The differential scanning calorimetry analysis of the murine major histocompatibility complex class II molecule, I-Ek, in complex with an antigenic peptide derived from mouse hemoglobin, showed that the thermal stability at the mildly acidic pH is higher than that at the neutral pH. Although the thermal unfolding of I-Ek-hemoglobin was irreversible, we extracted the equilibrium thermodynamic parameters from the kinetically controlled heat capacity curves. Both the denaturation temperatures and the enthalpy changes were almost independent of the heating rate over 1 °C per min. The linear relation between the denaturation temperature and the calorimetric enthalpy change provided the heat capacity changes, which are classified into one for the mildly acidic pH region and another for the neutral pH region. The equilibrium thermodynamic parameters showed that the increased stability at the mildly acidic pH is because of the entropic effect. These thermodynamic data provided new insight into the current structural model of a transition to an open conformation at the mildly acidic pH, which is critical for the peptide exchange function of major histocompatibility complex class II in the endosome. The differential scanning calorimetry analysis of the murine major histocompatibility complex class II molecule, I-Ek, in complex with an antigenic peptide derived from mouse hemoglobin, showed that the thermal stability at the mildly acidic pH is higher than that at the neutral pH. Although the thermal unfolding of I-Ek-hemoglobin was irreversible, we extracted the equilibrium thermodynamic parameters from the kinetically controlled heat capacity curves. Both the denaturation temperatures and the enthalpy changes were almost independent of the heating rate over 1 °C per min. The linear relation between the denaturation temperature and the calorimetric enthalpy change provided the heat capacity changes, which are classified into one for the mildly acidic pH region and another for the neutral pH region. The equilibrium thermodynamic parameters showed that the increased stability at the mildly acidic pH is because of the entropic effect. These thermodynamic data provided new insight into the current structural model of a transition to an open conformation at the mildly acidic pH, which is critical for the peptide exchange function of major histocompatibility complex class II in the endosome. major histocompatibility complex class II associated invariant chain-derived peptide hemoglobin differential scanning calorimetry calorimetric enthalpy change entropy change denaturation temperature 3-(N-morpholino)propanesulfonic acid van't Hoff enthalpy change molar excess heat capacity molar excess heat capacity change activation energy of irreversible unfolding temperature at which the rate constant from denatured to irreversibly arrived state is unity Gibbs free energy of unfolding as a function of temperature The major histocompatibility complex (MHC)1 class II is expressed by professional antigen-presenting cells, which present peptide antigens to CD4 T cells. The newly synthesized MHC class II is transported from the endoplasmic reticulum to acidic compartments as the complex with an invariant chain (Ii). In that complex, the peptide binding groove of MHC class II is occupied by the CLIP, which is part of Ii (1Cresswell P. Annu. Rev. Immunol. 1994; 12: 259-293Crossref PubMed Google Scholar, 2Cresswell P. Cell. 1996; 84: 505-507Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). The antigenic peptides derived from endocytosed proteins, which have been digested by cathepsins into 15–20-amino acid segments, are then loaded onto the peptide-binding groove of the MHC class II in exchange for the CLIP, at an endosomal pH (3Mellman I. Fuchs R. Helenius A. Annu. Rev. Biochem. 1986; 55: 663-700Crossref PubMed Google Scholar, 4Engelhard V.H. Annu. Rev. Immunol. 1994; 12: 181-207Crossref PubMed Scopus (357) Google Scholar, 5Villadangos J.A. Ploegh H.L. Immunity. 2000; 12: 233-239Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). The role of the acidic pH is considered to enhance the peptide exchange reaction rate as well as to provide a suitable reaction condition for cathepsins. Finally, the peptide-MHC class II complex is transported from the acidic compartments to the cell surface for the interaction with T cell receptors (6Davis M.M. Boniface J.J. Reich Z. Lyons D. Hampl J. Arden B. Chien Y. Annu. Rev. Immunol. 1998; 16: 523-544Crossref PubMed Scopus (778) Google Scholar). The acidic pH can change the properties of MHC class II molecules and accelerate the peptide exchange, because of the faster association and/or dissociation reactions (7Sette A. Southwood S. O'Sullivan D. Gaeta F.C. Sidney J. Grey H.M. J. Immunol. 1992; 148: 844-851PubMed Google Scholar, 8Reay P.A. Wettstein D.A. Davis M.M. EMBO J. 1992; 11: 2829-2839Crossref PubMed Scopus (83) Google Scholar, 9Kasson P.M. Rabinowitz J.D. Schmitt L. Davis M.M. McConnell H.M. Biochemistry. 2000; 39: 1048-1058Crossref PubMed Scopus (48) Google Scholar). To understand the pH-dependent functions of MHC class II molecules, it is essential to study their thermodynamic and structural properties. Previous thermal stability analyses have determined the energetic consequences of an alteration in the pH (10Reich Z. Altman J.D. Boniface J.J. Lyons D.S. Kozono H. Ogg G. Morgan C. Davis M.