Abstract
In the present study, a novel chimeric peptide was derived from camel lactoferrin designed with a considerable anti-HCV activity and its neutralization mechanism was predicted by molecular modelling tools. A novel anti-HCV peptide derived from camel lactoferrin (cLF36) was designed and expressed it recombinantly in HEK-293-T cells. Anti-viral activity of this peptide was evaluated against hepatitis C virus by Real-time PCR assay in vitro. Finally, to have a better insight into the mode of action of peptide on HCV entry inhibition, we examined the interaction of cLF36 with envelope glycoprotein E2 by molecular dynamic simulation. This chimeric peptide had significant inhibitory effects on both HCV entry (44 µg/mL) and viral replication (88 µg/mL) under in vitro (p > 0.01). Moreover, cLF36 peptide was not toxic to HEK cells as a normal cell at twofold of its anti-viral concentrations for HCV entry and even at concentrations as high as 250 µg/mL exhibited minimal hemolysis (2.5%) against human RBCs (red blood cells). The results of in silico analysis showed that cLF36 interacted with β-sandwich and front layer of E2 protein as two potential CD81 binding sites. We generated and characterized a new camel lactoferrin derived HCV inhibitors. This peptide blocked HCV entry and also intracellular HCV replication in cell culture experiment.
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References
Abe K, Nozaki A, Tamura K, Ikeda M, Naka K, Dansako H, Hoshino HO, Tanaka K, Kato N (2007) Tandem repeats of lactoferrin-derived anti-hepatitis C virus peptide enhance antiviral activity in cultured human hepatocytes. Microbiol Immunol 51(1):117–125. https://doi.org/10.1111/j.1348-0421.2007.tb03882.x
Albar AH, El-Fakharany EM, Almehdar HA, Uversky VN, Redwan EM (2017) In vitro exploration of the anti-HCV potential of the synthetic spacer peptides derived from human, bovine, and camel lactoferrins. Protein Pept Lett 24(10):909–921. https://doi.org/10.2174/0929866524666161111111320
Bellamy W, Takase M, Wakabayashi H, Kawase K, Tomita M (1992) Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin. J Appl Bacteriol 73(6):472–479. https://doi.org/10.1111/j.1365-2672.1992.tb05007.x
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
Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Cassarino TG, Bertoni M, Bordoli L, Schwede T (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42(1):252–258. https://doi.org/10.1093/nar/gku340
Bolscher JG, Adao R, Nazmi K, van den Keybus PA, van’t Hof W, Amerongen AVN, Bastos M, Veerman EC (2009) Bactericidal activity of LFchimera is stronger and less sensitive to ionic strength than its constituent lactoferricin and lactoferrampin peptides. Biochimie 91(1):123–132. https://doi.org/10.1016/j.biochi.2008.05.019
Bräu N (2012) Evaluation of the hepatitis C virus-infected patient: the initial encounter. Clin Infect Dis 56(6):853–860
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
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622. https://doi.org/10.1373/clinchem.2008.112797
Chang CC, Hsu HJ, Yen JH, Lo SY, Liou JW (2017) A sequence in the loop domain of hepatitis C virus E2 protein identified in silico as crucial for the selective binding to human CD81. PLoS ONE 12(5):e0177383. https://doi.org/10.1371/journal.pone.0177383
Comeau SR, Gatchell DW, Vajda S, Camacho CJ (2004) ClusPro: an automated docking and discrimination method for the prediction of protein complexes. Bioinformatics 20(1):45–50
