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 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,aswellasfor 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) Eur. J. Biochem. 270, 4587–4593 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03860.x Experimental procedures Proteins CPY was prepared from baker’s yeast as described previ- ously [1] or was obtained from Oriental Yeast Co. (Lot 21003805) (Osaka, Japan) and proCPY was prepared as the same manner as CPY, with minor modifications. Dgly CPY and Dgly proCPY, in which the asparagine residues at positions 13, 87, 168 and 368 (sequence number of CPY) had been replaced by alanine residues, were expressed in the proteinase A, B, and CPY-deficient strain, BJ2168, of S. cerevisiae transformed 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 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 where m i is the wave-number and F i is the fluorescence intensity at m i . 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 0.1 M phosphate 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 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 > 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 DG T and T m were derived from Eqns (3 and 4), respectively: DG T ¼ DH À TDS ð3Þ T m ¼ DH=DS ð4Þ Plots of <m> against pressure were similarly fitted according to Eqn (5): <m> ¼ð<m n > À <m d >Þ=½1 þ e À½ðDGpþPDVÞ=RT þ <m d > ð5Þ where DG p 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 were 585 and 557 kJÆmol )1 , respectively). In contrast, a DSC analysis of the mature form (CPY) revealed an apparently symmetrical single peak but the ratio of DH cal / DH v wasdeterminedtobe1.74(DH cal and DH v values were 765 and 440 kJÆmol )1 , respectively) and the peak was deconvoluted into two peaks with T m1 of 57.0 and T m2 of 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 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 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 600 cm )1 . This large difference in P m between 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, P m1 , of 50 MPa or lower. The second transition could be fitted to a theoretical curve of a two-state transition with a half transition, P m2 , 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, P m3 ,of500MPaorhigher.The <m> values of CPY and Dgly CPY decreased from 29 160 cm )1 to 29 010 cm )1 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 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>1haftercoolingfromthe highest temperature to 25 °C. m and n indicate glycosylated and unglycosylated forms, respectively. See Experimental procedures. Ó FEBS 2003 Propeptide of proCPY ensures cooperative unfolding (Eur. J. Biochem. 270) 4589 700 MPa (Fig. 3B, open circles). However, the P m2 value of Dgly CPY (P m , 302 MPa) was lower by 43 MPa than the corresponding value for the glycosylated CPY (P m , 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). Discussion Structural properties of mature form (CPY) As far as can be seen in the experiments involving the stepwise increase in temperature and pressure, the heat- induced unfolding of CPY and DglyCPYshowedatwo- 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) 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)andDgly proCPY (s)at25°C(B)CPY(d)and Dgly CPY (s)at25°C, and (C) CPY at 60 °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. 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. Contributions of the carbohydrate moiety The specific activities of glycosylated and unglycosylated CPY are the same as previously reported [13] with the same <m>valuesat20°C and 0.1 MPa (Fig. 2B). However, the <m> values of the precursor were higher by 100 cm )1 than 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 T m values for CPY and Dgly CPY were nearly the same, DG T and 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. Ó FEBS 2003 Propeptide of proCPY ensures cooperative unfolding (Eur. J. Biochem. 270) 4591 unfavourable. The T 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 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>forN-acetyl tryptophanamide is increased by 100 cm )1 in 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. References 1. Hayashi, R. (1976) Carboxypeptidase Y. Methods Enzymol. 45, 568–587. 2. Valls, L.A., Hunter, C.P., Rothman, J.H. & Stevens, T.H. (1987) Protein sorting in yeast: The localization determinant of yeast vacuolar carboxypeptidase Y residues in the propeptide. Cell. 48, 887–897. 3. Jung, G., Ueno, H. & Hayashi, R. 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Peptidase Temperature-induced transition at 0.1 MPa Pressure-induced transition at 25 °C T m (°C) DH (kJÆmol )1 ) DS (kJÆmol )1 ÆK )1 ) DG T (kJÆmol )1 ) P m (MPa) DV (mlÆmol )1 ) 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. Second transition 302, 282 b )72.3, ) 80 b 21.9, 22.9 b Third transition > 500 n.d. n.d. proCPY 54.5 185 0.57 16.9 253 )42.8 10.8 Dgly proCPY 51.0 176 0.54 12.8 164 )49.3 8.1 a Obtained at 60 °C. b Obtained at 25 °C (Dumoulin et al. [25]). 4592 M. Kato et al. (Eur. J. Biochem. 270) Ó FEBS 2003 13. Shimizu, H., Ueno, H. & Hayashi, R. (1999) Role of carbohydrate moiety in carboxypeptidase Y: Structural study of mutant enzyme lacking carbohydrate moiety. Biosci. Biotechnol. Biochem. 63, 1045–1050. 14. Zhou, J.M., Zhu, L. & Balny, C. 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(1999) Con- tribution of the carbohydrate moiety to conformational stability of the carboxypeptidase Y: High pressure study. Eur. J. Biochem. 262, 475–483. 26. Kunugi, S., Yanagi, Y., Kitayaki, M., Tanaka, N. & Uehara- Kunugi, Y. (1997) Effects of high-pressure on the activity and spectroscopic properties of carboxypeptidase Y. Bull. Chem. Soc. Jpn. 70, 1459–1463. 27. Masson, P. & Cle ´ ry, C. (1996) Pressure-induced molten globule states of proteins. In High Pressure Bioscience and Biotechnology (Hayashi, R. & Balny, C., eds), pp. 117–126. Elsevier Science B.V., the Netherlands. 28. Ptitsyn, O.B. (1995) Molten globule and protein folding. Adv. Prot. Chem. 47, 83–229. 29. Trovaslet, M., Dallet-Choisy, S., Meersman, F., Heremans, K., Balny, C. & Legoy, M D. (2003) Fluorescence and FTIR study of pressure-induced structural modifications of horse liver alcohol dehydrogenase (HLADH). Eur. J. Biochem. 270, 119–128. Ó FEBS 2003 Propeptide of proCPY ensures cooperative unfolding (Eur. J. Biochem. 270) 4593 . The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against. France The heat- and pressure- induced unfolding of the glycosyl- ated and unglycosylated forms of mature carboxypeptidase Y and the precursor procarboxypeptidase