Who Is Mr. HAMLET? Interaction of Human R-Lactalbumin with Monomeric Oleic Acid

A specific state of the human milk Ca2+ binding protein R-lactalbumin (hLA) complexed with oleic acid (OA) prepared using an OA-pretreated ion-exchange column (HAMLET) triggers several cell death pathways in various tumor cells. The possibility of preparing a hLA-OA complex with structural and cytotoxic properties similar to those of the HAMLET but under solution conditions has been explored. The complex was formed by titration of hLA by OA at pH 8.3 up to OA critical micelle concentration. We have shown that complex formation strongly depends on calcium, ionic strength, and temperature; the optimal conditions were established. The spectrofluorimetrically estimated number of OA molecules irreversibly bound per hLA molecule (after dialysis of the OA-loaded preparation against water followed by lyophilization) depends upon temperature: 2.9 at 17 °C (native apo-hLA; resulting complex referred to as LA-OA-17 state) and 9 at 45 °C (thermally unfolded apo-hLA; LA-OA-45). Intrinsic tryptophan fluorescence measurements revealed substantially decreased thermal stability of Ca2+-free forms of HAMLET, LA-OA-45, and OA-saturated protein. The irreversibly bound OA does not affect the Ca2+ association constant of the protein. Phase plot analysis of fluorimetric and CD data indicates that the OA binding process involves several hLA intermediates. The effective pseudoequilibrium OA association constants for Ca2+-free hLA were estimated. The far-UV CD spectra of Ca2+-free hLA show that all OA-bound forms of the protein are characterized by elevated content of R-helical structure. The various hLA-OA complexes possess similar cytotoxic activities against human epidermoid larynx carcinoma cells. Overall, the LA-OA-45 complex possesses physicochemical, structural, and cytotoxic properties closely resembling those of HAMLET. The fact that the HAMLET-like complex can be formed in aqueous solution makes the process of its preparation more transparent and controllable, opening up opportunities for formation of active complexes with specific properties. R-Lactalbumin (R-LA) is a well-studied small (molecular mass of 14 kDa, 123 residues) acidic (pI 4-5) globular calcium metalloprotein (1) from milk. It is one of the nutrients of milk which is believed to play a neoprotective role (for review, see ref 2). In the lactating mammary gland, R-LA functions as a noncatalytic regulatory subunit of the lactose synthase enzyme complex (E.C. 2.4.1.22) (3). The protein has a single strong calcium binding site (apparent binding constant 3 × 108 M-1 at 20 °C (4)) and is able to bind other physiologically significant cations such as Mg2+, Na+, K+, and Mn2+ (5). The binding of the metal ions causes pronounced changes in the thermodynamic properties of R-LA (6). It is highly destabilized in the apo state (halftransition temperature 10-30 °C (7)), while calcium association increases its unfolding transition temperature by more than 40 °C. The calciumand temperature-induced structural transitions in R-LA are adequately described on the basis of a four-state scheme (8). Native R-lactalbumin consists of two domains: a large R-helical domain (composed of three major R-helices and two 310 helices) and a small !-sheet domain (contains a small three-stranded antiparallel !-pleated sheet and a 310 helix) (9). The two domains are linked by a Ca2+ binding loop which contains two residues less than the classical EF-hand domain; moreover, the loop is cycled by characteristic Cys73-Cys91 disulfide bond. The significance of calcium ions for the functioning of R-LA remains to be elucidated; nevertheless, calcium binding is required for proper disulfide bond formation during the protein folding (10). The cation binding to R-LA modulates its interaction with both fatty acids (11) and model lipid membranes (12-15), which may be of physiological significance. Although R-LA has been studied for at least 3 decades, a new wave of interest in this protein appeared in 1995 after the report on its selective antitumor activity. The group of C. Svanborg found that a unique multimeric form of R-LA, † This work was supported by grants to P.E.A. from the Programs of the Russian Academy of Sciences, Molecular and Cellular Biology and Fundamental Sciences for Medicine. * Author to whom correspondence should be addressed. Tel: +7(4967) 73 41 35. Fax: +7(4967) 33 05 22. E-mail: [email protected]. ‡ Institute for Biological Instrumentation of the Russian Academy of Sciences. § Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences. | Pushchino State University. Biochemistry 2008, 47, 13127–13137 13127 10.1021/bi801423s CCC: $40.75  2008 American Chemical Society Published on Web 11/12/2008 isolated from the casein fraction of milk, induces apoptosis of transformed, embryonic, and lymphoid cells but spares mature and healthy cells (16). This form of R-LA, named MAL, was shown to possess molten globule-like properties (17). MAL crosses the plasma membrane and cytosol and enters the cell nucleus, where it induces DNA fragmentation through a direct effect at the nuclear level (18). Moreover, MAL interacts with mitochondria, which induces the release of cytochrome c and activation of the caspase cascade (19). Later on, it was reported that a complex of oleic acid (C18:1) with human or bovine R-LA (called HAMLET (20) and BAMLET (21), respectively) demonstrates similar cytotoxic activity. The Ca2+-free protein was complexed with oleic acid by means of ion-exchange chromatography on a DEAETrisacryl M column preconditioned with oleic acid and was subsequently eluted at high salt concentration (0.8 M NaCl). Neither saturated C18 fatty acids nor unsaturated C18:1 trans conformers or fatty acids with shorter or longer carbon chains can form complexes with apo-R-LA (22). Remarkably, unsaturated cis fatty acids other than C18:1:9cis are able to form stable complexes with apo-R-LA, but they are not active in