Skip to main content

Advertisement

SpringerLink
Log in

Functional analysis of recombinant buffalo lactoferrin and monoferric lobes and their cytotoxic effect on buffalo mammary epithelial cells

  • Published:
BioMetals Aims and scope Submit manuscript

Abstract

Lactoferrin (Lf) has been involved in diverse type of cellular activities and its biochemical properties are species specific. Lf is a bilobal molecule in which each lobe binds with one Fe2+/Fe3+ ion. A lot of physiological effects of Lf are regulated by its iron binding and release properties; however these properties are species-specific. To understand the iron-binding, thermal stability and cytotoxic effect of buffalo Lf (buLf) and contribution of individual N- and C-terminal lobes therein, buLf and the truncated monoferric lobes were expressed in Kluyveromyces lactis or Pichia pastoris yeast expression systems. The iron-uptake/release behavior and thermal stability of recombinant buLf was observed similar to the Lf purified from buffalo milk. Supplementation of recombinant buLf to the buffalo mammary epithelial cells (BuMEC) culture decreased their proliferation and the cell viability in a dose dependent manner. The cell growth decreased by 37% at 1.0 mg/ml Lf. C-lobe decreased the viability of BuMEC by 15% at 1 mg/ml. The C-lobe showed greater cytotoxic effect against BuMEC in comparison to N-lobe. buLf caused a reduced expression of the casein in BuMEC. At 1.0 mg/ml of buLf, CSN2 transcript level was reduced by 74% and 78% in the normal and hormone free media, respectively. The expression of IL-1β gene in BuMEC increased by 4–5 fold in the presence of 1.0 mg/ml of Lf. The effect was similar to that observed in the involutory mammary gland, suggesting the role of elevated level of Lf in remodeling of buffalo mammary tissue during involution.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Anand V, Dogra N, Singh S, Kumar SN, Jena MK, Malakar D, Dang AK, Mishra BP, Mukhopadhyay TK, Kaushik JK, Mohanty AK (2012) Establishment and characterization of a buffalo (Bubalus bubalis) mammary epithelial cell line. PLoS ONE 7:e40469

    Article  CAS  Google Scholar 

  • Bai X, Teng D, Tian Z, Zhu Y, Yang Y, Wang J (2010) Contribution of bovine lactoferrin inter-lobe region to iron binding stability and antimicrobial activity against Staphylococcus aureus. Biometals 23:431–439

    Article  CAS  Google Scholar 

  • Baker HM, Baker CJ, Smith CA, Baker EN (2000) Metal substitution in transferrins: specific binding of cerium(IV) revealed by the crystal structure of cerium-substituted human lactoferrin. J Biol Inorg Chem 5:692–698

    Article  CAS  Google Scholar 

  • Barrett AJ, Rawlings ND, Davies M, Machleidt W, Salvesen G, Turk V (1986) Proteinase inhibitors, 1st edn. Elsevier, Holland, pp 515–569

    Google Scholar 

  • Baumrucker C, Erondu N (2000) Insulin-like growth factor (IGF) system in the bovine mammary gland and milk. J Mammary Gland Biol Neoplasia 5:53–64

    Article  CAS  Google Scholar 

  • Beaton A, Broadhurst M, Wilkins R, Wheeler T (2003) Suppression of b-casein gene expression by inhibition of protein synthesis in mouse mammary epithelial cells is associated with stimulation of NF-kb activity and blockage of prolactin-Stat5 signaling. Cell Tissue Res 311:207–215

    CAS  PubMed  Google Scholar 

  • Bennett RM, Davis J (1982) Lactoferrin interacts with deoxyribonucleic acid: a preferential reactivity with double-stranded DNA and dissociation of DNA-anti-DNA complexes. J Lab Clin Med 99:127–138

    CAS  PubMed  Google Scholar 

  • Boehmer JL (2011) Proteomic analyses of host and pathogen responses during bovine mastitis. J Mammary Gland Biol Neoplasia 16:323–338

    Article  Google Scholar 

  • Chen GH, Yin LJ, Chiang IH, Jiang ST (2007) Expression and purification of goat lactoferrin from Pichia pastoris expression system. J Food Sci 72:67–71

    Article  Google Scholar 

  • Damiens E, Yazidi EI, Mazurier J, Rochard E, Duthille I, Spik G (1998) Role of heparan sulphate proteoglycans in the regulation of human lactoferrin binding and activity in the MDA-MB-231 breast cancer cell line. Eur J Cell Biol 77:344–351

