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J Biol Chem, Vol. 274, Issue 10, 6388-6396, March 5, 1999
From the This study characterized a protein complex in
human milk that induces apoptosis in tumor cells but spares healthy
cells. The active fraction was purified from casein by anion exchange
chromatography. Unlike other casein components the active fraction was
retained by the ion exchanger and eluted after a high salt gradient.
The active fraction showed N-terminal amino acid sequence identity with
human milk Human milk provides the newborn child with exquisite nutrition,
and a mucosal immune system. Breastfeeding protects against respiratory
and gastrointestinal infections, due to the presence in milk of
molecules with anti-microbial activity: antibodies, potentially
bactericidal molecules like lysozyme and lactoferrin (1), fatty acids
that lyse bacteria and viral particles (2, 3), and glycoconjugates that
inhibit bacterial adherence to epithelial cells (4, 5). Epidemiological
studies have shown that breastfeeding protects also against cancer (6),
suggesting that milk contains molecules with anti-tumor activity. We
recently observed that a protein fraction of human milk induces
apoptosis in tumor cells but not in mature healthy cells (7).
Surprisingly, the main protein constituent of this fraction was
Monomeric Unlike monomeric Chemicals--
ANS1
ammonium salt was from Fluka, Buchs, Switzerland. Ammonium sulfate,
Tris, calcium chloride, HCl, sodium chloride, methanol, acetic acid,
glycine, sodium barbitone, acetonitrile, trifluoroacetic acid,
sinapinic acid, and potassium phosphate were from Merck, Darmstadt,
Germany. EDTA, SDS, bromphenol blue, and glycerol were from Sigma.
Potassium oxalate was from Riedel-de Haen, Seelze, Germany. Agarose
(Sea Kem GTG) was from Bioproducts, Rockland, MI. PAGE ready gels were
from Bio-Rad. RPMI 1640 cell culture media, fetal calf serum,
nonessential amino acids, sodium pyruvate. and gentamicin were from
Life Technologies, Paisley, United Kingdom. The Biotin Labeling kit was
from Boehringer Mannheim, GmbH, Germany. All chemicals were of the
highest grade commercially available.
The classical molten globule state was obtained by lowering the pH of a
solution of native monomeric Casein Precipitation--
Frozen human milk was thawed and
centrifuged (Sorvall RC-5B refrigerated superspeed centrifuge, Du Pont
Instruments) at 2500 × g for 15 min; the upper fat
layer was removed. Casein was isolated by an overnight incubation at
4 °C with 10% potassium oxalate followed by a second overnight
incubation at 4 °C after lowering the pH to 4.3 using 1 M hydrochloric acid and heating the solution to 32 °C
for 2 h. The casein precipitate was harvested by centrifugation at
5000 × g for 15 min, washed by 3-5 cycles of
centrifugation, and resuspension in distilled water and lyophilized.
Anion Exchange Chromatography--
Casein was fractionated on
DEAE-Trisacryl M (Biosepra, Villeneuve la Garenne, France) using an
FPLC instrument (Pharmacia Biotech Inc.) with increasing NaCl gradient.
The sample was loaded in buffer A (0.01 M Tris-HCl, pH 8.5)
at 25 °C and eluted by increasing proportions of buffer B (buffer A
containing 1 M NaCl). Gradient program: start 15% B; from
0 to 60 ml: linear gradient from 60 to 90 ml: 30% B at 90 ml: 100% B;
for 10 min; thereafter 100% A. Flow rate: 1 ml/min, recorder: 0.1 cm/min. The buffers were degassed and filtered through 0.22-µm
filters before use. The peaks were monitored at 280 nm, and the
fraction size was 3 ml. The eluate was desalted by dialysis
(Spectra/Por, Spectrum Medical Industries, Laguna Hills, CA, membrane
cut off 3.5 kDa) against distilled water for at least 48 h and lyophilized.
Size Exclusion Chromatography--
The active fraction from
anion exchange chromatography was subjected to size exclusion
separation on Sephadex G-50 column (Pharmacia Biotech, Uppsala, Sweden,
93 × 2.5 cm) equilibrated with 0.06 M sodium
phosphate buffer, pH 7.0, 25 °C. The flow rate was 0.5 ml/min, peaks
were monitored at 280 nm and 3-ml fractions were collected and pooled.
The pools were desalted by dialysis (membrane cut off 3.5 kDa) against
distilled water for at least 48 h and lyophilized before further
analysis in subsequent experiments.
Further gel filtration of the active fraction from the anion exchange
chromatography was performed on a Superose 12 column (Pharmacia
Biotech, 30 × 1.0 cm) in 10 mM Tris/HCl, pH 7.5, 25 °C with 0.15 M NaCl. The flow rate was 0.3 ml/min,
the fraction size was 0.5 ml and peaks were monitored at 280 nm.
Observed peaks were collected, desalted by dialysis against distilled
water, and lyophilized.
PAGE--
Analytical PAGE was performed using 4-20%
polyacrylamide precast gels on a Bio-Rad Mini Protean II cell. To 10 µl of the lyophilized fractions from anion exchange chromatography or
gel filtrations dissolved in distilled water (5-10 mg/ml), an equal
volume of sample buffer (13.1% 0.5 M Tris-HCl, pH 6.8, 10.5% glycerol, and 0.05% bromphenol blue) was added. Samples (20 µl) were then loaded onto the gel, which was run in Tris glycine
buffer, pH 8.3, with 0.1% SDS at 200 V constant voltage for 40 min.
