Keywords apoptosis; HAMLET; oleic acid; protein fragments; a-lactalbumin Correspondence P.. These complexes were named human HAMLET or bovine BAMLET alpha-lactalbumin made lethal to tumo
Trang 1a-lactalbumin display apoptotic activity
Serena Tolin1, Giorgia De Franceschi1, Barbara Spolaore1, Erica Frare1, Marcella Canton2,
Patrizia Polverino de Laureto1and Angelo Fontana1
1 CRIBI Biotechnology Centre, University of Padua, Italy
2 Department of Experimental Biomedical Sciences, University of Padua, Italy
Introduction
a-Lactalbumin (a-LA) is a small, acidic, Ca2+
-bind-ing protein involved in the biosynthesis of lactose,
being a component of the lactose synthase complex
[1] For a few decades, a-LA has been the subject of
intensive structural investigations, and it has served
as a model system in many protein folding studies
[2,3] The 123 residue chain of a-LA is organized into
a discontinuous a-helical domain composed of
residues 1–39 and 81–123, and a small b-domain
comprising the rest of the polypeptide chain [4,5] (Fig 1) A noteworthy property of a-LA is its ability
to adopt a partly folded or molten globule (MG) state under a variety of conditions, including low pH This state, lacking the specific interactions of the ter-tiary structure of the native protein, but maintaining
a high degree of secondary structure, has been exten-sively analyzed with a variety of techniques and approaches [6–9]
Keywords
apoptosis; HAMLET; oleic acid; protein
fragments; a-lactalbumin
Correspondence
P Polverino de Laureto, CRIBI
Biotechnology Centre, University of Padua,
Viale G Colombo 3, 35121 Padua, Italy
Fax: +39 049 827 6159
Tel: +39 049 827 6157
E-mail: patrizia.polverinodelaureto@unipd.it
(Received 7 August 2009, revised 9 October
2009, accepted 27 October 2009)
doi:10.1111/j.1742-4658.2009.07466.x
The complexes formed by partially folded human and bovine a-lactalbumin with oleic acid (OA) have been reported to display selective apoptotic activity against tumor cells These complexes were named human (HAMLET) or bovine (BAMLET) alpha-lactalbumin made lethal to tumor cells Here, we analyzed the OA complexes formed by fragments of bovine a-lactalbumin obtained by limited proteolysis of the protein Speci-fically, the fragments investigated were 53–103 and the two-chain fragment species 1–40⁄ 53–123 and 1–40 ⁄ 104–123, these last being the N-terminal fragment 1–40 covalently linked via disulfide bridges to the C-terminal fragment 53–123 or 104–123 The OA complexes were obtained by mixing the fatty acid and the fragments in solution (10-fold and 15-fold molar excess of OA over protein fragment) or by chromatography of the frag-ments loaded onto an OA-conditioned anion exchange column and salt-induced elution of the OA complexes Upon binding to OA, all fragments acquire an enhanced content of a-helical secondary structure All OA com-plexes of the fragment species showed apoptotic activity for Jurkat tumor cells comparable to that displayed by the OA complex of the intact pro-tein We conclude that the entire sequence of the protein is not required
to form an apoptotic OA complex, and we suggest that the apoptotic activity of a protein–OA complex does not imply specific binding of the protein
Abbreviations
a-LA, a-lactalbumin; BAMLET, bovine a-lactalbumin made lethal to tumor cells; CAC, critical aggregate concentration; HAMLET, human a-lactalbumin made lethal to tumor cells; MG, molten globule; OA, oleic acid; [h], mean residue ellipticity; TNS,
6-(p-toluidino)-2-naphthalenesulfonate.
