Proteolysis of bovine b-lactoglobulin during thermal treatmentin subdenaturing conditions highlights some structural features of the temperature-modified protein and yields fragments wit
Trang 1Proteolysis of bovine b-lactoglobulin during thermal treatment
in subdenaturing conditions highlights some structural features
of the temperature-modified protein and yields fragments
with low immunoreactivity
Stefania Iametti1, Patrizia Rasmussen1,2, Hanne Frøkiær2, Pasquale Ferranti3, Francesco Addeo3
and Francesco Bonomi1
1 Dipartimento di Scienze Molecolari Agroalimentari, University of Milan, Italy; 2 Biocentrum, Technical University of Denmark, Lyngby, Denmark;3Istituto di Scienze dell’Alimentazione, CNR, Avellino, Italy
Bovine b-lactoglobulin was hydrolyzed with trypsin or
chymotrypsin in the course of heat treatment at 55, 60 and
65°C at neutral pH At these temperatures b-lactoglobulin
undergoes significant but reversible structural changes In
the conditions used in the present study, b-lactoglobulin was
virtually insensitive to proteolysis by either enzyme at room
temperature, but underwent extensive proteolysis when
either protease was present during the heat treatment
High-temperature proteolysis occurs in a progressive manner
Mass spectrometry analysis of some large-sized breakdown
intermediates formed in the early steps of hydrolysis
indicated that both enzymes effectively hydrolyzed some
regions of b-lactoglobulin that were transiently exposed during the physical treatments and that were not accessible
in the native protein The immunochemical properties of the products of b-lactoglobulin hydrolysis were assessed by using various b-lactoglobulin-specific antibodies, and most epitopic sites were no longer present after attack of the partially unfolded protein by the two proteases
Keywords: bovine b-lactoglobulin; limited proteolysis; partial unfolding; thermal treatment; reduced immunoreac-tivity
The globular protein b-lactoglobulin is found in the whey
fraction of the milk of many mammals, but is absent from
human milk In spite of numerous physical and biochemical
studies, its function is still not clearly understood [1,2] The
crystal structure of bovine b-lactoglobulin (BLG) shows a
marked similarity with the plasma retinol binding protein
and the odorant binding protein, that all belong to the
lipocalin superfamily [2–5]
Denaturation of BLG by physical means is a complex
phenomenon, that occurs through a series of intermediate
steps, whose kinetics and equilibrium depend on the
treatment conditions, on the protein concentration, and
on the interaction with other components when complex
systems such as milk and whey are considered Most of the
steps occurring below a given intensity threshold of physical
treatment (temperatures below 60–65°C, or pressures
below 600 MPa [6,7]) are fully reversible in solutions of the pure protein at neutral pH Transient BLG conformers are formed by either physical treatment in the same conditions, and the properties of these conformers have been investigated in some detail [7–10]
Limited proteolysis represents a common and powerful tool for the investigation of protein structure, including transient conformational states of proteins generated during folding or unfolding (reviewed in [11]) This approach has not been popular for use with BLG in view of its structural toughness, which makes native BLG quite insensitive to most proteases under nondenaturing conditions [12–16], in particular at pH values lower than 7.5, where the well-known Tanford transition of the protein structure occurs Most proteolytic studies on unfolded BLG only addressed the products of severe thermal treatment, i.e above the temperature threshold for irreversible structural modifica-tion of the protein [17,18]
Proteolysis has been used to lower or to eliminate the antigenicity of milk proteins, including BLG Indeed, BLG
is among the major causes of intolerance and/or allergenic response to cow’s milk in humans, that represent a major challenge to paediatricians, to nutritionists, and to food technologists High-temperature heat denaturation is most commonly used in the processes for producing extensively hydrolyzed formulae starting from whey proteins, because denaturation by itself leads to the removal of conforma-tional epitopes [19], and because the thermal precipitation of heat-denatured BLG allows to minimize the amount of residual intact protein in the preparation Similar processes rely on extensive hydrolysis of the partially modified form of
Correspondence to Francesco Bonomi, Dipartimento di Scienze
Molecolari Agroalimentari, Via G Celoria 2 20133 Milano, Italy.
Fax: + 39 02 58356801, Tel.: + 39 02 58356819,
E-mail: francesco.bonomi@unimi.it
Abbreviations: BLG, b-lactoglobulin; BAPA, benzoyl- L
-arginine-p-nitroanilide; SUNA, N-succinyl-Ala-Ala-Pro-Phe arginine-p-nitroanilide;
SE-HPLC, size-exclusion high performance liquid chromatography;
CP1 and CP2 (or TP1 and TP2), the large-sized proteolytic fragments
isolated after chymotryptic (or tryptic) digestion of BLG at
temperatures above 50 °C.
Enzymes: trypsin (EC 3.4.21.4); chymotrypsin (EC 3.4.21.1).
