Advertisement

Structural-functional characterization of recombinant Apolipoprotein A-I from Labeo rohita demonstrates heat-resistant antimicrobial activity

  • Sweta Karan
  • Amruta Mohapatra
  • Pramod Kumar Sahoo
  • Lalit C. Garg
  • Aparna DixitEmail author
Biotechnologically relevant enzymes and proteins
  • 84 Downloads

Abstract

Apolipoprotein A-I is an anti-inflammatory, antioxidative, cardioprotective, anti-tumorigenic, and anti-diabetic in mammals. Apolipoprotein A-I also regulates innate immune defense mechanisms in vertebrates and invertebrates. Apolipoproteins A-I from mammals and several teleosts display antibacterial activities against Gram negative and Gram positive bacteria. The present study describes strategies to obtain high amounts of soluble purified recombinant Apolipoprotein A-I of Labeo rohita, an Indian major carp (rLrApoA-I). The study also reports its detailed structural and functional characterization i.e. antimicrobial activity against a number of important marine and fresh water bacterial pathogens. The rLrApoA-I was expressed in Escherichia coli BL21(DE3) pLysS expression host as a soluble protein under optimized conditions. The yield of purified rLrApoA-I was ~ 75 mg/L from soluble fraction using metal ion affinity chromatography. The authenticity of the rLrApoA-I was confirmed by MALDI-TOF-MS analysis. The secondary structure analysis showed rLrApoA-I to be predominantly alpha helical, an evolutionary conserved characteristic across mammals and teleosts. The purified rLrApoA-I exhibited antimicrobial activity as evident from inhibition of growth of a number of bacteria namely Aeromonas hydrophila, A. liquefaciens, A. culicicola, A. sobria, Vibrio harveyi, V. parahaemolyticus and Edwardsiella tarda in a dose–dependent manner. Minimum bactericidal concentration for A. liquefaciens, A. culicicola, and A. sobria, was determined to be 25 μg/ml or 0.81 μM whereas for A. hydrophila, E. tarda, V. parahaemolyticus and V. harveyi, it was determined to be 100 μg/ml or 3.23 μM. These data strongly suggest that recombinant ApoA-I from Labeo rohita could play a role in primary defense against fish pathogen. Further, at temperature ≥ 55 °C, though a loss in secondary structure was observed, no effect on its antibacterial activity was observed. This is of significance as the antibacterial activity is not likely to be lost even if the protein is subjected to high temperatures during transport.

Keywords

Apolipoprotein A-I Antimicrobial activity Gram-negative bacteria Aeromonas species Vibrio species and Edwardsiella tarda Labeo rohita 

Notes

Acknowledgments

AD acknowledges the PURSE grant from the Department of Science and Technology [SR/PURSE/Phase2/11(C) 2015], New Delhi, India, to the Jawaharlal Nehru University (JNU), New Delhi, India, and the financial support from the University with Potential for Excellence grant (UPE-II – Project ID#43) from the University Grants Commission, New Delhi to JNU, New Delhi. LCG acknowledges the Indian National Science Academy, New Delhi for Senior Scientist Fellowship SK thanks the ICMR for providing research fellowship.

Funding information

The present study has been carried out with the financial support from the Department of Science and Technology PURSE grant to the JNU (SR/PURSE/Phase2/11(C) 2015) and the University with Potential for Excellence grant (UPE-II–Project ID#43) from the University Grants Commission, New Delhi, India, to the JNU.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human or animal participants performed by any of the authors.

Supplementary material

253_2019_10204_MOESM1_ESM.pdf (1.4 mb)
ESM 1 (PDF 1.37 mb)

