Conversion of alpha-lactalbumin to a protein inducing apoptosis

Abstract

In this study alpha-lactalbumin was converted from the regular, native state to a folding variant with altered biological function. The folding variant was shown to induce apoptosis in tumor cells and immature cells, but healthy cells were resistant to this effect. Conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells) required partial unfolding of the protein and a specific fatty acid, C18:1, as a necessary cofactor. Conversion was achieved with alpha-lactalbumin derived from human milk whey and with recombinant protein expressed in Escherichia coli. We thus have identified the folding change and the fatty acid as two key elements that define HAMLET, the apoptosis-inducing functional state of alpha-lactalbumin. Although the environment in the mammary gland favors the native conformation of alpha-lactalbumin that serves as a specifier in the lactose synthase complex, the conditions under which HAMLET was formed resemble those in the stomach of the nursing child. Low pH is known to release Ca(2+) from the high-affinity Ca(2+)-binding site and to activate lipases that hydrolyze free fatty acids from milk triglycerides. We propose that this single amino acid polypeptide chain may perform vastly different biological functions depending on its folding state and the in vivo environment. It may be speculated that molecules like HAMLET can aid in lowering the incidence of cancer in breast-fed children by purging of tumor cells from the gut of the neonate.

Figure 1

α-Lactalbumin has been studied extensively as a model of protein-folding intermediates (–15). On lowering the pH the acidic side chains are protonated and the protein adopts the A state or molten globule state (–18). A similar partially unfolded state, the apo state, is formed at neutral pH if the Ca²⁺ ion is removed (19, 20). Although these conformations essentially have retained secondary structure, they have fluctuating tertiary structure (18), exposed hydrophobic surfaces, and tryptophan residues accessible to solvent. (A) Three-dimensional structure of native human α-lactalbumin. α-Lactalbumin (14 kDa) is shown with four α-helices (red and yellow, residues 1–34 and 86–123) and an antiparallel β-sheet (blue, residues 38–82). The high-affinity Ca²⁺-binding site (green) is coordinated by the side chain carboxylates of Asp-82, Asp-87, and Asp-88, the carbonyl oxygens of Lys-79 and Asp-84, and two water molecules. Four disulfide bonds (cyan) are indicated with roman numerals: I, 61–77; II, 73–91; III, 28–111; and IV, 6–120. Crystal structure coordinates are from Acharya et al. (11), and the structure was created with molmol 2.6.1 (21). (B) SDS/PAGE of whey-derived and recombinant α-lactalbumin; SDS/PAGE on 4–20% polyacrylamide precast gels in a Bio-Rad Mini Protean II cell. Lanes: 1, molecular mass standard (Multimark Multicolored Standard; NOVEX, San Diego); 2, recombinant α-lactalbumin (14 and 30 kDa); 3, whey-derived α-lactalbumin (14 and 30 kDa). (C) Near-UV CD spectra of α-lactalbumin. Native whey-derived (solid, black line) or recombinant (solid, green line) α-lactalbumin showed the characteristic 270-nm tryptophan maximum and the 294-nm tyrosine minimum. The EDTA-treated whey-derived (dashed, black line) and recombinant (dashed, green line) apo α-lactalbumin controls showed the characteristic loss of signal in the tyrosine and tryptophan region. (D) ANS fluorescence spectra of α-lactalbumin. Whey-derived (solid, black line) or recombinant (solid, green line) α-lactalbumin in the native state did not bind ANS, but after EDTA treatment (whey: dashed, black line; recombinant: dashed, green line) ANS binding increased and the intensity maximum shifted to 480 nm.