Trang 2An interesting property of a-LA is its capacity to
interact with membranes and lipid bilayers [10–14], as
well as fatty acids [15] In particular, a complex
formed by Ca2+-depleted a-LA in its partly folded
state with oleic acid (OA) has been extensively studied
This OA complex, named human a-LA made lethal to
tumor cells (HAMLET), was initially isolated from
human milk, and was shown to selectively induce
apoptosis in tumor and immature cells, but not in
healthy cells [16–19] It was proposed that the
condi-tions required to induce HAMLET formation in vivo
are those of the stomach of the breastfed child, where
low pH may partially unfold a-LA by releasing its
protein-bound Ca2+ [19–21], and free OA can be
produced by lipases that hydrolyze milk triglycerides
[22,23] HAMLET can also be prepared in vitro, by
application of the apo form of a-LA to an anion
exchange column equilibrated with OA and elution of
the OA complex with high salt concentration, followed
by dialysis and lyophilization [18] It seems that the
formation of the OA complex relies on the fact that
the apo form of a-LA is more hydrophobic than the
holo form, and thus is prone to bind the hydrophobic
fatty acid [20] The conformational features of a-LA in the OA complex are those of a protein MG, and upon binding to the protein, OA can probably stabilize this altered protein conformation [21] HAMLET-like com-plexes with similar biological activity can be obtained with a-LA from different species, including bovine, equine, porcine, ovine and caprine a-LA [24] The OA complex of bovine a-LA was named bovine a-LA made lethal to tumor cells (BAMLET) [21] Moreover,
it was also shown that a-LA mutants with amino acid replacements at the level of the Ca2+-binding loop were capable of producing active OA complexes [21] Despite the intensive research on HAMLET and BAMLET, the molecular features of the active OA complex in terms of protein⁄ fatty acid stoichiometry and monomeric⁄ oligomeric state of the protein in the complex are still not clarified, and are a matter of debate in the current literature [20,25–27] The molecu-lar mechanism of interaction and physicochemical properties of the OA complex are not understood, and neither are the mechanisms and cellular events involved in the toxicity of HAMLET It was shown by using labeled a-LA that the OA complex is able to
104
1 5–11H1 h1b 23–34 S1 S2 S3 123
86–98 105–110 h3c
123 123
(1 −40/53−123)/OA
0.0 0.5
1.0 BAMLET
(53 −103)/OA
0.0 0.5
1.0 (1 −40/104−123)/OA
Volume (mL)
α-LA
EDTA
EDTA
a
A
B
c
b
d
Fig 1 (A) Top: scheme of the secondary structure of the 123 residue chain of a-LA [4] The four a-helices (H1–H4) are indicated
by large boxes, and the corresponding chain segments are given above them The three b-strands (S1, 41–44; S2, 47–50; S3, 55–56) and the 310helices (h1b, 18–20; h2, 77–80; h3c, 115–118) are indicated by small boxes Bottom: schematic representation of the three a-LA fragments investigated The four disulfide bridges (6–120, 28–111, 61–77, and 73–91) are represented by thin lines, and the gray box indicates the segment encompassing the Ca 2+ -binding loop (B) Preparation of the OA complexes of a-LA (a) and its fragments (b–d) by chromatography
on an OA-conditioned anion exchange col-umn [18] The protein material was applied
to a DEAE-Trisacryl M column conditioned with OA, and the OA complexes were eluted with a gradient of 1 M NaCl (dashed lines) The solid bars indicate the fractions
of the effluent from the column that were collected for further studies.
Trang 3bind to tumor cells and accumulate in the cell nuclei
[28] These authors identified specific histone proteins
as nuclear targets for HAMLET However, it was also
shown that a-LA in the absence of OA can interact
with histones and charged, disordered poly-a-amino
acids (i.e poly-Lys and poly-Arg) This a-LA
interac-tion was shown to be driven by electrostatic forces
[29,30] Therefore, the active species in the cell may be
the whole protein–fatty acid complex, the protein
alone, or even the OA by itself In this last case, the
fatty acid aggregation state should be considered, as it
can be significantly influenced by the presence of a
protein in the same solution
In this study, we analyzed the propensity of three
fragment species of bovine a-LA, obtained by limited
proteolysis of the protein [9,31,32], to bind OA and to
form biologically active OA complexes As shown in
Fig 1, the fragments have different structural
charac-teristics, with fragment 1–40⁄ 104–123 encompassing
three of the four a-helices of the native protein,
frag-ment 53–103 containing the chain segfrag-ment that binds
Ca2+in the native protein, and fragment 1–40⁄ 53–123
being able to adopt, at neutral pH, an MG
conforma-tion resembling that of the MG conformaconforma-tion adopted
by intact a-LA at pH 2.0 [31,32] The conformational
properties of the OA complexes of these fragments were
analyzed by far-UV CD measurements, and it was
shown that the fragments acquire an enhanced content
of a-helical secondary structure upon binding OA The
physical and aggregation state of OA at physiological