Proteins were stained by immersing the gel in 0.1% Coomassie Blue
solution in water/methanol/acetic acid (5:4:1) for 0.5 h.
Destaining was by several changes in 40% methanol, 10% acetic acid
until a clear background was obtained.
N-terminal Amino Acid Analysis--
After PAGE, the protein
bands of peak K from the G-50 column were transferred by Western
blotting onto polyvinylidene difluoride membranes. The protein bands
were visualized by Coomassie Blue staining and the stained bands were
cut out for protein sequencing. Protein sequencing was also performed
on an aliquot of each of the peaks 1-4 from the Superose-12 column
directly. All samples were subjected to Edman degradation performed in
an automated pulse-liquid sequencer (Applied Biosystems model 477A).
ESI-MS--
Peak K was analyzed on a VG Bio-Q ESI-MS (Fisons/VG,
Manchester, UK) equipped with an atmospheric pressure electrospray ion source and a quadruple mass analyzer with a maximum mass range of 4000. The mass spectrometer was scanned from m/z 600 to 2000 in
10 s. The mass resolution was set to 500. The data system was operated as a multichannel analyzer and 5 scans were averaged to obtain
the final spectrum. The electrospray carrier solvent was 1% acetic
acid in acetonitrile/water, 1:1, and the flow rate was 2-4 µl/min.
The sample was dissolved at a concentration of 10-20 pmol/µl in the
carrier solvent and 5 µl was injected. The molecular weight of sample
components was estimated from the m/z values of series of
ions as described earlier.
MALDI-TOF--
Peak K was analyzed by MALDI mass spectrometry on
an LDI 1700 time of flight mass spectrometer equipped with a pulsed
nitrogen laser (337 nm) (Biomolecular Separations Inc., Reno, NE). The laser power was set to 8.6 microjoule and the spectrum was the sum of
140 laser shots. Sinapinic acid was used as a matrix and bovine serum
albumin was used as the external standard. About 100 µg of the
protein was dissolved in 50 µl of water and 0.1% trifluoroacetic
acid. 10 µl of this solution was mixed with 10 µl of 50 mM sinapinic acid. The probe was loaded with 0.8 µl of the sample mixture, vacuum dried, loaded with another 0.8 µl of sample, and vacuum dried again before being inserted into the mass spectrometer.
Spectroscopic Analysis--
Prior to spectroscopic analysis, the
proteins or protein fractions were dialyzed against doubly distilled
water and lyophilized. Stock solutions of each sample were prepared by
dissolving the lyophilized material in 10 mM potassium
phosphate buffer at pH 7.5. The concentrations of the stock solutions
were determined using amino acid analysis after acid hydrolysis. The
spectra were recorded on solutions prepared by diluting aliquots of
stock solution into 10 mM potassium phosphate buffer at pH
7.5. All spectra were recorded at 25 °C.
UV Absorbance Spectroscopy--
UV absorbance spectra were
recorded at room temperature on a GBC UV/VIS 920 spectrophotometer, in
a quartz cuvette with 1-cm path length.
Fluorescence Spectroscopy--
Fluorescence spectra were
recorded at 25 °C on a Perkin-Elmer LS-50B spectrometer using a
quartz cuvette with 1-cm excitation path length. Intrinsic (tryptophan)
fluorescence emission spectra were recorded between 305 and 530 nm
(step 1 nm) with excitation at 295 nm. The excitation band width was 3 nm and the emission band width was 5 nm. ANS fluorescence emission
spectra were recorded between 400 and 600 nm (step 1 nm) with
excitation at 385 nm. Both the excitation and emission bandpass were
set to 5 nm.
Circular Dichroism Spectroscopy--
Circular dichroism (CD)
spectra were obtained using a JASCO J-720 spectropolarimeter with a
JASCO PTC-343 Peltier-type thermostated cell holder. Quartz cuvettes
were used with 1-cm path length in the near UV range and 1 and 0.1-mm
path length in the far UV range. Near UV spectra were recorded between
320 and 240 nm, and far UV spectra between 250 and 182 nm. The
wavelength step was 1 nm, the response time was 4 s, and the scan
rate was 10 nm/min. 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. The mean residue
ellipticity Tumor Cell Line--
The L1210 cell line was obtained from the
American Type Culture Collection (ATCC, Manassas, VA), cultured in
25-cm2 flasks (Falcon, Becton Dickinson, NJ) in RPMI 1640 supplemented with 10% fetal calf serum, nonessential amino acids,
sodium pyruvate, 50 µg of gentamicin/ml, kept at 37 °C in a
humidified atmosphere containing 5% CO2, with change of
medium every 3 days. The cells were harvested from the culture flasks
by centrifugation (200 × g for 10 min). The cell
pellet was resuspended in medium and seeded into 24-well plates, 2 × 106/well (Falcon, Becton Dickinson, NJ).
Cells were exposed to the different forms of Cell Viability--
For analysis, 30 µl of the washed cell
suspension was mixed with 30 µl of a 0.2% trypan blue solution and
the number of stained cells (dead cells) per 100 cells was determined
by interference contrast microscopy (Ortolux II, Leitz Wetzlar, Germany).
