Conversion of a-lactalbumin to a protein
`inducing apoptosis
`
`M. Svensson*, A. Håkansson*, A.-K. Mossberg*, S. Linse†, and C. Svanborg*‡
`
`*Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, So¨ lvegatan 23, S-223 62 Lund,
`Sweden; and †Department of Physical Chemistry 2, Lund University, P. O. Box 124, S-221 00 Lund, Sweden
`
`Edited by Lennart Philipson, Karolinska Institute, Stockholm, Sweden, and approved January 18, 2000 (received for review October 14, 1999)
`
`MEDICALSCIENCES
`
`inducing form and define the requirements for this structural
`and functional change. HAMLET (human a-lactalbumin made
`lethal to tumor cells) is shown to consist of partially unfolded
`a-lactalbumin that has integrated a cofactor, which stabilizes the
`conformation. The cofactor has been identified as a specific fatty
`acid.
`
`Materials and Methods
`Purification of Human a-Lactalbumin. Native a-lactalbumin was
`purified from human milk whey by ammonium sulfate precipi-
`tation followed by phenyl-Sepharose chromatography (8) and
`size-exclusion chromatography on a Sephadex G-50 column. The
`purity of the protein was controlled by SDSyPAGE and agarose
`gel electrophoresis and by spectroscopic techniques.
`Recombinant a-lactalbumin was purified from Escherichia
`coli (BL21 DE3 pLysS), carrying the vector pALA with the
`entire human a-lactalbumin gene inserted between the NdeI
`(site 100) and EcoRI (site 499) sites of the pAED4 vector (pALA
`was a kind gift from P. S. Kim, Howard Hughes Medical
`Institute, Cambridge, MA), after induction with isopropyl b-D-
`thiogalactoside (1 mM). Inclusion bodies were isolated from 1
`liter of culture medium, dissolved in 40 ml of buffer (8 M ureay10
`mM TriszHCly10 mM reduced glutathione, pH 8.0), and applied
`to a DEAE cellulose column. The protein was eluted with 10 mM
`Trisy7 M ureay1 M NaCly1 mM CaCl2, pH 8.0. The protein was
`reduced with 10 mM reduced glutathione added dropwise (2
`mlyh) to 500 ml of folding buffer (10 mM TriszHCly1 mM
`CaCl2y100 mM KCly10 mM reduced glutathioney1 mM oxi-
`dized glutathioney20% glycerol, pH 8.0, at room temperature)
`(9). When folding was complete, 10 mM EDTA was added, the
`folding suspension was applied to a phenyl-Sepharose column,
`and a-lactalbumin was eluted with 1 mM CaCl2 as described (8).
`The native fold was confirmed by 8-anilinonaphthalene-1-
`sulfonic acid (ANS) fluorescence and near-UV CD spectros-
`copy, with the characteristic 270-nm tyrosine minimum and the
`294-nm tryptophan maximum (Fig. 1C).
`Apo a-lactalbumin was generated from 25 mg of native
`a-lactalbumin dissolved at 1.8 mM in Tris (10 mM TriszHCl, pH
`8.5) by adding 3.5 mM EDTA to remove bound Ca21. The
`conformational change was confirmed by near-UV CD and ANS
`spectroscopy. The near-UV CD spectrum showed the charac-
`teristic loss of signal in the tyrosine and tryptophan regions, and
`the apo a-lactalbumin-bound ANS, as shown by increased
`intensity and an intensity maximum, shifted to 480 nm (Fig. 1 C
`and D).
`
`Spectroscopic Analyses. CD spectra were obtained by using a
`JASCO J-720 spectropolarimeter with a JASCO PTC-343 Pel-
`
`This paper was submitted directly (Track II) to the PNAS office.
`
`Abbreviations: HAMLET, human a-lactalbumin made lethal to tumor cells; ANS, 8-anili-
`nonaphthalene-1-sulfonic acid.
`‡To whom reprint requests should be addressed. E-mail: Catharina.Svanborg@mig.lu.se.
`
`The publication costs of this article were defrayed in part by page charge payment. This
`article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
`§1734 solely to indicate this fact.