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2495-2500Crossref PubMed Scopus (55) Google Scholar, 11Wilson N. Fremont D. Marrack P. Kappler J. Immunity. 2001; 14: 513-522Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In the present study, we characterized the thermal stability of the murine MHC class II molecule, I-Ek, in complex with the peptide, 64–76, of the d allele of mouse hemoglobin (Hb), using differential scanning calorimetry (DSC) measurements. As compared with other MHC class II molecules, the peptide binding to I-Ek is most affected by pH (7Sette A. Southwood S. O'Sullivan D. Gaeta F.C. Sidney J. Grey H.M. J. Immunol. 1992; 148: 844-851PubMed Google Scholar). The binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonic acid, a probe for exposed nonpolar sites, to I-Ek was enhanced by an acidic pH, indicating that the acidic environment is associated with an increase in the exposed hydrophobicity in class II molecules (12Boniface J.J. Lyons D.S. Wettstein D.A. Allbritton N.L. Davis M.M. J. Exp. Med. 1996; 183: 119-126Crossref PubMed Scopus (52) Google Scholar, 13Runnels H.A. Moore J.C. Jensen P.E. J. Exp. Med. 1996; 183: 127-136Crossref PubMed Scopus (56) Google Scholar). The analysis of the migration of I-Ek by SDS-PAGE showed that the “floppy” form is observed at pH 4.5 in addition to the “compact” form (14Sadegh-Nasseri S. Germain R.N. Nature. 1991; 353: 167-170Crossref PubMed Scopus (275) Google Scholar). The crystal structures of I-Ek-Hb and its mutants have been determined, and the structural difference between the acidic and neutral pH values has also been discussed in relation with the peptide exchange mechanism (11Wilson N. Fremont D. Marrack P. Kappler J. Immunity. 2001; 14: 513-522Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 15Fremont D.H. Hendrickson W.A. Marrack P. Kappler J. Science. 1996; 272: 1001-1004Crossref PubMed Scopus (335) Google Scholar, 16Kersh G.J. Miley M.J. Nelson C.A. Grakoui A. Horvath S. Donermeyer D.L. Kappler J. Allen P.M. Fremont D.H. J. Immunol. 2001; 166: 3345-3354Crossref PubMed Scopus (96) Google Scholar). Together with the biochemical analyses, the conformational change from a “closed” to an “open” form is considered to occur at low pH and to be critical for the function. Thus, I-Ek may be a good target for further thermodynamic characterizations and for studying their relation to the structural features. DSC analyses can provide accurate thermodynamic parameters, such as the calorimetric enthalpy change (ΔHcal) and the entropy change (ΔS), in addition to the denaturation temperature (Td) and the van't Hoff enthalpy change (ΔHvH), which are also obtained in circular dichroism (CD) measurements. It is interesting to determine the energetic contribution to the increased stability of MHC class II molecules at low pH relative to that at neutral pH (10Reich Z. Altman J.D. Boniface J.J. Lyons D.S. Kozono H. Ogg G. Morgan C. Davis M.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2495-2500Crossref PubMed Scopus (55) Google Scholar), which can explain the structural and functional differences. Additionally, to analyze the irreversible thermal denaturation of I-Ek-Hb, the kinetically controlled heat capacity functions were used for the extraction of equilibrium parameters (17Sanchez-Ruiz J.M. Lopez-Lacomba J.L. Cortijo M. Mateo P.L. Biochemistry. 1988; 27: 1648-1652Crossref PubMed Scopus (457) Google Scholar). Because reversible denaturation is limited to small, compact proteins, the evaluation of irreversible transitions will be more important to analyze structure-function relationships in the present post-genome era. Under the same conditions used for DSC measurements, with little interference from the irreversible process, the thermodynamic parameters can be quantitatively determined and compared as a function of pH. Baculovirus-infected insect cells, Sf9, were used to produce soluble I-Ek molecules with the Hb peptide, 64–76, linked to the N terminus of the β subunit via flexible linker, as described previously (18Kozono H. Parker D. White J. Marrack P. Kappler J. Immunity. 1995; 3: 187-196Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 19Kozono H. White J. Clements J. Marrack P. Kappler J. Nature. 1994; 369: 151-154Crossref PubMed Scopus (268) Google Scholar). The I-Ek-Hb molecule was secreted into the medium and was purified by immunoaffinity column chromatography with 14-4-4S, a monoclonal anti-MHC class II antibody, followed by gel filtration chromatography using a Superdex 200 column (16 mm × 60 cm, Amersham Biosciences). The purified fractions were pooled, and the buffer was exchanged to an appropriate buffer. The protein concentrations were determined from UV absorption at 280 nm and were calculated by using an absorption coefficient of 1.23 cm2 mg−1, which was estimated from the amino acid composition of I-Ek-Hb. Far-UV CD spectra of I-Ek-Hb were measured on a Jasco J-600 spectropolarimeter. The protein concentration was 0.36 mg/ml, and the optical path length was 0.1 cm. Spectra for CD between 200 and 250 nm were obtained in 10 mm phosphate buffer containing 150 mm NaCl at 20 °C using a scanning speed of 10 nm min−1, a time response of 2 s, a band width of 1 nm, and an average over 5 scans. DSC experiments were carried out on a Microcal MCS DSC calorimeter. All solutions (10 mmphosphate buffer containing 150 mm NaCl or 10 mm MOPS buffer containing 150 mm NaCl) were carefully degassed before the measurements. Data were collected in the temperature range between 20 and 90 °C at various heating rates, 0.2, 1.0, 1.5, and 2.0 °C min−1. The protein concentration was in the range from 0.08 to 0.43 mg ml−1. The analyses were done by a non-linear least-square method, as described in the DSC analysis software Microcal Origin version 4.1. The ΔHcal was calculated by integrating the area in each heat capacity curve. The van't Hoff enthalpy change (ΔHvH) was calculated by the next equation for assuming the two-state transition, ΔHvH(Td)=4RTd2Cp(Td)ΔHcal(Td)Eq. 1 where Cp is the molar excess heat capacity, and R is the gas constant. Using the ΔCp values with the ΔHcaland Td values, the Gibbs free energy of unfolding as a function of temperature, ΔGd(T), could be calculated from the following equation.