DNA Fragmentation--
Oligonucleosome length DNA fragments were
detected by agarose gel electrophoresis. The remainder of the washed
cell suspension (970 µl, 2 × 106/ml) was lysed in 5 mM Tris, 20 mM EDTA, 0.5% Triton X-100, pH 8.0, at 4 °C for 1 h and centrifuged at 13,000 × g for 15 min. DNA was ethanol precipitated overnight in
Intracellular Distribution of Biotinylated Fraction
VI--
L1210 cell suspension (4 × 106 cells/ml, 95 µl) were incubated at room temperature with 5 µl of biotinylated
fraction VI or native monomeric Isolation of an Apoptosis-inducing Complex from Human
Milk--
Human milk samples from four donors were separated into
casein and whey and the fractions were tested for apoptosis induction in L1210 leukemia cells. The activity precipitated with casein and
further purification was from the casein fraction. Following anion-exchange chromatography, six fractions were collected and tested
for apoptosis induction. Fractions I-V completely lacked activity.
Fraction VI that eluted with 1 M NaCl contained all of the
apoptosis inducing activity (Fig.
3A). PAGE of
fraction VI revealed one major band in the 14-kDa region, and
additional bands in the molecular mass range of 30, 60, and 100 kDa
(Fig. 2A, inset). After heat treatment (100 °C, 5 min),
fraction VI lost its activity (data not shown).
Size Exclusion Chromatography--
Fraction VI was applied on a
Sephadex G-50 column. Two well separated peaks (K and
L, Fig. 2B) were obtained. Peak K eluted near the
void volume and contained all of the apoptotic activity, peak L eluted
at the position of a 14-kDa protein and had no effect on cell
viability. PAGE of peak K showed one major band at about 14 kDa, and
additional bands of about 30, 60, and 100 kDa. Peak L gave only one
band at 14 kDa (Fig. 2B, inset).
N-terminal Amino Acid Sequence Analysis--
The different bands
of peak K were subjected to N-terminal amino acid sequence analysis
(Table I). The first 30 residues of the
band at 14 kDa and the first 9 residues of the 30-kDa band were
identical to the N-terminal sequence of human Induction of Apoptosis--
The effect on cell viability and
induction of DNA fragmentation was compared between the different milk
fractions (Fig. 3A). A rapid
reduction of cell viability from >95% to <10% occurred after
exposure of the L1210 cells to casein, fraction VI, and peak K (Fig.
3A), and oligonucleosome length DNA fragments were formed in
those cells. Whey, casein fractions I-V, and native monomeric
Nuclear Uptake of Fraction VI--
The localization of fraction VI
in the L1210 cells was examined using biotinylated material in confocal
microscopy (Fig. 3B). Fraction VI was shown to bind to the
cell surface, to enter the cytoplasm and finally to accumulate in the
nuclei of the L1210 cells (Fig. 3B). Native monomeric
Mass Spectrometry--
The results above suggested that the active
molecular form of
These results suggested that the apoptosis inducing activity was
dependent on a high molecular weight complex, containing Size Exclusion Chromatography--
To facilitate spectroscopic
analysis of the different oligomeric states of
The stability of the peaks was investigated by reinjection on the
Superose-12 column, after lyophilization and resuspension in 10 mM Tris/HCl, pH 7.5. No dissociation into monomers was
detected for the multimers in peaks 1-3. Peak 4 remained a stable
monomer (data not shown). N-terminal amino acid sequence analysis of
peaks 1-4 from the Superose-12 column showed the presence of
Spectroscopic Characterization--
Fraction VI and peaks 1-4
from the Superose-12 column were analyzed by spectroscopic techniques.
Monomeric
By UV absorption spectroscopy
Tryptophan fluorescence spectra were recorded to compare the degree of
folding between the molecular forms of
ANS was used as a probe of solvent accessible hydrophobic surfaces. ANS
was titrated into each sample in steps of 0.25 equivalents, relative to
the monomer concentration. The spectra at 1.5 equivalents of ANS are
shown (Fig. 7, A and
B). Native monomeric
The secondary structure content in the different samples was compared
by far UV CD spectroscopy. Native
Near UV CD spectroscopy was used to study rigidity versus
flexibility of aromatic side chains. Native Fraction VI had interesting and unusual effects on the L1210 leukemia
cells. Like casein, it induced apoptosis, as shown by a drastic
reduction in tumor cell viability and by the fragmentation of DNA. The
whey fraction and the other casein fractions lacked this activity, as
did the While there was no difference in cell surface binding between
native The term "molten globule" was introduced to describe a stable
folding variant of In this study, spectroscopic analysis of native monomeric
The spectroscopic characterization of fraction VI showed a large
contribution of the native, but there were, however, distinct spectral
differences compared with the native Protein folding variants have recently been proposed to differ in
biologic function. The conformational switch of the prion protein leads
to the formation of the disease causing isoform (31-33). The two
isoforms have the same amino acid sequence and no post-translational
modifications distinguish the two (34, 35). The prion protein first
changes to the molten globule state and then proceeds to a
non-reversible We thank Dr. Bryan Finn for help with
preparing Fig. 1 and Eva Thulin for discussions and help with
purification of whey *
This work was supported by Swedish Cancer Society Grant
3807-B97-01XAB (to C. S.), American Cancer Society Grant RPG
97-157-01 (to C. S.), Swedish Medical Research Council Grant
K97-03X-11552-02BK (to S. L.), and Swedish Natural Science
Research Council Grant K-AA/KU 10178-300 (to S. L.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Institute of Laboratory
Medicine, Section of Microbiology, Immunology, and Glycobiology, Lund
University, Sölvegatan 23, S-223 62 Lund, Sweden. Tel.: 46-46-173-933; Fax: 46-46-137-468; E-mail:
Malin.Svensson{at}mig.lu.se.