`
`PNAS u April 11, 2000 u vol. 97 u no. 8 u 4221– 4226
`
`In this study a-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 a-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 a-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 a-lactalbumin. Although the environment in the mammary
`gland favors the native conformation of a-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 Ca21 from the high-
`affinity Ca21-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 bio-
`logical 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.
`
`Proteins adopt a series of conformations during their synthesis
`
`and transport through human cells. According to the classical
`view, this process is determined by the amino acid sequence, and
`thermodynamics force the protein to adopt the conformation
`with the lowest free energy. During the last decade this classical
`view has been challenged, because proteins have been shown to
`adopt several stable conformations, depending on which kinetic
`pathway is followed (1). The high-energy barriers between the
`different conformations can be overcome with the help of
`chaperones (2, 3), but in many systems, the factors that cause or
`relieve such kinetic barriers remain to be specified.
`Stable conformational variants of a protein may expose dis-
`tinct functional regions and thus differ in biological activity. The
`most striking example is the prion protein that can change from
`the normal a-helix-rich to a b-sheet-rich, disease-causing iso-
`form (4, 5). In a previous study we proposed a-lactalbumin as a
`second example of a protein that can acquire different functions
`depending on its folding state (6). An a-lactalbumin complex
`from acid-precipitated human milk casein was shown to induce
`apoptosis in tumor cells and immature cells, but not in mature,
`differentiated cells (7). Surprisingly, native a-lactalbumin (Fig.
`1 A) isolated from its traditional source, human milk whey, did
`not induce apoptosis. The difference in activity between the two
`forms of the protein was not caused by a change in secondary
`structure, but the active complex was found to have undergone
`a change in tertiary structure, with a conformational switch
`toward a molten globule-like state (6).
`If folding variants of a-lactalbumin differ in biologic activity,
`it should be possible to convert native a-lactalbumin to the
`active, apoptosis-inducing form by changing its conformation. In
`the present study, we prove that a-lactalbumin, indeed, can be
`converted from the native conformation to the apoptosis-
`
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`tier-type thermostated cell holder. Quartz cuvettes were used
`with 1-cm path length, and spectra were recorded between 320
`and 240 nm. The wavelength step was 1 nm, the response time
`was 4 s, and the scan rate was 10 nm per minute. Six scans were
`recorded and averaged for each spectrum. Baseline spectra were
`recorded with pure buffer in each cuvette and subtracted from
`the protein spectra.
`ANS fluorescence emission spectra were recorded at 25°C on
`a Perkin–Elmer LS-50B spectrometer by using a quartz cuvette
`with 1-cm excitation path length, between 400 and 600 nm (step,
`1 nm), with excitation at 385 nm. Both the excitation and
`emission bandpass were set to 5 nm.
`Stock solutions were prepared by dissolving lyophilized sam-
`ples in 10 mM potassium phosphate buffer at pH 7.5. The
`concentrations were determined by amino acid analysis after
`acid hydrolysis, and spectra were recorded on aliquots diluted in
`10 mM potassium phosphate buffer at pH 7.5.
`
`1H NMR Spectra. 1H NMR spectra were recorded by using an
`Omega 500 spectrometer at 500 MHz in D2O with 0.15 M NaCl
`at 37°C for 1–2 mM solutions of protein. Lyophilized HAMLET
`or native or apo a-lactalbumin (15 mg) was dissolved in 500 ml
`of D2O, and the pH was set to 7.0 by using NaOD. Oleic acid
`(4 mg) was dissolved in 75 ml of ethanol-d6, and 10 ml was added
`to 500 ml of D2O. Apo a-lactalbumin for 1H NMR was generated
`by dissolving a-lactalbumin in doubly distilled water containing
`10-fold molar excess of EGTA at pH 8.0. The sample was applied
`to a G-25 gel-filtration column after an aliquot of saturated NaCl
`(calcium-depleted) and eluted by doubly distilled water. The
`sample was passed through the saturated NaCl to reduce binding
`of EGTA to the protein, and EGTA-free protein was eluted in
`the water.