ΔGd(T)=ΔHcal(Td)1−TTd−ΔCpTlnTTd−ΔCp(Td−T)Eq. 2 A rate-limiting irreversible unfolding is assumed to follow a reversible transition (20Lumry R. Eyring H. J. Phys. Chem. 1954; 58: 110-120Crossref Scopus (730) Google Scholar). k1k2N (native)↔ D(denatured)→I (irreversible arrived)k−1Eq. 3 The temperature dependence of the first-order rate constant,k2, is given by the Arrhenius equation,k2=exp−EaR1T−1T*Eq. 4 where Ea is the activation energy of irreversible unfolding, and T* is the temperature at whichk2 is unity. A detailed theoretical analysis for irreversible denaturation has been reported previously (17Sanchez-Ruiz J.M. Lopez-Lacomba J.L. Cortijo M. Mateo P.L. Biochemistry. 1988; 27: 1648-1652Crossref PubMed Scopus (457) Google Scholar). The four methods, A, B, C, and D, were applied to determine the activation parameters for denaturation of I-Ek-Hb in this study. The rate constant, k2, is obtained at each temperature from the next equation, k2=rCpΔHcal−ΔHEq. 5 where r is the heating rate in kelvin per second, and ΔH is the corresponding enthalpy change at a given temperature. The activation energy is obtained from the Arrhenius plot,k2=Aexp−EaRT*Eq. 6 where A = exp (Ea/RT*). The variation of Td withr is given by the following equation. r(Td)2=AREaexp(−EaRTd)Eq. 7 The dependence of the enthalpy evolved with temperature is expressed by the following equation. lnlnΔHcalΔHcal−ΔH=EaR1Td−1TEq. 8 The activation energy can be calculated from the heat capacity at Td,Cp(Td), by the following equation. Ea=eRCp(Td)Td2ΔHcalEq. 9 To eliminate the binding of endogenous peptides to I-Ek during its expression, the Hb peptide was attached by a flexible linker to the N terminus of the I-Ek β subunit, and the soluble molecule, I-Ek-Hb, was purified as a single peak on gel filtration high performance liquid chromatography analysis, corresponding to the αβ heterodimer (Fig.1A). The SDS-PAGE analysis also revealed the purity of the I-Ek-Hb molecule to be over 95%. The far-UV CD spectra of I-Ek-Hb were similar to those of I-Ek, reported previously (12Boniface J.J. Lyons D.S. Wettstein D.A. Allbritton N.L. Davis M.M. J. Exp. Med. 1996; 183: 119-126Crossref PubMed Scopus (52) Google Scholar), and showed that the secondary structure was not grossly altered as a function of pH (Fig. 1B). For DSC measurements at mildly acidic and neutral pH values, the I-Ek-Hb molecule was first dissolved in phosphate buffer, because of its lower enthalpy change for the deprotonation and its lower temperature dependence (21Fukada H. Takahashi K. Proteins. 1998; 33: 159-166Crossref PubMed Scopus (298) Google Scholar). Fig.2 shows the excess heat capacity curves as a function of pH, at a heating rate of 1 °C per min. All curves were irreversible as shown by the lack of reproduced excess heat capacity in the second scanning, as described below. The small transition ranges of about 10 °C indicated the high cooperativity of this transition. Assuming the two-state transition, the thermodynamic parameters for denaturation of I-Ek-Hb were determined, and are summarized in Table I. It should be noted that the stability at the mildly acidic pH is higher than that at the neutral pH. The correlation between Td and ΔHcal could be classified into two groups, one for the mildly acidic pH and the other for the neutral pH (Fig.3). The heat capacity change (ΔCp), determined from this correlation, is 11.1 kJ mol−1 K−1 for the mildly acidic pH and that of the neutral pH is 15.9 kJ mol−1K−1.Table IThermodynamic parameters for denaturation of I-Ek-Hb as a function of pHpHConcentrationTdΔHcalΔHvHΔHcal/ΔHvHmg ml−1°CkJ mol−15.00.4374.87166821.05.50.3475.47237021.06.00.3374.97186651.16.50.3272.88136521.27.40.3367.27446001.28.00.3362.76515651.2The heating rate was 1.0 °C per min. Open table in a new tab Figure 3Correlation betweenTd and ΔHcal of I-Ek-Hb.The Td and ΔHcal values were taken from Table I. The slopes were determined by the linear least square method for the mildly acidic and neutral pH regions, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The heating rate was 1.0 °C per min. To evaluate the protonation effects on the buffer and the protein (22Baker B.M. Murphy K.P. Biophys. J. 1996; 71: 2049-2055Abstract Full Text PDF PubMed Scopus (275) Google Scholar,23Petrosian S.A. Makhatadze G.I. Protein Sci. 2000; 9: 387-394Crossref PubMed Scopus (30) Google Scholar), the thermodynamic stability of I-Ek-Hb, in a buffer with a large heat of ionization, MOPS, was also analyzed using DSC. Because the pH of MOPS buffer is largely dependent on the temperature (21Fukada H. Takahashi K. Proteins. 