α-Lactalbumin, Engineered to be Nonnative and Inactive, Kills Tumor Cells when in Complex with Oleic Acid: A New Biological Function Resulting from Partial Unfolding |
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Authors: | Jenny Pettersson-Kastberg Maria Trulsson Soyoung Min John E O'Brien K Hun Mok |
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Institution: | 1 Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Faculty of Medicine, Lund University, 223 62 Lund, Sweden 2 School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland 3 Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, South Korea 4 School of Chemistry, Trinity College Dublin, Dublin 2, Ireland 5 Singapore Immunology Network (SIgN), Biomedical Sciences Institutes, Agency for Science, Technology, and Research (A?STAR), 8A Biomedical Grove, Immunos, Biopolis, Singapore 138648, Singapore |
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Abstract: | HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex consisting of partially unfolded protein and fatty acid and was first identified in casein fractions of human breast milk. The complex can be produced from its pure components through a modified chromatographic procedure where preapplied oleic acid binds with partially unfolded α-lactalbumin on the stationary phase in situ. Because native α-lactalbumin itself cannot trigger cell death, HAMLET's remarkable tumor-selective cytotoxicity has been strongly correlated with the conformational change of the protein upon forming the complex, but whether a recovery to the native state subsequently occurs upon entering the tumor cell is yet unclear. To this end, we utilize a recombinant variant of human α-lactalbumin in which all eight cysteine residues are substituted for alanines (rHLAall-Ala), rendering the protein nonnative and biologically inactive under all conditions. The HAMLET analogue formed from the complex of rHLAall-Ala and oleic acid (rHLAall-Ala-OA) exhibited equivalent strong tumoricidal activity against lymphoma and carcinoma cell lines and was shown to accumulate within the nuclei of tumor cells, thus reproducing the cellular trafficking pattern of HAMLET. In contrast, the fatty acid-free rHLAall-Ala protein associated with the tumor cell surface but was not internalized and lacked any cytotoxic activity. Structurally, whereas HAMLET exhibited some residual native character in terms of NMR chemical shift dispersion, rHLAall-Ala-OA showed significant differences to HAMLET and, in fact, was found to be devoid of any tertiary packing. The results identify α-lactalbumin as a protein with strikingly different functions in the native and partially unfolded states. We posit that partial unfolding offers another significant route of functional diversification for proteins within the cell. |
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Keywords: | HLA human α-lactalbumin apo-HLA calcium ion depleted-human α-lactalbumin HAMLET human alpha-lactalbumin made lethal to tumor cells rHLAall-Ala recombinant human α-lactalbumin with all eight cysteines substituted with alanines (C6A C28A C61A C73A C77A C91A C111A C120A) rHLAall-Ala-OA rHLAall-Ala complexed with oleic acid (C18:1:9cis) rHLAD87A recombinant human α-lactalbumin with Asp87 substituted with alanine PBS phosphate-buffered saline ANS 8-anilinonaphthalene-1-sulfonic acid TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling EDTA ethylenediaminetetraacetic acid |
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