`
`Anion-Exchange Chromatography. a-Lactalbumin in the native or
`apo state was subjected to ion-exchange chromatography as
`described (6). Briefly, the column (14 cm 3 1.6 cm) was packed
`with DEAE-Trisacryl M (BioSepra, Villeneuve-la-Garonne,
`France) attached to a Bio-Logic chromatography system (Bio-
`Rad) and eluted with a NaCl gradient (buffer A: 10 mM
`TriszHCl, pH 8.5; buffer B: buffer A containing 1 M NaCl).
`The eluted protein fractions were desalted by dialysis (Spec-
`trayPor; Spectrum Medical Industries, Laguna Hills, CA;
`membrane cut-off, 3.5 kDa) against distilled water, with at
`least four changes of water, and lyophilized.
`
`Casein Conditioning of the Ion-Exchange Matrix. Casein was isolated
`from human milk as described (6). The casein (50 mg) was
`dissolved in 10 ml of 10 mM TriszHCl (pH 8.5), applied to the
`column, and run as described. This procedure yielded apopto-
`sis-inducing material (6), and the matrix thereafter was denoted
`as casein-conditioned. a-Lactalbumin in the native or in the apo
`state was applied to the casein-conditioned matrix (25 mg
`dissolved in 10 ml of 10 mM TriszHCl, pH 8.5, with or without
`EDTA) and eluted as described above.
`
`Identification of the Cofactor on the Casein-Conditioned Column
`Matrix. To elute protein cofactors, the casein-conditioned matrix
`was sequentially washed with 10 mM EDTA, 4 M urea, and 20%
`ethanol. Eluted protein fractions were analyzed by SDSyPAGE.
`This procedure yielded residual a-lactalbumin, but no other
`proteins were detected.
`Lipids were eluted from the ion-exchange matrix using organic
`solvents (10). Casein-conditioned matrix (2 ml) was dissolved in
`chloroformymethanolywater (1:2:0.8 volyvolyvol) and incubated
`at 37°C for 1.5 h. Chloroformywater (1:1 volyvol) was added, the
`solution was mixed thoroughly, the two phases were separated
`overnight, and the organic phase was collected and dried
`under nitrogen. Lipids were dissolved in chloroform
`
`(3 mgy30 ml) and 10 ml was applied in triplicates to silica gel glass
`plates (1 mg on each lane) and developed by using petroleum
`etherydietyl etheryacetic acidymethanol (80:20:1:2 volyvolyvoly
`vol). The lipids were visualized by iodine vapor, and defined lipid
`species were scraped off the plates and eluted from the silica gel
`with chloroformymethanol (2:1, 1:1, 1:2 volyvol). The fatty acids
`were analyzed further on a Varian gas chromatograph (model
`3500) equipped with a splitysplitless injector and a flame ion-
`ization detector. The fatty acids were separated on a fused silica
`capillary column (0.25-mm inner diameter) (Chrompack, Stock-
`holm). The column temperature was programmed from 140°C to
`240°C at 8°C per min. Chromatograms were evaluated by using
`a Varian integrator model 4290.
`
`Fatty Acid Conditioning of the Ion-Exchange Matrix. Ten milligrams
`of palmitic acid (16:0), steric acid (18:0), myristic acid (14:0), or
`oleic acid (18:1) was dissolved in 500 ml of 99.5% ethanol by
`sonication (3 min using a Branson 2200 bath sonicator; Bran-
`son). After addition of 10 ml of 10 mM TriszHCl, pH 8.5, the
`different lipid solutions were applied to four separate, newly
`packed DEAE-Trisacryl M matrices. Native or apo a-lactalbu-
`min isolated from milk or E. coli was applied to each matrix and
`eluted with NaCl, as described above.
`
`Bioassays of Apoptosis. The L1210 (ATCC, CCL 219), A549
`(ATCC, CLL 185), A-498 (ATCC, HTB 44) and Jurkat (Euro-
`pean Cell Culture Collection, no. 88042803) cell lines were
`cultured as described (7). The cells were harvested by centrifu-
`gation (200 3 g for 10 min), resuspended in cell culture medium,
`and seeded into 24-well plates (FalconyBecton Dickinson) at a
`density of 2 3 106 per well. The different molecular forms of
`a-lactalbumin,
`lipid extracts, or individual fatty acids were
`dissolved in cell culture medium, without FCS, and added to the
`cells (final volume, 1 ml per well). Plates were incubated at 37°C
`in 5% CO2 atmosphere with addition of 100 ml of FCS to each
`well after 30 min. Cell culture medium served as a control. Cell
`viability and DNA fragmentation were determined after 6 h of
`incubation, as described (7).
