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Two major bovine milk whey proteins induce distinct responses in IEC-6 intestinal cells

Published online by Cambridge University Press:  23 September 2024

Mizuho Inagaki ,
Midori Nohara ,
Chihiro Kobayashi ,
Mayuko Fukuoka ,
Xijier ,
Keisuke Matsumura ,
Kiyomi Ohashi ,
Haru Enomoto ,
Tomio Yabe  and
Yoshihiro Kanamaru
Mizuho Inagaki*
Affiliation:
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan
Midori Nohara
Affiliation:
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan
Chihiro Kobayashi
Affiliation:
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan
Mayuko Fukuoka
Affiliation:
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan
Xijier
Affiliation:
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan
Keisuke Matsumura
Affiliation:
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan
Kiyomi Ohashi
Affiliation:
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan
Haru Enomoto
Affiliation:
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan
Tomio Yabe
Affiliation:
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan Institute for Glyco-core Research (iGCORE), Gifu University, Gifu, 501-1193, Japan Preemptive Food Research Center (PFRC), Gifu University Institute for Advanced Study, Gifu, 501-1193, Japan
Yoshihiro Kanamaru
Affiliation:
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan
*
Corresponding author: Mizuho Inagaki; Email: inagaki.mizuho.u9@f.gifu-u.ac.jp
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Abstract

α-Lactalbumin (α-LA) and β-lactoglobulin (β-LG) are major whey proteins in bovine milk. We studied the effects of these molecules on the intestinal cell response by comparing the native form with the denatured form containing oligomers obtained by treatment with 2,2,2-trifluoroethanol (TFE). We previously reported that proteins in native and TFE-treated forms exhibited cell growth stimulation and cytotoxicity, respectively, in undifferentiated rat crypt IEC-6 and human colon Caco-2 cells. However, neither whey protein showed cytotoxicity even in the TFE-treated form in differentiated Caco-2 cells. Only undifferentiated immature intestinal cells can distinguish between these native and denatured proteins. Moreover, α-LA and β-LG exhibited different oligomer formation characteristics during the TFE treatment. In the present study, we compared the effects of native and TFE-treated whey proteins on IEC-6 cells in more detail. The native forms of both whey proteins exhibited cell proliferative effects in a concentration-dependent manner. For the TFE-treated forms, α-LA showed rapid and potent cytotoxicity, whereas β-LG altered cell responses depending on its concentration and exposure time; lower concentration/shorter exposure and higher concentration/longer exposure induced cell growth stimulation and cytotoxicity, respectively. Pre-treatment of the cell membrane with cholesterol suppressed the effects on the cell response only in TFE-treated β-LG (TFE-β-LG). In a preliminary examination using inhibitors of signal transduction, TFE-treated α-LA acted on the intrinsic apoptosis pathway via Bcl-2-associated X and p53, whereas the action of TFE-LG did not require this pathway. Tyrosine phosphorylation is necessary for the cell proliferation effect of both native whey proteins; however, native α-LA, but not native β-LG, also required activation of the pathway with selective epidermal growth factor receptor tyrosine kinase and Janus kinase 2/3. In summary, the two major bovine milk whey proteins induced similar yet discrete responses in undifferentiated intestinal cells. Even when oligomers are formed, β-LG may be much less hazardous to immature intestinal cells than α-LA.

Keywords

α-Lactalbuminβ-lactoglobulinapoptosiscell proliferationmisfolding

Information

Type
Research Article
Information
Journal of Dairy Research , Volume 91 , Issue 2 , May 2024 , pp. 239 - 245
Copyright
Copyright © The Author(s), 2024. Published by Cambridge University Press on behalf of Hannah Dairy Research Foundation