pH was analyzed by fluorescence and turbidimetric
analyses It was shown that the fragments, as well as
the entire protein, depress the critical concentration for
aggregate formation [critical aggregate concentration
(CAC)] of OA and induce the formation of small and
water-soluble OA aggregates All OA complexes
dis-played apoptotic activity for tumor cells, and the extent
of their activity was comparable to that observed with
the OA complex of the intact protein, i.e BAMLET
Our results indicate that the entire 123 residue chain of
a-LA is not required for forming a cytotoxic OA
com-plex, and raise the possibility that the cell-damaging
effects of the various OA complexes could result from
an enhanced solubility of the otherwise poorly soluble
and inherently toxic fatty acid [33]
Results
Preparation of complexes of a-LA fragments
with OA
Two procedures were followed to prepare the
OA–fragment complexes, namely by simple mixing the
two components in solution, or by chromatography using an OA-conditioned anion exchange column, as described by Svensson et al [18] for the preparation of HAMLET The two procedures were used here, as it is not clear whether a mixing procedure results in a less active or inactive complex [18,20,34] Instead, we [35] and others [26,36,37] have shown that it is indeed pos-sible to prepare an OA complex displaying similar structural properties to HAMLET or BAMLET, i.e
to an OA complex prepared by chromatography Nev-ertheless, here we preferred to use and compare both procedures in preparing the OA complexes
Bovine a-LA and its fragments were loaded on an anion exchange column conditioned with OA The chromatographic profile obtained with intact a-LA was similar to that previously reported [18] Salt and EDTA were eluted first from the column The free protein was eluted from the column at low salt concen-tration, whereas the OA complex was eluted at 1 m salt The three fragments strongly bound to the OA-conditioned matrix, and their OA complexes could
be eluted by high salt (Fig 1B) The amounts of pro-tein fragment in the eluted OA complex, calculated on the basis of the material loaded onto the column, were
50% for fragments 1–40 ⁄ 53–123 and 1–40 ⁄ 104–123, and 25% for fragment 53–103, as estimated from
UV absorption measurements This indicated that a proportion of the protein fragment material remained bound to the column In the case of fragment 53–103, aggregated species were eluted at a higher retention time than that of the OA–fragment 53–103 complex Aggregation of the fragment was deduced from the turbidity of the last eluted fraction (Fig 1Bd) This would account for the low recovery of soluble OA complex of fragment 53–103 For the sake of compari-son, the OA complexes were also prepared in solution
by direct mixing of the a-LA fragments with OA at a molar ratio of 1 : 10 or 1 : 15 (see below)
Conformational properties of protein fragment complexes with OA
The conformational properties of the OA complexes formed by a-LA fragments prepared by chromatogra-phy or by direct mixing in solution were analyzed by far-UV CD spectroscopy in NaCl⁄ Pi (pH 7.4) Figure 2A shows the far-UV CD spectra of fragment 1–40⁄ 104–123 in the presence of increasing concentra-tions of OA The CD spectrum of this fragment appeared to be that of a largely disordered polypep-tide, but upon addition of OA the spectrum acquired the characteristics of a-helical secondary structure It is
of interest that, in the presence of OA (protein⁄ fatty
Trang 4acid molar ratio of 1 : 10), the CD spectrum of
frag-ment 1–40⁄ 104–123 was quite similar to that of the
corresponding OA complex prepared by
chromato-graphy, implying that the OA complexes prepared by
the two procedures displayed similar conformational
features Analogous conformational effects of OA
binding were observed with fragment 53–103 in the
presence of 15 equivalents of OA (Fig 2B) Thus,
frag-ment species 1–40⁄ 104–123 and 53–103 both appeared
to be rather disordered in solution at pH 7.4, but upon
binding OA they acquired a folded structure
character-ized by a significant content of a-helical structure, as
the OA complexes displayed far-UV CD spectra with
the typical minima of ellipticity at 208 and 222 nm of
the a-helical secondary structure [38]
The fragment species 1–40⁄ 53–123 comprises almost
all of the 123 residue chain of a-LA (Fig 1), and
adopted a significantly folded structure in solution, as
shown by the characteristics of its far-UV CD
spec-trum (Fig 2C) In this case, the addition of OA
induced a conformational change, but not as dramatic
as seen with the other two fragments Here, we used
fragment solutions devoid of Ca2+, because we have
previously shown that the conformational features of
fragments 53–103 and 1–40⁄ 53–123, containing the
Ca2+-binding loop of the intact protein (Fig 1), are
affected by Ca2+[31,32]
Determination of the aggregation state of OA
The phase behavior of OA is strongly dependent on
pH and fatty acid concentration [39] In NaCl⁄ Pi(pH
7.4), OA forms oil droplets and vesicles of variable size [40–42] To understand the effect of protein fragments