    Article  CAS  Google Scholar 

  • Dauria E, Agostoni C, Giovannin M, Riva E, Zetterstrom R, Fortin R, Greppi GF, Bonizzi L (2005) Proteomic evaluation of milk from different mammalian species as a substitute for breast milk. Acta Paediatr Int J Paediatr 12:1708–1713

    Article  Google Scholar 

  • Day CL, Stowell KM, Baker EN, Tweedie JW (1992) Studies of the N-terminal half of human lactoferrin produced from the cloned cDNA demonstrate that interlobe interactions modulate iron release. J Biol Chem 267:13857–13862

    CAS  PubMed  Google Scholar 

  • El-Fattah AMA, Rabo FH, El-Dieb SM, El-Kashef HA (2012) Changes in composition of colostrum of Egyptian buffaloes and Holstein cows. BMC Vet Res 8:1–7

    Article  Google Scholar 

  • Fakharany EM, Sanchez L, Mehdar HA, Redwan EM (2013) Effectiveness of human, camel, bovine and sheep lactoferrin on the hepatitis C virus cellular infectivity: comparison study. Virol J. https://doi.org/10.1186/1743-422X-10-199

    Article  PubMed  Google Scholar 

  • Farnaud S, Evans RW (2005) Lactoferrin: a multifunctional protein with antimicrobial properties. Mol Immunol 40:395–405

    Article  Google Scholar 

  • Flintegaard TV, Thygesen P, Rahbek-Nielsen H, Levery SB, Kristensen C, Clausen H, Bolt G (2010) N-Glycosylation increases the circulatory half-life of human growth hormone. Endocrinol 151:5326–5336

    Article  CAS  Google Scholar 

  • Fujita K, Matsuda E, Sekine K, Iigo M, Tsuda H (2004) Lactoferrin enhances Fas expression and apoptosis in the colon mucosa of azoxymethane-treated rats. Car-cinogen 25:1961–1966

    CAS  Google Scholar 

  • Furlong SJ, Mader JS, Hoskin DW (2006) Lactoferricin-induced apoptosis in estrogen-nonresponsive MDA-MB-435 breast cancer cells is enhanced by C6 ceramide or tamoxifen. Oncol Rep 15:1385–1390

    CAS  PubMed  Google Scholar 

  • Guschina TA, Soboleva SE, Nevinsky GA (2013) Recognition of specific & nonspecific DNA by human lactoferrin. J Mol Recognit 26:136–148

    Article  CAS  Google Scholar 

  • Hettinga K, Van Valenberg H, Vries SD, Boeren S, Van Hooijdonk T, Van Arendonk J (2011) The host defense proteome of human and bovine milk. PLoS ONE 6:e19433

    Article  CAS  Google Scholar 

  • Hu L, Gao CH, Hong C, Zhong Q, Dong HL, Gao XM (2016) Expression, purification, and breast cancer cell inhibiting effect of recombinant human lactoferrin C-lobe. Biosci Biotechnol Biochem 80:257–263

    Article  CAS  Google Scholar 

  • Jefferis R (2009) Glycosylation as a strategy to improve antibody-based therapeutics. Nat Rev Drug Discov 8:226–234

    Article  CAS  Google Scholar 

  • Kanyshkova TG, Semenov DV, Buneve VN, Nevinsky GA (1999) Human milk lactoferrin binds two DNA molecules with different affinities. FEBS Lett 451:235–237

    Article  CAS  Google Scholar 

  • Karthikeyan S, Paramasivam M, Yadav S, Srinivasan A, Singh TP (1999) Structure of buffalo lactoferrin at 2.5 Å resolution using crystals grown at 303 K shows different orientations of the N and C lobes. Acta Crystallogr Sect D 55:1805–1813

    Article  CAS  Google Scholar 

  • Kaushik JK, Iimura S, Ogasahara K, Yamagata Y, Segawa S, Yutani K (2006) Completely-buried, non-ion-paired glutamic acid contributes favourably to the conformational stability of pyrrolidone carboxylic peptidases from hyperthermophiles. Biochemistry 45:7100–7112

    Article  CAS  Google Scholar 

  • Khan JA, Kumar P, Paramasivam M, Yadav RS, Sahani MS, Sharma S, Srinivasan A, Singh TP (2001) Camel lactoferrin a transferrin-cum-lactoferrin: crystal structure of camel apolactoferrin at 2.6 Å resolution and structural basis of its dual role. J Mol Biol 309:751–761

