Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
권호 표지
DOI QR코드

DOI QR Code

Effects of Bovine α-Lactalbumin Added with Oleic Acid and Microbial Transglutaminase on Cancer Cell Apoptosis

올레산과 미생물 유래 트랜스글루타미나제를 첨가한 우유 알파-락트알부민의 암세포사멸 효과

  • Jeong, Ji-Eun (Dept. of Food and Nutrition.Human Ecology Research Institute, College of Human Ecology, Chonnam National University) ;
  • Hong, Youn-Ho (Dept. of Food and Nutrition.Human Ecology Research Institute, College of Human Ecology, Chonnam National University)
  • 정지은 (전남대학교 생활과학대학 식품영양학과.생활과학연구소) ;
  • 홍윤호 (전남대학교 생활과학대학 식품영양학과.생활과학연구소)
  • Received : 2011.12.09
  • Accepted : 2012.01.08
  • Published : 2012.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

In order to investigate the biological effects of conformational changes in the folding state of bovine ${\alpha}$-lactalbumin (${\alpha}$-La), the protein was prepared and classified as apo form, microbial transglutaminase (MTGase) added form, or bovine ${\alpha}$-La made lethal to tumor cell (BAMLET) form. Apo ${\alpha}$-La form showed weaker cancer cell inhibitory activity (apoptosis) than native ${\alpha}$-La, which suggests that the metal ion-like $Ca^{2+}$ had a positive effect, whereas BAMLET form showed strong cancer cell apoptotic activity. The BAMLET form seemed to be a molten globule structure that increased hydrophobicity. MTGase added to apo ${\alpha}$-La polymer showed similar anti-cancer activity as native ${\alpha}$-La, and it was well hydrolyzed by digestive enzymes. NMR results showed that BAMLET interacted with oleic acid and produced a complex.

본 연구에서는 기능적 특성이 우수한 우유 단백질 소재를 개발하여 식품산업에 응용하고자 우유 ${\alpha}$-La의 영양생리학적 특성에 대하여 분석하였다. 우유 ${\alpha}$-La의 기능성을 향상 시키려는 시도로써 MTGase의 첨가가 이 단백질의 이화학적 및 면역학적 특성에 미치는 영향을 조사하고, 수종의 암세포주의 생육에 대한 억제 효과를 탐색하였으며, ${\alpha}$-La의 구조적 상태에 따라 항암활성의 관련성을 검토하였다. ${\alpha}$-La에 반응한 MTGase에 의해 생성된 중합체는 인체 내 가수분해효소에 의해 분해가 용이한 것으로 확인되었다. 위암 세포주에 ${\alpha}$-La을 10 mg/mL 농도로 처리한 경우 높은 세포독성을 나타냈다. ${\alpha}$-La과 올레산의 복합체인 BAMLET은 간암 세포주에서 ${\alpha}$-La에 비해 더 강한 활성을 보였다. BAMLET은 apo 상태로 상당량의 이차구조를 지니고 있지만, 소수성 중심부는 느슨하게 형성된 MG와 같은 형태를 지니고 있을 것으로 추정되었다. NMR 분석 결과, BAMLET은 1.2 ppm과 0.8 ppm에서 지방산 보조인자인 올레산과 상호 작용하는 것으로 분석되었다.

