Novel Recombinant Human Lactoferrin: Differential Activation of Oxidative Stress Related Gene Expression

Overview

Journal J Biotechnol

Specialties Biomedical Engineering
Biotechnology

Date 2013 Sep 28

PMID 24070904

Citations 32

Authors

Marian L Kruzel

Jeffrey K Actor

Michal Zimecki

Jasen Wise

Paulina Ploszaj

Shaper Mirza

Mark Kruzel

Shen-An Hwang

Xueqing Ba

Istvan Boldogh

Affiliations

Soon will be listed here.

Abstract

Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins. The aim of this study was to scale-up expression and purification of rhLF in a CHO expression system, verify its glycan primary structure, and assess its biological properties in cell culture models. A stable CHO cell line producing >200mg/L of rhLF was developed and established. rhLF was purified by a single-step cation-exchange chromatography procedure. The highly homogenous rhLF has a molecular weight of approximately 80 kDa. MALDI-TOF mass spectrometric analysis revealed N-linked, partially sialylated glycans at two glycosylation sites, typical for human milk LF. This novel rhLF showed a protective effect against oxidative stress in a similar manner to its natural counterpart. In addition, rhLF revealed a modulatory effect on cellular redox via upregulation of key antioxidant enzymes. These data imply that the CHO-derived rhLF is fully compatible with the native molecule, thus it has promise for human therapeutic applications.

Citing Articles

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References

Mirza S, Wilson L, Benjamin Jr W, Novak J, Barnes S, Hollingshead S . Serine protease PrtA from Streptococcus pneumoniae plays a role in the killing of S. pneumoniae by apolactoferrin. Infect Immun. 2011; 79(6):2440-50. PMC: 3125832. DOI: 10.1128/IAI.00489-10. View

Font-Nieves M, Sans-Fons M, Gorina R, Bonfill-Teixidor E, Salas-Perdomo A, Marquez-Kisinousky L . Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes. J Biol Chem. 2012; 287(9):6454-68. PMC: 3307308. DOI: 10.1074/jbc.M111.327874. View

Marth J, Grewal P . Mammalian glycosylation in immunity. Nat Rev Immunol. 2008; 8(11):874-87. PMC: 2768770. DOI: 10.1038/nri2417. View

Huffman S, Combest C . Role of breast-feeding in the prevention and treatment of diarrhoea. J Diarrhoeal Dis Res. 1990; 8(3):68-81. View

Hossler P, Khattak S, Li Z . Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology. 2009; 19(9):936-49. DOI: 10.1093/glycob/cwp079. View

Boldogh I, Bacsi A, Choudhury B, Dharajiya N, Alam R, Hazra T . ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation. J Clin Invest. 2005; 115(8):2169-79. PMC: 1180538. DOI: 10.1172/JCI24422. View

Davies J, Jiang L, Pan L, LaBarre M, Anderson D, Reff M . Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FC gamma RIII. Biotechnol Bioeng. 2001; 74(4):288-94. View

Matsumoto A, Shikata K, Takeuchi F, Kojima N, Mizuochi T . Autoantibody activity of IgG rheumatoid factor increases with decreasing levels of galactosylation and sialylation. J Biochem. 2000; 128(4):621-8. DOI: 10.1093/oxfordjournals.jbchem.a022794. View

Freeze H, Aebi M . Altered glycan structures: the molecular basis of congenital disorders of glycosylation. Curr Opin Struct Biol. 2005; 15(5):490-8. DOI: 10.1016/j.sbi.2005.08.010. View

10.

Morelle W, Faid V, Chirat F, Michalski J . Analysis of N- and O-linked glycans from glycoproteins using MALDI-TOF mass spectrometry. Methods Mol Biol. 2009; 534:5-21. DOI: 10.1007/978-1-59745-022-5_1. View

11.

Ding W, Nothaft H, Szymanski C, Kelly J . Identification and quantification of glycoproteins using ion-pairing normal-phase liquid chromatography and mass spectrometry. Mol Cell Proteomics. 2009; 8(9):2170-85. PMC: 2742440. DOI: 10.1074/mcp.M900088-MCP200. View

12.

Hwang S, Wilk K, Kruzel M, Actor J . A novel recombinant human lactoferrin augments the BCG vaccine and protects alveolar integrity upon infection with Mycobacterium tuberculosis in mice. Vaccine. 2009; 27(23):3026-34. PMC: 2680785. DOI: 10.1016/j.vaccine.2009.03.036. View

13.

Sanchez L, Calvo M, Brock J . Biological role of lactoferrin. Arch Dis Child. 1992; 67(5):657-61. PMC: 1793702. DOI: 10.1136/adc.67.5.657. View

14.

Baynes R, Bezwoda W . Lactoferrin and the inflammatory response. Adv Exp Med Biol. 1994; 357:133-41. DOI: 10.1007/978-1-4615-2548-6_13. View

15.

Loignon M, Perret S, Kelly J, Boulais D, Cass B, Bisson L . Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells. BMC Biotechnol. 2008; 8:65. PMC: 2538527. DOI: 10.1186/1472-6750-8-65. View

16.

Conesa C, Calvo M, Sanchez L . Recombinant human lactoferrin: a valuable protein for pharmaceutical products and functional foods. Biotechnol Adv. 2010; 28(6):831-8. DOI: 10.1016/j.biotechadv.2010.07.002. View

17.

Moguilevsky N, Retegui L, Masson P . Comparison of human lactoferrins from milk and neutrophilic leucocytes. Relative molecular mass, isoelectric point, iron-binding properties and uptake by the liver. Biochem J. 1985; 229(2):353-9. PMC: 1145067. DOI: 10.1042/bj2290353. View

18.

Kruzel M, Harari Y, Mailman D, Actor J, Zimecki M . Differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on LPS-induced inflammatory responses in mice. Clin Exp Immunol. 2002; 130(1):25-31. PMC: 1906493. DOI: 10.1046/j.1365-2249.2002.01956.x. View

19.

Bradford M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54. DOI: 10.1016/0003-2697(76)90527-3. View

20.

Shental-Bechor D, Levy Y . Folding of glycoproteins: toward understanding the biophysics of the glycosylation code. Curr Opin Struct Biol. 2009; 19(5):524-33. DOI: 10.1016/j.sbi.2009.07.002. View