References

  1. Abraham TJ, Mallick PK, Adisesavalu H, Banerjee S (2015) Pathology of Edwardsiella tarda infection in African catfish, Clarias gariepinus IBurchell 1822, fingerlings. Arch Pol Fish 23:141–148CrossRefGoogle Scholar
  2. Ajees AA, Anantharamaiah GM, Mishra VK, Hussain MM, Murthy HMK (2006) Crystal structure of human Apolipoprotein A-I: insights into its protective effect against cardiovascular diseases. Proc Natl Acad Sci USA 103:2126–2131PubMedCrossRefPubMedCentralGoogle Scholar
  3. Alonso-Villaverde C, Segués T, Coll-Crespo B, Pérez-Bernalte R, Rabassa A, Gomila M, Parra S, González-Esteban MA, Jiménez-Expósito JM, Masana L (2003) High-density lipoprotein concentrations relate to the clinical course of HIV viral load in patients undergoing antiretroviral therapy. AIDS 17:1173–1177PubMedCrossRefPubMedCentralGoogle Scholar
  4. Bashtovyy D, Jones MK, Anantharamaiah GM, Segrest JP (2011) Sequence conservation of apolipoprotein A-I affords novel insights into HDL structure-function. J lipid Res 52:435–450PubMedPubMedCentralCrossRefGoogle Scholar
  5. Beck WHJ, Adams CP, Biglang-awa IM, Patel AB, Vincent H, Haas-Stapleton EJ, Weers PMM (2013) Apolipoprotein A-I binding to anionic vesicles and lipopolysaccharides: role for lysine residues in antimicrobial properties. Biochim Biophys Acta 1828:1503–1510PubMedPubMedCentralCrossRefGoogle Scholar
  6. Benli ACK, Yildiz HY (2004) Blood parameters in Nile tilapia (Oreochromisniloticus L.) spontaneously infected with Edwardsiella tarda. Aquacult Res 35:1388–1390CrossRefGoogle Scholar
  7. Biedzka-Sarek M, Metso J, Kateifides A, Meri T, Jokiranta TS, Muszyński A, Radziejewska-Lebrecht J, Zannis V, Skurnik M, Jauhiainen M (2011) Apolipoprotein A-I exerts bactericidal activity against Yersinia enterocolitica serotype O:3. JBiol Chem 286:38211–38219CrossRefGoogle Scholar
  8. Chroni A, Liu T, Gorskova I, Kan HY, Uehara Y, von Eckardstein A, Zannis VI (2003) The central helices of ApoA-I can promote ATP-binding cassette transporter A1(ABCA1)-mediated lipid efflux. Amino acid residues 220-231 of the wild-type ApoA-I are required for lipid efflux in vitro and high density lipoprotein formation in vivo. J Biol Chem 278:6719–6730PubMedCrossRefPubMedCentralGoogle Scholar
  9. Concha MI, Smith VJ, Castro K, Bastıas A, Romero A, Amthauer RJ (2004) Apolipoproteins A-I and A-II are potentially important effectors of innate immunity in the teleost fish Cyprinus carpio. Eur J Biochem 271:2984–2990PubMedCrossRefPubMedCentralGoogle Scholar
  10. Costa AC, Brandão HM, da Silva SR, Bentes-Sousa AR, Diniz JA Jr, Viana Pinheiro Jde J, deMeloMde F, Silva JO Jr, Matos ER, Ribeiro-Costa RM (2016) Mucoadhesive nanoparticles: a new perspective for fish drug application. J Fish Dis 39:503–506PubMedCrossRefPubMedCentralGoogle Scholar
  11. Denslow ND, Chow MM, Folmar LC (1994) Isoforms of Apolipotein A-I in the serum of brown bullheads (Ameiurus nebulosus) with liver cancer. Can J Zool 72:1522–1527CrossRefGoogle Scholar
  12. Dietrich MA, Adamek M, Bilińska B, Hejmej A, Steinhagen D, Ciereszko A (2014) Characterization, expression and antibacterial properties of apolipoproteins A from carp (Cyprinus carpio L.) seminal plasma. Fish Shellfish Immunol 41:389–401PubMedCrossRefPubMedCentralGoogle Scholar
  13. Duggan A, Callard IP (2001) Phylogenetic distribution of Apolipoproteins A-I and E in vertebrates as determined by western blot analysis. J Exp Zool 290:255–264PubMedCrossRefPubMedCentralGoogle Scholar