Cudic M, Condie BA, Weiner DJ, Lysenko ES, Xiang ZQ, Insug O, Bulet P, Otvos L Jr (2002) Development of novel antibacterial peptides that kill resistant isolates. Peptides 23(12):2071–2083. https://doi.org/10.1016/S0196-9781(02)00244-9
Daneshmand A, Kermanshahi H, Sekhavati MH, Javadmanesh A, Ahmadian M (2019) Antimicrobial peptide, cLF36, affects performance and intestinal morphology, microflora, junctional proteins, and immune cells in broilers challenged with E. coli. Sci Rep 9(1):1–9
Drummer HE, Boo I, Maerz AL, Poumbourios P (2006) A conserved Gly436-Trp-Leu-Ala-Gly-Leu-Phe-Tyr motif in hepatitis C virus glycoprotein E2 is a determinant of CD81 binding and viral entry. J Virol 80(16):7844–7853. https://doi.org/10.1128/JVI.00029-0
El-Awady MK, Tabll AA, Redwan ERM, Youssef S, Omran MH, Thakeb F, El-Demellawy M (2005) Flow cytometric detection of hepatitis C virus antigens in infected peripheral blood leukocytes: binding and entry. World J Gastroenterol 11(33):5203–5208. https://doi.org/10.3748/wjg.v11.i33.5203
El-Fakharany EM, Abedelbaky N, Haroun BM, Sánchez L, Redwan NA, Redwan EM (2012) Anti-infectivity of camel polyclonal antibodies against hepatitis C virus in Huh7. 5 hepatoma. Virol J 9(1):201. https://doi.org/10.1186/1743-422X-9-201
Flint M, McKeating JA (2000) The role of the hepatitis C virus glycoproteins in infection. Rev Med Virol 10(2):101–117. https://doi.org/10.1002/(SICI)1099-1654(200003/04)10:2%3c101
Gogela NA, Lin MV, Wisocky JL, Chung RT (2015) Enhancing our understanding of current therapies for hepatitis C virus (HCV). Current HIV/AIDS Reports 12(1):68–78. https://doi.org/10.1007/s11904-014-0243-7
Gower E, Estes C, Blach S, Razavi-Shearer K, Razavi H (2014) Global epidemiology and genotype distribution of the hepatitis C virus infection. J Hepatol 61(1):45–57. https://doi.org/10.1016/j.jhep.2014.07.027
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
Iyengar S, Tay-Teo K, Vogler S, Beyer P, Wiktor S, de Joncheere K, Hill S (2016) Prices, costs, and affordability of new medicines for hepatitis C in 30 countries: an economic analysis. PLoS Med 13(5):e1002032. https://doi.org/10.1371/journal.pmed.1002032
Jordan M, Schallhorn A, Wurm FM (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res 24(4):596–601
Kang JH, Lee MK, Kim KL, HAHM KS (1996) Structure–biological activity relationships of 11-residue highly basic peptide segment of bovine lactoferrin. Int J Pept Protein Res 48(4):357–363. https://doi.org/10.1111/j.1399-3011.1996.tb00852.x
Karplus M, McCammon JA (2002) Molecular dynamics simulations of biomolecules. Nat Struct Mol Biol 9(9):646–652. https://doi.org/10.1021/ar020082r
Keck ZY, Angus AG, Wang W, Lau P, Wang Y, Gatherer D, Patel AH, Foung SK (2014) Non-random escape pathways from a broadly neutralizing human monoclonal antibody map to a highly conserved region on the hepatitis C virus E2 glycoprotein encompassing amino acids 412–423. PLoS Pathog 10(8):e1004297. https://doi.org/10.1371/journal.ppat.1004297
Khan AG, Whidby J, Miller MT, Scarborough H, Zatorski AV, Cygan A, Price AA, Yost SA, Bohannon CD, Jacob J, Grakoui A (2014) Structure of the core ectodomain of the hepatitis C virus envelope glycoprotein 2. Nature 509(7500):381. https://doi.org/10.1038/nature13117
Kong L, Giang E, Nieusma T, Kadam RU, Cogburn KE, Hua Y, Dai X, Stanfield RL, Burton DR, Ward AB, Wilson IA (2013) Hepatitis C virus E2 envelope glycoprotein core structure. Science 342(6162):1090–1094. https://doi.org/10.1126/science.1243876
