The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure Michiko Kato1, Y
Trang 1The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts
a protective effect against heat and pressure
Michiko Kato1, Yasuhiro Sato1, Kumiko Shirai1, Rikimaru Hayashi1,*, Claude Balny2and Reinhard Lange2 1
Laboratory of Biomacromolecular Chemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan;2INSERM U128, IFR 122, Montpellier, France
The heat- and pressure-induced unfolding of the
glycosyl-ated and unglycosylglycosyl-ated forms of mature carboxypeptidase
Y and the precursor procarboxypeptidase Y were analysed
by differential scanning calorimetry and/or by their intrinsic
fluorescence in the temperature range of 20–75°C or the
pressure range of 0.1–700 MPa Under all conditions, the
precursor form showed a clear two-state transition from a
folded to an unfolded state, regardless of the presence of the
carbohydrate moiety In contrast, the mature form, which
lacks the propeptide composed of 91 amino acid residues,
showed more complex behaviour: differential scanning
calorimetry and pressure-induced changes in fluorescence
were consistent with a three-step transition These results show that carboxypeptidase Y is composed of two structural domains, which unfold independently but that procarb-oxypeptidase Y behaves as a single domain, thus ensuring cooperative unfolding The carbohydrate moiety has a slightly protective role in heat-induced unfolding and a highly protective role in pressure-induced unfolding Keywords: carboxypeptidase Y; fluorescence spectrometry; pressure unfolding; procarboxypeptidase Y; thermal unfolding
Carboxypeptidase Y (CPY), a member of the serine
carboxypeptidase family, is a 61-kDa vacuolar enzyme
obtained from Saccharomyces cerevisiae [1] This enzyme is
synthesized in the form of procarboxypeptidase Y
(pro-CPY) and sorted to the vacuole via the Golgi apparatus
where it undergoes carbohydrate modification ProCPY has
an N-terminal extension (propeptide) of 91 residues [2,3],
compared to the mature CPY This propeptide structure is
essential for folding both in vivo and in vitro, as well as for
maintaining CPY in an inactive form [4–6] The mature and
precursor forms are glycoproteins [7], which contain 16%
carbohydrates [8]: the four carbohydrate chains are of
similar sizes and are bound to asparagine residues at
Asn-Xaa-Thr glycosylation sites [9–12] The genetic
replace-ment of these asparagine residues by alanine residues
produces unglycosylated (Dgly) CPY [13] and proCPY with
no change in their activities
The reason why the presence of the propeptide is important for the correct folding of CPY and role that the large amount of carbohydrate moiety plays on the stability and function of CPY has not been fully clarified To answer these questions, we examined the folding/unfolding of mature and precursor CPY as well as their unglycosylated forms using temperature and pressure as the structural perturbant Compared to heat, pressure studies have been used to obtain complementary information concerning protein–solvent interactions [14,15], the unfolded states of proteins [16,17], and protein folding pathways [18] Our analytical techniques involved the use of differential scanning calorimetry (DSC) and protein fluorescence as a function of temperature and pressure The intrinsic fluor-escence of CPY is due mainly to tryptophan and, to a lesser extent, tyrosine residues [19] The shape and the wavelength
of the emission maximum reflects the polarity of the environment of these residues, which can be conveniently assessed by the centre of spectral mass, <m>, which corresponds to the wave-number of the emission maximum, normalized by the fluorescence intensity [17] CPY contains
10 tryptophan and 24 tyrosine residues, which are distri-buted evenly throughout the entire protein molecule, and the propeptide of proCPY contains two tryptophan and two tyrosine residues Upon protein unfolding, these residues come into contact with solvent water and the increase in polarity is evidenced by the observed decrease in <m> The present study leads to the conclusion that the propeptide plays a role in the unfolding mechanism, ensuring cooperative structural transitions, and that the carbohydrate moiety serves to stabilize the protein structure, especially against pressure These results imply the biologi-cal significance of the CPY maturation process
Correspondence to M Kato, Division of Applied Life Sciences,
Graduate School of Agriculture, Kyoto University, Kyoto 606-8502,
Japan Fax: +81 75 7536128, Tel.: + 81 75 7536495,
E-mail: mk@kais.kyoto-u.ac.jp
Abbreviations: CPY, carboxypeptidase Y; Dgly, unglycosylated
carboxypeptidase Y; DSC, differential scanning calorimetry;
proCPY, procarboxypeptidase Y.