(Received 28 August 2001, revised 7 December 2001, accepted
8 January 2002)
Trang 2BLG produced at pH > 8.0 [13,20] However, several
studies have reported a residual antigenic activity in
hydrolysed milk formulae [21–26] Residual allergenicity in
these preparations could stem from the inability of the
hydrolytic reaction to address all the sequential epitopes
even in the denatured protein In fact, despite the apparent
simplicity of the approaches described above, heat
denatur-ation and aggregdenatur-ation of BLG upon heat treatment may
hide putative sites of attack from the action of proteases,
therefore leaving intact some regions of the protein that may
be relevant to its allergenic properties
The BLG conformers transiently formed during a
physical treatment of subdenaturing intensity may represent
ideal substrates for the action of proteases, as ample regions
of the hydrophobic protein core are unfolded, contrarily to
what happens in the very compact native protein or in the
aggregated products of extensive thermal denaturation of
BLG [6,8], thus making even the most inner parts of the
protein accessible, at least in principle, to enzymatic
hydrolysis In more advanced steps of physical
denatura-tion, collapse of the hydrophobic portion of the structure
may occur [6], possibly making the same enzyme attack sites
once again as they were inaccessible in the native folded
protein
In previous studies on limited proteolysis of partially
unfolded BLG, we used high-pressure as the physical
denaturant, as the intensity threshold of pressure treatment
appears less critical than temperature with respect to the
aggregation behavior of BLG and of the sensitivity of the
aggregation process to protein concentration [10,27] In
those studies, several enzymes were tested Trypsin and
chymotrypsin gave the best results, both in terms of
interpretation of the hydrolysis pattern and of reduced
immunoreactivity [28] Trypsin and chymotrypsin were used
in the present study, also in view of a possible comparison
with the results obtained under pressure In this work we
used short time periods (10 min) for the combined
proteo-lytic/thermal treatment of BLG at relatively high enzyme/
BLG ratios (1 : 10 and 1 : 20) and at the highest
tempera-ture compatible with retention of enzyme activity and with
the reversibility of structural modifications of BLG Limited
proteolysis studies on BLG are significant not only to
understanding its unfolding mechanism, but may have
practical relevance as for decreasing the immunoreactivity
of the protein Therefore, we also tested some of the
hydrolysis products obtained in this study for their
immu-noreactivity towards different sets of various BLG-specific
antibodies
E X P E R I M E N T A L P R O C E D U R E S
Proteins
BLG was from Sigma Each protein batch was tested as
received for its content in multimeric forms or in
disulfide-linked dimers, by using HPLC gel-permeation and SDS/
PAGE under nonreducing conditions Bovine pancreatic
trypsin (N-a-tosyl-L-phenylalanylchloromethane treated,
type XIII) and chymotrypsin (N-a-tosyl-L
-lysylchloro-methane treated, type VII) and the synthetic substrates
benzoyl-L-arginine-p-nitroanilide (BAPA, trypsin) or
N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SUNA,
chymo-trypsin), also were from Sigma
BLG-specific monoclonal antibodies were prepared according to [29]
Proteolytic experiments during thermal treatment Thermal treatments at fixed temperature were carried out
as reported in [6] with minor modifications Proper aliquots
of concentrated solutions of the appropriate proteolytic enzyme were added at 0°C to 1 mL of BLG (2.5 mgÆmL)1
in 50 mMphosphate buffer, pH 6.8) to a final mass ratio enzyme/BLG of 1 : 20 or of 1 : 10 The protein/protease mixture was then placed in a thermostatted water bath for the required amount of time At the end of the heat treatment the mixtures were placed in an ice/water bath, and the enzymatic activity was stopped by lowering the pH
of the reaction mixture to 3 by addition of 0.2 mL of 50% (v/v) acetic acid in water All these manipulations were carried out within 1–2 min from the end of the thermal treatment
Analytical measurements Enzyme activities were determined at 37°C in 0.1MTris/ HCl, pH 8.1, by following the increase in absorbance at
405 nm due to p-nitroanilide released from BAPA or SUNA, as appropriate Although the supplier gives nominal specific activities on synthetic substrates of 10.000 lmolÆmin)1Æmg)1(trypsin, on benzoyl-arginine ethyl ester), and 50 lmolÆmin)1Æmg)1(chymotrypsin, on benzoyl-tyro-sine ethyl ester), we measured specific activities in the range
of 2 lmolÆmin)1Æmg)1 (trypsin, 0.5 mM BAPA) and
50 lmolÆmin)1Æmg)1 (chymotrypsin, 0.2 mM SUNA) Residual enzyme activity was measured after heat treatment
of each enzyme in the same conditions and concentrations used for proteolysis experiments, in the presence or in the absence of BLG
RP-HPLC analysis of the proteolyzed samples was performed directly on aliquots of the acidified material after 10-fold dilution with 0.1% trifluoroacetic acid and centrifugation for 5 min at 10 000 g to remove minor amounts of materials made insoluble by the addition of trifluoroacetic acid A Deltapak C18 column (3.9· 150 mm, Waters), fitted to a Waters 625 HPLC equipped with a Waters 490E dual wavelength detector was used Elution of the hydrolytic products and of the residual intact protein was performed with a linear gradient from 20
to 60% acetonitrile (in 0.1% trifluoroacetic acid) in 30 min Flow was 0.8 mLÆmin)1, detection was at 220 nm Residual intact BLG was quantitated by on-line integration, using native BLG as a standard