Keywords

References

  1. Brew K. 2003. $\alpha-lactalbumin$. In Advanced in Dairy Chemistry. 3rd ed. Fox PF, McSweeney PLH, eds. Kluwer Academic/Plenum Press, New York, USA. Vol I, p 387- 421.
  2. Barbana C, Perez MD, Sanchez L, Dalgalarrondo M, Chobert JM, Haertle T, Calvo M. 2006. Interaction of bovine a-lactalbumin with fatty acids as determined by partition equilibrium and fluorescence spectroscopy. Int Dairy J 61: 52-81.
  3. Kamijima T, Ohmura A, Sato T, Akimoto K, Itabashi M, Mizuguchi M, Kamiya M, Kikukawa T, Aizawa T, Takahashi M, Kawano K, Demura M. 2008. Heat-treatment method for producing fatty acid-bound α-lactalbumin that induces tumor cell death. Biochem Biophys Res Commun 376: 211-214. https://doi.org/10.1016/j.bbrc.2008.08.127
  4. Svensson M, Hakansson A, Mossberg AK, Linse S, Svanborg C. 2000. Conversion of $\alpha$-lactalbumin to a protein inducing apoptosis. Proc Natl Acad Sci USA 97: 4221-4226. https://doi.org/10.1073/pnas.97.8.4221
  5. Wijesinha-Bettoni R, Dobson CM, Redfield C. 2001. Comparison of the structural and dynamical properties of holo and apo bovine $\alpha$-lactalbumin by NMR spectroscopy. J Mol Biol 307: 885-898. https://doi.org/10.1006/jmbi.2001.4530
  6. Barbana C, Sanchez L, Perez MD. 2011. Bioactivity of $\alpha$- lactalbumin related to its interaction with fatty acids: a review. Crit Rev Food Sci Nutr 51: 783-794. https://doi.org/10.1080/10408398.2010.481368
  7. Svensson M, Fast J, Mossberg AK, Dueringer C, Gustafsson L, Hallgren O, Brooks CL, Berliner L, Linse S, Svanborg C. 2003. $\alpha-lactalbumin$ unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells). Protein Sci 12: 2794-2804. https://doi.org/10.1110/ps.0231003
  8. FitzGerald RJ, Murray BA, Walsh DJ. 2004. Hypotensive peptides from milk proteins. J Nutr 134: 980S-988S.
  9. Mills S, Ross RP, Hill C, Fitzgerald GF, Stanton C. 2011. Milk intelligence: mining milk for bioactive substances associated with human health. Int Dairy J 21: 377-401. https://doi.org/10.1016/j.idairyj.2010.12.011
  10. Nagendra PS. 2000. Effects of milk-derived bioactives: an overview. Brit J Nutr 84: S3-S10.
  11. Pellegrini A, Thomas U, Bramaz N, Hunziker P, von Fellenberg R. 1999. Isolation and identification of three bactericidal domains in the bovine $\alpha-lactalbumin$ molecule. Biochem Biophys Acta 1426: 439-448. https://doi.org/10.1016/S0304-4165(98)00165-2
  12. Bonisch MP, Heidebach TC, Kulozik U. 2008. Influence of transglutaminase protein cross-linking on the rennet coagulation of casein. Food Hydrocoll 22: 288-297. https://doi.org/10.1016/j.foodhyd.2006.11.015
  13. Motoki M, Nio N. 1983. Crosslinking between different food proteins by transglutaminase. J Food Sci 48: 561-566. https://doi.org/10.1111/j.1365-2621.1983.tb10790.x
  14. Motoki M, Segura K. 1998. Transglutaminase and its uses for food processing. Trends Food Sci Technol 9: 204-210. https://doi.org/10.1016/S0924-2244(98)00038-7
  15. Jeong JE, Hong YH. 2005. Properties of transglutamunase treated milk product powders. Korean J Food Sci Technol 37: 345-351.
  16. Jeong JE, Hong YH. 2006. Elecrophoretical properties of transglutamunase treated milk product powders. Korean J Food Sci Technol 38: 304-308.
  17. Jeong JE, Hong YH. 2009. Effect of transglutaminase addition on the physicochemical properties of sodium caseinate and whey proteins. Korean J Food Sci Ani Resour 29: 415-422. https://doi.org/10.5851/kosfa.2009.29.4.415
  18. O'Sullivan MM, Kelly AL, Fox PF. 2002. Influence of transglutaminase treatment on some physico-chemical properties of milk. J Dairy Res 69: 433-442.
  19. Sibbel A. 2007. The sustainability of functional foods. Soc Sci Medic 64: 554-561. https://doi.org/10.1016/j.socscimed.2006.08.042
  20. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. https://doi.org/10.1038/227680a0
  21. Houghton P, Fang R, Techatanawat I, Steventon G, Hylands PJ, Lee CC. 2007. The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity. Methods 42: 377-387. https://doi.org/10.1016/j.ymeth.2007.01.003
  22. Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P, Vaigro-Wolff A, Gray-Goodrich M, Campbell H, Mayo J, Boyd M. 1991. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 83: 757-766. https://doi.org/10.1093/jnci/83.11.757
  23. Skehan P. 1999. Cell growth and cytotoxicity assays. In Cell Growth, Differentiation and Senescence. Oxford University Press, Oxford, UK. p 41-42.
  24. Fast J, Mossberg AK, Nilsson H, Svanborg C, Akke M, Linse S. 2005. Compact oleic acid in HAMLET. FEBS 579: 6095-6100. https://doi.org/10.1016/j.febslet.2005.08.089
  25. Hakansson A. 1999. Apoptosis induced by a human milk protein complex. PhD Dissertation. Lund University, Lund, Sweden. p 1-98.
  26. Gustafsson L, Hallgren O, Mossberg AK, Pettersson J, Fischer W, Aronsson A, Svanborg C. 2005. HAMLET kills tumor cells by apoptosis: structure, cellular mechanisms, and therapy. J Nutr 135: 1299-1303.
  27. Park SH. 2003. Spectroscopic analysis of partially folded state of ubiquitin. J Korean Soc Agric Chem Biotechnol 46: 305-310.
  28. Svensson M, Sabharwal H, Hakansson A, Mossberg AK, Lipniunas P, Leffler H, Svanborg C, Linse S. 1999. Molecular characterization of α-lactalbumin folding variants that induce apoptosis in tumor cells. J Biol Chem 274: 6388- 6396. https://doi.org/10.1074/jbc.274.10.6388
  29. Nieuwenhuizen WF, Dekker HL, De Koning L, Groneveld T, De Koster CG, De Jong GA. 2003. Modification of glutamine and lysine residues in holo and apo α-lactalbumin with microbial transglutaminase. J Agric Food Chem 51: 7132-7139. https://doi.org/10.1021/jf0300644
  30. Ptitsyn CB. 1995. Molten globule and protein folding. Adv Protein Chem 47: 83-229. https://doi.org/10.1016/S0065-3233(08)60546-X
  31. Bonfils C, Bec N, Larroque C, Rio MD, Gongora C, Pugniere M, Martineau P. 2010. Cyclophilin A as negative regulator of apoptosis by sequestering cytochrome C. Biochem Biophys Res Commun 393: 325-330. https://doi.org/10.1016/j.bbrc.2010.01.135
  32. Jiang J, Serinkan BF, Tyurina YY, Borisenko GG, Mi Z, Robbins PD, Schroit AJ, Kagan VE. 2003. Peroxidation and externalization of phosphatidylserine associated with release of cytochrome C from mitochondria. Free Radic Biol Med 35: 814-825. https://doi.org/10.1016/S0891-5849(03)00429-5