**
Present address: Astra Draco, P. O. Box 34, 221 00 Lund, Sweden.
The abbreviations used are:
ANS, 8-anilinonaphtalene-1-sulfonic acid;
PAGE, polyacrylamide gel
electrophoresis;
FPLC, fast protein liquid chromatography;
ESI-MS, electrospray ionization mass spectrometry;
MALDI-TOF, matrix assisted
laser desorbtion ionization-time of flight mass spectrometry;
PBS, phosphate-buffered saline.
Molecular Characterization of 
-Lactalbumin Folding
Variants That Induce Apoptosis in Tumor Cells*
§,,,
**,,, and
Institute of Laboratory Medicine, Section of
Microbiology, Immunology, and Glycobiology (MIG), Lund University,
Sölvegatan 23, S-223 62 Lund, Sweden, the ¶ Department of
Clinical Microbiology and Immunology, Rockefeller University, New York,
New York 10021, the Department of Physical
Chemistry 2, Lund University, P. O. Box 124, S-221 00 Lund,
Sweden, and the Department of Pharmaceutical Chemistry,
University of California, San Francisco, California 94143
ABSTRACT
Top
Abstract
Introduction
References
-lactalbumin and mass spectrometry ruled out post-translational modifications. Size exclusion chromatography resolved monomers and oligomers of -lactalbumin that were
characterized using UV absorbance, fluorescence, and circular dichroism
spectroscopy. The high molecular weight oligomers were kinetically
stable against dissociation into monomers and were found to have an
essentially retained secondary structure but a less well organized
tertiary structure. Comparison with native monomeric and molten globule -lactalbumin showed that the active fraction contains oligomers of
-lactalbumin that have undergone a conformational switch toward a
molten globule-like state. Oligomerization appears to conserve -lactalbumin in a state with molten globule-like properties at physiological conditions. The results suggest differences in biological properties between folding variants of -lactalbumin.
INTRODUCTION
Top
Abstract
Introduction
References
-lactalbumin.
-lactalbumin is secreted by the mammary epithelium and is
the major whey protein of human milk (8). Its main known function is to
change the acceptor specificity of 
-galactosyltransferase from
GlcNAc to Glc, thus enabling the synthesis of lactose in milk (9, 10).
The crystal structure of -lactalbumin has been solved (11) (Fig.
1). It is a metalloprotein with high affinity for Ca2+ and other divalent cations (12, 13), and
Ca2+ is essential for the folding and structural stability
of -lactalbumin (14, 15). At low pH, -lactalbumin forms a
relatively stable protein folding variant (16). This form, the molten
globule, has native-like secondary structure but less well defined
tertiary structure, and larger stokes radius (17). Similar states are formed at elevated temperatures, by reduction of disulfide bonds or by
removal of calcium at neutral pH (18-20).
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Fig. 1.
Three-dimensional structure of native
human -lactalbumin. The backbone, the
three tryptophan side chains, and the tightly bound Ca2+
ion are shown. The disulfide bonds are indicated with roman
numerals as follows: I, 61-77; II, 73-91;
III, 28-111; IV, 6-120. This figure was
generated using UCSF software MidasPlus (37). -lactalbumin is a
14-kDa protein with four -helices formed by residues 1-37 and
85-123. The triple-stranded -sheet is found at one side of the
protein (residue 38-84) and unfolds more easily than the -helical
region (38). A high affinity calcium-binding site is formed by the
carboxylates of Asp-82, Asp-87, and Asp-88, the carbonyl oxygens of
Lys-79 and Asp-84, and two water molecules (11).
-lactalbumin from whey, the
apoptosis-inducing component was purified from the casein
fraction (precipitated at pH 4.3), and behaved as a multimeric protein
rather than a monomer. Furthermore, native monomeric -lactalbumin
from human milk whey was inactive in the apoptosis assay. These
observations suggested that the active fraction contains an
alternative molecular form of -lactalbumin. Here we show,
using mass spectrometry, gel filtration, UV absorption, fluorescence,
and CD spectroscopy, that the apoptosis-inducing form of
-lactalbumin is a mixture of monomeric and multimeric forms with
molten globule-like properties and suggest that folding variants of
-lactalbumin differ in biologic activity.