1998; 33: 159-166Crossref PubMed Scopus (298) Google Scholar), the pH at the Td of the buffer used in this study was determined and applied to compare the thermodynamics in the phosphate buffer described above. TableII summarizes the thermodynamic parameters obtained in MOPS buffer. The thermal stability at an acidic pH is higher than that at a neutral pH, which is similar to the stability in the phosphate buffer. The ΔHcalvalues in MOPS buffer are relatively smaller than those in phosphate buffer. This difference should be because of the protonation effects on the buffer and the protein.Table IIThermodynamic parameters for denaturation of I-Ek-Hb in MOPS bufferpHpHConcentrationTdΔHcalat 25 °Cat Tdmg ml−1°CkJ mol−16.55.50.3375.26827.06.00.3373.66817.46.50.3370.67947.77.00.5068.5677The heating rate was 1.0 °C per min. Open table in a new tab The heating rate was 1.0 °C per min. To analyze whether the protein concentration used in the present DSC measurements has an effect on the thermal denaturation, mainly because of the irreversibility, the thermodynamic parameters were determined as a function of concentration (Table III). Within the range from 0.08 to 0.33 mg ml−1 at pH 7.4, both Td and ΔH values were independent of the protein concentration.Table IIIThermodynamic parameters for denaturation of I-Ek-Hb at pH 7.4 as a function of protein concentrationConcentrationTdΔHcalΔHvHΔHcal/ΔHvHmg ml−1°CkJ mol−10.0866.97446081.20.1865.97565931.30.333-aData were taken from Table I.67.27446001.2The heating rate was 1.0 °C per min.3-a Data were taken from Table I. Open table in a new tab The heating rate was 1.0 °C per min. To determine the temperature range in which the unfolding of I-Ek-Hb is predominantly irreversible, the heating was stopped at various temperatures and the solution was subsequently cooled to 20 °C, followed by the next heating (Fig.4). These heat capacity curves showed that the irreversible step occurs early in the DSC scans at both pH 5.5 and 7.4. Thus, the calorimetric traces have to be analyzed according to an irreversible model. We carried out DSC experiments at different heating rates, 0.2, 1.0, 1.5, and 2.0 °C per min, at pH 5.5 and 7.4, to analyze the kinetically controlled denaturation of I-Ek-Hb (Fig. 5). The thermodynamic parameters obtained from each transition curve are summarized in Table IV. Whereas theTd value at the rate of 0.2 °C per min was significantly lower than the others, those at the rate over 1 °C per min were similar at both pH values, indicating that the transition temperatures at high heating rates reach their maximum values with little effect from the irreversibility. All of the ΔHcal values obtained with the various heating rates were similar, within experimental error.Figure 5Variation with heating rate of I-Ek-Hb unfolding at pH 5.5 (A) and 7.4 (B). The heating rate is indicated at each transition curve. The protein concentration for the respective measurements was 0.4 mg/ml.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IVThermodynamic parameters for denaturation of I-Ek-Hb at pH 5.5 and 7.4 as a function of heating ratepHHeating rateTdΔHcalΔHvHΔHcal/ΔHvH°C min−1°CkJ mol−15.50.271.67666471.21.075.47237021.01.576.96567270.92.077.86187250.97.40.260.46505711.11.067.27446001.21.567.47055791.22.066.27755561.4The protein concentration was in the range between 0.33 and 0.38 mg ml−1. Open table in a new tab The protein concentration was in the range between 0.33 and 0.38 mg ml−1. Sanchez-Ruiz et al. (17Sanchez-Ruiz J.M. Lopez-Lacomba J.L. Cortijo M. Mateo P.L. Biochemistry. 1988; 27: 1648-1652Crossref PubMed Scopus (457) Google Scholar) proposed four methods to evaluate the activation energy from the excess heat capacity curves, using the scan rate dependence of irreversible denaturation. The graphical presentations of the evaluations according to methods A, B, and C, described under “Experimental Procedures,” are shown in Fig. 6, and the analyzed activation parameters, including method D, are summarized in TableV. Although the activation energies derived from the various methods differed, the average value was larger than the previously reported values for the denaturation of other proteins, which are in the range between 280 and 360 kJ mol−1 (24Vogl T. Jatzke C. Hinz H.J. Benz J. Huber R. Biochemistry. 1997; 36: 1657-1668Crossref PubMed Scopus (79) Google Scholar). The large activation energy suggests that most of the native structure of I-Ek-Hb should be denatured before the irreversible transition occurs, and the thermodynamic parameters for the denaturation of I-Ek-Hb could be analyzed as an equilibrium quantity, as described above. Similar to the activation energy, the T* values were almost independent of the scan rate, and were higher than the Td values, except for those from the scan rate of 2 °C per min (Tables IV andV). Together with the results of the scan rate experiments, this result supports the validity of the approximation to determine the equilibrium thermodynamics from DSC experiments with the scan rate of 1 °C per min.Table VActivation parameters for denaturation of I-Ek-Hb at pH 5.5 and 7.4pHHeating rateT*EaMethod AMethod BMethod CMethod D°C min−1°CkJ mol−15.50.277.04514914361.076.84374834761.577.74925004902.077.1535545489478 ± 44371 ± 23504 ± 27482 ± 547.40.266.13993683851.069.14304204011.568.74854303912.066.6395389375427 ± 41311 ± 81401 ± 28385 ± 10The protein concentration was in the range between 0.33 and 0.38 mg ml−1. Open table in a new tab The protein concentration was in the range