Enzymes: carboxypeptidase Y (EC 3.4.12.1).
*Present address: Department of Food Science and Technology,
College of Bioresource Science, Nippon University, Fujisawa,
Kanagawa 252-8510, Japan.
(Received 22 May 2003, revised 12 August 2003,
accepted 1 October 2003)
Trang 2had been replaced by alanine residues, were expressed in
the proteinase A, B, and CPY-deficient strain, BJ2168, of
S cerevisiaetransformed by plasmid pTSY3 for CPY and
mutated pTSY3 for proCPY, and purified as described
previously [13]
Measurement of fluorescence
Fluorescence under pressure or temperature was recorded
with an Aminco-Bowmann Series 2 luminescence
spectro-meter (SLM Co.), equipped with a thermostated
high-pressure resistant cell accommodating a round quartz
cuvette (5 mm inner diameter) [20,21] or with a Shimadzu
RF-5300PC spectrofluorimeter accommodating a
thermo-stated square quartz cuvette (5-mm light path) The
excitation wavelength was 280 nm (4-nm bandpass)
Emis-sion spectra were recorded between 310 and 410 nm (4-nm
bandpass, in steps of 1 nmÆs)1) The fluorescence intensities
were corrected for volume contraction of the sample due to
solvent compressibility [22] The protein concentration was
0.1 mgÆmL)1 in 50 mM Mops buffer (pH 7.0) for all
experiments, as the pK of the Good’s buffer, that includes
Mops, is relatively independent of pressure [23] Spectral
changes were quantified by determining the centre of
spectral mass, <m>, as defined by Weber and coworkers
in Eqn (1) [24]
<m>¼ RmiFi=RFi ð1Þ where mi is the wave-number and Fi is the fluorescence
intensity at mi
Temperature or pressure change
Temperature or pressure was increased in steps of 5°C
or 50 MPa, respectively The sample was allowed to
equilibrate for 5 min prior to each spectral recording
Reversibility was measured 1 h after cooling the sample
from the highest temperature to 25°C, or after releasing
the pressure from the highest pressure to ambient
pressure
DSC
DSC was performed by using a VP-DSC microcalorimeter
(MicroCal Inc.) with a scan rate of 1.0°CÆmin)1 Protein
(1.0 mgÆmL)1), dissolved in 0.1Mphosphate buffer pH 7.0,
was dialysed against the same buffer overnight The
solutions inside and outside the dialysis tube were used as
the protein and the reference solutions, respectively The
solutions were degassed under vacuum prior to applying to
the DSC cell Heating curves were corrected for the baseline
DH and DH (van’t Hoff enthalpy) were determined from
<m>¼ ð<mn> <md>Þ=½1 þ e½ðDHTDSÞ=RTg
where <m>, <mn>, and <md> are the observed
<m>, <m> for the native state, and <m> of the denatured state, respectively The correlation coefficient
of the fitting was 0.999 or higher in all cases DH and DS were determined from Eqn (2) and DGT and Tm were derived from Eqns (3 and 4), respectively:
Plots of <m> against pressure were similarly fitted according to Eqn (5):
<m>¼ ð<mn> <md>Þ=½1 þ e½ðDGpþPDVÞ=RTþ <md>
ð5Þ where DGpand DV are the Gibbs free energy change at T (298 K) and 0.1 MPa and the volume change at T, respectively The correlation coefficient of the fitting was 0.999 or higher in all cases
DGpand DV were determined from Eqn (5) and Pmwas derived from Eqn (6):
Results
Temperature-induced unfolding of CPY and proCPY DSC analysis of Dgly proCPY revealed a perfectly sym-metrical single peak (Fig 1A), indicating that the thermal unfolding process of the precursor form follows a two-state transition The ratio of the unfolding enthalpy (DHcal) to the van’t Hoff enthalpy (DHv) was 1.05 (DHcaland DHvvalues were 585 and 557kJÆmol)1, respectively) In contrast, a DSC analysis of the mature form (CPY) revealed an apparently symmetrical single peak but the ratio of DHcal/