  14. Duggan A, Paolucci M, Tercyak A, Gigliotti M, Small D, Callard I (2000) Seasonal variation in plasma lipids, lipoproteins, Apolipoproteins A-I and vitellogenin in the freshwater turtle, Chrysemys picta. Comp Biochem Physiol Part A Mol Integr Physiol 130:253–269CrossRefGoogle Scholar
  15. Harikrishnan R, Nisha RM, Balasundaram C (2003) Hematological and biochemical parameters in common carp, Cyprinus carpio, following herbal treatment for Aeromonas hydrophila infection. Aquaculture 221:41–50CrossRefGoogle Scholar
  16. Henning MF, Herlax V, Bakás L (2011) Contribution of the C-terminal end of apolipoprotein AI to neutralization of lipopolysaccharide endotoxic effect. Innate Immun 17:327–337PubMedCrossRefPubMedCentralGoogle Scholar
  17. Hubsch AP, Casas AT, Doran JE (1995) Protective effects of reconstituted high-density lipoprotein in rabbit gram-negative bacteremia models. J Lab Clin Med 126:548–558PubMedPubMedCentralGoogle Scholar
  18. Jenssen H, Hamill P, Hancock RE (2006) Peptide antimicrobial agents. Clin Microbiol Rev 19:491–511PubMedPubMedCentralCrossRefGoogle Scholar
  19. Johnston LD, Brown G, Gauthier D, Reece K, Kator H, VanVeld P (2008) ApoA-I lipoproteins A-I from striped bass (Morone saxatilis) demonstrates antibacterial activity in vitro. Comp Biochem Physiol Part B Biochem Mol Biol 151:167–175CrossRefGoogle Scholar
  20. Kaconis Y, Kowalski I, Howe J, Brauser A, Richter W, Razquin-Olazaran I, Inigo-Pestana M, Garidel P, Rossle M, Martinez de Tejada G, Gutsmann T, Brandenburg K (2011) Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides. Biophys J 100:2652–2661PubMedPubMedCentralCrossRefGoogle Scholar
  21. Karan S, Dash P, Kaushik H, Sahoo PK, Garg LC, Dixit A (2016) Structural and functional characterization of recombinant interleukin-10 from Indian major carp Labeo rohita. J Immunol Res 3962596:11.  https://doi.org/10.1155/2016/3962596 CrossRefGoogle Scholar
  22. Kaushik H, Dixit A, Garg LC (2018) Synthesis of peptide based epsilon toxin vaccine by covalent anchoring to tetanus toxoid. Anaerobe 53:50–55PubMedCrossRefPubMedCentralGoogle Scholar
  23. Khalili M, Soleyman MR, Baazam M, Beyer C (2015) High-level expression and purification of soluble bioactive recombinant human heparin-binding epidermal growth factor in Escherichia coli. Cell Biol Int 39:858–864PubMedCrossRefPubMedCentralGoogle Scholar
  24. Kim MN, Bang HJ (2008) Detection of marine pathogenic bacterial Vibrio species by multiplex polymerase chain reaction (PCR). J Environ Biol 29:543–546PubMedPubMedCentralGoogle Scholar
  25. Kim TD, Ryu HJ, Cho HI, Yang CH, Kim J (2000) Thermal behaviour of proteins: heat-resistant proteins and their heat-induced secondary structural changes. Biochemistry 39:14839–14846PubMedCrossRefPubMedCentralGoogle Scholar
  26. Kondo H, Morinaga K, Misaki R, Nakaya M, Watabe S (2005) Characterization of the puffer fish Takifugu rubripes apolipoprotein multigene family. Gene 346:257–266PubMedCrossRefPubMedCentralGoogle Scholar
  27. Koyama M, Tanaka M, Dhanasekaran P, Lund-Katz S, Phillips MC, Saito H (2009) Interaction between the N- and C-terminal domains modulates the stability and lipid binding of Apolipoprotein A–I. Biochemistry 48:2529–2537PubMedPubMedCentralCrossRefGoogle Scholar
  28. Kulkarni P, Chaudhari GH, Sripuram V, Banote RK, Kirla KT, Sultana R, Rao P, Qruganti S, Chatti K (2014) Oral dosing in adult zebrafish: proof-of-concept using pharmacokinetics and pharmacological evaluation of carbamazepine. Pharmacol Rep 66:179–183PubMedCrossRefPubMedCentralGoogle Scholar