between 0.33 and 0.38 mg ml−1. Irreversible thermal denaturation is observed in many proteins, including MHC molecules, which makes it difficult to analyze the precise thermodynamics. In the present study, we found the interesting phenomenon of MHC stability as a function of pH, although the thermal denaturation process was irreversible. The thermodynamic origin of the increased stability at the mildly acidic pH could be because of the dynamic properties of MHC molecules, which are important for the function of MHC class II molecules. To determine the thermodynamics and to evaluate their validity, the effects of the irreversibility should first be analyzed under various conditions. Because the method to extract thermodynamic equilibrium parameters from kinetically controlled heat capacity curves has been applied successfully to several systems (17Sanchez-Ruiz J.M. Lopez-Lacomba J.L. Cortijo M. Mateo P.L. Biochemistry. 1988; 27: 1648-1652Crossref PubMed Scopus (457) Google Scholar, 24Vogl T. Jatzke C. Hinz H.J. Benz J. Huber R. Biochemistry. 1997; 36: 1657-1668Crossref PubMed Scopus (79) Google Scholar, 25Freire E. van Osdol W.W. Mayorga O.L. Sanchez-Ruiz J.M. Annu. Rev. Biophys. Biophys. Chem. 1990; 19: 159-188Crossref PubMed Scopus (208) Google Scholar), we used this method in the present study. The Td and ΔH values were almost independent at a protein concentration of around 0.3 mg ml−1 and a heating rate over of 1 °C per min (TablesIII and IV). Additionally, the large activation energy and the highT* value indicated that the thermal denaturation process under these conditions could be analyzed as the equilibrium thermodynamics, with little interference from the irreversible process (Table V). Therefore, the thermodynamic parameters as a function of pH (Table I), obtained with a heating rate of 1 °C per min and a protein concentration of about 0.3 mg ml−1 are meaningful. In the previous CD analyses of the thermal denaturation of MHC class II molecules, the temperature was increased in a stepwise mode (10Reich Z. Altman J.D. Boniface J.J. Lyons D.S. Kozono H. Ogg G. Morgan C. Davis M.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2495-2500Crossref PubMed Scopus (55) Google Scholar, 11Wilson N. Fremont D. Marrack P. Kappler J. Immunity. 2001; 14: 513-522Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Because it takes a few minutes at each temperature for equilibrium attainment and recording, the Td values should be lower than those obtained with the method of continuous heating used in this study. This is supported by the fact that the stability of I-Ek-Hb at pH 7.4 in the previous CD measurements was similar to that at the heating rate of 0.2 °C per min in this study (Table IV) (11Wilson N. Fremont D. Marrack P. Kappler J. Immunity. 2001; 14: 513-522Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Under the conditions of a stepwise mode or a low heating rate for the analysis of irreversible denaturation, theTd value is largely dependent on the heating rate. In the thermal denaturation process of I-Ek-Hb, three transitions should be involved: 1) peptide dissociation from the MHC class II molecule, 2) dissociation of the αβ heterodimer to each subunit, and 3) denaturation of each subunit. The excess heat capacity curve of I-Ek-Hb should be the sum of these transitions. Although the ratio ΔHcal/ΔHvH under the various conditions of the DSC measurements is around 0.9 to 1.4 (TablesI, III, and IV), we cannot exclude the possibility of the existence of intermediate states and/or the coupling of respective transitions. Because the bound peptide contributes to the stability of MHC molecules (26Simon A. Dosztanyi Z. Rajnavolgyi E. Simon I. Biophys. J. 2000; 79: 2305-2313Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), the peptide dissociation should occur first, to cause the subsequent denaturation. This is also supported by the results that the thermal denaturation profiles of I-Ek in complex with mutant peptides of Hb seemed to be cooperative, similar to that of I-Ek-Hb, although their Td values differed from each other. 2K. Saito, A. Sarai, M. Oda, T. Azuma, and H. Kozono, unpublished results.These results indicate that the apparent stability of the MHC-peptide complex is largely dependent on the binding kinetics and/or the affinity of the bound peptide. The empty MHC class II molecule, I-Ek without the peptide, showed apparent size heterogeneity, including the αβ heterodimer and the high molecular weight aggregates, and the addition of peptide changed this heterogeneous molecule into the homogeneous αβ heterodimer (19Kozono H. White J. Clements J. Marrack P. Kappler J. Nature. 1994; 369: 151-154Crossref PubMed Scopus (268) Google Scholar,27Stern L.J. Wiley D.C. Cell. 1992; 68: 465-477Abstract Full Text PDF PubMed Scopus (286) Google Scholar), indicating that the irreversible transition is followed by the dissociation of each subunit and its denaturation. Increased stability at a mildly acidic pH relative to that at a neutral pH was also observed in a murine MHC class II molecule, I-Ab, in complex with CLIP and the antigenic peptide derived from the α subunit of the I-E molecule, Eα (28Tobita T. Oda M. Morii H. Kuroda M. Yoshino A. Azuma T. Kozono H. Immunol. Lett. 2003; 85: 47-52Crossref PubMed Scopus (23) Google Scholar). In contrast, the thermal stabilities of I-Ad-Eα and I-Ek complexed with the moth cytochrome cpeptide at a mildly acidic pH are similar to those at neutral pH (10Reich Z. Altman J.D. Boniface J.J. Lyons D.S. Kozono H. Ogg G. Morgan C. Davis M.