DHvwas determined to be 1.7 4 (DHcaland DHvvalues were
765 and 440 kJÆmol)1, respectively) and the peak was deconvoluted into two peaks with Tm1of 57.0 and Tm2of 62.1°C, as shown by the dashed lines of Fig 1B This strongly suggests that the thermal unfolding of CPY involves a multistate transition
The temperature dependent fluorescence data for pro-CPY and pro-CPY, as well as their unglycosylated forms showed two-state transitions The carbohydrate moiety appeared to increase the heat stability of proCPY slightly but had no effect on mature CPY: the temperature of half transition, T , of proCPY and Dgly proCPY were 54.5 and
Trang 351.0°C, respectively (Fig 2A) Moreover, even in the native
state, Dgly proCPY exhibited a <m> value lower by
150 cm)1 than its glycosylated form (Fig 2A,
double-headed arrow a) Interestingly, the Tmvalues of CPY and
Dgly CPY, which were almost identical, were higher by 4
and 7°C, respectively, than the corresponding values of
proCPY and Dgly proCPY (Fig 2B), indicating that the precursor form was less thermally stable than the mature form, regardless of the carbohydrate moiety
After the temperature was lowered from the highest temperature tested to 25°C, the <m> values for CPY, Dgly CPY, proCPY, and Dgly proCPY were partially reversible (open and closed triangles, Fig 2)
Pressure-induced unfolding of CPY and proCPY The pressure-induced changes in <m> of proCPY and Dgly proCPY up to 700 MPa at 25°C were perfectly cooperative (Fig 3A) These precursor forms showed simple two-state transitions characterized by a Pm of
253 MPa for proCPY and 164 MPa for Dgly proCPY with
a parallel change in the <m> values of approximately
600 cm)1 This large difference in Pmbetween proCPY and Dgly proCPY ( 90 MPa) indicates that the carbohydrate moiety contributes to the effective stabilization of proCPY against pressure
In contrast to the two-state transition of the precursor form, mature CPY showed a multistate transition: a first transition in the 0.1–150 MPa range, a second from 150
to 450 MPa, and a third at pressures above 500 MPa (Fig 3B) The first transition was small with a half transition, Pm1, of 50 MPa or lower The second transition could be fitted to a theoretical curve of a two-state transition with a half transition, Pm2, of 345 MPa (Fig 3B, inset showing a magnified change in <m>) The third transition was incomplete, even at 700 MPa, with an estimated half transition, Pm3, of 500 MPa or higher The
<m> values of CPY and Dgly CPY decreased from
29 160 cm)1to 29 010 cm)1as the pressure increased to
700 MPa at 25°C (Fig 3B) This pressure-induced decrease in <m> of 150 cm)1 was significantly smaller than that observed for the thermal-induced unfolding reaction (400 cm)1) This suggests that pressure does not induce the complete unfolding of the structures of mature CPY even at 700 MPa and 25°C However, the pressure-induced unfolding of the mature CPY clearly showed a multistep transition at 60°C with Pm1, Pm2, and Pm3 of
50 MPa or lower, 194 MPa, and 492 MPa, respectively (Fig 3C)
The pressure-induced transition of Dgly CPY also showed at least a three-step transition for pressures up to
Fig 1 DSC profiles of (A) Dgly proCPY and
(B) CPY Solid and dashed lines indicate
observed and deconvoluted curves,
respect-ively.