`Subcellular Localization Studies. L1210 cells (2 3 106 cellsyml, 490
`ml) were incubated at 25°C with 12 ml of the biotinylated protein
`preparations, and surface-bound protein was visualized after
`counterstaining with FITC-conjugated streptavidin. Intracellu-
`lar protein was detected after fixation in 4% paraformaldehyde
`and permeabilization with 0.1% saponin to allow entry of
`FITC-conjugated streptavidin. Cells were analyzed in Bio-Rad
`1024 laser-scanning confocal equipment attached to a Nikon
`Eclipse E800 microscope.
`
`Results
`Apo a-Lactalbumin Can Be Converted to a Folding Variant with
`Apoptosis-Inducing Activity. Whey-derived or recombinant a-lact-
`albumin was used as starting material in experiments aiming to
`convert the inactive, native protein to the active form (Fig. 1B).
`As a first step, the native proteins were partially unfolded by
`EDTA treatment, and the conformational change to the apo-
`state was confirmed by UV CD and ANS fluorescence spec-
`troscopy (Fig. 1 C and D).
`Apo a-lactalbumin then was subjected to ion-exchange chro-
`matography on a column previously exposed to human milk
`casein. Eluted material was tested for its ability to kill tumor cells
`and to induce DNA fragmentation and was examined by spec-
`troscopic techniques. Apo a-lactalbumin bound strongly to the
`ion-exchange matrix and eluted only after 1 M NaCl (Fig. 2A).
`Eluted protein remained in a partially unfolded conformation as
`shown by loss of signal
`in the near-UV CD spectrum and
`increased binding of ANS (Fig. 2 C and D), and it induced
`apoptosis with DNA fragmentation in tumor cells (Fig. 2E).
`
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`Fig. 1. a-Lactalbumin has been studied extensively as a model of protein-folding intermediates (12–15). On lowering the pH the acidic side chains are
`protonated and the protein adopts the A state or molten globule state (16 –18). A similar partially unfolded state, the apo state, is formed at neutral pH
`if the Ca21 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 a-lactalbumin.
`a-Lactalbumin (14 kDa) is shown with four a-helices (red and yellow, residues 1–34 and 86 –123) and an antiparallel b-sheet (blue, residues 38 – 82). The
`high-affinity Ca21-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) SDSyPAGE of whey-derived and recombinant
`a-lactalbumin; SDSyPAGE 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 a-lactalbumin (14 and 30 kDa); 3, whey-derived a-lactalbumin (14 and 30 kDa). (C) Near-UV
`CD spectra of a-lactalbumin. Native whey-derived (solid, black line) or recombinant (solid, green line) a-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
`a-lactalbumin controls showed the characteristic loss of signal in the tyrosine and tryptophan region. (D) ANS fluorescence spectra of a-lactalbumin.
`Whey-derived (solid, black line) or recombinant (solid, green line) a-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.
`
`a-Lactalbumin in the native conformation was used as a negative
`control. It could not be activated by these procedures (Fig. 2).
`
`Does Conversion Require a Cofactor? The conversion of apo a-lact-
`albumin to the apoptosis-inducing form involved a cofactor from
`
`casein. This was first suspected when conversion could not be
`achieved on a clean column matrix (Fig. 2B). Furthermore, the
`eluted protein differed from the previously described partially
`denatured form of a-lactalbumin as Ca21-depleted or acid-
`denatured protein revert to the native state when solvent con-
`
`MEDICALSCIENCES
`
`Conversion of a-lactalbumin to the apoptosis-inducing form requires partial unfolding of the protein and the presence of a cofactor. (A) Apo
`Fig. 2.
`a-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 a-lactalbumin was not retained (solid, blue line). The NaCl gradient is shown by the dotted line.