Law M, Maruyama T, Lewis J, Giang E, Tarr AW, Stamataki Z, Gastaminza P, Chisari FV, Jones IM, Fox RI, Ball JK (2008) Broadly neutralizing antibodies protect against hepatitis C virus quasi species challenge. Nat Med 14(1):25. https://doi.org/10.1038/nm1698
Liao Y, El-Fakkarany E, Lönnerdal B, Redwan EM (2012) Inhibitory effects of native and recombinant full-length camel lactoferrin and its N and C lobes on hepatitis C virus infection of Huh7. 5 cells. J Med Microbiol 61(3):375–383. https://doi.org/10.1099/jmm.0.033894-0
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
Majumdar A, Kitson MT, Roberts SK (2016) Systematic review: current concepts and challenges for the direct-acting antiviral era in hepatitis C cirrhosis. Aliment Pharmacol Ther 43(12):1276–1292. https://doi.org/10.1111/apt.13633
Maupetit J, Derreumaux P, Tuffery P (2009) PEP-FOLD: an online resource for de novo peptide structure prediction. Nucleic Acids Res 37(2):498–503. https://doi.org/10.1093/nar/gkp323
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
Meng XY, Zhang HX, Mezei M, Cui M (2011) Molecular docking: a powerful approach for structure-based drug discovery. Curr Comput Aided Drug Des 7(2):146–157
Nozaki A, Ikeda M, Naganuma A, Nakamura T, Inudoh M, Tanaka K, Kato N (2003) Identification of a lactoferrin-derived peptide possessing binding activity to hepatitis C virus E2 envelope protein. J Biol Chem 278(120):10162–10173. https://doi.org/10.1074/jbc.M207879200
Owsianka AM, Timms JM, Tarr AW, Brown RJ, Hickling TP, Szwejk A, Bienkowska-Szewczyk K, Thomson BJ, Patel AH, Ball JK (2006) Identification of conserved residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for CD81 binding. J Virol 80(17):8695–8704. https://doi.org/10.1128/JVI.00271-06
Owsianka AM, Tarr AW, Keck ZY, Li TK, Witteveldt J, Adair R, Foung SK, Ball JK, Patel AH (2008) Broadly neutralizing human monoclonal antibodies to the hepatitis C virus E2 glycoprotein. J Gen Virol 89(3):653–659. https://doi.org/10.1128/JVI.01138-09
Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca R, Weiner AJ, Houghton M, Rosa D, Grandi G, Abrignani S (1998) Binding of hepatitis C virus to CD81. Science 282(5390):938–941. https://doi.org/10.1126/science.282.5390.938
Pirkhezranian Z, Tanhaeian A, Mirzaii M, Sekhavati MH (2019) Expression of Enterocin-P in HEK platform: evaluation of its cytotoxic effects on cancer cell lines and its potency to interact with cell-surface glycosaminoglycan by molecular modeling. Int J Pept Res Ther. https://doi.org/10.1007/s10989-019-09956-7
Qin ZL, Ju HP, Liu Y, Gao TT, Wang WB, Aurelian L, Zhao P, Qi ZT (2013) Fetal bovine serum inhibits hepatitis C virus attachment to host cells. J Virol Methods 193(2):261–269. https://doi.org/10.1016/j.jviromet.2013.06.024
Redwan EM, EL-Fakharany EM, Uversky VN, Linjawi MH (2014) Screening the anti-infectivity potentials of native N-and C-lobes derived from the camel lactoferrin against hepatitis C virus. BMC Complement Altern Med 14(1):219. https://doi.org/10.1186/1472-6882-14-219
Sabahi A (2009) Hepatitis C virus entry: the early steps in the viral replication cycle. Virol J 6(1):1–11. https://doi.org/10.1186/1743-422X-6-117
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (No. Ed. 2). Cold Spring Harbor Laboratory Press, Cold Spring Harbor
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
Shah N, Pierce T, Kowdley KV (2013) Review of direct-acting antiviral agents for the treatment of chronic hepatitis C. Expert Opin Investig Drugs 22(9):1107–1121. https://doi.org/10.1517/13543784.2013.806482
