowadays, cancer remains a major cause of death affecting millions of people. Currently, the antimicrobial peptides (AMPs) as potent anticancer therapeutic agents offer specificity and low levels of side effects in cancer therapy. In the present study, a cationic chimeric peptide (cLFchimera), derived from camel lactoferrin, was expressed as a secretory peptide using P170 expression system in L. lactis. Peptide purification was carried out using Ni-NTA agarose column from culture medium with 21 μ/mL concentration. The recombinant peptide was investigated for its activity against four tumor and one normal cell line. The cLFchimera was more active against two tumor cell lines (chondrosarcoma and colorectal cancer cells), but the activity against two other tumor cell lines (hepatoma and breast cancer cell line) and normal cells was low. Finally, to have better insight into the mode of action of the peptide on cytotoxic activity, we examined the interaction of cationic peptide with two glycosaminoglycans (GAGs), heparan sulfate (HS) and chondroitin sulfate (CS), as the two most anionic molecules on the cell surface by molecular dynamic simulation. The results of in silico analysis showed that the cLFchimera interacted with HS and CS with a totally different amino acid profile. Hydrogen bonding screening in GAGs-peptide complexes revealed K21, V23 and I3, R16 are the dominant amino acids involved in peptide-HS and CS interaction, respectively. Overall, the results of this investigation showed the P170 expression system successfully expressed a cationic peptide with potent anticancer activity. Moreover, molecular docking analysis revealed the pattern of peptide interaction with negatively charged membrane molecules.

Keywords

P170 expression system Lactococcus lactis Cytotoxic activity Glycosaminoglycans Molecular dynamic simulation 

Notes

Acknowledgements

We are very grateful to Miss Marjan Azghandi and Dr. Reza Majidzade for their outstanding technical assistance.

Authors’ Contributions

MHS and MRJ planed the experiment. Cloning, protein expression, and western blotting analysis were performed by AT and FSA. Cytotoxicity assay was carried out by RV. MHS and AB did the data collection. MHS performed MD simulation. MHS, AB, and MRJ interpreted the data. MHS prepared the manuscript.

