ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)
Biotechnologia Acta V. 15, No. 5, 2022
P. 31-40 . Bibliography 35, Engl.
UDC: 577.27:606.61:617.7
https://doi.org/10.15407/biotech15.05.031
1 Palladin Institute of Biochemistry of NAS of Ukraine, Kyiv, Ukraine
2 Bogomolets National Medical University, Kyiv, Ukraine
3 Bingöl University, Bingöl, Türkiye
Lactoferrin is a ubiquitous and multifunctional protein, which has antimicrobial and immunomodulatory activities. Lactoferrin plays an important role in the maintenance of ocular health.
The aim of the study was to produce polyclonal antibodies against human lactoferrin in order to apply them in evaluation of lactoferrin levels in tear fluid collected from healthy eye and after corneal injury.
Materials and methods. Affine chromatography on Protein A-sepharose was applied in order to isolate immunoglobulin G (IgG) fraction from the blood serum of lactoferrin-immunized rabbits. Each step of protein purification was monitored by denaturing gel electrophoresis (SDS-PAGE). Target antigen recognition by produced antibodies was established by western blot analysis with the use of diluted IgG fraction. Lactoferrin levels in the tear fluids collected from healthy individuals (n = 4) and patients with non-penetrating corneal injures (n = 6) were determined immunochemically with the use of purified antibodies. The results of western blot of lactoferrin levels in the tear fluids of healthy individuals and patients with corneal wounds were analysed using Mann-Whitney U-test. The difference between group mean values was considered significant at P<0.05.
Results. Using affine chromatography on Protein A-sepharose, antibodies against human lactoferrin were purified as IgG fraction from blood serum of lactoferrin-immunized rabbits. Western blot analysis showed that obtained antibodies recognize the antigen as a 75-kDa band, which corresponds to the intact human lactoferrin polypeptide. The same major polypeptide band was visualized by western blot with enhanced chemiluminescence detection in the tear fluid samples. Densitometry analysis of 75-kDa lactoferrin band showed 3.2-fold decrease in lactoferrin level in the tear fluid samples obtained from patients with non-penetrating corneal traumas as compared with samples collected from healthy persons (P<0.05). Besides, tear fluid of patients with injured corneas contained large amounts of truncated lactoferrin immunoreactive polypeptides as well as high molecular weight bands, which could correspond to lactoferrin complexes with other proteins occurring during inflammation.
Conclusions. According to our data, obtained anti-lactoferrin antibodies can be used as a valuable tool for development of advanced tests and procedures for diagnostics of eye diseases associated with the corneal lesions. Reduced lactoferrin concentration might represent a potential prognostic biomarker for diagnosis of ocular diseases including non-penetrating corneal injuries in a simple and non-invasive way.
Key words: lactoferrin, antibodies, western blot analysis, corneal wounds, tear fluid
© Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine, 2022
References
1. Kell D. B., Heyden E. L., Pretorius E. The biology of lactoferrin, an iron-binding protein that can help defend against viruses and bacteria. Front. Immunol. 2020, 11, 1221. https://doi/org/10.3389/fimmu.2020.01221
2. Kowalczyk P., Kaczyńska K., Kleczkowska P., Bukowska-Ośko I., Kramkowski K., Sulejczak D. The lactoferrin phenomenon - a miracle molecule. Molecules. 2022, 27 (9), 2941. https://doi.org/10.3390/molecules27092941