on OA aggregation state, we measured the OA CAC
We use this term because a complete morphological characterization of the aggregate state of OA is not yet available The CAC of OA at pH 7.4 was estimated by using the fluorescent dye 6-(p-toluidino)-2-napthalene-sulfonate (TNS) [43] In NaCl⁄ Pi(pH 7.4), the CAC of
OA was calculated as 19.8 ± 0.3 lm (Fig 3A, insert) This value is similar to that previously determined for
OA [40] The same measurements using TNS were con-ducted in the presence of a-LA (Fig 3A) or its frag-ments (Fig 3B) All protein species were able to reduce by 20-fold the CAC value of OA Estimated values of the CAC of OA were 0.94 ± 0.24 lm in the presence of a-LA, and 0.86 ± 0.29, 1.22 ± 0.24 and 0.93 ± 0.56 lm, respectively, in the presence of frag-ment species 1–40⁄ 53–123, 1–40 ⁄ 104–123 and 53–103
We also conducted turbidity analysis of OA solu-tions and mixtures, as this method is often used for measuring the critical vesicular concentration of lipids [44] Figure 3C shows the variation of absorbance (A)
at 400 nm of samples containing increasing amounts
of OA in the absence or presence of a-LA The strik-ing observation derivstrik-ing from these measurements is that the added protein was able to completely inhibit the formation of large aggregates that caused light scattering at 400 nm (Fig 3C, open circles) Fragment species 1–40⁄ 53–123 and 1–40 ⁄ 104–123 were also able
to similarly depress the OA aggregation Fragment 53–103 also caused a reduction in the aggregation of
OA, but to a minor extent (Fig 3D)
–15 –10 –5 0 5
2·dmol
–15
–10
–5
0
5
1 : 1
1 : 3
1 : 5
1 : 7
1 : 10
1 : 15
by column
1 −40/104−123
–15 –10 –5 0 5
(53 −103)/OA (by mix 1 : 15) (53 −103)/OA (by column)
53−103
B
(1 −40/53−123)/OA (by column)
1 −40/53−123
Wavelength (nm)
(1−40/53−123)/OA (by mix 1 : 15)
Fig 2 Far-UV CD spectra of a-LA fragments in NaCl ⁄ P i (pH 7.4) (A) Far-UV CD spectra of fragment 1–40 ⁄ 104–123 in the absence (dotted line) or presence (continuous line) of increasing amounts of OA Numbers near the CD spectra refer to fragment ⁄ OA molar ratios of 1 : 1,
1 : 3, 1 : 5, 1 : 7, 1 : 10, and 1 : 15 The CD spectrum of the OA complex of the fragment obtained by chromatography is also shown (dashed line) (B) CD spectra of the OA complex of fragment 53–103 obtained by chromatography (dashed line) or by mixing in solution (continuous line) The spectrum of the OA free fragment is reported as a reference (dotted line) (C) CD spectra of fragment 1–40 ⁄ 53–123 (dotted line) and its OA complex obtained by chromatography (dashed line) and by mixing the fragment and OA in solution at a fragment ⁄
OA molar ratio of 1 : 15 (continuous line).
Trang 5Cellular toxicity
The ability of the OA complexes of the a-LA
frag-ments to induce apoptosis-like cell death was
exam-ined Assays were conducted on Jurkat cells, using the
OA complexes prepared by direct mixing or by
chro-matography using an OA-conditioned column (see
Experimental procedures) Cells treated with the
fragment–OA complexes suffered considerable loss of
viability through apoptosis-like death, whereas the
OA-free fragments displayed negligible toxicity
(Fig 4) The OA complexes of the various fragment
species were also tested at a fragment⁄ OA molar ratio
of 1 : 3, a condition that caused only a slight
confor-mational change in the fragment’s secondary structure,
as deduced from far-UV CD spectra In this case, the
OA complexes did not display cellular toxicity (not
shown) The extent of apoptotic activity of the OA
complexes of the fragments was comparable to that
observed with the OA complex of the intact protein
prepared by chromatography, i.e BAMLET, or by
mixing the intact protein with 15 equivalents of OA in
solution In the absence of added protein species, OA
alone and at the concentration used for formation of
OA complexes displayed negligible toxicity, similarly
to NaCl⁄ Pi or the control sample (culture medium)
Conversely, as previously shown, OA can be toxic to Jurkat cells via an apoptosis mechanism at higher con-centrations and with a much longer duration of incu-bation [33]
Fragment 1–40⁄ 104–123 is a two-chain species cross-linked by the two disulfide bridges 6–120 and 28–123
of intact a-LA Reduction of this fragment with tris(2-carboxyethyl)phosphine, followed by S-alkylation with iodoacetamide and RP-HPLC chromatography, allowed us to prepare the single-chain, S-carboxami-domethylated fragments 1–40 and 104–123 The inter-action of OA with these fragments was monitored by far-UV CD measurements As shown in Fig S1, OA induced a-helical secondary structure in both frag-ments, which were otherwise largely unfolded in the absence of the fatty acid It is of note that fragment 1–40 encompasses helix H1 (5–11) and helix H2 (23–34), and fragment 104–123 encompasses helix H4 (105–110), in native a-LA [4] The OA complexes of the two fragments, as obtained by mixing them with
15 equivalents of the fatty acid, displayed significant apoptotic activity on Jurkat cells (Fig S1, bottom) It