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2495-2500Crossref PubMed Scopus (55) Google Scholar). Despite the stability difference at both pH regions, all of the MHC class II molecules are resistant to a lower pH, that is in contrast to the stability of the MHC class I molecule (10Reich Z. Altman J.D. Boniface J.J. Lyons D.S. Kozono H. Ogg G. Morgan C. Davis M.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2495-2500Crossref PubMed Scopus (55) Google Scholar). The increased stability at a mildly acidic pH of the other MHC class II molecules may also be because of entropic effects, similar to I-Ek-Hb as described below, which generally correlates with their functions in an acidic compartment. In addition to the difference in the Td values, other thermodynamic parameters for denaturation at the mildly acidic pH differed from those at the neutral pH. The correlation ofTd with ΔHcal at various pH values indicated that the folding of I-Ek-Hb could be classified into two groups (Fig. 3), which is consistent with the difference observed in the structural analyses, as described below. The ΔCp values obtained from the slope ofTdversus ΔHcalin phosphate buffer were 11.1 kJ mol−1 K−1for the mildly acidic pH and 15.9 kJ mol−1K−1 for the neutral pH. To compare the obtained values with those of other proteins with a similar size, the empirical method proposed by Oobatake and Ooi (29Oobatake M. Ooi T. Prog. Biophys. Mol. Biol. 1993; 59: 237-284Crossref PubMed Scopus (199) Google Scholar) was applied and the ΔCp value of a 420-residue (NR) protein was calculated to be 30.2 kJ mol−1 K−1 from the following equation.ΔCp(cal mol−1K−1)=−512+18.39NREq. 10 This value, 30.2 kJ mol−1 K−1, is larger than the experimentally determined values of I-Ek-Hb at either pH region, indicating that the surface of non-polar residues of I-Ek-Hb is more accessible to the solvent than that of other proteins. Furthermore, it should be noted that the ΔCp value at the mildly acidic pH is lower than that at the neutral pH. This is consistent with the previous results, in which the acidification changed I-Ek into a more fluctuating state with an increase in the exposed hydrophobicity, like a molten globule state (12Boniface J.J. Lyons D.S. Wettstein D.A. Allbritton N.L. Davis M.M. J. Exp. Med. 1996; 183: 119-126Crossref PubMed Scopus (52) Google Scholar, 13Runnels H.A. Moore J.C. Jensen P.E. J. Exp. Med. 1996; 183: 127-136Crossref PubMed Scopus (56) Google Scholar). The analyses of the protonation effects showed that the ΔHcal values in the buffer with a larger heat of ionization, MOPS, are smaller than those in the buffer with a lower heat of ionization, phosphate, whereas the thermal stability at an acidic pH is higher than that at a neutral pH in both buffers. The difference of the ΔHcal values should be because of the enthalpy of buffer ionization and the linked protonation effects (21Fukada H. Takahashi K. Proteins. 1998; 33: 159-166Crossref PubMed Scopus (298) Google Scholar, 22Baker B.M. Murphy K.P. Biophys. J. 1996; 71: 2049-2055Abstract Full Text PDF PubMed Scopus (275) Google Scholar). Petrosian and Makhatadze (23Petrosian S.A. Makhatadze G.I. Protein Sci. 2000; 9: 387-394Crossref PubMed Scopus (30) Google Scholar) successfully evaluated the contribution of linked protonation effects on the stability of a 69-amino acid residue protein, with consideration of the isoelectric point (pI). In the case of a 420-residue protein, I-Ek-Hb, the pI value in the denatured state is estimated to be 4.8 from the amino acid composition, and that in the native state is estimated to be 5.0 by the isoelectric focusing experiments (data not shown). Amino acid residues such as Asp and His, with charges that are affected at the pH region analyzed in this study, are thought to be involved in the structural and functional characters of MHC class II molecules (16Kersh G.J. Miley M.J. Nelson C.A. Grakoui A. Horvath S. Donermeyer D.L. Kappler J. Allen P.M. Fremont D.H. J. Immunol. 2001; 166: 3345-3354Crossref PubMed Scopus (96) Google Scholar, 30McFarland B.J. Katz J.F. Beeson C. Sant A.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9231-9236Crossref PubMed Scopus (45) Google Scholar, 31Rötzschke O. Lau J.M. Hofstätter M. Falk K. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16946-16950Crossref PubMed Scopus (50) Google Scholar). Therefore, the present thermodynamic results obtained in phosphate buffer, which has lower enthalpy change for the deprotonation, could be the comparable data to analyze the effects of pH on the stability difference of I-Ek-Hb. Assuming the enthalpy derived from linked protonation effects is compensated by the large ionization enthalpy of MOPS buffer, as observed in the CspA unfolding (23Petrosian S.A. Makhatadze G.I. Protein Sci. 2000; 9: 387-394Crossref PubMed Scopus (30) Google Scholar), the conformational enthalpy change can be estimated to be about 700 kJ/mol. Still on this assumption, the increased stability of I-Ek-Hb at acidic pH is because of a favorable ΔS, as described below. To analyze the thermodynamic origin of the stability difference, the thermodynamic parameters at 75.4 °C, the denaturation temperature at pH 5.5, were calculated using the correlation of ΔG and ΔH with temperature (TableVI). Within the narrow range of temperature around the Td values, the errors of the calculated ΔG and ΔH values at the reference temperature should be small, even if the ΔCpvalues used contain some errors, because of the temperature dependence and the linked protonation effects. This result clearly indicates that the higher stability at the mildly acidic pH than that at the neutral pH is because of the difference in the entropic contributions. One possible explanation for this entropic difference as a function of pH is that the native structure of I-Ek-Hb at the mildly acidic pH is more flexible, which can facilitate the peptide exchange. This is consistent with the previous SDS-PAGE and structural analyses, in which I-Ek gained flexibility at low pH (11Wilson N. Fremont D. Marrack P. Kappler J. Immunity. 