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References

Akiyama, T and Ogawara, H (1991) Use and specificity of genistein as inhibitor of protein-tyrosine kinases. Methods in Enzymology 201, 362–370.CrossRefGoogle ScholarPubMed
Akiyama, T, Ishida, J, Nakagawa, S, Ogawara, H, Watanabe, S, Itoh, N, Shibuya, M and Fukami, Y (1987) Genistein, a specific inhibitor of tyrosine-specific protein kinases. The Journal of Biological Chemistry 262, 5592–5595.CrossRefGoogle ScholarPubMed
Bucciantini, M, Giannoni, E, Chiti, F, Baroni, F, Formigli, L, Zurdo, J, Taddei, N, Ramponi, G, Dobson, CM and Stefani, M (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416, 507–511.CrossRefGoogle ScholarPubMed
Buck, M (1998) Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptide and proteins. Quarterly Reviews of Biophysics 31, 297–355.CrossRefGoogle Scholar
Cagnol, S and Chambard, JC (2010) ERK and cell death: mechanisms of ERK-induced cell death – apoptosis, autophagy and senescence. The FEBS Journal 277, 2–21.CrossRefGoogle ScholarPubMed
Cecchi, C, Baglioni, S, Fiorillo, C, Pensalfini, A, Liguri, G, Nosi, D, Rigacci, S, Bucciantini, M and Stefani, M (2005) Insights into the molecular basis of the differing susceptibility of varying cell types to the toxicity of amyloid aggregates. Journal of Cell Science 118, 3459–3470.CrossRefGoogle Scholar
Dahlgren, KN, Manelli, AM, Stine, WB Jr, Baker, LK, Krafft, GA and LaDu, MJ (2002) Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability. The Journal of Biological Chemistry 277, 32046–32053.CrossRefGoogle ScholarPubMed
Dobson, CM (2003) Protein folding and misfolding. Nature 426, 884–890.CrossRefGoogle ScholarPubMed
Hambling, SG, McAlpine, AS and Sawyer, L (1992) β-Lactoglobulin. In Fox, PF (ed.), Advanced Dairy Chemistry I: Proteins. Amsterdam: Elsevier, pp. 141–190.Google Scholar
Hashimoto, K, Takeda, K, Nakayama, T and Shimizu, M (1995) Stabilization of the tight junction of the intestinal Caco-2 cell monolayer by milk whey proteins. Bioscience, Biotechnology, and Biochemistry 59, 1951–1952.CrossRefGoogle ScholarPubMed
Hashimoto, K, Nakayama, T and Shimizu, M (1998) Effects of beta lactoglobulin on the tight-junctional stability of Caco-2-SF monolayer. Bioscience, Biotechnology, and Biochemistry 62, 1819–1821.CrossRefGoogle ScholarPubMed
Hill, RL and Brew, K (1975) Lactose synthetase. Advances in Enzymology and Related Areas of Molecular Biology 43, 411–490.Google ScholarPubMed
Hubbard, SR and Till, JH (2000) Protein tyrosine kinase structure and function. Annual Review of Biochemistry 69, 373–398.CrossRefGoogle ScholarPubMed
Inagaki, M, Kawai, S, Xijier, , Fukuoka, M, Yabe, T, Iwamoto, S and Kanamaru, Y (2017) Effects of heat treatment on conformation and cell growth activity of alpha-lactalbumin and beta-lactoglobulin from market milk. Biomedical Research (Tokyo, Japan) 38, 53–59.CrossRefGoogle ScholarPubMed
Karnell, FG, Brezski, RJ, King, LB, Silverman, MA and Monroe, JG (2005) Membrane cholesterol content accounts for developmental differences in surface B cell receptor compartmentalization and signaling. The Journal of Biological Chemistry 280, 25621–25628.CrossRefGoogle ScholarPubMed
Kobayashi, C, Inagaki, M, Nohara, M, Fukuoka, M, Xijier, , Yabe, T and Kanamaru, Y (2021) The effects of denatured major bovine whey proteins on the digestive tract, assessed by Caco-2 cell differentiation and on viability of suckling mice. The Journal of Dairy Research 88, 221–225.CrossRefGoogle ScholarPubMed
Komarova, EA and Gudkov, AV (2000) Suppression of p53: a new approach to overcome side effects of antitumor therapy. Biochemistry. Biokhimii︠a︡ 65, 41–48.Google ScholarPubMed