Fig 2 Temperature-induced changes in the centre of the spectral mass
< m>of (A) proCPY (d) and Dgly proCPY (s) and (B) CPY (d) and
Dgly CPY (s) Fluorescence of the enzymes (concentration of
enzymes, 0.1 mgÆmL)1) was measured at 310–410 nm and excited at
280 nm Solid lines show the best-fit curves for the two-state transition
model (Eqn 2) Triangles indicate <m> 1 h after cooling from the
highest temperature to 25 °C m and n indicate glycosylated and
unglycosylated forms, respectively See Experimental procedures.
Trang 4700 MPa (Fig 3B, open circles) However, the Pm2value
of Dgly CPY (Pm, 302 MPa) was lower by 43 MPa than
the corresponding value for the glycosylated CPY (Pm,
345 MPa), indicating that the carbohydrate moiety has a
slight protective effect on the pressure-induced unfolding of
CPY This finding is consistent with results reported by
Dumoulin et al [25]
After the pressure was released from the highest pressure
tested, the <m> values for CPY, Dgly CPY, proCPY and
Dgly proCPY were partially reversible (open and closed
triangles in Fig 3B)
change in <m> induced at relatively low pressures of up
to 150 MPa is small with no increase in ANS-binding fluorescence, with approximately 80% of the catalytic activity being retained [25]; a large conformational change induced by higher pressures at 150–500 MPa (Fig 3B, solid line) shows a two-state transition, accompanied by an increase in ANS-binding fluorescence and a loss of enzymatic activity [25], indicating exposure of the hydro-phobic core to the solvent; further conformational change induced by higher pressures of 500–700 MPa is not complete, even at 700 MPa Such a complex pressure-induced transition has been observed and interpreted as a reflection of multiple molten globule-like state transitions [26–29]
The difference between the heat- and pressure-unfold-ing of CPY described above may be due to its two domains (the b-sheet-rich and the helix-rich domains [9]) (Fig 4) The fact that a DSC peak of CPY was deconvoluted into two peaks (Fig 1B) suggests that CPY contains two domains, which are differently heat sensitive Thus, it can be concluded that the mature form
of CPY essentially unfolds in a multistate transition by temperature and pressure, regardless of the presence of the carbohydrate moiety Probably the two domains unfold with similar activation energies but with different activation volumes
Structural properties of the precursor form (proCPY) The temperature-induced unfolding of proCPY and Dgly proCPY followed a two-state transition even in the DSC experiments (Fig 1A), showing a cooperative unfolding (Fig 2A) Their pressure-induced unfoldings also clearly followed a two-state transition (Fig 3A)
Although the X-ray crystal structure of proCPY has not yet been solved, it is naturally anticipated that the cleft of the active site will be located in the interface between the two structural domains of CPY and would be filled by the propeptide, thus uniting the two domains in a body, as if the entire structure of proCPY were composed of a single domain Although the change in fluorescence for the unfolded CPY and proCPY were partially reversible in the present experiments (Figs 2 and 3), it has been reported that neither changes in the secondary structure nor the activity of CPY are irreversible but those of proCPY are reversible [5] These results support the view that the mature form, CPY, is composed of two independent domains the sensitivities of which to temperature and pressure are different from those of each other In contrast, the precursor form, proCPY, consists of a single domain, which exhibits a two-state transition for heating and high pressure
Fig 3 Pressure-induced changes of the centre of the spectral mass of
(A) proCPY (d) and Dgly proCPY (s) at 25 °C (B) CPY (d) and
Dgly CPY (s) at 25 °C, and (C) CPY at60 °C (d) Insert shows an
enlargement of the ordinate Triangles indicate <m> 1 h-later after
pressure-release from highest pressure to 0.1 MPa m and n indicate
glycosylated and unglycosylated forms, respectively Solid lines show
the best-fit curves for the two-state transition model (Eqn 5) Three
fit curves are applied in C See Experimental procedures for other
details.