Size-exclusion HPLC separations of the proteolyzed samples was performed directly on aliquots of the acidified material after centrifugation for 5 min at 10 000 g to remove insoluble materials A Superdex Peptide 10/30 column (Pharmacia) was used, fitted to a Waters 625 HPLC equipped with a Waters 490E dual wavelength detector The eluant was 20% acetonitrile in water containing 0.1% trifluoroacetic acid, at 0.5 mLÆmin)1 Detection was at 220 and 280 nm
Electrospray mass spectrometry (ES/MS) analysis was performed using a Platform single-quadrupole mass spec-trometer (Micromass), after liophylization of the original materials Peptide samples (10 lL, 50 pmol protein in
Trang 3water) were injected into the ion source at a flow rate of
10 lLÆmin)1; the spectra were scanned from 1400 to 600 at
10 s per scan Mass scale calibration was carried out using
the multiple-charged ions of a separate introduction of
myoglobin Mass values are reported as average masses
Quantitative analysis of individual components was
performed by integrating the signals from the multiple
charged ions of the single species [30] The peptide identity
was determined by analysis of the spectral data using a
computer software developed by the instrument
manufac-tureer (Biolynx, Micromass), and confirmed by MS analysis
of the samples prior and after a reduction step in 10 mM
dithiothreitol for 2 h at 37°C
Immunochemistry
ELISA tests were performed as competitive capture
ELISA by using BLG specific monoclonal or polyclonal
antibodies as capture antibodies All steps were carried
out at 30°C Polyclonal rabbit anti-BLG Ig was diluted
4000–10000 times in carbonate buffer (15 mM Na2CO3;
35 mMNaHCO3, pH 9.6) and coated directly to the wells
of microtiter plates, at 0.1 mL per well When using
monoclonal antibodies, polyclonal antimouse antibodies
(Z109, 1 : 1000, 0.1 mL per well, DAKO) were used for
coating followed by incubation of 0.1 mL per well of
monoclonal antibody diluted to approximately
250 ngÆmL)1 in KCl/NaCl/Pi buffer containing 0.1%
Triton X-100 (KCl/NaCl/Pi/Triton; 1.5 mM KH2PO4;
6.5 mM Na2HPO4; 0.5M NaCl; 2,7 mM KCl; 1 mLÆL)1
Triton X-100) Plates were washed four times with KCl/
NaCl/Pi/Triton buffer diluted 1 : 10, and the various
samples in KCl/NaCl/Pi/Triton were applied to the wells
in serial twofold dilutions and incubated for 1 h After
four washes, the plates were incubated with a fixed
concentration of biotinylated BLG (10–200 ngÆmL)1,
depending on the antibody) After incubation and another
four washes, plates were incubated with horseradish
peroxidase-labeled streptavidin (HRP-streptavidin,
DAKO, diluted 1 : 5000 in KCl/NaCl/Pi/Triton), and
washed four times Bound HRP activity was measured by
using a substrate-containing buffer (0.2M potassium
citrate pH 5.0; 3 mM H2O2; 0.6 mM
3,3¢,5,5¢-tetram-ethylbenzidine) The reaction was terminated by addition
of 2M H3PO4, 0.1 mL per well The absorbance at
450 nm was determined on a microtiter plate reader
R E S U L T S
Thermal stability of enzymes Trypsin and chymotrypsin were chosen for this study for the following reasons: (a) neither enzyme is capable of attacking BLG significantly at room temperature [14,15,18]; (b) both enzymes are available at very high purity; (c) both enzymes are highly specific; and (d) they act on complementary sets
of amino acids (hydrophobic, chymotrypsin; basic, trypsin) Other enzymes were tested, but their action was not further investigated in that they did not comply with all the requirements listed above, as reported in other studies [12–14,17] Furthermore, the results obtained with trypsin and chymotrypsin on transiently temperature-unfolded BLG could be compared with those we obtained on transiently pressure-unfolded BLG [28]
The only major drawback in the use of trypsin and chymotrypsin in the experiments reported here was their limited thermostability As shown in Table 1, both enzymes had very little residual activity after 5 min at 65°C, also in the presence of a 20-fold mass excess of the substrate protein Contrarily to what expected for a generic protective effect of added proteins, the residual activity after heat treatment in the presence of BLG was lower than in the absence of BLG
One explanation is that residual BLG (or BLG hydrolysis products) in the enzyme assays performed on the heated BLG/protease mixtures competed with the artificial sub-strates for binding to the enzymes In our conditions, the assay mixtures for residual protease activity contained from 0.125 to 0.25 mgÆmL)1BLG (or BLG hydrolysis products), equivalent to 0.075 and 0.15 mMBLG, respectively To test this possibility in a simple way, we performed assays in which native BLG was added to a protease assay mixture containing synthetic substrates at 37°C and pH 8.1 and the same amounts of enzyme present in the heated mixtures We found 25% and 35% inhibition of trypsin activity on 0.5 mMBAPA when native BLG was added at 0.075 mM and 0.15 mM, respectively Under similar conditions, inhi-bition figures for chymotrypsin (0.2 mM SUNA as sub-strate) were 8 and 15%, respectively These figures do not fully account for the differences shown in Table 1, that apparently are better explained by assuming an inhibitory effect of the peptides produced by hydrolysis of BLG at high temperature
Table 1 Thermal stability of trypsin and chymotrypsin Proteins (0.125 mgÆmL)1in 50 m M phosphate buffer, pH 6.8) were heated for 5 min at the given temperatures in the absence or in the presence of BLG (2.5 mgÆmL)1in 50 m M phosphate buffer, pH 6.8, corresponding to a 1 : 20 mass ratio enzyme/BLG) Residual enzyme activity after heat treatment was measured spectrophotometrically at 37 °C with 0.02–0.05 mL enzyme in 1 mL of the synthetic substrates BAPA (trypsin), or SUNA (chymotrypsin) Substrates (0.5 m M BAPA and 0.2 m M SUNA) were in 100 m M Tris/HCl,
pH 8.1 Activity is given as percentage of that of control enzymes kept at 37 °C in the absence of BLG.