3. Alexander D. B., Iigo M., Yamauchi K., Suzui M., Tsuda H. Lactoferrin: an alternative view of its role in human biological fluids. Biochem. Cell Biol. 2012, 90 (3), 279-306. https://doi.org/10.1139/o2012-013
4. Flanagan J. L., Willcox M. D. Role of lactoferrin in the tear film. Biochimie. 2009, 91 (1), 35-43. https://doi.org/10.1016/j.biochi.2008.07.007
5. Rageh A. A., Ferrington D. A., Roehrich, H., Yuan, C., Terluk, M. R., Nelson, E. F., Montezuma, S. R. Lactoferrin expression in human and murine ocular tissue. Curr Eye Res. 2016, 41 (7), 883-889. https://doi.org/10.3109/02713683.2015.1075220
6. Li Y.-Q. Guo C. A Review on Lactoferrin and Central Nervous System Diseases. Cells. 2021, 10, 1810. https://doi.org/10.3390/cells10071810
7. Bielecka M., Cichosz G., Czeczot H. Antioxidant, antimicrobial and anticarcinogenic activities of bovine milk proteins and their hydrolysates - A review. Int. Dairy J. 2022, 127, 105208. https://doi.org/10.1016/j.idairyj.2021.105208
8. Zwirzitz A., Reiter M., Skrabana R., Ohradanova-Repic A., Majdic O., Gutekova M., Cehlar O., Petrovčíková E., Kutejova E., Stanek G., Stockinger H., Leksa, V. Lactoferrin is a natural inhibitor of plasminogen activation. J. Biol. Chem. 2018, 293 (22), 8600–8613. https://doi.org/10.1074/jbc.RA118.003145
9. Matsumura-Takeda K., Ishida T., Sogo S., Isakari Y., Taki T., Sudo T., Kiwada H. Lactoferrin inhibits platelet production from human megakaryocytes in vitro. Biol. Pharm. Bull. 2008, 31 (4), 569–573. https://doi.org/10.1248/bpb.31.569
10. Vagge A., Senni C., Bernabei F., Pellegrini M., Scorcia V., Traverso C. E., Giannaccare G. Therapeutic effects of lactoferrin in ocular diseases: from dry eye disease to infections. Int. J. Mol. Sci. 2020, 21 (18), 6668. https://doi.org/10.3390/ijms21186668
11. Rusciano D., Pezzino S., Olivieri M., Cristaldi M., Gagliano C., Lupo G., Anfuso C.D. Age-related dry eye lactoferrin and lactobionic acid. Ophthalmic Res. 2018, 60 (2), 94–99. https://doi.org/10.1159/000489093
12. Williams T. J., Schneider R. P., Willcox M. D. The effect of protein-coated contact lenses on the adhesion and viability of gram-negative bacteria. Curr. Eye Res. 2003, 27 (4), 227–235. https://doi.org/10.1076/ceyr.27.4.227.16602
13. Hanstock H. G., Edwards J. P., Walsh, N. P. Tear lactoferrin and lysozyme as clinically relevant biomarkers of mucosal immune competence. Front. Immunol. 2019, 10, 1178. https://doi.org/10.3389/fimmu.2019.01178
14. Wakabayashi H., Oda H., Yamauchi K., Abe F. Lactoferrin for prevention of common viral infections. J. Infect. Chemother. 2014, 20 (11), 666–671. https://doi.org/10.1016/j.jiac.2014.08.003
15. Sen M., Honavar S. G., Sharma N., Sachdev M. S. COVID-19 and eye: a review of ophthalmic manifestations of COVID-19. Indian J. Ophthalmol. 2021, 69 (3), 488-509. https://doi.org/10.4103/ijo.IJO_297_21
16. Briana D. D., Papadopoulou A., Syridou G., Marchisio E., Kapsabeli E., Daskalaki A., Papaevangelou V. Early human milk lactoferrin during SARS-CoV-2 infection. J. Matern. Fetal. Neonatal. Med. 2021, 1–4. https://doi.org/10.1080/14767058.2021.1920010
17. Zimecki M., Actor J. K., Kruzel M. L. The potential for Lactoferrin to reduce SARS-CoV-2 induced cytokine storm. Int. Immunopharmacol. 2021, 95, 107571. https://doi.org/10.1016/j.intimp.2021.107571
18. Yolton D. P., Mende S., Harper A., Softing, A. Association of dry eye signs and symptoms with tear lactoferrin concentration. J. Am. Optom. Assoc. 1991, 62 (3), 217–223.