Funding

This work was supported by Ferdowsi University of Mashhad, Iran with grant No.38736.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Harris F, Dennison SR, Singh J, Phoenix DA (2013) On the selectivity and efficacy of defense peptides with respect to cancer cells. Med Res Rev 33(1):190–234.  https://doi.org/10.1002/med.20252 Google Scholar
  2. 2.
    Riedl S, Rinner B, Asslaber M, Schaider H, Walzer S, Novak A, Zweytick D (2011) In search of a novel target-phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy. Biochim Biophys Acta 1808(11):2638–2645.  https://doi.org/10.1016/j.bbamem.2011.07.026 Google Scholar
  3. 3.
    Kalyanaraman BJSSES, Joseph J, Kalivendi S, Wang S, Konorev E, Kotamraju S (2002) Doxorubicin-induced apoptosis: implications in cardiotoxicity. Mol Cell Biochem 234(1):119–124.  https://doi.org/10.1023/A:101597643 Google Scholar
  4. 4.
    Al-Benna S, Shai Y, Jacobsen F, Steinstraesser L (2011) Oncolytic activities of host defense peptides. Int J Mol Sci 12(11):8027–8051.  https://doi.org/10.3390/ijms12118027 Google Scholar
  5. 5.
    Gatti L, Zunino F (2005) Overview of tumor cell chemoresistance mechanisms. In: Chemosensitivity, vol II. Humana Press, Totowa NJ, pp 127–148.  https://doi.org/10.1385/1-59259-889-7:127 Google Scholar
  6. 6.
    Deslouches B, Di YP (2017) Antimicrobial peptides with selective antitumor mechanisms: prospect for anticancer applications. Oncotarget 8(28):46635–46651.  https://doi.org/10.18632/oncotarget.16743 Google Scholar
  7. 7.
    Felício MR, Silva ON, Gonçalves S, Santos NC, Franco OL (2017) Peptides with dual antimicrobial and anticancer activities. Front Chem 5:5.  https://doi.org/10.3389/fchem.2017.00005 Google Scholar
  8. 8.
    Linde A, Ross CR, Davis EG, Dib L, Blecha F, Melgarejo T (2008) Innate immunity and host defense peptides in veterinary medicine. J Vet Intern Med 22(2):247–265.  https://doi.org/10.1111/j.1939-1676.2007.0038.x Google Scholar
  9. 9.
    Skalickova S, Heger Z, Krejcova L, Pekarik V, Bastl K, Janda J, Kizek R (2015) Perspective of use of antiviral peptides against influenza virus. Viruses 7(10):5428–5442.  https://doi.org/10.3390/v7102883 Google Scholar
  10. 10.
    Sang Y, Blecha F (2009) Porcine host defense peptides: expanding repertoire and functions. Dev Comp Immunol 33(3):334–343.  https://doi.org/10.1016/j.dci.2008.05.006 Google Scholar
  11. 11.
    Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3(3):238–250.  https://doi.org/10.1038/nrmicro1098 Google Scholar
  12. 12.
    Yang N, Lejon T, Rekdal Ø (2003) Antitumor activity and specificity as a function of substitutions in the lipophilic sector of helical lactoferrin-derived peptide. J Pept Sci 9(5):300–311.  https://doi.org/10.1002/psc.457 Google Scholar
  13. 13.
    Fadnes B, Rekdal Ø, Uhlin-Hansen L (2009) The anticancer activity of lytic peptides is inhibited by heparan sulfate on the surface of the tumor cells. BMC Cancer 9(1):183.  https://doi.org/10.1186/1471-2407-9-183 Google Scholar
  14. 14.
    Kjellén L, Lindahl U (1991) Proteoglycans: structures and interactions. Annu Rev Biochem 60(1):443–475.  https://doi.org/10.1146/annurev.bi.60.070191.002303 Google Scholar
  15. 15.
    Tanhaiean A, Azghandi M, Razmyar J, Mohammadi E, Sekhavati MH (2018) Recombinant production of a chimeric antimicrobial peptide in E. coli and assessment of its activity against some avian clinically isolated pathogens. Microb Pathog 122:73–78.  https://doi.org/10.1016/j.micpath.2018.06.012 Google Scholar
  16. 16.
    Bermúdez-Humarán LG, Kharrat P, Chatel JM, Langella P (2011) Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines. Microb Cell Factories 10(1):S4). BioMed Central.  https://doi.org/10.1186/1475-2859-10-S1-S4 Google Scholar
  17. 17.
    Jørgensen CM, Vrang A, Madsen SM (2014) Recombinant protein expression in Lactococcus lactis using the P170 expression system. FEMS Microbiol Lett 351(2):170–178.  https://doi.org/10.1111/1574-6968.12351 Google Scholar
  18. 18.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (No. Ed. 2). Cold spring harbor laboratory press.  https://doi.org/10.1016/0167-7799(91)90068-S
  19. 19.
    Holo H, Nes IF (1989) High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol 55(12):3119–3123Google Scholar
  20. 20.
    Wells JM, Wilson PW, Norton PM, Gasson MJ, Le Page RW (1993) Lactococcus lactis: high-level expression of tetanus toxin fragment C and protection against lethal challenge. Mol Microbiol 8(6):1155–1162.  https://doi.org/10.1111/j.1365-2958.1993.tb01660.x Google Scholar
  21. 21.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254.  https://doi.org/10.1016/0003-2697(76)90527-3 Google Scholar
  22. 22.