is of note that the OA–fragment 104–123 complex was even more active than BAMLET, i.e the OA–a-LA complex prepared by column chromatography (see Experimental procedures) Therefore, OA complexes of
Relative fluorescence1.0 1.2 1.4 1.6 1.8
[OA] μ M
0 20 40 60 80 100
Rel fluorescence0.8
1.0 1.2 1.4 1.6 1.8
[OA] (μM)
D
A400 nm
0.0 0.1 0.2
0.3
C
Fig 3 Characterization of the physical state
of OA solutions by TNS fluorescence
emis-sion (A, B) and turbidity (C, D) All
measure-ments were conducted in NaCl ⁄ P i (pH 7.4),
in the absence (d) or presence of a-LA (s),
fragment 1–40 ⁄ 53–123 ( ), fragment 1–
40 ⁄ 104–123 (n), or fragment 53–103 ()).
(A, B) Aliquots of OA (from 0 to 10 l M )
were added to a solution of TNS (20 l M ),
and the intensity of fluorescence emission
at 460 nm was recorded, after excitation at
360 nm The CAC is defined as the lipid
concentration at which the two linear
por-tions of the lines of fluorescence intensities
intersect [61] The TNS fluorescence of OA
(up to 100 l M ) solutions was also measured
in the absence of intact a-LA (A, insert) (C,
D) Turbidimetric analysis of OA solutions in
the absence (filled circles) or presence of
10 l M protein (open circles) or fragment
species (symbols as above) Measurements
of absorbance were conducted at 400 nm
on samples containing OA up to 500 l M
Trang 6peptide fragments even shorter than the three
frag-ments shown in Fig 1 can display cellular toxicity
Discussion
The data presented here indicate a strong mutual
inter-action between OA and a-LA fragments Indeed, the
addition of OA induces enhancement of secondary
structure of the fragment species, and these
signifi-cantly modify the physical state of the fatty acid in
solution The increase in the a-helical structure in the
fragment species upon addition of OA (Fig 2) derives
from the fact that fatty acids, lipids and detergents can
provide a hydrophobic environment that is able to
induce and stabilize secondary structure of
polypep-tides [45–47] The interaction of protein species with
OA is also simply shown by the fact that a turbid OA
suspension in water at neutral pH becomes clear after
the addition of protein⁄ fragment species
The OA complexes of the a-LA fragments have been
prepared by direct mixing in solution and by the
chro-matographic method of Svensson et al [18], utilizing
an OA-conditioned anion exchange column, followed
by extensive dialysis of the OA complex eluted from
the column at high salt concentration, and then
lyoph-ilization As the conformational and biological
proper-ties of the OA complexes prepared here by the two
methods are very similar, we consider the mixing
procedure in solution to be suitable, being easier,
reproducible and less cumbersome than the
chromato-graphic one Furthermore, we have previously shown that the mixing procedure can be effectively utilized for the preparation of an a-LA–OA complex with comparable structural features to BAMLET [35], and other reports have more recently documented its successful use [26,36,37]
The phase behavior of OA is critically dependent on the ionization degree of the fatty acid, and is thus affected by the pH and ionic strength of the aqueous solution [40–42] In NaCl⁄ Pi (pH 7.4), OA forms aggregates of different sizes (diameter 25–250 nm), as deduced by transmission electron microscopy (not shown) The a-LA fragment species, as well as the intact protein, strongly affect these structures Both turbidimetric and transmission electron microscopy analyses show the disappearance of the large, aggre-gated structures, and by means of fluorescence emis-sion measurements, a 20-fold decrease in CAC was found, indicating the formation of smaller aggregates
at a lower OA concentration in the presence of protein species As the formation of OA micelles requires the complete ionization of the fatty acid molecules, and micelles are formed at pH > 9 [39], it is likely that, under the experimental conditions described here, small vesicles or oil droplets are induced in OA in the presence of the protein or its fragments
A depression of the CAC of anionic detergents simi-lar to that observed here with OA aggregates was reported to occur also with other proteins, and this effect was explained by considering both electrostatic
1−
123
104
−123
n
3−
m
3−
n
)/O
x
1−
/OA by
n BAML
E
NaCl/P
i
CT 0
20
40
60
80
100
Late apoptosis Early apoptosis
Fig 4 Cytotoxicity of OA complexes Jur-kat cells (10 6 cells per mL) were incubated
at 37 C with the OA complexes of a-LA or its fragments prepared by column chroma-tography or direct mixing in solution (see Experimental procedures) All protein ⁄ frag-ment samples were tested at 7 l M As a control, OA was tested at 100 l M After incubation for 6 h, cell death by apoptosis was evaluated by appropriate changes of nuclei stained with Hoechst-33258 (early apoptotic cells, gray bars) and propidium iodide (late apoptotic ⁄ necrotic cells, open bars) The test was also conducted on a-LA,
OA, NaCl ⁄ P i , and the medium (CT) as a con-trol Data are shown as percentage of BAM-LET activity Values are means ± standard deviation of at least three experiments.