19. Pattamatta U., Willcox M., Stapelton, F., Garrett, Q. Bovine lactoferrin promotes corneal wound healing and supresses IL-1 expression in alkali wounded mouse cornea. Curr. Eye Res. 2013, 38 (11), 1110–1117. https://doi.org/10.3109/02713683.2013.811259
20. Schägger H., Von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 1987, 166 (2), 368–379. https://doi.org/10.1016/0003-2697(87)90587-2
21. Stoscheck C. M. Quantitation of protein. Methods Enzymol. 1990, 182, 50–68. https://doi.org/10.1016/0076-6879(90)82008-p
22. Zhang Y., Lu C., Zhang J. Lactoferrin and its detection methods: a review. Nutrients. 2021, 13 (8), 2492. https://doi.org/10.3390/nu13082492
23. Mruk D. D., Cheng C. Y. Enhanced chemiluminescence (ECL) for routine immunoblotting: an inexpensive alternative to commercially available kits. Spermatogenesis. 2011, 1 (2), 121–122. https://doi.org/10.4161/spmg.1.2.16606
24. Hopp T. P., Spiewak K., Matthews M. H., Athanasiou Z., Blackmore R. S., Gelbfish, G. A. Characterization of proteolytic degradation products of vaginally administered bovine lactoferrin. PloS One. 2022, 17 (5), e0268537. https://doi.org/10.1371/journal.pone.0268537
25. Massucci M. T., Giansanti F., Di Nino G., Turacchio M., Giardi M. F., Botti D., Ippoliti R., De Giulio B., Siciliano R. A., Donnarumma G., Valenti P., Bocedi A., Polticelli F., Ascenzi P., Antonini, G. Proteolytic activity of bovine lactoferrin. Biometals. 2004, 17 (3), 249–255. https://doi.org/10.1023/b:biom.0000027700.90
26. Sabatucci A., Vachette P., Vasilyev V. B., Beltramini M., Sokolov A., Pulina, M. Salvato B., Angelucci C. B., Maccarrone M., Cozzani I., Dainese, E. Structural characterization of the ceruloplasmin: lactoferrin complex in solution. J. Mol. Biol. 2007, 371 (4), 1038–1046. https://doi.org/10.1016/j.jmb.2007.05.089
27. Pulina M. O., Zakharova E. T., Sokolov A. V., Shavlovski M. M., Bass M. G., Solovyov K. V., Kokryakov V. N., Vasilyev, V. B. Studies of the ceruloplasmin-lactoferrin complex. Biochem. Cell. Boil. 2002, 80 (1), 35–39. https://doi.org/10.1139/o01-206
28. Jones G., Lee T.J., Glass J., Rountree G., Ulrich L., Estes A., Sezer M., Zhi W., Sharma S., Sharma A. Comparison of different mass spectrometry workflows for the proteomic analysis of tear fluid. Int. J. Mol. Sci. 2022, 23 (4), 2307. https://doi.org/10.3390/ijms23042307
29. Valenti P., Antonini G. Lactoferrin: an important host defense against microbial and viral attack. Cell. Mol. Life Sci. 2005, 62 (22), 2576–2587. https://doi.org/10.1007/s00018-005-5372-0
30. Wotring J. W., Fursmidt R., Ward L., Sexton J. Z. Evaluating the in vitro efficacy of bovine lactoferrin products against SARS-CoV-2 variants of concern. J. Dairy Sci. 2022, 105 (4), 2791–2802. https://doi.org/10.3168/jds.2021-21247
31. Alpogan O., Karakucuk S. Lactoferrin: the natural protector of the eye against coronavirus-19. Ocul. Immunol. Inflamm. 2021, 29(4), 751–752. https://doi.org/10.1080/09273948.2021.1954202
32. Kuo M. T., Fang P. C., Chao T. L., Chen A., Lai Y. H., Huang Y. T., Tseng C.Y. Tear proteomics approach to monitoring sjögren syndrome or dry eye disease. Int. J. Mol. Sci. 2019, 20 (8), 1932. https://doi.org/10.3390/ijms20081932
33. Ohashi Y., Ishida R., Kojima T., Goto E., Matsumoto Y., Watanabe K., Ishida N., Nakata K., Takeuchi T., Tsubota K. Abnormal protein profiles in tears with dry eye syndrome. Am. J. Ophthalmol. 2003, 136 (2), 291–299. https://doi.org/10.1016/s0002-9394(03)00203-4
34. Ponzini E., Scotti L., Grandori R., Tavazzi S., Zambon A. Lactoferrin concentration in human tears and ocular diseases: a meta-analysis. Invest. Ophthalmol. Vis. Sci. 2020, 61 (12), 9. https://doi.org/10.1167/iovs.61.12.9
35. Zhang Y, Lu C, Zhang J. Lactoferrin and its detection methods: a review. Nutrients. 2021, 13 (8), 2492. https://doi.org/10.3390/nu13082492