title: alpha-Lactalbumin, engineered to be nonnative and inactive, kills tumor cells when in complex with oleic acid: a new biological function resulting from partial unfolding.
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 alpha-lactalbumin on the stationary phase in situ. Because native alpha-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 alpha-lactalbumin in which all eight cysteine residues are substituted for alanines (rHLA(all-Ala)), rendering the protein nonnative and biologically inactive under all conditions. The HAMLET analogue formed from the complex of rHLA(all-Ala) and oleic acid (rHLA(all-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 rHLA(all-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, rHLA(all-Ala)-OA showed significant differences to HAMLET and, in fact, was found to be devoid of any tertiary packing. The results identify alpha-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.
authors.affiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Faculty of Medicine, Lund University, 223 62 Lund, Sweden.
authors.forename: Jenny
authors.initials: J
authors.lastname: Pettersson-Kastberg
authors.forename: Ann-Kristin
authors.initials: AK
authors.lastname: Mossberg
authors.forename: Maria
authors.initials: M
authors.lastname: Trulsson
authors.forename: Yeon Joong
authors.initials: YJ
authors.lastname: Yong
authors.forename: Soyoung
authors.initials: S
authors.lastname: Min
authors.forename: Yoongho
authors.initials: Y
authors.lastname: Lim
authors.forename: John E
authors.initials: JE
authors.lastname: O'Brien
authors.forename: Catharina
authors.initials: C
authors.lastname: Svanborg
authors.forename: K Hun
authors.initials: KH
authors.lastname: Mok
mesh_terms: D019943:Amino Acid Substitution; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry; D002470:Cell Survival / Q000187:drug effects; D003545:Cysteine / Q000235:genetics; D004847:Epithelial Cells / Q000187:drug effects; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008214:Lymphocytes / Q000187:drug effects; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D009829:Oleic Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D017434:Protein Structure, Tertiary
chemical_list: D000970:Antineoplastic Agents; C506422:HAMLET complex, human; D009829:Oleic Acids; D007768:Lactalbumin; D003545:Cysteine