Trang 7and hydrophobic interactions [48–50] A reduction of
the electrostatic repulsion between negative charges at
the surface of the detergent aggregates and positively
charged amino acid side chains of a protein allows the
formation of aggregates at a lower concentration
However, considering that the apo form of a-LA is
negatively charged, it may well be that the interaction
with OA aggregates of this protein in its Ca2+-free
form is mostly mediated by hydrophobic interactions,
as the apo-a-LA is more hydrophobic than the holo
form [51,52] Nonetheless, even the negatively charged
protein molecule may possess positively charged
clus-ters or areas that mediate the interaction with the
neg-atively charged head groups of OA aggregates, as, for
example, indicated by the fact that the negatively
charged a-synuclein contributes to CAC depression of
anionic surfactants [48] In the 123 residue chain of
a-LA at neutral pH, Lys and Arg residues are
clus-tered at the level of helical segments A, C and D of
the native protein, whereas the central region of the
protein contains many negatively charged carboxylates
of Asp and Glu residues Therefore, it could be that
fragment 53–103 interacts with the negatively charged
OA aggregates less effectively than the other fragments
investigated here, as shown by the results of
turbidi-metric analyses (Fig 3D)
The mechanisms of biological activity of the OA
complexes of a-LA are not yet understood and, in fact,
a variety of diverse biological effects have been
described for HAMLET [20,25,27] For this reason,
HAMLET was metaphorically named ‘Hydra’ [25]
Besides the cytotoxicity via an apoptotic mechanism,
an OA complex of human a-LA was shown to also
possess bactericidal activity against Streptococcus
pneu-moniaeand Haemophilus influenzae [53] It is of interest
that digestion of a-LA with trypsin and chymotrypsin
yields three peptides displaying bactericidal activity
against Gram-positive bacteria These bactericidal
spe-cies are peptide 1–5 and the two-chain peptides linked
by a disulfide bridge, 17–31⁄ 109–114 and 61–68 ⁄ 75–80
[54] However, the structural features responsible for
their bactericidal action were not clarified Probably,
bactericidal action of the LA–OA complex requires a
different molecular mechanism than that occurring in
apoptosis Hence, HAMLET-like complexes can be
detrimental by various cellular pathways, and exert
their actions by different molecular mechanisms
The proteolytic fragments of a-LA investigated here
have widely differing chain lengths and amino acid
sequences (Fig 1), and it therefore does not seem
possible to explain their cytotoxicity in terms of their
specific structural features The variability in
struc-ture of the polypeptide chain in forming active OA
complexes seems to indicate instead that a generic poly-peptide chain can eventually interact with OA, and thus that the toxic action of an OA complex resides in the fatty acid rather than in the protein moiety The pres-ent results show that OA displays new physicochemical and aggregation properties in the presence of a-LA or its fragments With a decrease in the CAC of the fatty acid in the presence of the protein or its fragments, soluble and smaller aggregates of protein–OA or frag-ment–OA complexes are easily formed and stabilized
In previous studies, the tumor-selective cytotoxicity
of HAMLET or BAMLET was correlated with the conformational properties of a-LA upon formation of the OA complex [17,18] In particular, it was proposed that the fatty acid acts as a stabilizer of a partially folded or MG conformation of the protein under phys-iological conditions [21] In a very recent paper, it was reported that a recombinant mutant a-LA with all eight Cys residues replaced by Ala residues (named all-Ala mutant), and thus devoid of the four disulfide bridges of the native protein, formed a cytotoxic OA complex equivalent to HAMLET [55] Even if the con-formation of the all-Ala mutant at neutral pH is simi-lar to the MG of a-LA at low pH [6–9], the addition
of OA to the all-Ala protein is required in order to form a cytotoxic species, indicating that the fatty acid