Treatment temp.
(°C)
Residual activity (%)
Trang 4Extent of proteolysis as a function of temperature, time,
and enzyme concentration
BLG appeared to be virtually insensible to hydrolysis by
either trypsin or chymotrypsin in the absence of a thermal
unfolding step, as less than 10% of the native protein was
degraded by either enzyme in 20 min at 37°C at an enzyme/
BLG mass ratio of 1 : 10 Essentially the same results (i.e
less than 10% degradation of the total protein) were
obtained under the same conditions with BLG that was
previously heated for 10 min at 65°C This confirms earlier reports on the remarkable resistance of BLG to most proteases [13,17], and the reversibility of heat-induced structural modifications in these conditions
As shown in Fig 1, that reports the RP-HPLC profiles obtained after proteolysis at 60°C, hydrolysis of BLG became significant when either protease were allowed to act
on BLG during exposure at temperatures between 55 and
65°C As summarized in Table 2, the amount of residual BLG decreased with time and with the amount of added
Fig 1 RP-HPLC analysis of the products obtained upon enzymatic digestion of BLG at 60 °C Proper volumes of concentrated solutions of each given enzyme were added at 0 °C to separate aliquots of a BLG solution (1 mL, 2.5 mgÆmL)1in 50 m M phosphate buffer, pH 6.8) to a final mass ratio enzyme/BLG of 1 : 20 or of 1 : 10 Each BLG/protease mixture was then placed in a water bath thermostatted at 60 °C for the given amount
of time At the end of the heat treatment the mixtures were placed on ice, and the enzymatic activity was stopped by adding 0.2 mL of 50% (v/v) acetic acid in water RP-HPLC separations on the proteolytic samples were performed directly on aliquots of the acidified material after 10-fold dilution with 0.1% trifluoroacetic acid in 20% acetonitrile and centrifugation for 5 min at 1100 g to remove some trifluoroacetic acid- insoluble material A Deltapak C 18 column (3.9 · 150 mm, Waters), fitted to a Waters 625 HPLC equipped with a Waters 490E detector was used Elution was performed by applying a linear gradient from 20 to 60% acetonitrile (v/v) in 0.1% trifluoroacetic acid in 30 min Flow was 0.8 mLÆmin)1, detection was at 220 nm.
Table 2 Residual intact BLG after proteolysis under different conditions The amount of residual intact BLG after proteolysis in the given conditions was determined by integration of the intact BLG peaks from RP-HPLC separations similar to those reported in Fig 1, and is given as percentage of the signal produced by the native protein as a standard.