MATERIALS AND METHODS
-Lactalbumin--
Native, monomeric -lactalbumin was
purified from human milk by ammonium sulfate precipitation. The
ammonium sulfate was added as a salt, 264 g/liter milk, and the mixture
was incubated overnight at 4 °C. The mixture was centrifuged
(Sorvall RC-5B refrigerated superspeed centrifuge, Du Pont Instruments,
Wilmington, DE) at 5000 × g for 15 min. The whey
fraction was collected, lyophilized, and dissolved in 50 mM
Tris/HCl with 35 mM EDTA, pH 7.5. A 400-ml phenyl-Sepharose
column (Pharmacia Biotech, Uppsala, Sweden) was packed in 50 mM Tris/HCl with 1 mM EDTA, pH 7.5, 25 °C
and a 500-ml sample was loaded onto the column. The column was first
washed with 50 mM Tris/HCl with 1 mM EDTA, pH
7.5, and -lactalbumin was then eluted from the column with 50 mM Tris/HCl with 1 mM CaCl2, pH
7.5, thus yielding the native, Ca2+ bound form of
-lactalbumin.
-lactalbumin to 2.0 by adding 0.1 M HCl. This material was used as a control sample in
subsequent spectroscopic studies. In the cellular experiments, EDTA
(0.14 mM/mg) was added to -lactalbumin to remove
Ca2+ and thus form the apo state, which is molten
globule-like.
m (mdeg × cm2 × dmol
1) was calculated from the recorded ellipticity, ,
as m = /(c·n·l),
where c is the protein concentration in M, n the number of residues in the protein (123 in this case), l the
path length in m, and the ellipticy in degrees.
-lactalbumin, with
medium as a control. At time 0, 100 µl of medium was aspirated from
each well, replaced by 100 µl of the different experimental solutions
and incubated at 37 °C in an atmosphere of 5% CO2 for 6 h. Cells were harvested from the 24-well plates by aspiration, resuspended in PBS (5 ml), washed, and resuspended in 1 ml of PBS.
20 °C, treated with RNase proteinase K, and loaded on 1.8%
agarose gels, and electrophoresed with constant voltage set at 50 V
overnight. DNA fragments were visualized with ethidium bromide using a
305-nm UV-light source and photographed using Polaroid type 55 positive-negative film.
-lactalbumin (5 mg/ml, biotinylated
according to the manufacturer's instructions), and then washed in PBS
with centrifugation at 320 × g for 10 min to remove
unbound protein. To detect intracellular protein, cells exposed to
biotinylated protein were permeabilized with saponin to allow entry of
fluorescein isothiocyanate-conjugated streptavidin. Cells harvested by
centrifugation at 320 × g were fixed by suspension in
phosphate-buffered paraformaldehyde (4%) (21) for 5 min at room
temperature, washed in PBS, and permeabilized with 0.1% saponin in
PBS. After washing in 0.1% saponin, fluorescein
isothiocyanate-conjugated streptavidin (1:100 in 0.1% saponin, 100 µl) was added and the cells were incubated for 30 min at room
temperature. The cells were washed twice in PBS/saponin and once in
PBS, mounted on a glass slide and analyzed in a Bio-Rad 1024 laser
scanning confocal equipment (Bio-Rad, Hemel-Hempstead, UK) attached to
a Nikon Diaphot inverted microscope (Nikon, Japan).
RESULTS
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Fig. 2.
Anion exchange chromatography and gel
filtration. Panel A, ion-exchange chromatogram of human
casein. 30 mg of casein was fractionated using a DEAE-Trisacryl column
attached to a FPLC system using a stepwise NaCl gradient, at 25 °C.
The apoptosis-inducing fraction eluted after 1 M NaCl
indicated by the arrow. Inset, PAGE of the
apoptosis inducing fraction VI and monomeric -lactalbumin
(ALA). The molecular weight standards (lane S)
are: myosin (Mr = 200,000), -galactosidase
(Mr = 116, 250), phosphorylase b
(Mr = 97, 400), bovine serum albumin
(Mr = 66, 200), ovalbumin
(Mr = 45,000), carbonic anhydrase
(Mr = 31,000), soybean trypsin inhibitor
(Mr = 21,500), and lysozyme
(Mr = 14,400), respectively. Panel B,
size exclusion chromatogram of fraction VI from the ion-exchange
column. 100 mg was applied to a Sephadex G-50 column (93 × 2.5 cm) equilibrated with 0.06 M sodium phosphate buffer, pH
7.0, 25 °C. The flow rate was 30 ml/h and the fraction size was 3 ml. Two well separated peaks were obtained. Inset, PAGE of
peaks K and L. The molecular weight standards are as in panel
A. Panel C, size exclusion chromatogram of fraction VI
on a Superose 12 column. Fraction VI (10 mg) was suspended in 500 µl
of 10 mM potassium phosphate buffer, pH 7.5, 25 °C (flow
rate 0.3 ml/min, fraction size 0.5 ml). Peak 1 eluted with the void
volume (8 ml), peak 2 eluted at 9.4-11.6 ml corresponding to a
molecular mass of 150-400 kDa, peak 3 eluted at 13.8-14.2
ml corresponding to a mass of 30-42 kDa, and peak 4 eluted at
15.0-15.2 ml, corresponding to monomeric -lactalbumin, 14 kDa.
Inset, PAGE of peaks 1-4, monomeric -lactalbumin
(ALA) and fraction VI. The molecular weight standards
(S) are as in panel A.
-lactalbumin, except
for residue 6, which was not detected. The main N-terminal sequence of
the 60-kDa band and the two 100-kDa bands was also identical to human
-lactalbumin, but some sequencing cycles showed heterogeneity. These
results suggested that peak K contained -lactalbumin complexes of
increasing molecular size
N-terminal amino acid sequence of the protein bands of peaks K and 1-4
and human -lactalbumin (HLA)
-lactalbumin is cysteine. Residues shown below the 60- and 100-kDa
sequence of peak K are other possible candidates.