is needed for the development of cytotoxicity [55] Here, we show that a variety of a-LA fragments can mimic the action of the entire 123 residue chain of the protein in forming OA complexes displaying cytotoxic-ity A reasonable deduction from this and previous studies is that the protein⁄ peptide moiety can act as a carrier of the inherently toxic fatty acid [33], and there-fore that OA itself is the active species of a cytotoxic protein⁄ peptide complex This view is in line with the fact that all variants of a-LA of human, bovine, equine, porcine and caprine origin, as well as recombi-nant mutants of a-LA devoid of Ca2+-binding proper-ties, were all able to form HAMLET-like complexes with little difference in biological activity [21,24] Inter-estingly, it was recently reported that the OA complex
of lysozyme displays cellular toxicity similar to that of HAMLET [56] In our laboratory, we have performed initial experiments indicating that even the 153 residue chain of apomyoglobin can form cytotoxic complexes when combined with OA [57]
In summary, the results of this study indicate that, besides substantial variation in amino acid sequence of the polypeptide chain of a-LA, severe truncation of the polypeptide chain of the protein is also tolerated in the formation of biologically active OA complexes Therefore, we are inclined to conclude that the poly-peptide moiety can serve mainly as a carrier of the
Trang 8fatty acid We have shown here that the addition of a
protein⁄ fragment species strongly influences the
aggre-gation behavior of OA, in particular making it more
water-soluble and thus enhancing its intrinsic apoptotic
effects [33] Nevertheless, we cannot exclude the
possi-bility that the protein itself can act in a synergic way
in the observed cytotoxicity of the OA complexes This
could be particularly true in the case of OA complexes
of a-LA, considering that: (a) a-LA itself can display
an inherent apoptotic activity [58,59]; (b) a-LA alone
can interact with histones at the cellular level and thus
display cytotoxic effects [29,30]; and (c) even various
fragments of a-LA have been shown to have
bacterici-dal activity, and a-LA fragments can therefore be toxic
[54] Despite these caveats regarding the specific role of
the protein moiety in HAMLET-like or BAMLET-like
species, it should be emphasized that the beneficial
effects of these OA complexes in selective killing a
variety of tumor cells appear to be remarkable and will
prompt additional studies on OA–protein⁄ peptide
complexes as possible new anticancer agents
Experimental procedures
Materials
Bovine a-LA and DEAE-Trisacryl M resin were purchased
from Sigma (St Louis, MO, USA); OA and the fluorescent
dye TNS were from Fluka (Buchs, Switzerland) All other
chemicals were of analytical reagent grade and were Sigma
or Fluka products
Preparation of the OA complexes of a-LA
fragments
The a-LA fragments investigated, 1–40⁄ 53–123, 1–40 ⁄ 104–
123 and 53–103 (Fig 1A), were produced by limited
prote-olysis of the protein with pepsin at pH 2.0 [9,31] The OA
complexes of a-LA and its fragments were prepared
follow-ing two procedures, column chromatography and mixfollow-ing in
solution
Column chromatography
The protein material was loaded onto an OA-conditioned
anion exchange chromatographic column (1.0· 7.0 cm),
following the procedure reported by Svensson et al [18] A
DEAE-Trisacryl M resin was employed, equilibrated with
10 mm Tris⁄ HCl and 0.1 m NaCl (pH 8.5) An aliquot of
the protein or fragment material ( 2 mgÆmL)1) was
dis-solved in 10 mm Tris⁄ HCl (pH 8.5), containing 1 mm
EDTA, and then loaded onto the anion exchange column,
which was eluted with a gradient of 10 mm Tris⁄ HCl and
1 m NaCl (pH 8.5) The absorbance of the effluent from the column was monitored at 214 nm The high-salt eluates from the column containing the OA complexes were
desalt-ed by dialysis against water, using a membrane of 3.5 kDa cut-off, and then lyophilized
Mixing in solution The OA complexes were prepared by direct mixing of pro-tein species with 10 or 15 equivalents of OA dissolved (20 mgÆmL)1), in ethanol and then diluted with NaCl⁄ Pi