`(B) Apo a-lactalbumin (solid, red line) and native a-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 a-lactalbumin eluate (solid, red line) from the casein-conditioned matrix did not differ from the apo a-lactalbumin
`control (dashed, black line). The active fraction from casein was used as a control (solid, red line with solid circle). Native a-lactalbumin had similar spectra before
`(solid, black line) and after (solid, blue line) elution from the column. (D) ANS spectra of the apo a-lactalbumin eluate (solid, red line) from the casein-conditioned
`matrix did not differ from the apo a-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 mgyml); C, native a-lactalbumin (1.0 mgyml); D,
`void peak vol from the clean matrix (1.0 mgyml); E, native a-lactalbumin eluate from the casein-conditioned matrix (1.0 mgyml); and F, apo a-lactalbumin eluate
`from the casein-conditioned matrix (0.2 mgyml). The proteins in lanes B and F were active.
`
`Svensson et al.
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`PNAS u April 11, 2000 u vol. 97 u no. 8 u 4223
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`C18:1 is the fatty acid needed to convert apo a-lactalbumin to the apoptosis-inducing form. (A) Whey-derived or recombinant a-lactalbumin was
`Fig. 3.
`subjected to ion-exchange chromatography by using a matrix preconditioned with C18:1 fatty acid. Whey-derived a-lactalbumin was added to the column in
`its native (solid, blue line) or apo state (solid, red line). The apo a-lactalbumin bound to the C18:1-conditioned matrix and eluted as a sharp peak after 1 M NaCl.
`Native a-lactalbumin bound poorly to the matrix, with .50% in the void. Recombinant apo a-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 a-lactalbumin control (dashed, black line). Native a-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 a-lactalbumin control (dashed, black line). The native a-lactalbumin
`eluate off the C18:1-conditioned column (solid, blue line) and the native a-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 a-lactalbumin (1.0 mgyml); C, recombinant, native a-lactalbumin
`(1.0 mgyml); D, HAMLET (0.2 mgyml); E, recombinant HAMLET (0.2 mgyml); F, native a-lactalbumin eluate off a C18:1-conditioned column (5 mgyml); G, lipids
`extracted from casein-conditioned matrix (0.05 mgyml); and H, 18:1 fatty acid (0.025 mgyml). 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 a-lactalbumin bound weakly to the cell surface and did not enter the cells (Upper). The cellular
`outline is shown in blue reflection mode (Lower).
`
`ditions are brought back to normal, but the converted protein
`was preserved in a partially unfolded state even at neutral pH
`and in the presence of Ca21. We also observed that the conver-
`sion efficiency of the casein-conditioned column gradually de-
`creased with repeated runs. Thus, a cofactor from casein ap-
`peared to be required to maintain the protein in its altered state
`with apoptosis-inducing activity.
`
`Identification of the Cofactor. The cofactor was identified by
`chemical extraction of the casein-conditioned matrix under
`conditions suitable for proteins or lipids. Solvents known to elute
`proteins (1 M NaCl, 10 mM EDTA, 4 M urea, or 20% ethanol)
`released only residual a-lactalbumin; no other proteins were
`detected. Organic solvents (chloroform, methanol), on the other
`hand, released lipids from the column matrix. Individual lipid
`species were identified by GCyMS as C18:1, C16:0, and C14:0
`fatty acids (data not shown).
`New column matrices then were conditioned with each of the
`fatty acids or with C18:0 and exposed to whey-derived or
`recombinant a-lactalbumin in the native or apo states. Fractions
`eluting after 1 M NaCl were tested for apoptosis-inducing
`activity. a-Lactalbumin from human milk whey and recombinant
`protein was shown to convert to the active complex only on the
`C18:1 fatty acid-preconditioned column and only when applied
`in the apo form. The proteins were retained on the C18:1-
`
`conditioned column, and each eluted as a sharp peak after 1 M
`NaCl, with a yield of about 90% (Fig. 3A). By near-UV CD and
`ANS spectroscopy (Fig. 3 B and C), the whey-derived and
`recombinant activated complexes strongly resembled the apo
`a-lactalbumin control, and both complexes induced apoptosis in
`L1210 cells (Fig. 3D). The C18:1-converted apo a-lactalbumin
`from human milk whey was named HAMLET, and the con-
`verted recombinant apo a-lactalbumin was named recombinant
`HAMLET.