    Article  CAS  Google Scholar 

  • Kumar P, Khan JA, Yadav S, Singh TP (2002) Crystal Structure of equine apolactoferrin at 303 K providing further evidence of closed conformations of N and C lobes. Acta Crystallogr D 58:225–332

    Article  Google Scholar 

  • Legrand D (2012) Lactoferrin, a key molecule in immune and inflammatory processes. Biochem Cell Biol 3:252–268

    Article  Google Scholar 

  • Lin T, Chiou S, Chen M, Kuo C (2005) Human lactoferrin exerts bi-directional actions on PC12 cell survival via ERK1/2 pathway. Biochem Biophys Res Commun 337:330–336

    Article  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔ C T method. Methods 25:402–408

    Article  CAS  Google Scholar 

  • Lonnerdal B, Iyer S (1995) Lactoferrin: molecular structure and biological function. Annu Rev Nutr 15:93–110

    Article  CAS  Google Scholar 

  • Masson PL, Heremans JF (1971) Lactoferrin in milk from different species. Comp Biochem Physiol B 39:119–129

    Article  CAS  Google Scholar 

  • Mazurier J, Spik G (1980) Comparative study of the iron-binding properties of human tranferrins. I. Complete and sequential iron saturation and desaturation of the lactotransferrin. Biochem Biophys Acta 629:399–408

    Article  CAS  Google Scholar 

  • Mazurier J, Legrand D, Hu WL, Montreuil J, Spik G (1989) Expression of human lactotransferrin receptors in phytohemagglutinin-stimulated human peripheral blood lymphocytes: isolation of the receptors by antiligand-affinity chromatography. Eur J Biochem 179:481–487

    Article  CAS  Google Scholar 

  • Montoya-G IA, Cendon TS, Gallegos AS, Cruz-R Q (2012) Lactoferrin a multiple bioactive protein. An overview. Biochim Biophys Acta 1820:226–236

    Article  Google Scholar 

  • Ohashi A, Murata E, Yamamoto K, Majima E, Sano E, Le QT, Katunumaa N (2003) New functions of lactoferrin and β-casein in mammalian milk as cysteine protease inhibitors. Biochem Biophysic Res Commun 306:98–103

    Article  CAS  Google Scholar 

  • Rahman M, Kim WS, Kumura H, Shimazaki K (2009) Bovine lactoferrin region responsible for binding to bifidobacterial cell surface proteins. Biotechnol Lett 31:863–868

    Article  CAS  Google Scholar 

  • Redwan EM, 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 M. https://doi.org/10.1186/1472-6882-14-219

    Article  Google Scholar 

  • Rejman JJ, Payne KD, Lewis MJ, Torre PM, Muenchen RA, Oliver SP (1992) Influence of apo- and iron-saturated lactoferrin and transferrin, immunoglobulin G and serum albumin on proliferation of bovine peripheral blood mononuclear cells. Food Agric Immunol 4:253–257

    Article  CAS  Google Scholar 

  • Riley LG, Williams P, Wynn PC, Sheehy PA (2008) Lactoferrin decreases primary bovine mammary epithelial cell viability and casein expression. J Dairy Res 75:135–141

    Article  CAS  Google Scholar 

  • Rochard E, Legrand D, Lecocq M, Hamelin R, Crepin M, Montreuil J, Spik G (1992) Characterization of lactotransferrin receptor in epithelial cell lines from non-malignant human breast, benign mastopathies and breast carcinomas. Anticancer Res 12:2047–2051

    CAS  PubMed  Google Scholar 

  • Roy M, Kuwabara Y, Hara K, Watanabe Y, Tamai Y (2002) Peptides from the N-terminal end of bovine lactoferrin induce apoptosis in human leukemic (HL-60) cells. J Dairy Sci 85:2065–2074

    Article  CAS  Google Scholar 

  • Sakai T, Banno Y, Kato Y, Nozawa Y, Kawaguchi M (2005) Pepsin-digested bovine lactoferrin induces apoptotic cell death with JNK/SAPK activation in oral cancer cells. J Pharmacol Sci 98:41–48

    Article  CAS  Google Scholar 

  • Sano E, Miyauchi R, Takakura N, Yamauchi K, Murata E, Le QT, Katunuma N (2005) Cysteine protease inhibitors in various milk preparations and its importance as a food. Food Res Int 38:427–433

    Article  CAS  Google Scholar 

  • Senda A, Fukuda K, Ishii T, Urashima T (2011) Changes in the bovine whey proteome during the early lactation period. Anim Sci J 82:698–706