the apoptosis assay (22). HAMLET (human alpha-lactalbumin made lethal to tumor cells) triggers several cell death pathways in various tumor cell lines and undifferentiated cells while healthy and mature cells are spared (for review, see ref 23). First, it causes mitochondrial damage followed by the release of cytochrome c (24). One more cell death pathway is the reduction of proteasome activity (25). Moreover, HAMLET induces macroautophagy in tumor cells, which also might contribute to cell death (23). Interestingly, the HAMLET complex passes through the cytoplasm to the nucleus where it accumulates and interacts with histones disrupting chromatin assembly and interfering with intact chromatin, thus preventing the cell from transcription, replication, and recombination (26). It was shown that this interaction has electrostatic nature and the modification of R-LA by oleic acid actually is not necessary for effective binding to histones (27). The molecular details of the above-mentioned cell events caused by HAMLET remain mostly unclear. Molecular characterization of the HAMLET complex showed that it represents mostly monomeric protein with a single bound oleic acid molecule and possesses decreased thermal stability but native-like calcium affinity. Gel-filtration and magnetic resonance dispersion measurements showed the presence of maximum 5% of dimers or higher order oligomers in the HAMLET preparations (28), in accord with the report by Casbara et al. (29). At the same time, much higher population of multimeric forms of HAMLET was detected using gel-filtration chromatography; prolonged incubation of the complex at elevated temperatures caused conversion of the mostly monomeric protein into mostly multimeric forms (30). According to gas chromatography and mass spectrometry data, the average number of oleic acid molecules bound per HAMLET complex is 0.9, although the real values depend upon the particular batch (0.6-1.3) (22). The thermal stability of apo-HAMLET is decreased (ca. 15 °C) compared to the intact apoprotein (31). The effective Ca2+ binding constant (K) for HAMLET measured at 25 °C (5.9 × 108 M-1) is within experimental error of that for intact R-LA (K ) 1.8 × 109 M-1) (21). The nearUV CD spectrum of HAMLET at 25 °C (pH 7.5) is similar to that of the classical molten globule of R-LA (22), i.e., a state with lost rigid tertiary but native-like secondary structure. Since apo-R-LA at 25 °C represents a mixture of the native and thermally denatured forms, adequate interpretation of the data obtained is complicated. Distinct structural differences between HAMLET and intact protein were detected (29). H/D exchange experiments showed that HAMLET incorporates a greater number of deuterium atoms than does intact apoprotein. HAMLET and the intact apoprotein are both accessible to proteases in the !-domain but have substantial differences in accessibility to proteases at specific sites. Nuclear Overhauser enhancement spectroscopy showed that the two ends of the oleic acid molecule in HAMLET are in close proximity to each other and close to the double bound, which demonstrates that OA is bound in a compact conformation (28). Notably, the complex between R-LA and oleic acid can be formed by simply adding OA to the protein solution (without the conventional use of ion-exchange chromatography). The 1H NMR investigation of calcium-independent OA binding to 1 mM hLA at pH 7.0 and 37 °C gave an apparent dissociation constant (Kd) of 0.1 mM (31). Since oleic acid has a very low CMC under these conditions, the reported value of Kd seems to be affected by the process of micelle formation. According to partition equilibria and fluorescent spectroscopy data, bovine R-lactalbumin has one binding site for oleic acid with a binding constant of (4.6-3.3) × 106 M-1 (32). The addition of 7.5 equiv of oleic acid to bovine apo-R-lactalbumin (pH 8.3, 5 mM EDTA) at 20 °C induces CD changes resembling those observed in the formation of the molten globule state of R-LA (30). According to gel-filtration data, the resulting complex is fully monomeric, in contrast to HAMLET (30). At the same time, despite the effective association of OA with the protein under solution conditions, the resulting R-LA-OA complex is believed to possess lower antitumor activity than HAMLET at similar protein and lipid concentrations (20). It should be noted that complete systematic studies of the process of the R-LA-OA complex formation under solution conditions have not been carried out to date. In this work we present optimal temperature, calcium, and monovalent salt conditions for formation of complexes showing maximal OA binding capacity. The numbers of oleic acid molecules irreversibly bound per one R-lactalbumin molecule in these complexes were estimated. Characterization of physicochemical, conformational, and cytotoxic properties of the resulting R-LA-OA complexes formed in aqueous buffers showed that they share many common features with HAMLET. Although the protein states reported here exhibit certain differences from HAMLET, their cytotoxic activities against tumor cells are close to that of HAMLET, which makes them well suited for further cytotoxic characterization. The fact that hLA-OA complexes having well-defined cytotoxic 1 Abbreviations: OA, oleic acid (C18:1:9cis); CMC, critical micelle concentration; hLA or intact hLA, R-lactalbumin isolated from human milk; OA-saturated hLA, human R-lactalbumin titrated by oleic acid up to its critical micelle concentration; MAL, multimeric form of human R-LA extracted from casein milk fraction as described in ref 16; HAMLET (BAMLET), specific complex of human (bovine) R-lactalbumin with oleic acid as described in ref 20; LA-OA-17 and LA-OA45, complexes of human R-lactalbumin with oleic acid, prepared at 17 and 45 °C, respectively, as described in Materials and Methods; CD, circular dichroism; HEp-2, human epidermoid larynx carcinoma cells. 13128 Biochemistry, Vol. 47, No. 49, 2008 Knyazeva et al.