  29. Laccotripe M, Makrides SC, Jonas A, Zannis VI (1997) The carboxyl-terminal hydrophobic residues of Apolipoprotein A-I affect its rate of phospholipid binding and its association with high density lipoprotein. J Biol Chem 272:17511–17522PubMedCrossRefPubMedCentralGoogle Scholar
  30. Law SW, Brewer HB Jr (1984) Nucleotide sequence and the encoded amino acids of human apolipoprotein A-I mRNA. Proc Natl Acad Sci USA 81:66–70PubMedCrossRefPubMedCentralGoogle Scholar
  31. Lewis GF, Rader DJ (2005) New insights into the regulation of HDL metabolism and reverse cholesterol transport. Circ Res 96:1221–1232PubMedCrossRefPubMedCentralGoogle Scholar
  32. Llewellyn L, Ramsurn VP, Wigham T, Sweeney GE, Power DM (1998) Cloning, characterisation and expression of the apolipoprotein A-I gene in the sea bream (Sparus aurata). Biochim Biophys Acta 1442:399–404PubMedCrossRefPubMedCentralGoogle Scholar
  33. Lyssenko NN, Hata M, Dhanasekaran P, Nickel M, Nguyen D, Chetty PS, Phillips MC (2012) Influence of C-terminal α-helix hydrophobicity and aromatic amino acid content on apolipoprotein A-I functionality. Biochim Biophys Acta 1821:456–463PubMedCrossRefPubMedCentralGoogle Scholar
  34. Magnadottir B, Lange S (2004) Is Apolipoprotein A-I a regulating protein for the complement system of cod (Gadus morhua). Fish Shelfish Immunol 16:265–269CrossRefGoogle Scholar
  35. Marr AK, Gooderham WJ, Hancock REW (2006) Antibacterial peptides for therapeutic use: obstacles and realistic outlook. Curr opin pharmacol 6:468–472PubMedCrossRefPubMedCentralGoogle Scholar
  36. Mathews PD, Fernandes Patta ACM, Goncalves JV, Gama GDS, Garcia ITS, Mertins O (2018) Targeted Drug Delivery and Treatment of Endoparasites with Biocompatible Particles of pH-Responsive Structure. Biomacromolecules 19:499–510PubMedCrossRefPubMedCentralGoogle Scholar
  37. Mohapatra A, Karan S, Kar B, Garg LC, Dixit A, Sahoo PK (2016) Apolipoprotein AI in Labeo rohita: cloning and functional characterisation reveal its broad spectrum antimicrobial property, and indicate significant role during ectoparasitic infection. Fish Shellfish Immunol 55:717–728PubMedCrossRefPubMedCentralGoogle Scholar
  38. Nagao K, Hata M, Tanaka K, Takechi Y, Nguyen D, Dhanasekaran P, Lund-Katz S, Phillips MC, Saito H (2014) The roles of C-terminal helices of human apolipoprotein A-I in formation of high-density lipoprotein particles. Biochim Biophys Acta 1841:80–87PubMedCrossRefPubMedCentralGoogle Scholar
  39. Oram JF (2003) HDL Apolipoproteins and ABCA1: partners in the removal of excess cellular cholesterol. Arterioscler Thromb Vasc Biol 23:720–727PubMedCrossRefPubMedCentralGoogle Scholar
  40. Page NM, Butlin DJ, Lomthaisong K, Lowry PJ (2001) The human apolipoprotein L gene cluster: identification, classification and sites of distribution. Genomics 74:71–78PubMedCrossRefPubMedCentralGoogle Scholar
  41. Palgunachari MN, Mishra VK, Lund-Katz S, Phillips MC, Adeyeye SO, Alluri S, Anantharamaiah GM, Segrest JP (1996) Only the two end helixes of eight tandem amphipathic helical domains of human ApoA-I have significant lipid affinity. Implications for HDL assembly. Arterioscler ThrombVasc Biol 16:328–338CrossRefGoogle Scholar