Enzyme
Mass ratio enzyme/BLG
Hydrolysis time (min)
Temperature (°C)
Trang 5enzyme As for the effects of temperature, inactivation of
proteases (Table 1) came into play at the highest
temper-atures and at the lowest ratio enzyme/BLG
Besides the decrease in intact protein, the tracings in
Fig 1 show that some hydrolysis products were formed
during the early phases of hydrolysis, and their
concentra-tion remained constant with time Other hydrolysis products
were only formed in significant amounts during the late
stages of proteolysis, suggesting a possible sequential
mechanism for hydrolysis
From the data in Table 2 it is evident that chymotrypsin
gave an extent of proteolysis not very different from that of
trypsin on transiently heat-unfolded BLG This is
some-what puzzling, as both enzymes were added in the same
weight ratio to BLG, but they had very different specific
activities (at least on synthetic substrates) Appropriate
control experiments did not provide evidence for a
transient increase in catalytic activity on synthetic
sub-strates at temperatures above 50°C for either enzyme (not
shown) Both enzymes rather underwent a marked
decrease in activity above 50°C, chymotrypsin being much
less thermostable than trypsin, as reported in the
equilib-rium data shown in Table 1 Thus, our observations (and
the specific activity data on different synthetic substrates,
as reported in Materials and methods) confirm that the
accessibility of cleavage sites on the substrate, rather than
the intrinsic catalytic ability of a given protease, is what
limits the effectiveness of the enzyme action on actual
protein substrates When proteases were added during the
thermal treatment, hydrolysis of BLG was extensive, in
spite of significant inactivation of the enzymes This
indicates that the transient conformers originating in the
course of thermal treatment had exposed novel access sites
for either enzyme
After thermal treatment in these conditions, there were no
major irreversible changes in all structural levels of BLG, at
least as detectable by spectroscopic and separation tech-niques [6,8,9,20,27] The increased accessibility of thermally treated BLG could indicate that some of the residues specifically recognized by each protease were exposed to the enzyme action in the treated protein even in the absence of spectroscopically detectable irreversible structural modifica-tions Thermal treatment was shown to promote transient modifications of the BLG structure at neutral pH, inducing transient dimer dissociation with concomitant exposure of previously buried hydrophobic sites [7–9,31] Physically induced reversible dimer dissociation at temperatures below
65°C [9] results in the exposure of hydrophobic residues along the ÔIÕ strand of the b fold, and of positively charged residues on the edge of the large a helix in each monomer [5] Evidence has been provided that heat treatment in this temperature range may affect a heat-labile domain of the protein [32], that was hypothesized to be relevant also for the stabilization of associated forms of BLG in solution [20]
In this context, it seems significant that chymotrypsin (specific for aromatic residues) gave the same hydrolysis levels obtained with trypsin, in spite of its lower thermal stability This could confirm that buried, compact hydro-phobic regions may be transiently unfolded and exposed by the thermal treatment
The RP-HPLC tracings in Fig 2 indicate that the major peaks in the hydrolysis products obtained with each protease (added in a 1 : 10 ratio to BLG) after 10 min at various temperatures from 37 to 60°C had similar elution times, suggesting that the same sites of attack were accessible to either protease in this temperature range Some differences among the RP-HPLC tracings were only evident when BLG was hydrolyzed at 65°C It is not clear whether the different proteolysis patterns obtained at
65°C with either enzyme related to the inaccessibility of some cleavage sites in the BLG conformer that is predom-inant at this temperature, or rather to the fact that the rapid
Fig 2 RP-HPLC analysis of the products obtained upon enzymatic digestion of BLG for
10 min at various temperatures Proper volumes of concentrated solutions of each given enzyme were added at 0 °C to separate aliquots of a BLG solution (1 mL,
2.5 mgÆmL)1in 50 m M phosphate buffer,
pH 6.8) to a final mass ratio enzyme/BLG of
1 : 10 Individual BLG/protease mixtures were placed in a water bath thermostatted at the given temperature for 10 min Further sample processing and RP-HPLC separation were performed as detailed in the legend to Fig 1.
Trang 6thermal inactivation of both enzymes at this temperature
prevented further degradation of some hydrolysis
interme-diates formed in the earliest steps of proteolysis The
peculiar nature of the hydrolysis products obtained at 65°C
with either enzyme will be discussed in the following section
Molecular characterization of the major hydrolysis
products and intermediates
The proteolysis products obtained in the conditions
repor-ted above were separarepor-ted on the basis of their molecular size
by SE-HPLC As shown in the different panels of Fig 3,
that presents data obtained during treatment at 60°C, the
SE-HPLC patterns obtained at different times show
progressive digestion of the intact protein, and significant
accumulation of hydrolytic fragments of appreciable size
(that is, between 3 and 10 kDa) The largest hydrolysis
fragments separated by SE-HPLC were named after the
enzyme used (T, trypsin; C, chymotrypsin) and after their
elution order from a Superdex Peptide column (hence, the P
in their names), and correspond to the peaks labeled CP1
and CP2 (or TP1 and TP2) in the chromatograms presented
in Fig 3
Analysis of the proteolyzed samples by SDS/PAGE (data
not shown) was consistent with the figures reported in
Table 2 The extensive proteolysis observed with
chymo-trypsin resulted in the formation of appreciable amounts of
proteolytic products capable of being retained by the gel,
according to the SE-HPLC data shown in Fig 3
Confirm-ing previous reports, temperature-induced formation of covalently linked BLG aggregates in this temperature range
as detected by SDS/PAGE was modest [6] No formation of covalently linked aggregates was observed in the enzyme-treated samples, indicating that proteolysis took place more rapidly than protein aggregation even at 65°C [27,28] The time-progressive change in the size distribution of hydrolytic products obtained after treatment with either enzyme at different temperatures is reported in Fig 4 Both the SE-HPLC tracings in Fig 3 (obtained with 0.1% trifluoroacetic acid in 20% acetonitrile as eluant) and the time courses in Fig 4 clearly indicate that a limited number
of large fragments constituted a set of intermediate hydro-lysis products, suggesting a progressive hydrohydro-lysis mechan-ism with either enzyme More specifically, it appears that the concentration of TP1 remained constant during progressive hydrolysis of heated BLG by trypsin, whereas the concen-tration of the intact protein decreased with an accompany-ing increase in TP2 The pattern of events observed with chymotrypsin is made somewhat more complicated by the more extensive thermal inactivation of this enzyme How-ever, also in this case, formation of the larger CP1 fragment occurred in the early phases of hydrolysis, and this intermediate was further degraded to the smaller CP2 intermediate (and to even smaller peptides) when enough enzyme activity was present (that is, at relatively low
Fig 4 Time course of the formation of fragments having different size during proteolysis of BLG at 55 and 65 °C Data are taken from integration of the chromatograms shown in Fig 3 Full symbols,
55 °C; open symbols, 65 °C Excluded peptides (M r app > 10 000), circles and full lines, TP1 and TP2 (or CP1 and CP2, as appropri-ate), triangles and dotted lines; low molecular weight material (M < 3000), squares and dashed lines.