Skalickova S, Heger Z, Krejcova L, Pekarik V, Bastl K, Janda J, Kostolansky F, Vareckova E, Zitka O, Adam V, Kizek R (2015) Perspective of use of antiviral peptides against influenza virus. Viruses 7(10):5428–5442. https://doi.org/10.3390/v7102883
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(4):787–793. https://doi.org/10.1007/s12602-018-9416-9
Tanhaeian A, Jaafari MR, Ahmadi FS, Vakili-Ghartavol R, Sekhavati MH (2019) 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. Probiotics Antimicrob Proteins 11(3):1034–1041
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
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
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
Tomita M, Bellamy W, Takase M, Yamauchi K, Wakabayashi H, Kawase K (1991) Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin. J Dairy Sci 74(12):4137–4142. https://doi.org/10.1016/j.peptides.2003.12.006
Van der Kraan MI, Groenink J, Nazmi K, Veerman EC, Bolscher JG, Amerongen AVN (2004) Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin. Peptides 25(2):177–183. https://doi.org/10.1016/j.peptides.2003.12.006
Van der Kraan MI, Nazmi K, Teeken A, Groenink J, van’t Hof W, Veerman EC, Bolscher JG, Amerongen AVN (2005) Lactoferrampin, an antimicrobial peptide of bovine lactoferrin, exerts its candidacidal activity by a cluster of positively charged residues at the C-terminus in combination with a helix-facilitating N-terminal part. Biol Chem 386(2):137–142. https://doi.org/10.1515/BC.2005.017
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
Vogel HJ, Schibli DJ, Jing W, Lohmeier-Vogel EM, Epand RF, Epand RM (2002) Towards a structure–function analysis of bovine lactoferricin and related tryptophan- and arginine-containing peptides. Biochem Cell Biol 80(1):49–63. https://doi.org/10.1139/o01-213
Vorland LH, Ulvatne H, Andersen J, Haukland HH, Rekdal Ø, Svendsen JS, Gutteberg TJ (1999) Antibacterial effects of lactoferricin B. Scand J Infect Dis 31(2):179–184. https://doi.org/10.1080/003655499750006245
Warren L (2002) The PyMOL molecular graphics system. DeLano Scientific LLC, San Carlos, CA, USA
Yin P, Zhang L, Ye F, Deng Y, Lu S, Li YP, Zhang L, Tan W (2017) A screen for inhibitory peptides of hepatitis C virus identifies a novel entry inhibitor targeting E1 and E2. Sci Rep 7(1):1–10. https://doi.org/10.1038/s41598-017-04274-8
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
Zaiou M (2007) Multifunctional antimicrobial peptides: therapeutic targets in several human diseases. J Mol Med 85(4):317–329. https://doi.org/10.1007/s00109-006-0143-4
Acknowledgements
The authors would also like to express their gratitude to the Ferdowsi University of Mashhad for their support. The present study was funded by INFS (infs.gov.ir) of I.R.I with Grant No. 93,025,031.
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Approvals to conduct experimental protocols to study hemolysis on human red cells were approved by the Committee on Publication Ethics (COPE), where this work was done. Human red cells were from volunteer Mohammad Hadi Sekhavati, who signed the informed consent for this study and is also author of this report.
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Tahmoorespur, M., Azghandi, M., Javadmanesh, A. et al. A Novel Chimeric Anti-HCV Peptide Derived from Camel Lactoferrin and Molecular Level Insight on Its Interaction with E2. Int J Pept Res Ther 26, 1593–1605 (2020). https://doi.org/10.1007/s10989-019-09972-7
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DOI: https://doi.org/10.1007/s10989-019-09972-7