(8 mm Na2HPO4, 137 mm NaCl, 2 mm KH2PO4, 2.7 mm KCl, pH 7.4) [35] The fatty acid was added to the protein solution, and the mixture was analyzed after 1 h of incuba-tion in the dark
CD spectroscopy
CD spectra were recorded on a Jasco J-710 spectropolarim-eter (Tokyo, Japan) The spectra were recorded in NaCl⁄ Pi
(pH 7.4), in the absence or presence of OA, at a pro-tein⁄ fragment concentration of 0.05–0.1 mgÆmL)1, using
1 mm quartz cells The interaction of OA with protein spe-cies was followed by far-UV CD measurements by adding aliquots of an OA solution to the protein fragment samples (10 lm) in NaCl⁄ Pi(pH 7.4) Mean residue ellipticity [h] is reported as degÆcm2Ædmol)1 Protein fragment concentra-tions were determined by absorption measurements at
280 nm on a double-beam Lambda-25 spectrophotometer (Perkin-Elmer, Norwalk, CT, USA) The molar extinction coefficients at 280 nm for a-LA fragments were 1.22 mg)1Æcm)1 for fragment 53–103, 2.89 mg)1Æcm)1 for fragment 1–40⁄ 104–123, and 2.23 mg)1Æcm)1 for fragment 1–40⁄ 53–123, as calculated according to Gill and von Hippel [60]
Determination of the aggregation state of OA The CAC of OA was determined by using the fluorescent dye TNS [43] The TNS (20 lm) fluorescence emission at
460 nm, after excitation at 360 nm, was measured at 25C
in the presence of increasing concentrations of OA in NaCl⁄ Pi (pH 7.4) The analyses were conducted in the absence or presence of a-LA or its fragments at 10 lm Three readings were taken, and the average fluorescence intensities relative to blanks were plotted The first and the last five data points were joined separately by statistically fitted straight lines The CAC is defined as the lipid concen-tration at which the two linear portions of the fluorescence emission intensity lines intersect [61] The aggregation state
of OA, in the absence or presence of a-LA or its fragments (10 lm), was also analyzed by turbidity measurements at
400 nm of different samples containing increasing amounts
of OA (from 0 to 500 lm) in NaCl⁄ Pi(pH 7.4) [44]
Trang 9Apoptosis assays
Cell culture T-lymphoblastoid Jurkat cells were cultured in
RPMI-1640 medium supplemented with 10%
heat-inacti-vated fetal bovine serum, 2 mm glutamine, 100 IUÆmL)1
penicillin and 100 lgÆmL)1 streptomycin in 5% CO2⁄ 95%
air at 37C The Jurkat cells (106
cells per mL) were incu-bated with the protein⁄ fragment samples in serum-free
medium for 6 h at 37C These samples were tested at
7 lm, and the OA complexes were prepared by mixing 10
molar equivalents of OA for fragment 1–40⁄ 104–123 and
15 equivalents for fragments 1–40⁄ 53–123 and 53–103, as
well as intact a-LA In order to assess cell viability, Jurkat
cells were stained with 10 lm Hoechst-33258 and 1 lm
pro-pidium iodide for 5 min, in order to allow visualization of
early and late apoptotic⁄ necrotic cells, respectively Cells
were then washed with Hanks’ balanced salt solution, and
visualized with an Olympus IMT-2 inverted microscope
equipped with a xenon lamp and a 12-bit digital, cooled,
charge-coupled device camera (Princeton Instruments,
Monmouth Junction, NJ, USA) Excitation⁄ emission cubes
of 340⁄ 440 ± 25 nm and a 568 ⁄ 585 ± 25 nm long-pass
fil-ter were used for Hoechst-33258 and propidium iodide,
respectively Three randomly selected fields were acquired
for each treatment The corresponding bright field images
were also acquired, and the three channels were overlaid
using the appropriate function of metamorph software
(Universal Imaging, West Chester, PA, USA) The
percent-age of cell death and the standard deviation were calculated
from three acquisitions of each treatment The data are
reported as percentage of BAMLET activity
Acknowledgements
We gratefully acknowledge the financial support of the
Italian Ministry of University and Research (MIUR)
through PRIN-2004, PRIN-2006 and the FIRB Project
on Protein Misfolding and Aggregation (Project No
RBNEOPX83) We thank M Zambonin for his
excel-lent technical assistance This work was presented at
the Symposium on HAMLET (12–14 May 2009, Lund,
Sweden) and at the XXI Symposium of the Protein
Society (21–25 July, 2007, Boston, MA, USA) [Protein
Sci 16 (Suppl 1), Commun 257]
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