    Teimourpour R, Meshkat Z, Gholoubi A, Nomani H, Rostami S (2015) Viral load analysis of hepatitis C virus in Huh7. 5 cell culture system. Jundishapur J Microbiol 8(5):e19279.  https://doi.org/10.5812/jjm.8(5)2015.19279 Google Scholar
  23. 23.
    Maupetit J, Derreumaux P, Tuffery P (2009) PEP-FOLD: an online resource for de novo peptide structure prediction. Nucleic Acids Res 37(suppl_2):W498–W503.  https://doi.org/10.1093/nar/gkp323 Google Scholar
  24. 24.
    Maupetit J, Derreumaux P, Tufféry P (2010) A fast method for large-scale de novo peptide and miniprotein structure prediction. J Comput Chem 31(4):726–738.  https://doi.org/10.1002/jcc.21365 Google Scholar
  25. 25.
    Mulloy B, Forster MJ, Jones C, Davies DB (1993) Nmr and molecular-modelling studies of the solution conformation of heparin. Bioch J 293(3):849–858Google Scholar
  26. 26.
    Winter WT, Arnott S, Isaac DH, Atkins EDT (1978) Chondroitin 4-sulfate: the structure of a sulfated glycosaminoglycan. J Mol Biol 125(1):1–19.  https://doi.org/10.1016/0022-2836(78)90251-6 Google Scholar
  27. 27.
    Sapay N, Cabannes E, Petitou M, Imberty A (2011) Molecular modeling of the interaction between heparan sulfate and cellular growth factors: bringing pieces together. Glycobiology 21(9):1181–1193.  https://doi.org/10.1093/glycob/cwr052 Google Scholar
  28. 28.
    Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJ (2005) GROMACS: fast, flexible, and free. J Comput Chem 26(16):1701–1718.  https://doi.org/10.1002/jcc.20291 Google Scholar
  29. 29.
    Hermans J, Berendsen HJ, Van Gunsteren WF, Postma JP (1984) A consistent empirical potential for water–protein interactions. Biopolymers 23(8):1513–1518.  https://doi.org/10.1002/bip.360230807 Google Scholar
  30. 30.
    York DM, Darden TA, Pedersen LG (1993) The effect of long-range electrostatic interactions in simulations of macromolecular crystals: a comparison of the Ewald and truncated list methods. J Chem Phys 99(10):8345–8348.  https://doi.org/10.1063/1.465608 Google Scholar
  31. 31.
    Berendsen HJ, Postma JV, van Gunsteren WF, DiNola ARHJ, Haak JR (1984) Molecular dynamics with coupling to an external bath. J Chem Phys 81(8):3684–3690.  https://doi.org/10.1063/1.448118 Google Scholar
  32. 32.
    Warren L (2002) The PyMOL molecular graphics system. DeLano Scientific LLC, San Carlos, CA.  https://doi.org/10.1371/journal.pone.0133011.g002 Google Scholar
  33. 33.
    Song AAL, In LL, Lim SHE, Rahim RA (2017) A review on Lactococcus lactis: from food to factory. Microb Cell Factories 16(1):55.  https://doi.org/10.1186/s12934-017-0669-x Google Scholar
  34. 34.
    Tanhaieian A, Sekhavati MH, Ahmadi FS, Mamarabadi M (2018) Heterologous expression of a broad-spectrum chimeric antimicrobial peptide in Lactococcus lactis: its safety and molecular modeling evaluation. Microb Pathog 125:51–59.  https://doi.org/10.1016/j.micpath.2018.09.016 Google Scholar
  35. 35.
    Tanhaeian A, Ahmadi FS, Sekhavati MH, Mamarabadi M (2018) Expression and purification of the main component contained in camel milk and its antimicrobial activities against bacterial plant pathogens. Probiotics Antimicrob Proteins 10:1–7.  https://doi.org/10.1007/s12602-018-9416-9 Google Scholar
  36. 36.
    Salanti A, Clausen TM, Agerbæk MØ, Al Nakouzi N, Dahlbäck M, Oo HZ, Mao Y et al (2015) Targeting human cancer by a glycosaminoglycan binding malaria protein. Cancer Cell 28(4):500–514.  https://doi.org/10.1016/j.ccell.2015.09.003 Google Scholar
  37. 37.
    Yamada H, Kiyohara H (2007) Cell glycobiology and development health and disease in glycomedicine. Elsevier, Oxford, pp 664–693Google Scholar
  38. 38.
    Christianson HC, Belting M (2014) Heparan sulfate proteoglycan as a cell-surface endocytosis receptor. Matrix Biol 35:51–55.  https://doi.org/10.1016/j.matbio.2013.10.004 Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Abbas Tanhaeian
    • 1
  • Mahmoud Reza Jaafari
    • 2
    • 3
  • Farajollah Shahriari Ahmadi
    • 1
  • Roghayyeh Vakili‐Ghartavol
    • 4
  • Mohammad Hadi Sekhavati
    • 5
    Email author
  1. 1.Department of Biotechnology and Plant Breeding, Faculty of AgricultureFerdowsi University of MashhadMashhadIran
  2. 2.Nanotechnology Research Center, Pharmaceutical Technology InstituteMashhad University of Medical SciencesMashhadIran
  3. 3.Department of Pharmaceutical Nanotechnology, School of PharmacyMashhad University of Medical SciencesMashhadIran
  4. 4.Department of Medical Nanotechnology, School of Advanced Technologies in MedicineTehran University of Medical SciencesTehranIran
  5. 5.Department of Animal Sciences, Faculty of AgricultureFerdowsi University of MashhadMashhadIran

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