-lactalbumin did not reduce cell viability or induce DNA
fragmentation (Fig. 3A).
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Fig. 3.
Induction of apoptosis and cellular
localization. Panel A, the different milk fractions
were dissolved in cell culture medium and added to L1210 cells, the
viability and induction of DNA fragmentation was measured after 6 h of incubation at 37 °C, 5% CO2. Cell viability was
measured using trypan blue exclusion and oligonucleosome length DNA
fragments were detected using gel electrophoresis. The inactive
fractions were tested at 5 mg/ml (whey, casein fraction I-V, G-50 peak
L, and native and molten globule-like monomeric -lactalbumin). The
active fractions were tested at 0.5 mg/ml (casein, casein fraction VI,
and G-50 peak K). The cell viability in % of the control is shown
under each lane (mean of three separate experiments).
Panel B, nuclear localization of fraction VI. The
localization of fraction VI and native -lactalbumin in the L1210
cells was examined using biotinylated material in confocal microscopy.
Cells were exposed to the biotinylated material for 6 h and
stained with fluorescein isothiocyanate-conjugated streptavidin.
Fraction VI readily accumulated in the nuclei of L1210 cells
(c), whereas native -lactalbumin bound to the surface of
the cells but was not located to the nuclei (b). No staining
was seen with streptavidin alone (a).
-lactalbumin did not accumulate in the cell nuclei, and only showed
surface binding (Fig. 3B). The apoprotein did not induce DNA
fragmentation in L1210 cells.
-lactalbumin may have different post-translational
modifications and/or different conformation as compared with native
-lactalbumin. Peak K was therefore analyzed by ESI-MS and MALDI-TOF,
native -lactalbumin was used as a control. The estimated molecular
mass of the major component (14.088 kDa) was close to the molecular mass of -lactalbumin calculated from the amino acid sequence (14.078 kDa). The small mass differences ruled out post-translational modifications such as phosphorylation and glycosylation. MALDI-TOF of
peak K showed a major peak close to 14 kDa consistent with monomeric
-lactalbumin, but peaks consistent with dimeric and trimeric forms
(28 and 42 kDa) were also seen (Fig. 4).
These results support the conclusion from ESI-MS of lack of
post-translational modifications, and in addition showed the
possibility of formation of multimeric forms of -lactalbumin.
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Fig. 4.
Mass spectrometry. MALDI-MS of peak K. 100 µg of peak K was dissolved in 50 µl of water and 0.1%
trifluoroacetic acid. 10 µl of this solution was mixed with 10 µl
of 50 mM sinapinic acid and the probe was loaded twice with
0.8 µl before insertion into the mass spectrometer.
-lactalbumin. Mass spectrometry ruled out post-translational modifications which indicated that the difference between native, monomeric -lactalbumin and the apoptosis inducing complex was conformational.
-lactalbumin,
fraction VI was applied on a Superose-12 column. Four peaks were
obtained (Fig. 2C). Peak 1 eluted with the void volume, peak
2 eluted as 150-400 kDa, peak 3 as 30-42 kDa, and peak 4 eluted as a
14-kDa protein, at the same position as the native, native
-lactalbumin control. SDS-PAGE of peak 1 showed a faint band larger
than 200 kDa, peak 2 had a faint band at 90 kDa, peak 3 had bands at
14, 30, and 66 kDa, and peak 4 gave one band at 14 kDa and one at 31 kDa (Fig. 2C, inset).
-lactalbumin in all peaks. Peak 4 was >95% pure. Peaks 1, 2, and 3 showed a higher background (steps 1-10), but no dominating second
sequence suggestive of another protein (Table I).
-lactalbumin in the native or the acid-induced molten
globule state were included as controls.
-lactalbumin and fraction VI showed
virtually identical spectra (Fig.
5A), except for the elevated background absorbance for fraction VI indicating that the complex scattered light more than native -lactalbumin. Peaks 1, 2, 3, and 4 strongly resembled native, monomeric -lactalbumin, but with elevated
backgrounds. Peak 1 had the highest background absorbance as expected
for very large complexes (Fig. 5B).
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Fig. 5.
UV spectra. UV absorbance spectra were
recorded at 25 °C on a GBC UV/VIS 920 spectrophotometer, in a quartz
cuvette with 1-cm path length. Panel A shows fraction VI
(solid black line) and native, monomeric -lactalbumin
(thin black line). Panel B shows peaks 1-4 (peak
1, 
; peak 2, 
; peak 3, 
; peak 4, 
) and pH 2 molten globule
-lactalbumin (dashed line). All spectra were recorded for
1 mg/ml protein solutions, corresponding to 70 µM
monomer.
-lactalbumin. Native human
-lactalbumin had its intensity maximum at 335 nm and a shoulder at
320 nm, indicative of tryptophan residues in a folded hydrophobic core.
The pH 2 molten globule and fraction VI had an intensity maximum at 340 nm and a shoulder at 355 nm, indicating that the tryptophans in
fraction VI and the pH 2 molten globule are more accessible to the
solvent compared with native -lactalbumin (Fig.