`Conditioning of the ion-exchange matrix with the other fatty
`acids (C18:0, C16:0, and C14:0) did not convert apo a-lactalbu-
`min to HAMLET, and native a-lactalbumin could not be
`activated on the C18:1-conditioned column. Most of the material
`eluted in the void volume with a small peak after 1 M NaCl that
`showed native three-dimensional structure and lacked apopto-
`sis-inducing activity (Fig. 3D). These results identified the C18:1
`fatty acid as the responsible cofactor required to convert a-lact-
`albumin to HAMLET.
`There was no direct cellular effect of the C18:1 fatty acid or
`of lipid extracts from the casein-conditioned matrix at concen-
`trations found in HAMLET (Fig. 3D). Furthermore, simple
`mixing experiments with C18:1 fatty acid and apo a-lactalbumin
`showed that the mixtures had lower activity than HAMLET at
`similar protein and lipid concentrations.
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`MEDICALSCIENCES
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`human and animal origin (7). HAMLET was shown to induce
`apoptosis in several human and murine tumor cell lines, includ-
`ing the Jurkat and L1210 leukemia cell lines, the A549 lung
`carcinoma line, and the A-498 kidney carcinoma line. Native
`a-lactalbumin had no effects on these cells.
`The apoptosis-inducing activity of the converted fractions
`was compared by using the L1210 mouse leukemia cell line.
`The loss of cell viability and induction of DNA fragmentation
`were used as end points (Figs. 2E and 3D). The L1210 cells
`died rapidly when exposed to whey-derived or recombinant
`HAMLET (200 mgyml), and DNA fragmentation was induced.
`The L1210 cells survived exposure to native a-lactalbumin
`both before and after the protein had been eluted from the
`ion-exchange column.
`By confocal microscopy we observed striking differences in
`subcellular localization and nuclear uptake depending on the
`folding state of a-lactalbumin. Whey-derived and recombinant
`HAMLET bound to the cell surface, passed through the cyto-
`plasm to the nucleus, and accumulated in the cell nucleus (Fig.
`3E). Native a-lactalbumin bound weakly to the cell surface but
`was not seen to translocate into the cytoplasm or to reach the cell
`nuclei (Fig. 3E).
`
`Discussion
`The present study demonstrated that a-lactalbumin can alter its
`biological function depending on the conformational state. We
`also showed that a specific fatty acid, C18:1, was a necessary
`cofactor. We thus have identified the folding change and the
`fatty acid as the two key elements that define HAMLET, the
`apoptosis-inducing functional state of a-lactalbumin.
`The conversion to HAMLET was achieved first by changing
`the conformation of a-lactalbumin from the native to a partially
`unfolded state. EDTA treatment was chosen because it releases
`calcium and opens up the three-dimensional structure of the
`protein, and the conformational change was confirmed by
`spectroscopic techniques. By near-UV CD spectroscopy native
`a-lactalbumin had a minimum at 270 nm arising from tyrosine
`residues and a maximum at 294 nm arising from tryptophan
`residues. The EDTA-treated protein showed the characteristic
`loss of signal, indicating partial unfolding with less restrained
`tyrosines and tryptophans. Removal of Ca21 made the protein
`more hydrophobic as probed in ANS fluorescence spectroscopy,
`and HAMLET had increased ANS fluorescence intensity and a
`shift to a shorter wavelength compared with the native protein.
`The second requirement for the stable conversion of native
`a-lactalbumin was the presence of the C18:1 fatty acid on the
`ion-exchange matrix. In the absence of this cofactor, the apo
`form of the protein was unstable and reverted to the native,
`inactive form at neutral pH and in the presence of Ca21. The
`cofactor initially was identified by GCyMS in a lipid extract from
`the spent ion-exchange matrix. Several fatty acids present in
`casein eluted as part of the active complex, but only the
`unsaturated form of the C:18 fatty acid was able to precondition
`the column matrix for conversion of apo a-lactalbumin to
`HAMLET. Other structurally related fatty acids like the C18:0,
`the C16:0, and the C14:0 fatty acids were inactive, suggesting that
`only the unsaturated C18:1 could act as the necessary cofactor.
