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 Kato
1
, Yasuhiro Sato
1
, Kumiko Shirai
1
, Rikimaru Hayashi
1,
*, Claude Balny
2
and Reinhard Lange
2
1
Laboratory of Biomacromolecular Chemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Japan;
2
INSERM U128, IFR 122, Montpellier, France
The heat- and pressure-induced unfolding of the glycosyl-
ated and unglycosylated 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 °Corthe
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

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-

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 m
M
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 Rm
i
F
i
=RF
i
ð1Þ

the DSC cell. Heating curves were corrected for the baseline.
DH
cal
and DH
v
(van’t Hoff enthalpy) were determined from
the scanned data using the
ORIGIN
software program
(version 4.0) (MicroCal Inc.).
Qualitative thermodynamic parameters
for temperature- and pressure-induced unfoldings
The <m> values of the unfolding reaction were fitted
against temperature in the frame of simple two-state
transitions between the native and denatured states,
according to Eqn (2):
<m> ¼ð<m
n
> À <m
d
>Þ=½1 þ e
À½ðDHÀTDSÞ=RT
g
þ <m
d
> ð2Þ
where <m>, <m
n
>, and <m
d

and 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.
DG
p
and DV were determined from Eqn (5) and P
m
was
derived from Eqn (6):
P
m
¼ÀDG
p
=DV ð6Þ
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 (DH
cal
)tothe
van’t Hoff enthalpy (DH
v
) was 1.05 (DH
cal
and DH
v
values

transition, T
m
,ofproCPYandDglyproCPYwere54.5and
4588 M. Kato et al. (Eur. J. Biochem. 270) Ó FEBS 2003
51.0 °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 T
m
values 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>ofproCPYand
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 P
m
of
253 MPa for proCPY and 164 MPa for Dgly proCPY with
a parallel change in the <m> values of approximately

as the pressure increased to
700 MPa at 25 °C (Fig. 3B). This pressure-induced
decrease in <m>of150cm
)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 °CwithP
m1
, P
m2
,andP
m3
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

state transition which is typical of a cooperative unfolding
(Fig. 2B), but their pressure-induced unfoldings showed a
multistate transition (Fig. 3B). The pressure-induced
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 Ômultiple molten globule-like state transitionsÕ
[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).ThefactthataDSCpeakofCPYwas
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)

fit curves are applied in C. See Experimental procedures for other
details.
4590 M. Kato et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Thermodynamic 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 T
m
, P
m
, DG
T
and DG
P
values 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 vivo because 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.

m
of proCPY was higher by 4 °Cthan
that of Dgly proCPY, indicating that the carbohydrate
moiety exerts a slightly protective effect on the thermal
unfolding of proCPY.
The P
m
value for CPY was higher than that of its
unglycosylated form, though the DV and DG
P
values were
almost the same. The P
m
value 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 hydrated 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.
Thedifferencein<m> values for the glycosylated and

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)
DG
P
(kJÆmol
)1
)
CPY 58.6 405 1.22 41.4
First transition < 50, < 50
a
n.d. n.d.
Second transition 345, 334
b
, 194
a
)75.8, )61
b
, )117
a
26.2, 20.6
b
, 22.8
a
Third transition > 500, 492
a
)24.5
a
12.1
a
Dgly CPY 58.4 323 0.97 32.4
First transition < 50 n.d. n.d.

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