6, A and B). Peaks
1, 2, and 3 showed the same intensity maxima as fraction VI and the pH
2 molten globule -lactalbumin, but the intensity at 355 nm was
almost as high as at 340 nm. Peak 4 strongly resembled native
-lactalbumin but with an additional weak shoulder at 355 nm. (Fig.
6B). The results indicate that tryptophan residues are
shielded from solvent in the monomer and in peak 4, but are more
solvent exposed in the pH 2 molten globule, in fraction VI and in peaks
1-3.
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Fig. 6.
Intrinsic fluorescence spectra.
Panel A, fluorescence spectra for fraction VI (solid
line) and native, monomeric -lactalbumin (thin
line). Panel B, fluorescence spectra for peaks 1-4
(peak 1, ; peak 2, ; peak 3, ; peak 4, ) and pH 2 molten
globule -lactalbumin (dashed line). All spectra were
recorded for 0.3 mg/ml protein solutions, corresponding to 20 µM monomer, in 10 mM potassium phosphate
buffer at pH 7.5, 25 °C.
-lactalbumin did not bind ANS as
shown by the low intensity of the spectrum with a maximum at 515 nm
strongly resembling the spectrum of ANS added to pure buffer (Fig.
7A). The pH 2 molten globule -lactalbumin showed the most
significant ANS binding with the maximum at 475 nm and significantly
enhanced intensity (Fig. 7B). The fraction VI spectrum was
blue-shifted compared with native -lactalbumin with the intensity
maximum at 475 nm and increased quantum yield, indicating that ANS
binds to fraction VI (Fig. 7A). The ANS fluorescence spectra
of peaks 1-3 showed intensity maxima at 475 nm, and for peaks 2 and
3 a strongly enhanced quantum yield, indicating significant ANS
binding. Peak 4 was virtually identical to native -lactalbumin with
low intensity and a non-shifted maximum at 515 nm (Fig. 7B). The results indicate exposed hydrophobic surfaces in the pH 2 molten
globule, fraction VI and peaks 1-3, but not in peak 4 or the native
monomer.
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Fig. 7.
ANS fluorescence spectra. Panel
A, ANS titration of fraction VI (solid line) and
native, monomeric -lactalbumin (thin line). Panel
B, ANS titration of peaks 1-4 (peak 1, ; peak 2, ; peak 3, , peak 4, ) and pH 2 molten globule -lactalbumin (dashed
line). The figure shows spectra obtained with 1.5 µM
ANS. The protein concentration was 0.3 mg/ml, corresponding to 20 µM monomer, in 10 mM potassium phosphate
buffer at pH 7.5, 25 °C.
-lactalbumin showed a double dip
at 208 and 228 nm. The spectrum is broader than for many other proteins
composed of mixed -helices (double dip at 208 and 222 nm) and
-sheets (single dip at 215 nm). This may be due to influences from
aromatic groups at the higher wavelengths. The far UV CD spectra did
not indicate any major differences in secondary structure between
native -lactalbumin, the pH 2 molten globule, fraction VI, and peaks
1-4, although the analysis becomes uncertain for peaks 1 and 2 due to
the higher background observed in the amino acid analysis (Fig.
8, A and B). An
analysis of the spectra using the Selcon program (22) gave no
differences in secondary structure beyond the error limits when
comparing any of the samples studied.
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Fig. 8.
Far UV CD spectra. Panel A,
circular dichroism spectra in the far UV region of fraction VI
(solid line) and native, monomeric - lactalbumin
(thin line). Panel B, CD spectra of peaks 1-4
(peak 1, ; peak 2, ; peak 3, ; peak 4, ) and pH 2 molten
globule -lactalbumin. The protein concentration was 0.14 mg/ml,
corresponding to 10 µM monomer, in 10 mM
potassium phosphate buffer at pH 7.5, 25 °C.
-lactalbumin had a minimum at 270 nm arising from tyrosine residues and a maximum at 294 nm arising from tryptophan residues. The near UV CD spectrum of the pH
2 molten globule showed the characteristic loss of signal, indicating
less restrained tyrosines and tryptophans. Fraction VI had a spectrum
similar to native -lactalbumin but with less signal, indicating that
the motion of tyrosines and tryptophans is less restrained (Fig.
9A). Peaks 1, 2, and 3 showed
similar spectra as the pH 2 molten globule, but peak 4 was almost
identical to native -lactalbumin (Fig. 9B). The results
point to a higher degree of mobility of aromatic residues in fraction
VI and peaks 1-3 as compared with peak 4 and native -lactalbumin.
The results also confirm the presence of -lactalbumin in all
samples.
View larger version (19K):
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Fig. 9.
Near UV CD spectra. Panel A,
circular dichroism spectra in the near UV region for fraction VI
(solid line) and native, monomeric - lactalbumin
(thin line). Panel B, CD spectra in the near UV
region for peaks 1-4 (peak 1, ; peak 2, ; peak 3, ; peak 4, ) and pH 2 molten globule -lactalbumin (dashed line).
The protein concentration was 1.0 mg/ml, corresponding to 70 µM monomer, in 10 mM potassium phosphate
buffer at pH 7.5, 25 °C.