Fig 3 SEC-HPLC analysis of the products obtained upon enzymatic
digestion of BLG at 60 °C Size-exclusion chromatography (SEC) was
carried out on the acetic acid-treated materials obtained as detailed in
the legend to Fig 1, with no further processing A Superdex Peptide
column (10/30, Pharmacia Biotech) was used on the same
chromato-graphic system described in the legend to Fig 1 Eluant was
20% acetonitrile in aqueous 0.1% trifluoroacetic acid Flow was
0.5 mLÆmin)1, detection was at 220 nm.
Trang 7temperature, short reaction times and high enzyme
concen-tration) Indeed, no formation of CP2 was detected during
chymotryptic hydrolysis of BLG at 65°C, and significant
accumulation of TP2 only occurred at the longest hydrolysis
times at this temperature (data not shown)
The figures given in Fig 4 for peptides with a
Mr app> 10 000 are significantly higher than the amounts
of residual native protein detected by RP-HPLC (Table 2)
This discrepancy could indicate that formation of
proteo-lytic intermediates having a larger size than TP1/2 and CP1/2
may occur to a significant extent
To test the hypothesis of progressive hydrolysis, and to
assess the nature of the regions being attacked by proteases
in the thermally unfolded protein, fragments CP 1 and 2, as
well as fragments TP 1 and 2, were further purified by
RP-HPLC and SE-HPLC in the same conditions and with
the same equipment used for analysis of the whole
proteolyzate, and these chromatographically homogeneous
peptides were analyzed by ES-MS
The results obtained by MS are reported in Table 3, and
make it clear that most of the material that contributes to
the microheterogeneity of the isolated fragments originate
from further proteolytic degradation of a limited number of
primary hydrolysis products
All these fragments share a common feature, namely the
presence of the disulfide bridge connecting Cys66 and
Cys160 in the native protein
The position of these fragments in the primary sequence
of BLG is given in Fig 5 A nonspecific hydrolysis by
trypsin is observed between the two leucine residues 57 and
58 As expected, chymotrypsin also cut in the same position,
indicating that this region of the molecule was accessible to
enzyme action However, in general terms, the fact that
these fragments were only attacked at their ends under our
conditions (Table 3), in spite of the relative abundance
of protease-sensitive residues in their sequence (Fig 5),
suggests that these fragments could have retained (or could have assumed) a rather compact conformation even at the temperatures used in this study Indeed, recent studies based
Table 3 Molecular parameters for large-sized fragments obtained from hydrolysis of BLG Fragments were obtained by size-exclusion HPLC of digests carried out as in the legend to Fig 3 after hydrolysis at 55 °C ES-MS analysis was performed using a Platform single-quadrupole mass spectrometer (VG-Biotech), after liophylization of the original materials Peptide samples (10 lL, 50 pmol protein in water) were injected into the ion source at a flow rate of 10 lLÆmin)1; the spectra were scanned from 1400 to 600 at 10 s per scan Mass scale calibration was carried out using the multiple-charged ions of a separate introduction of myoglobin Actual mass values are reported as average masses For each fragment, the highest size precursor is listed first, and the products of its further proteolysis are listed in order of relative abundance in each chromatographic fraction, derived by comparison of the respective mass signal intensity.
Mass (Da) Individual peptides Total fragment
Val43-Phe82(Cys66-Cys160)Lys150-Ile162 4596.7 + 1607.9 6204.6 Arg40-Phe82(Cys66-Cys160)Lys150-Ile162 5015.2 + 1607.9 6623.1 Val43-Phe82(Cys66-Cys160)His146-Ile162 4596.7 + 1911.7 6508.4
Glu62-Phe82(Cys66-Cys160)Lys150-Ile162 2376.0 + 1607.9 3983.9 Lys60-Phe82(Cys66-Cys160)Lys150-Ile162 2690.4 + 1607.9 4298.3
Val41-Lys69(Cys66-Cys160)Leu149-Ile162 3418.0 + 1721.1 5239.1
Trp61-Lys69(Cys66-Cys160)Leu149-Ile162 1122.2 + 1721.1 2843.3 Trp61-Lys70(Cys66-Cys160)Leu149-Ile162 1250.1 + 1721.1 2971.5 Leu58-Lys69(Cys66-Cys160)Leu149-Ile162 1491.7 + 1721.1 3212.8
Fig 5 Position of proteolytic fragments CP1/2 and TP1/2 within the primary structure of BLG Residues susceptible to chymotrypsin and trypsin hydrolysis are labeled with ÔcÕ and ÔtÕ superscripts, respectively Cysteines 66 and 160 are underlined.