2001; 14: 513-522Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 14Sadegh-Nasseri S. Germain R.N. Nature. 1991; 353: 167-170Crossref PubMed Scopus (275) Google Scholar). The smaller ΔCp value of I-Ek-Hb at low pH, as described above, should also be because of this increased flexibility. Although the difference in the secondary and tertiary structures between the mildly acidic and neutral pH values detected in the CD measurements is subtle (Fig. 1B), similar to the previous reports (12Boniface J.J. Lyons D.S. Wettstein D.A. Allbritton N.L. Davis M.M. J. Exp. Med. 1996; 183: 119-126Crossref PubMed Scopus (52) Google Scholar, 32Lee J.M. Kay C.M. Watts T.H. Int. Immunol. 1992; 4: 889-897Crossref PubMed Scopus (23) Google Scholar), the molecule will be more dynamically fluctuating and the surface of non-polar residues will be more accessible to solvent at a mildly acidic pH.Table VIThermodynamic parameters for denaturation of I-Ek-Hb in phosphate buffer at the denaturation temperature at pH 5.5pHTd6-aTd values were taken from Table I.ΔTdΔHTΔS6-bTΔS values were calculated from the equation, ΔG(T) = ΔH(T) −TΔS(T).ΔG°CkJ mol−15.575.472372307.467.2−8.2874885−116-a Td values were taken from Table I.6-b TΔS values were calculated from the equation, ΔG(T) = ΔH(T) −TΔS(T). Open table in a new tab The kinetic and structural analyses of I-Ek-Hb have shown that the hydrogen bonding network, formed by the cluster of carboxylate groups around the P6 pocket, Asp66 and Glu11 of the α subunit, Asp73 of the Hb peptide, and water molecules, changes as a function of pH, which can regulate the conformation of I-Ek and the peptide exchange rate (11Wilson N. Fremont D. Marrack P. Kappler J. Immunity. 2001; 14: 513-522Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The protonation of these carboxylate groups at low pH, together with other residues such as His81 of the β subunit, facilitates the flexibility and the conformational change of the MHC class II molecule from the closed to the open form (30McFarland B.J. Katz J.F. Beeson C. Sant A.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9231-9236Crossref PubMed Scopus (45) Google Scholar). In addition, the recent mutational analyses of HLA-DR, the human homologue of I-Ek, have shown that His33 of the α subunit has the role of a pH-sensitive switch at low pH, which can regulate the conformational transition and the peptide exchange (31Rötzschke O. Lau J.M. Hofstätter M. Falk K. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16946-16950Crossref PubMed Scopus (50) Google Scholar). The decreased ΔH, TΔS, and ΔCp values for the thermal denaturation of I-Ek-Hb at the mildly acidic pH relative to those at the neutral pH should be because of the differences in the dynamic properties, which are closely related with the function of MHC class II molecules. The present DSC analyses provide thermodynamic insight into the increased stability at the acidic pH. We thank Dr. Atsuko Yoshino and Dr. Koji Furukawa, Tokyo University of Science, Dr. Haruki Nakamura, Osaka University, Dr. Hatsuho Uedaira, RIKEN, Dr. Shun-ichi Kidokoro, Nagaoka University of Technology, and Dr. Harumi Fukada, University of Osaka Prefecture, for technical support and helpful discussions. We gratefully acknowledge Dr. Motohisa Oobatake, who died in August 2002, and pay tribute to a great scientist.
Blockade of immune checkpoint receptors has shown outstanding efficacy for tumor immunotherapy. Promising treatment with anti-lymphocyte-activation gene-3 (LAG-3) has already been recognized as the next efficacious treatment, but there is still limited understanding of the mechanism of LAG-3-mediated immune suppression. Here, utilizing high-resolution molecular imaging, we find a mechanism of CD4 T cell suppression via LAG-3, in which LAG-3-bound major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) gather at the central region of an immunological synapse and are trans-endocytosed by T cell receptor-driven internalization motility toward CD4 and CD8 T cells expressing LAG-3. Downregulation of MHC class II molecules on APCs thus results in the attenuation of their antigen-presentation function and impairment of CD4 T cell activation. From these data, anti-LAG-3 treatment is suggested to have potency to directly block the inhibitory signaling via LAG-3 and simultaneously reduce MHC class II expression on APCs by LAG-3-mediated trans-endocytosis for recovery from T cell exhaustion.