DISCUSSION
-Lactalbumin is present in the milk of all mammals. Pig, sheep,
and goat milk contain multiple forms of -lactalbumin, that vary in
amino acid sequence (23), but only one molecular form of human
-lactalbumin has been described. This 14-kDa protein is a major
constituent of human milk whey, and occurs at concentrations around 2 mg/ml (8). The present study showed that -lactalbumin complexes of
larger molecular size could be purified from human milk casein. The
complexes were characterized as -lactalbumin multimers that induces
apoptosis in tumor cells. The apoptosis-inducing form was isolated
from casein by ion exchange chromatography. N-terminal amino acid
sequencing showed identity with -lactalbumin and gel filtration
demonstrated the presence of multimers that were kinetically stable
toward dissociation. Mass spectrometry excluded post-translational
modifications such as glycosylation and phosphorylation indicating that
activity was associated with changes in secondary or tertiary
structure. Spectroscopic analyses showed that the multimers in the
apoptosis-inducing fraction had properties strongly resembling those of
the molten globule state. These results show that the
apoptosis-inducing fraction consists of -lactalbumin in monomeric
and oligomeric states. The oligomers constitute novel folding variants
with molten globule-like properties.
-lactalbumin both in the native and in the molten
globule-like state. This might be explained by the presence of
Ca2+ in the cell culture medium reverting
apo--lactalbumin back to its native fold. Consequently, the
apoptosis inducing activity appeared to be specific for the molecular
complex in fraction VI. This activity was partly explained by cellular
localization studies. The distribution of the active fraction was
examined by confocal microscopy after biotinylation of the protein,
with native -lactalbumin as a negative control. Fraction VI was
shown to bind the cell surface, to enter the cytoplasm and accumulate in the cell nuclei. Nuclear uptake was rapid and was detected at the
time of DNA fragmentation. Furthermore, it was specific for the tumor
cells; primary cultures of non-transformed cells showed no nuclear
uptake (24). We propose that the interaction with the nucleus is
critical for the induction of DNA fragmentation, since inhibition of
nuclear uptake with wheat germ agglutinin rescued cells from DNA fragmentation.
-lactalbumin and fraction VI, native -lactalbumin
did not accumulate in the cell nuclei. This is consistent with previous studies suggesting that the molten globule state is important for
translocation of proteins across phospholipid bilayers (25). While
native -lactalbumin interacted with negatively charged membranes
only at pH values below the isoelectric point, partially unfolded,
molten globule-like conformers of -lactalbumin bind to
phospholipid membranes also at higher pH values (26). This may be a
consequence of the surface accessible hydrophobic residues in the
partially unfolded state. Based on the present study we propose that
nuclear uptake and DNA fragmentation require that -lactalbumin
is oligomerized and preserved in the molten globule-like state.
-lactalbumin, which has native-like secondary structure but less well defined tertiary structure (17, 27). Molten
globules are formed under acidic conditions, and similar states are
formed at neutral pH upon removal of the tightly bound Ca2+
ion, reduction of the disulfide bonds, or at elevated temperatures (18-20). Molten globules have also been proposed as kinetic
intermediates observed during protein folding (28-30).
-lactalbumin and the pH 2 molten globule control, agreed with
earlier reports. The fluorescence spectrum for native -lactalbumin
showed tryptophan residues in a folded hydrophobic core, and the small effects on the ANS spectrum suggested that no larger hydrophobic surfaces were exposed in the native monomer. The far UV CD spectrum was
typical for native -lactalbumin, and the near UV CD spectrum had the
characteristic tyrosine dip and tryptophan peak (17). In the pH 2 molten globule the tryptophan residues were more accessible to the
solvent and ANS binding was strong indicating interaction with
hydrophobic surfaces. The far UV CD spectrum showed no difference in
secondary structure between the pH 2 molten globule and native -lactalbumin, but the near UV CD spectrum showed the characteristic loss of signal, indicating less restrained tyrosines and tryptophans (17).
-lactalbumin. The intrinsic fluorescence spectrum indicated solvent accessible tryptophan residues in fraction VI, and the large intensity increase and wavelength decrease in the ANS spectrum of fraction VI suggested that
ANS bound to hydrophobic surfaces which had become accessible on
oligomerization. Peak 1 from the Superose-12 column contained the
largest multimers, and had intrinsic fluorescence, far UV CD, and near
UV CD spectra similar to those of -lactalbumin in the pH 2 molten
globule state. The ANS fluorescence spectrum showed very low intensity
compared with the pH 2 molten globule, possibly due to inaccessibility
of hydrophobic surfaces to ANS in the large aggregates or due to
fluorescence quenching arising from molecular collisions. Peaks 2 and 3 bound ANS strongly and the fluorescence and UV CD spectra resembled the
pH 2 molten globule -lactalbumin. Peak 4, that eluted at the same
volume as native, monomeric -lactalbumin behaved as native monomeric
-lactalbumin. The fraction VI spectra could be generated by a
weighted summation of the spectra for peaks 1-4.
-sheet rich form (33, 36). In this study, we
have identified a new example of a protein which acquires novel
functions after conformational switching. Like in the prion system, the
two molecular forms of -lactalbumin had identical amino acid
sequence, with no post-translational modifications as detected by mass
spectrometry. We propose that the relative folding instability of
-lactalbumin determines its ability to attain new essential functions.
ACKNOWLEDGEMENTS
-lactalbumin.
FOOTNOTES
ABBREVIATIONS
REFERENCES
Top
Abstract
Introduction
References
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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