Trang 8on completely different methodological approaches have
shown the existence of compact structural regions in BLG,
that are not affected by physical treatments [10,32]
Figure 6 presents some schematics of the structural
relationship of CP1/CP2 and TP1/TP2 with the remainder
of the BLG structure It is evident that all these fragments
include a generous portion of the b-barrel structure (strands
B, C and part of strand D), along with part of the distant I
strand in the C-terminus region that includes Cys160 and
connects to the leftovers of strand D via a disulfide bridge to
Cys66 [5]
Immunoreactivity of the products of BLG hydrolysis
As stated in the introduction, one of the goals of this work was to take advantage of the interplay of treatment conditions and enzyme action to produce fragments of sizable mass, but unable to be recognized by specific antibodies by standard immunochemical techniques ELISA was used to assess residual immunochemical reactivity in the unresolved digests and in the TP and CP fragments discussed in the previous section
The results obtained with a rabbit anti-BLG Ig on unresolved hydrolyzates obtained in conditions of maxi-mum BLG proteolysis (namely, 55°C, 20 min, 1 : 10 weight ratio of enzyme/BLG) are shown as an example in Fig 7 The decrease in immunoreactivity in the unresolved BLG hydrolysates obtained with either chymotripsin or trypsin at the various temperatures and treatment condi-tions parallels the decrease in intact BLG (Table 2) This also suggests that nonproteolyzed BLG retained its immuno-reactivity in spite of the thermal treatment, confirming that the structural changes in the temperature range investigated here were fully reversible
None of the isolated proteolytic fragments CP1/CP2 and TP1/TP2 was found to be immunoreactive against rabbit anti-BLG Ig even at very high fragment concentration (not shown) When epitope-specific monoclonal antibodies were used to test the same material, the immunoreactivity of the purified fragments was found to depend on the monoclonal antibody used for the assay Some of the ELISA curves obtained with different antibodies are reported in Fig 8 While most of the monoclonal antibodies did not recognize any of the large proteolytic fragments, as exem-plified by monoclonal 5G6 in Fig 8, monoclonals 9G10 and 1E3 recognized CP1 (although at 100-fold the concentration of native BLG), but neither CP2 nor TP1 and TP2 As the only relevant difference among the fragments is the presence of Arg40 in CP1 (Fig 5, Table 3),
it could be possible that this residue determined the recognizability of CP1 by these particular antibodies However, it remains to be assessed whether Arg40 is a
Fig 6 Position of the proteolytic fragments obtained at high
tempera-ture within the structempera-ture of the BLG monomer In both schemes, the
appropriate proteolytic fragments are as colored ribbons: red and
purple, CP1 (TP1); purple, CP2 (TP2) Residues attacked by proteases
are given as sticks (blue, basic; green, hydrophobic) The
disulfide-forming Cys66 and Cys160 are in yellow ball and stick Structures were
generated by using RASMOL [38], and coordinates in file 1B8E deposited
in the RCSB Protein Databank [34].
Fig 7 ELISA assay of unresolved BLG hydrolysates Hydrolysates were obtained after 20 min treatment at 55 °C with trypsin (triangles)
or chymotrypsin (squares) at an 1 : 10 enzyme/BLG ratio Native BLG, circles A rabbit anti-BLG Ig was used.