Levieux, D and Ollier, A (1999) Bovine immunoglobulin G, beta-lactoglobulin, alpha-lactalbumin and serum albumin in colostrum and milk during the early postpartum period. The Journal of Dairy Research 66, 421–430.CrossRefGoogle Scholar
Liu, H, Grosvenor, AJ, Li, X, Wang, XL, Ma, Y, Clerens, S, Dyer, JM and Day, L (2019) Changes in milk protein interactions and associated molecular modification resulting from thermal treatments and storage. Journal of Food Science 84, 1737–1745.CrossRefGoogle ScholarPubMed
Lönnerdal, B and Lien, EL (2003) Nutritional and physiologic significance of α-lactalbumin in infants. Nutrition Reviews 61, 295–305.CrossRefGoogle ScholarPubMed
Meydan, N, Grunberger, T, Dadi, H, Shahar, M, Arpaia, E, Lapidot, Z, Leeder, JS, Freedman, M, Cohen, A, Gazit, A, Levitzki, A and Roifman, CM (1996) Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature 379, 645–648.CrossRefGoogle ScholarPubMed
Naeem, A, Iram, A and Bhat, SA (2015) Anesthetic 2,2,2-trifluoroethanol induces amyloidogenesis and cytotoxicity in human serum albumin. International Journal of Biological Macromolecules 79, 726–735.CrossRefGoogle ScholarPubMed
Peña-Blanco, A and García-Sáez, AJ (2018) Bax, Bak and beyond – mitochondrial performance in apoptosis. The FEBS Journal 285, 416–431.CrossRefGoogle ScholarPubMed
Pérez, MD, Sanchez, L, Aranda, P, Ena, JM, Oria, R and Calvo, M (1990) Synthesis and evolution of concentration of beta-lactoglobulin and alpha-lactalbumin from cow and sheep colostrum and milk throughout early lactation. Cellular and Molecular Biology 36, 205–212.Google ScholarPubMed
Sawyer, L (2021) β-Lactoglobulin and glycodelin: two sides of the same coin? Frontiers in Physiology 12, 678080.CrossRefGoogle ScholarPubMed
Soto, C and Pritzkow, S (2018) Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases. Nature Neuroscience 21, 1332–1340.CrossRefGoogle ScholarPubMed
Stefani, M and Dobson, CM (2003) Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. Journal of Molecular Medicine (Berlin, Germany) 81, 678–699.CrossRefGoogle ScholarPubMed
Suzuki, K, Yanagihara, T, Yokoyama, T, Maeyama, T, Ogata-Suetsugu, S, Arimura-Omori, M, Mikumo, H, Hamada, N, Harada, E, Kuwano, K, Harada, T and Nakanishi, Y (2017) Bax-inhibiting peptide attenuates bleomycin-induced lung injury in mice. Biology Open 6, 1869–1875.Google ScholarPubMed
Xijier, , Mori, Y, Fukuoka, M, Cairangzhuoma, , Inagaki, M, Iwamoto, S, Yabe, T and Kanamaru, Y (2012) Comparison of the efficacy of alpha-lactalbumin from equine, bovine, and human milk in the growth of intestinal IEC-6 cells. Bioscience, Biotechnology, and Biochemistry 76, 843–846.CrossRefGoogle ScholarPubMed
Xu, M, Sugiura, Y, Nagaoka, S and Kanamaru, Y (2005a) IEC-6 intestinal cell death induced by bovine milk α-lactalbumin. Bioscience, Biotechnology, and Biochemistry 69, 1082–1089.CrossRefGoogle ScholarPubMed
Xu, M, Sugiura, Y, Nagaoka, S and Kanamaru, Y (2005b) Involvement of SDS-stable higher Mr forms of bovine normal milk α-lactalbumin in inducing intestinal IEC-6 cell death. Bioscience, Biotechnology, and Biochemistry 69, 1189–1192.CrossRefGoogle ScholarPubMed
Yoshida, T, Tomioka, I, Nagahara, T, Holyst, T, Sawada, M, Hayes, P, Gama, V, Okuno, M, Chen, Y, Abe, Y, Kanouchi, T, Sasada, H, Wang, D, Yokota, T, Sato, E and Matsuyama, S (2004) Bax-inhibiting peptide derived from mouse and rat Ku70. Biochemical and Biophysical Research Communications 321, 961–966.CrossRefGoogle ScholarPubMed
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