  42. Park SB, Aoki T, Jung TS (2012) Pathogenesis of and strategies for preventing Edwardsiella tarda infection in fish. Vet Res 43:67.  https://doi.org/10.1186/1297-9716 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Perez-Morga D, Vanhollebeke B, Paturiaux-Hanocq F, Nolan DP, Lins L, Homble F, Vanhamme L, Tebabi P, Pays A, Poelvoorde P, Jacquet A, Brasseur R, Pays E (2005) Apolipoprotein L-I promotes trypanosome lysis by forming pores in lysosomal membranes. Science 309:469–472PubMedCrossRefPubMedCentralGoogle Scholar
  44. Phillips MC (2013) New insights into the determination of HDL structure by apolipoproteins: thematic review series: high density lipoprotein structure, function, and metabolism. J Lipid Res 54:2034–2048PubMedPubMedCentralCrossRefGoogle Scholar
  45. Piotrowska M, Popowska M (2014) The prevalence of antibiotic resistance genes among Aeromonas species in aquatic environments. Ann Microbiol 64:921–934CrossRefGoogle Scholar
  46. Plotkin JB, Kudla G (2011) Synonymous but not the same: the causes and consequences of codon bias. Nature Rev Genet 12:32–42PubMedCrossRefPubMedCentralGoogle Scholar
  47. Pridgeon JW, Klesius PH (2013) Apolipoprotein A1 in channel catfish: transcriptional analysis, antimicrobial activity, and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection. Fish Shellfish Immunol 35:1129–1137PubMedCrossRefPubMedCentralGoogle Scholar
  48. Qu M, Huang X, Zhang X, Liu Q, land Ding S. (2014) Cloning and expression analysis of apolipoprotein A-I (ApoA-I) in the Hong Kong grouper (Epinephelus akaara). Aquaculture 432:85–96CrossRefGoogle Scholar
  49. Rahim S, Abdullah HM, Ali Y, Khan UI, Ullah W, Shahzad MA, Waleed M (2016) Serum Apo A-1 and its role as a biomarker of coronary artery disease. Cureus 8:e941.  https://doi.org/10.7759/cureus.941 CrossRefPubMedPubMedCentralGoogle Scholar
  50. Rico M, Bruix M, González C, Monsalve RI, Rodríguez R (1996) H1 NMR assignment and global fold of napin BnIb, are presentative2S albumin seed protein. Biochemistry 35:15672–15682PubMedCrossRefPubMedCentralGoogle Scholar
  51. Ryan RO, Forte TM, Oda MN (2003) Optimized bacterial expression of human apolipoprotein A-I. Protein Expr Purif 27:98–103PubMedCrossRefPubMedCentralGoogle Scholar
  52. Saito H, Lund-Katz S, Phillips MC (2004) Contributions of domain structure and lipid interaction to the functionality of exchangeable human apolipoproteins. Prog Lipid Res 43:350–380PubMedCrossRefPubMedCentralGoogle Scholar
  53. Schmidt HH, Genschel J, Haas R, Büttner C, Manns MP (1997) Expression and purification of recombinant human apolipoprotein A-I in Chinese hamster ovary cells. Protein Expr Purif 10:226–236PubMedCrossRefPubMedCentralGoogle Scholar
  54. Segrest JP, Jones MK, Mishra VK, Pierotti V, Young SH, Borén J, Innerarity TL, Dashti N (1998) Apolipoprotein B-100: conservation of lipid-associating amphipathic secondary structural motifs in nine species of vertebrates. J Lipid Res 39:85–102PubMedPubMedCentralGoogle Scholar
  55. Shaalan M, Saleh M, El-Mahdy M, El-Matbouli M (2016) Recent progress in applications of nanoparticles in fish medicine: a review. Nanomedicine 12:701–710PubMedCrossRefPubMedCentralGoogle Scholar
  56. Singh IP, Chopra AK, Coppenhaver DH, Ananatharamaiah GM, Baron S (1999) Lipoproteins account for part of the broad non-specific antiviral activity of human serum. Antiviral Res 42:211–218PubMedCrossRefPubMedCentralGoogle Scholar
  57. Smith LE, Yang J, Goodman L, Huang X, Huang R, Dressman J, Morris J, Silva RA, Davidson WS, Cavigiolio G (2012) High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli. J Lipid Res 53:1708–1715PubMedPubMedCentralCrossRefGoogle Scholar