`It may be speculated that the increased hydrophobicity of the apo
`a-lactalbumin-folding variant enhances binding of C18:1 to
`exposed hydrophobic regions of the molecule, but the molecular
`details of these interactions remain to be defined.
`a-Lactalbumin is the most abundant protein in human milk
`(22). The nursing child ingests around 2 g of a-lactalbumin,
`which travels from the mammary gland through the gastroin-
`testinal tract of the baby. The environmental conditions in the
`mammary gland favor the native state of the protein with the
`tightly bound Ca21 ion, and a-lactalbumin functions as a spec-
`ifier protein in lactose synthesis (23). Through lactose, the water
`
`1H NMR spectra of HAMLET, native a-lactalbumin, apo a-lactalbumin,
`Fig. 4.
`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 a-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.
`
`1H NMR Spectroscopy. 1H NMR spectra of the native a-lactalbu-
`mins were characteristic of folded and well-ordered proteins with
`narrow lines and significant shift dispersion, a large number of
`sharp signals in the aromatic region (around 7 ppm), and several
`out-shifted methyl signals (between 0.7 and 20.6 ppm) (Fig. 4).
`The spectra of the apoproteins displayed narrow lines and
`significant shift dispersion, with significant variations relative to
`the native state in the chemical shifts of a large number of
`resonances (Fig. 4).
`There were marked differences between HAMLET and na-
`tive a-lactalbumin. The spectrum of HAMLET showed broader
`lines and little shift dispersion. The lines in the aromatic region
`were more clustered, with no outshifted methyl signals below
`0.7 ppm (Fig. 4). The spectrum of HAMLET did not change
`between low and physiological salt concentrations. Despite the
`similarities seen by optical spectroscopy, there were, by NMR,
`distinct spectral differences between HAMLET and the apo
`form of a-lactalbumin, suggesting additional conformational
`changes because of the interaction with the lipid cofactor.
`Although C18:1 fatty acid was detected in the spectrum of
`HAMLET (as indicated by arrows), the signals were much
`broader compared with the signals of free oleic acid, suggesting
`that the fatty acid has become an integral part of HAMLET.
`
`HAMLET Induces Apoptosis in Tumor Cell Lines and Targets Cell Nuclei.
`When isolated from human milk casein the a-lactalbumin fold-
`ing variant has broad activity against transformed cells of both
`
`Svensson et al.
`
`PNAS u April 11, 2000 u vol. 97 u no. 8 u 4225
`
`Page 5
`
`

`

`content of milk is controlled. This is crucial for successful
`breast-feeding, and it has been shown that a-lactalbumin knock-
`out mice produce extremely viscous milk and fail to nurse their
`offspring (24).
`Quite different environmental conditions meet a-lactalbumin
`in the stomach of the breast-fed child. The low pH is known to
`favor the release of Ca21 (16–18), and casein is precipitated. The
`acid lipase hydrolyses triglycerides, and fatty acids are released
`(25, 26). By chance, we happened to purify the apoptosis-
`inducing form of a-lactalbumin from human milk after precip-
`itation of casein at low pH, thus mimicking the conditions in the
`stomach of the breast-fed child (7, 27). When these conditions
`were reproduced in vitro, a-lactalbumin was shown to change to
`the partially unfolded state and to form HAMLET in complex
`with the obligatory C18:1 fatty acid.
`The purification of proteins from biological fluids has a long
`history of uncertainty because of the potential for contamina-
`tion. We used two approaches to exclude that the activity of our
`complex was due to some contaminating component other than
`the predominant protein. First, we used a-lactalbumin from
`human milk whey rather than from casein, showed that it was
`inactive, and then activated it by partial unfolding and exposure
`to oleic acid. Second, we expressed the native protein in E. coli
`and showed that the product was inactive in the native state and
`that it could be converted equally to the active form through the
`same procedures. Thus, we feel confident that contamination
`with other biologically active milk ingredients does not explain
`our results.
`There are obvious parallels between a-lactalbumin and the
`prion system. Both proteins have multiple activities depending
`on their folding state, and both require a cofactor for the
`functional transition. The prion protein first changes to a molten
`globule-like state and then proceeds to a nonreversible b-sheet-
`rich form (4, 5), which i