    Article  CAS  Google Scholar 

  • Sharma S, Singh TP, Bhatia KL (1999a) Preparation and characterization of the N and C monoferric lobes of buffalo lactoferrin produced by proteolysis using proteinase K. J Dairy Res 66:81–90

    Article  CAS  Google Scholar 

  • Sharma AK, Rajashankar KR, Yadav MP, Singh TP (1999b) Structure of mare apolactoferrin: the N and C lobes are in the closed form. Acta Crystallogr Sect D 55:1152–1157

    Article  CAS  Google Scholar 

  • Sharma S, Sinha M, Kaushik S, Kaur P, Singh TP (2013) C-lobe of lactoferrin: the whole story of the half-molecule. Biochem Res Int 2013:271641

    Article  Google Scholar 

  • Shimazaki K, Tanaka T, Kon H, Oota K, Kawaguchi A, Maki Y, Sato T (1993) Separation and characterization of the Cterminal half molecule of bovine lactoferrin. J Dairy Sci 76:946–955

    Article  CAS  Google Scholar 

  • Smith KL, Schanbacher FL (1977) Lactoferrin as a factor of resistance to infection of the bovine mammary gland. J Am Vet Med Assoc 170:1224–1227

    CAS  PubMed  Google Scholar 

  • Son K, Park J, Chung C, Chung D, Yu D, Lee K, Kim J (2002) Human lactoferrin activates transcription of IL-1b gene in mammalian cells. Biochem Biophys Res Commun 290:236–241

    Article  CAS  Google Scholar 

  • Ward PP, Chu H, Zhou X, Conneely OM (1997) Expression and characterization of recombinant murine lactoferrin. Gene 204:171–176

    Article  CAS  Google Scholar 

  • Ward PP, Uribe-Luna S, Conneely OM (2002) Lactoferrin and host defense. Biochem Cell Biol 80:95–102

    Article  CAS  Google Scholar 

  • Welty F, Smith K, Schanbacher F (1975) Lactoferrin concentration during involution of the bovine mammary gland. J Dairy Sci 59:224–231

    Article  Google Scholar 

  • Wilde CJ, Knight CH, Flint DJ (1999) Control of milk secretion and apoptosis during mammary involution. J Mammary Gland Biol Neoplasia 4:129–136

    Article  CAS  Google Scholar 

  • Yadav P, Singh DD, Mukesh M, Kataria RS, Yadav A, Mohanty AK, Mishra BP (2012) Identification of suitable housekeeping genes for expression analysis in mammary epithelial cells of buffalo (Bubalus bubalis) during lactation cycle. Livest Sci 147:72–76

    Article  Google Scholar 

  • Zhang L, Van Dijk AD, Hetinga K (2017) An interactomics overview of the human and bovine milk proteome over lactation. Proteome Sci. https://doi.org/10.1186/s12953-016-0110-0

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We acknowledge the financial support provided by the Department of Biotechnology (Project BT/PR8960/01/323/2007), Ministry of Science and Technology and Indian Council of Agricultural Research under its Niche Area of Excellence project on buffalo production and reproduction genomics. We acknowledge the hardware support for sequence analysis at BTIS Sub-Distributed Information Centre, supported by DBT, Govt of India at the ICAR-National Dairy Research Institute, Karnal.

Author information

Author notes

  1. Parvesh Bubber

    Present address: School of Life Sciences, Biochemistry Discipline, Indira Gandhi National Open University, New Delhi, India

Authors and Affiliations

  1. Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, 132 001, India

    Surender Singh, Shalini Kalra, Tirtha K. Datta, Ashok K. Mohanty & Jai K. Kaushik

  2. Department of Bioscience & Biotechnology, Banasthali Vidyapith, Vanasthali, Rajasthan, India

    Surender Singh & Parvesh Bubber

Authors
  1. Surender Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shalini Kalra
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Parvesh Bubber
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tirtha K. Datta
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ashok K. Mohanty
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jai K. Kaushik
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jai K. Kaushik.

Ethics declarations

Conflicts of interest

The authors declare no conflicts of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 582 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Singh, S., Kalra, S., Bubber, P. et al. Functional analysis of recombinant buffalo lactoferrin and monoferric lobes and their cytotoxic effect on buffalo mammary epithelial cells. Biometals 32, 771–783 (2019). https://doi.org/10.1007/s10534-019-00209-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10534-019-00209-0

Keywords

Search

Navigation