  58. Solanki AK, Bhatia B, Kaushik H, Deshmukh SK, Dixit A, Garg LC (2017) Clostridium perfringens beta toxin DNA prime-protein boost elicits enhanced protective immune response in mice. Appl Microbiol Biotechnol 101:5699–5708PubMedCrossRefPubMedCentralGoogle Scholar
  59. Srinivas RV, Venkatachalapathi YY, Rui Z, Owens RJ, Gupta KB, Anantharamaiah GM, Segrest JP, Compans RW (1991) Inhibition of virus-induced cell fusion by apolipoprotein A-I and its amphipathic peptide analogs. J Cell Biochem 45:224–237PubMedCrossRefPubMedCentralGoogle Scholar
  60. Srinivasa Rao PS, Lim TM, Leung KY (2001) Opsonized virulent Edwardsiella tarda strains are able to adhere to and survive and replicate within fish phagocytes but fail to stimulate reactive oxygen intermediates. Infect Immun 69:5689–5697PubMedCrossRefPubMedCentralGoogle Scholar
  61. Ulevitch RJ, Johnston AR, Weinstein DB (1981) New function for high density lipoproteins. Isolation and characterization of a bacterial lipopolysaccharide-high density lipoproteincomplex formed in rabbit plasma. J Clin Invest 67:827–837PubMedPubMedCentralCrossRefGoogle Scholar
  62. van Oosten M, Rensen PC, van Amersfoort ES, van Eck M, van Dam AM, Breve JJ, Vogel T, Panet A, van Berkel TJ, Kuiper J (2001) Apolipoprotein E protects against bacterial lipopolysaccharide-induced lethality. A new therapeutic approach to treat gram-negative sepsis. J Biol Chem 276:8820–8824PubMedCrossRefPubMedCentralGoogle Scholar
  63. Verma R, Balaji BS, Dixit A (2018) Phytochemical analysis and broad spectrum antimicrobial activity of ethanolic extract of Jasminum mesnyi Hance leaves and its solvent partitioned fractions. Bioinformation 14:430–438PubMedPubMedCentralCrossRefGoogle Scholar
  64. Villarroel F, Bastias A, Casado A, Amthauer R, Concha MI (2007) Apolipoprotein A-I, an antimicrobial protein in Oncorhynchus mykiss: evaluation of its expression in primary defence barriers and plasma levels in sick and healthy fish. Fish Shellfish Immunol 23:197–209PubMedCrossRefPubMedCentralGoogle Scholar
  65. Wang L, Hua N, Atkinson D, Small DM (2007) The N-terminal (1–44) and C-terminal (198–243) peptides of Apolipoprotein A–I behave differently at the triolein/water interface. Biochemistry 46:12140–12151PubMedCrossRefPubMedCentralGoogle Scholar
  66. Wang W, Que Q, Chen J (2019) Identificaiton, expression analysis, and antibacterial activity of apolipoprotein A-I from amphioxus (Branchiostoma belcheri). Comp Biochem Physiol Part B Biochem Mol Biol 238:110329.  https://doi.org/10.1016/j.cbpb.2019.110329 CrossRefGoogle Scholar
  67. Wu T, Lee CG, Buckler-White A, Kozak CA (2002) Genetic control of a mouse serum lipoprotein factor that inactivates murine leukemia viruses: evaluation of apolipoprotein F as a candidate. J Virol 76:2279–2286PubMedPubMedCentralCrossRefGoogle Scholar
  68. Yadav SKR, Sahu T, Dixit A (2016) Structural and functional characterization of recombinant napin-like protein of Momordica charantia expressed in methylotrophic yeast Pichia pastoris. Appl Microbiol Biotechnol 100:6703–6713PubMedCrossRefPubMedCentralGoogle Scholar
  69. Zamanian-Daryoush M, DiDonato JA (2015) Apolipoprotein A-I and cancer. Front Pharmacol 6:265.  https://doi.org/10.3389/fphar.2015.00265 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Gene Regulation Laboratory, School of BiotechnologyJawaharlal Nehru UniversityNew DelhiIndia
  2. 2.Fish Health Management DivisionCentral Institute of Freshwater AquacultureBhubaneswarIndia
  3. 3.Gene Regulation LaboratoryNational Institute of ImmunologyNew DelhiIndia

Personalised recommendations