Escherichia coli B/SM, strain 1-1, was killed dose dependently by human hereditary C9-deficient serum (C9DHS), which was shown to contain no C9 Ag by an ELISA method. On the other hand, human hereditary C7-deficient serum did not kill the bacteria under similar conditions. The bactericidal activity of C9DHS was inhibited by rabbit anti-C5 antibody but not by murine anti-C9 mAb. The anti-C9 antibody decreased the bactericidal activity of normal human serum (NHS) to the level of that with C9DHS. Sheep anti-human lysozyme antibody did not affect the bactericidal activity of C9DHS or NHS even when added at more than twice the concentration required to block the serum lysozyme activity on Micrococcus luteus. After treatment with C9DHS and washing, surviving Escherichia coli were killed by C9, but not by lysozyme, transferrin, or both. Other strains of E. coli (K12 W3110, C600, and NIHJ) and Salmonella typhimurium (strain NCTC 74), all maintained in the laboratory, were also killed by C9DHS. However, pathogenic strains recently isolated from patients with traveler's diarrhea and some strains of S. typhimurium were resistant to both C9DHS and NHS, at least at the serum concentration tested. A concentration of 0.1 M Tris did not increase the susceptibility of serum-resistant strains of bacteria to C9DHS, but made one strain of S. typhimurium tested susceptible to NHS, but not to C9DHS. These results clearly showed that C9DHS kills bacteria that are sensitive to NHS through activation of C up to the step of C8 in the same way that C9-deficient C serum lyzed sensitized erythrocytes.
Ubiquitination is a process that dictates the lifespan of major histocompatibility complex class II (MHC II)/peptide complexes on antigen-presenting cells. This process is tightly controlled by the levels of ubiquitin ligases, and disruptions in the turnover of MHC II can lead to the improper development of CD4+ T cells within the thymus and hinder the formation of regulatory T cells in the peripheral tissue. To investigate the underlying mechanisms, we utilized dendritic cells lacking the Membrane-associated RING-CH (MARCH) I ubiquitin ligase. We discovered that the overexpression of MARCH I decreases the interaction with LAG-3. Moreover, the MHC II molecules tethered with ubiquitin also showed diminished binding to LAG-3. We employed Diffracted X-ray Blinking (DXB), a technique used for single-molecule X-ray imaging, to observe the protein movements on live cells in real time. Our observations indicated that the normal MHC II molecules moved more rapidly across the cell surface compared to those on the MARCH I-deficient dendritic cells or MHC II KR mutants, which is likely a result of ubiquitination. These findings suggest that the signaling from ubiquitinated MHC II to the T cell receptor differs from the non-ubiquitinated forms. It appears that ubiquitinated MHC II might not be quickly internalized, but rather presents antigens to the T cells, leading to a range of significant immunological responses.
Mouse liver RNA analyzed by Northern blotting with a full-length complement factor H cDNA probe demonstrates the 4.4-kilobase (kb) H mRNA as well as three additional hybridizing species of 3.5, 2.8, and 1.8 kb, respectively. Further characterization of these alternative transcripts was pursued by isolation of additional cDNAs from a liver library using a full-length H probe. Twelve clones homologous to but distinct from H were isolated, analyzed by restriction mapping, and divided into four classes, A, B, C, and D, based on their sequences. Clones from classes A, B, and C all contained nearly identical 5'-untranslated regions and leader sequences that differed from H at more than 50% of their nucleotide positions. The 5'-untranslated and leader sequences of the class D clone were unrelated to the corresponding regions of H or the class A, B, or C clones. The remaining portions of the H-related cDNAs were made up of short consensus repeats, 7 in class A, 4 in class B, 13 in class C, and 5 in class D. To determine the relationship between the H-related transcripts and the cDNA clones, Northern blots of liver RNA were analyzed by hybridization with two probes, one specific for the class D cDNA and the other reacting specifically with the class A, B, and C cDNAs. The class A/B/C probe detected transcripts of 3.5, 2.8, and 1.8 kb in liver RNA, and the class D probe hybridized to a distinct 1.8-kb message. Additionally, a cosmid genomic library was screened with H cDNA, and nine H-related clones were isolated. They spanned a region of approximately 120 kb, defining at least two discrete H-related gene loci. These results identify new members of the super-family of C3b/C4b binding protein genes.
To determine the energetic contribution of the hydrogen bond between betaHis81 of the major histocompatibility complex class II (MHC II) molecule, I-E(k), and the bound hemoglobin peptide (Hb), we analyzed the thermal stability of the hydrogen bond-disrupted mutant, I-E(k)-Hb betaH81Y, in which the betaHis81 residue was replaced with Tyr, by differential scanning calorimetry. The thermal stability of the I-E(k)-Hb betaH81Y mutant was lower than that of the I-E(k)-Hb wild-type, mainly due to the decreased enthalpy change. The difference in the denaturation temperature of the I-E(k)-Hb betaH81Y mutant as compared with that of the I-E(k)-Hb wild-type at pH 5.5 was only slightly smaller than that at pH 7.4, in agreement with the increased stability at an acidic pH, a unique characteristic of MHC II. Thus, the hydrogen bond contributed by betaHis81 is critical for peptide binding, and is independent of pH, which can alter the hydrophilicity of the His residue.