Trang 5Thermodynamic properties of CPY and proCPY
Thermodynamic parameters were calculated based on Eqns
(2–6) to compare qualitatively the temperature and pressure
effects of the four proteins, and summarized in Table 1
In both thermal and pressure unfolding, the Tm, Pm, DGT
and DGPvalues for CPY are higher than those of proCPY,
regardless of the extent of glycosylation (Table 1) This
indicates that the mature CPY is more stable to heating and
high pressure than the precursor proCPY This is supported
by the higher DH value of CPY, compared to that of
proCPY
It is interesting to note that protein stability is not
necessarily dependent on the number of structural domains,
but this issue may be extended to the biological meaning of
the structure of proCPY proCPY must be rather unstable
in vivobecause it is a precursor to an active enzyme, and the
in vivo structure is either the native or denatured form
(without the presence of intermediate structures) because
only the native form leads to an active enzyme, while others are effectively digested by intracellular proteases
Contributions of the carbohydrate moiety The specific activities of glycosylated and unglycosylated CPY are the same as previously reported [13] with the same
<m> values at 20°C and 0.1 MPa (Fig 2B) However, the
<m> values of the precursor were higher by 100 cm)1than that of Dgly proCPY (double-headed arrow a, Fig 2A), indicating that the carbohydrates in proCPY shield some of the tryptophan and tyrosine residues, which are exposed to the solvent This is evidence that high concentrations of glucose increase the <m> value of N-acetyl tryptophan-amide (see below)
Although the Tmvalues for CPY and Dgly CPY were nearly the same, DGTand DH for CPY were higher than the corresponding values for Dgly CPY (Table 1), indicating that glycosylation causes the unfolding to be energetically
Fig 4 Ribbon diagram of CPY showing two
structural domains [9] When the catalytic triad
shown by the CPK model is placed in the
centre of the model, the CPY structure is
divided into a b-sheet rich domain on the
left side and an a-helix rich domain on the
right side.
Trang 6unfavourable The Tmof proCPY was higher by 4°C than
that of Dgly proCPY, indicating that the carbohydrate
moiety exerts a slightly protective effect on the thermal
unfolding of proCPY
The Pm value for CPY was higher than that of its
unglycosylated form, though the DV and DGPvalues were
almost the same The Pmvalue of proCPY was higher than
that of its unglycosylated form This is due to the higher DV
value of the unglycosylated form, according to Eqn (6) (see
Experimental procedures) This is consistent with a more
pronounced conformational change and/or a more
pro-nounced hydration upon unfolding of the unglycosylated
form
At high pressure, in the glycosylated forms the
carbohy-drate moiety of CPY and proCPY would be hycarbohy-drated to
compensate the volume contraction and the protein portion
is minimally hydrated However, in the unglycosylated
forms the protein portion would be directly hydrated to
ensure the corresponding volume contraction Hence, the
protein portion of the unglycosylated forms would be
more heavily hydrated under high pressure, resulting in
instability
The difference in <m> values for the glycosylated and
unglycosylated forms of CPY and proCPY at 75–80°C
(double-headed arrows b, Fig 2A and a, Fig 2B,
respect-ively) is caused by the presence of the carbohydrate moiety,
because the <m> for N-acetyl tryptophanamide is increased
by 100 cm)1in a 16% glucose solution (T Maki, M Kato,
and R Hayashi, unpublished data) Tryptophan and
tyro-sine residues (Y17, Y20, Y82, W84, and W369) in CPY
would be perturbed by the carbohydrate moiety, thus
increasing their fluorescence, since they are in close proximity
to the carbohydrate-attachment sites, N13, 87, and 368
In conclusion, the mature enzyme, CPY, unfolds in a
multistate transition, but the precursor, proCPY, unfolds in
a two-state transition, indicating that CPY is composed of
two structural domains, while proCPY would be composed
of a single fragile domain The propeptide of the proenzyme
would be located at the interface of the two domains thus
combining them into one body, to ensure structural
cooperativity
Acknowledgements
We are grateful for the technical assistance of C Valentin in the high-pressure experiments The authors are grateful to G Jung for his initial work on the construction of the expression plasmid.
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