Trang 9relevant component of a sequential epitope, or is rather
important for proper structuring of a conformational
epitope
D I S C U S S I O N
Increasing temperature greatly facilitated proteolytic attack
of BLG by both trypsin and chymotrypsin However, no
ÔnovelÕ hydrolysis sites were made available at temperatures
in the 50–60°C range with respect to those already available
at 37°C, where little if any hydrolysis occurred at the times
and enzyme concentrations used here Rather, an increased
accessibility of attack sites in the ÔswollenÕ form of the BLG
monomer that represented the most abundant species in the
temperature range exploited here [6,8] should account for
increased proteolysis, in spite of thermal inactivation of
both trypsin and chymotrypsin at the highest temperatures
used here
More details on the structural modifications induced by
temperature may be derived from careful analysis of the
data presented in this paper A first point concerns tryptic attack on Arg148 This residues is located in the I strand that, as shown in Fig 9, is the closest contact point between monomers in the BLG dimer There are a number of interprotein hydrogen bonds in this region, in addition to hydrophobic interactions (such as those between Ile147 and Leu149 and the corresponding residues on the facing antiparallel strand) that all together contribute to make this region virtually inaccessible at neutral pH Both the network of H-bonds and the hydrophobic interactions are disrupted when the temperature is raised, so that the BLG dimer may dissociate [8,9], therefore exposing the polypep-tide backbone to the action of trypsin The nature of the interactions in this region, as pointed out above, also explains the reversibility of temperature-induced dissoci-ation of the BLG dimer Although this will have to be tested with true monomeric BLGs (such as the ones found in mare’s or sow’s milk), it is likely that dissociation of the bovine BLG dimer represents the primary event for facilitated proteolysis at high temperature The relevance
of dimer dissociation to facilitated proteolysis is also evident when considering the high hydrolysis yields obtained at
pH > 8.0 (i.e above the Tanford transition at pH 7.5 [13]), although the structural features of the BLG monomer obtained at high pH [20,33,34] appear different from those
of monomers obtained at low pH [35] or under pressure [10] Arg148 is not the only buried arginine in the structure of BLG The whole side chain of Arg40 (the other main point
of trypsin action) in the native structure of the protein is deeply buried inside a hydrophobic pocket that comprises several side chains, and provides an envelope for the guanidinium function (Fig 6) A number of spectroscopic studies have shown a reversible exposure of hydrophobic regions of BLG in the temperature range considered in this study [6,8,32] This may be instrumental in facilitating tryptic attack on Arg40, and the action of chymotrypsin on the adjacent Leu39 Given their position in the structure
Fig 8 ELISA assay of proteolytic fragments of BLG with various
monoclonal antibodies Fragments were purified as reported in the text
and in Fig 3, and are identified as in Fig 5 Native BLG, squares.
CP1, full circles; CP2, open circles; TP1, full triangles; TP2, open
tri-angles.
Fig 9 Relevance of protease-sensitive residues to the dimeric structure
of BLG Fragments CP1 and TP1 are as colored ribbons The orange regions correspond to the shorter TP1 fragments within the CP1 frag-ments (orange and brown) Residues attacked by proteases are iden-tified by their position in the sequence, and are given as sticks (blue, basic; green, hydrophobic) The disulfide-forming Cys66 and Cys160 are in yellow Structures were generated by using RASMOL [38], and coordinates in file 1BEB deposited in the RCSB Protein Databank [5].
Trang 10(Fig 9), both residues appear to be much more accessible to
proteases after proteolytic removal of the long exposed helix
and of strand I at the dimer interface
The disulfide-connected peptides that originate from
early proteolysis steps have some interesting features
Perhaps the most striking regards resistance to
chymotryp-sin of the three strands encompaschymotryp-sing residues 40–82, in
spite of the relative abundance of residues sensitive to this
protease Most of these hydrophobic residues are pointing
inwards in the native protein structure, in which they line the
hydrophobic cavity that characterizes all lipocalins The
insensitivity to chymotryptic attack suggests that some
degree of folding is retained in this region, although the
separation condition we used prevented the possibility of
producing direct evidence for retention of structural
organ-ization in CP1
Our observation on the resistance of this region to
physical denaturants is consistent with recent independent
observations on the different stability of secondary structure
elements with respect to physical denaturation in peculiar
regions of the BLG structure [10,32] The region at the
dimer interface is the most sensitive to heat or pressure, as
demonstrated by spectroscopic and chemical modification
studies [6–8,32], and it could be modified without sensible
modification in the remainder of the structure [36] On the
other hand, treatment in subdenaturing conditions has
revealed transient formation of a number of unfolding
intermediates that retain an Ôopen barrelÕ conformation [10]
In this frame, it should be noted that the protease-resistant
region of the barrel that constitutes most of our largest
fragments is located at the opposite site of the molecule with
respect to the dimerization interface (Fig 9)
One of the few hydrophobic residues that are not
pointing inwards in the region of native BLG
encompas-sing residues 39–70 is Leu57, which is attacked by both
proteases, but only after release of the primary proteolysis
products, CP1 and TP1, from the remainder of the
protein structure Further hydrolysis in this region is
however, slow enough to allow significant accumulation
of the hydrolysis intermediate even in conditions where no
intact BLG is left
Once the intact protein is removed from the system,
there is no residual immunochemical reactivity of the
hydrolysis products against monoclonal antibodies or
rabbit antisera Only fragment CP1 retains some faint
reactivity towards one of the monoclonals used in this
study We also obtained preliminary evidence that none of
the large-sized proteolysis products of BLG discussed
above was recognized by sera from allergic pediatric
patients in Western blot experiments [37] These findings
may be of practical relevance, in what hydrolysis at
temperatures that facilitate access to otherwise buried
structural regions of the protein may allow production of
hydrolysates with improved qualities with respect to those
presently on the market as for their sensory, nutritional
and technological properties
A C K N O W L E D G E M E N T S
Work supported by grants from the Ministry of University and
Scientific Research (MURST-FIRST, Rome, Italy, to S I.) and from
the Ministry of Policies for Agriculture and Forestry (MiPAF, Rome,
Italy, to F B.) This is publication no 1 of the Project ÔTOLLELATÕ.
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