Figure 2

Conversion of α-lactalbumin to the apoptosis-inducing form requires partial unfolding of the protein and the presence of a cofactor. (A) Apo α-lactalbumin was subjected to ion-exchange chromatography on a column previously exposed to human milk casein. More than 95% of the protein was retained on the column and eluted with 1 M NaCl (solid, red line). Native α-lactalbumin was not retained (solid, blue line). The NaCl gradient is shown by the dotted line. (B) Apo α-lactalbumin (solid, red line) and native α-lactalbumin (solid, blue line) eluted in the void volume when subjected to ion-exchange chromatography on a clean column. (C) CD spectra of the apo α-lactalbumin eluate (solid, red line) from the casein-conditioned matrix did not differ from the apo α-lactalbumin control (dashed, black line). The active fraction from casein was used as a control (solid, red line with solid circle). Native α-lactalbumin had similar spectra before (solid, black line) and after (solid, blue line) elution from the column. (D) ANS spectra of the apo α-lactalbumin eluate (solid, red line) from the casein-conditioned matrix did not differ from the apo α-lactalbumin control (dashed, black line). The native control did not bind ANS before (solid, black line) or after elution from the casein-conditioned matrix (solid, blue line). The active fraction from casein was used as a control (solid, red line with solid circle). (E) Loss of viability and DNA fragmentation of L1210 cells. Lanes: A, cell culture medium; B, the active fraction from human milk casein (0.2 mg/ml); C, native α-lactalbumin (1.0 mg/ml); D, void peak vol from the clean matrix (1.0 mg/ml); E, native α-lactalbumin eluate from the casein-conditioned matrix (1.0 mg/ml); and F, apo α-lactalbumin eluate from the casein-conditioned matrix (0.2 mg/ml). The proteins in lanes B and F were active.

Figure 3

C18:1 is the fatty acid needed to convert apo α-lactalbumin to the apoptosis-inducing form. (A) Whey-derived or recombinant α-lactalbumin was subjected to ion-exchange chromatography by using a matrix preconditioned with C18:1 fatty acid. Whey-derived α-lactalbumin was added to the column in its native (solid, blue line) or apo state (solid, red line). The apo α-lactalbumin bound to the C18:1-conditioned matrix and eluted as a sharp peak after 1 M NaCl. Native α-lactalbumin bound poorly to the matrix, with >50% in the void. Recombinant apo α-lactalbumin bound to the C18:1-conditioned matrix (solid, green line) and eluted after 1 M NaCl. (B) Near-UV CD spectra of proteins eluting from the C18:1-conditioned matrix. The spectrum of HAMLET (solid, red line) and recombinant HAMLET (solid, green line) strongly resembled the apo α-lactalbumin control (dashed, black line). Native α-lactalbumin before (solid, black line) and after (solid, blue line) passage over the column had native properties. (C) ANS fluorescence spectra of material eluted from the C18:1-conditioned column. HAMLET (solid, red line) and recombinant HAMLET (solid, green line) resembled the apo α-lactalbumin control (dashed, black line). The native α-lactalbumin eluate off the C18:1-conditioned column (solid, blue line) and the native α-lactalbumin control (solid, black line) showed low ANS binding. (D) DNA fragmentation and loss of cell viability in L1210 cells. Lanes: A, cell culture medium; B, whey-derived native α-lactalbumin (1.0 mg/ml); C, recombinant, native α-lactalbumin (1.0 mg/ml); D, HAMLET (0.2 mg/ml); E, recombinant HAMLET (0.2 mg/ml); F, native α-lactalbumin eluate off a C18:1-conditioned column (5 mg/ml); G, lipids extracted from casein-conditioned matrix (0.05 mg/ml); and H, 18:1 fatty acid (0.025 mg/ml). Material in lanes D and E induced apoptosis. (E) Subcellular distribution of HAMLET in L1210 cells. Cell surface binding of HAMLET and recombinant HAMLET was detected after 30 min, followed by translocation into the cytoplasm and accumulation in the cell nuclei (Upper). Native α-lactalbumin bound weakly to the cell surface and did not enter the cells (Upper). The cellular outline is shown in blue reflection mode (Lower).

Figure 4

¹H NMR spectra of HAMLET, native α-lactalbumin, apo α-lactalbumin, oleic acid, and a lipid extract from HAMLET. The aromatic and methylated regions are shown Left and Right, respectively. Virtually identical spectra were obtained for whey-derived and recombinant α-lactalbumin. The broad lines and lack of out-shifted methyl signals suggest that HAMLET is in a partially unfolded state that is significantly different from the native form of the protein, which displays narrow lines and a large shift dispersion. Furthermore, signals arising from oleic acid are much broader in the HAMLET spectrum (arrows) than in the oleic acid spectrum.