| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
2 Veterans Administration Medical Center, Buffalo, New York 14215–1129, USA
Reprint requests to: R.M. Epand, Department of Biochemistry, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5; e-mail: epand{at}mcmaster.ca; fax: 905–521–1397.
(RECEIVED November 20, 2000; FINAL REVISION February 26, 2001; ACCEPTED April 13, 2001)
Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ps.48601.
 |
Abstract |
|---|
 TOP Abstract IntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Keywords: ATP; DSC; thermal denaturation; Trp fluorescence; acrylamide quenching
Abbreviations: DSC, differential scanning calorimetry • GLUT 1, the predominant isoform of the passive glucose transporter found in erythrocytes • 
HvH, van't Hoff enthalpy
|
Introduction |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Studies of thermal denaturation of the protein can provide information about the conformational properties of proteins. A technique that is useful for measuring the thermal denaturation of proteins is differential scanning calorimetry (DSC). We recently reported on the DSC of GLUT 1 and the stabilization of its conformation by glucose (Epand et al. 1999). The presence of 500 mM D-glucose increased by 4°C the thermal denaturation temperature of GLUT 1 reconstituted in vesicles of erythrocyte lipids, while 500 mM L-glucose and 10 µM cytochalasin B each had no significant effect. The calorimetric enthalpy, 150 kcal/mol, was found to be independent of the presence of D-glucose and is comparable to values obtained with other membrane proteins. The van't Hoff enthalpy and the calorimetric enthalpy agree within the experimental error, and therefore the transition is not likely to be cooperative. The presence of 500 mM D-glucose also decreased the activation energy, indicating that the higher denaturation temperature was not the result of increased kinetic stability.
The activity of the glucose transporter is increased in response to hypoxia and other increased metabolic demands (Wood and Morgan 1969; Ismail-Beigi 1993). The regulation may be through the binding of an allosteric ligand. Ligands which affect the binding of glucose as well as the functioning of GLUT 1 include ATP (Diamond and Carruthers 1993; Levine et al. 1998; Heard et al. 2000), ADP (Sofue et al. 1993) and adenosine (M. Lachaal et al., 2001). ATP required low pH to inhibit cytochalasin B binding (M. Lachaal et al., in prep.). In addition, it was recently shown that tricyclic antidepressants bind to GLUT 1 and block the activity of the transporter (Pinkofsky et al. 2000). Tricyclic antidepressants may bind to the same site on GLUT 1 as does ATP, since both ligands are aromatic. Loss of ATP production with mitochondrial uncoupling agents leads to a rapid stimulation of glucose transport (Khayat et al. 1998). The region comprised of residues 301–364 of GLUT 1 has been shown to bind ATP on the basis of azido-ATP photo labeling and peptide mapping (Levine et al. 1998). Within this region, residues 332–343 with the sequence GRRTIHLIGLAG, which lie at the interface between the fourth cytoplasmic loop and the putative ninth transmembrane segment, are homologous to sequences in other ATP-binding proteins. This segment has been suggested to be the ATP binding site (Levine et al. 1998). In addition, a search for a known ATP/GTP binding site motif within GLUT 1, using the GenomeNet Motif Search, allowed us to identify residues 111 to 118 of GLUT 1 as a possible additional putative ATP binding site, with the sequence GFSKLGKS. These residues also lie at the interface between the putative third transmembrane segment and the second extracellular loop (Fig. 1
). These two putative ATP binding sites in the GLUT 1 molecule fall at the interface of the two outermost packed transmembrane helices (third and ninth) relative to those which line the channel, according to a model recently proposed by Hruz and Mueckler (2000) and also suggested from molecular modeling studies (Zeng et al. 1996). This contrasts with the positioning of those helices currently identified as participating in the glucose permeation pathway, which line the interior of an aqueous channel.
|
Malignant cells have been known to exhibit increased rates of glycolysis and glucose uptake. Reports on the overexpression of GLUT 1 in several human cancers (Younes et al., 1996), including thyroid cancer (Haber et al., 1997) led to GLUT 1 becoming clinically useful as a marker in human cancers. More recently, overexpression of GLUT 1 was found in endometrial adenocarcinomas (Wang et al. 2000) and in patients with colorectal carcinoma associated with poor prognosis (Haber et al. 1998). This highlights the clinical importance of this protein and its possible role in promoting nutrient uptake by tumors.
Crystallographic data of transmembrane proteins is generally not available due to the difficulty of obtaining good diffraction quality crystals. To obtain information about the tertiary structure of GLUT 1, several different approaches, including molecular modeling (Zeng et al., 1996), chemical modification, and cysteine scanning mutagenesis have been used (Hruz and Mueckler 1999; Mueckler and Makepeace 1999; Hruz and Mueckler 2000).
Here we studied, using DSC and fluorescence quenching, the effects that ATP binding has on the conformational stability of GLUT 1, a membrane protein for which limited structural information has thus far been obtained.
|
Results |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
) or 0.4 M NaCl at pH 7.4 (not shown). We previously showed by DSC that the thermal denaturation temperature of GLUT 1 is increased by 4°C in the presence of 0.5 M D-glucose at pH 7.4 (Epand et al. 1999). The addition of 4.8 mM ATP at acidic pH, however, shifts the transition temperature (Tm) dramatically (Table 1), lowering it by 12°C. Although the transition of GLUT 1 is irreversible, such protein denaturation transitions are frequently analyzed assuming that there is a rapid, reversible step, followed by an irreversible transition. Although the transition temperature was not invariant with the scan rate, we have used this assumption to obtain an estimate of the calorimetric transition enthalpy (Hcal).
|
). From the scan rate dependence, the value of the van't Hoff enthalpy (HvH) is calculated to be 99 ± 10 kcal/mol, and the value of the Arrhenius energy of activation of thermal unfolding, E, is calculated as 67 ± 7 kcal/mol. The addition of 0.4 M NaCl was used to shield electrostatic interactions. As with the addition of ligands, the mixture was taken through three cycles of freezing and thawing (see the Materials and Methods section). In the case of the addition of NaCl, the freeze/thaw cycles will dissipate any osmotic gradient. We found that 0.4 M NaCl does not significantly affect the denaturation temperature of GLUT 1 at pH 4.3, but it has the effect of partially reversing the destabilization introduced by ATP at pH 4.3 (Fig. 3). This indicates that at least part of the ATP binding to GLUT 1 is electrostatic in nature. The addition of 0.5 M D-glucose to 4.8mM ATP at pH 4.3 also partly reverses the effect of ATP (Fig. 3). Thus glucose stabilizes the protein in the presence of ATP, as well as in its absence (Epand et al. 1999).
|
|
Hvh and of E (Table 1
). The fits we obtained to the experimental curves in a particular analysis were highly precise, to within about 1%. However, because of the broadness of the transitions, uncertainties about the correct choice of baseline lead to a more realistic estimate of about 15% for the uncertainty. Nevertheless, the values given above for estimates made from the scan rate dependence agree quite well with those given in Table 1, which were calculated from the shape of the DSC curve. This provides support for the validity of the values we have obtained using these analyses. In general, the activation energy at pH 4.3 is not greatly affected by the presence of ligands or salt, except for the case of GLUT in the presence of ATP and D-glucose being somewhat higher (Table 1). This suggests that the observed shifts in denaturation temperature at pH 4.3 are generally a result of a thermodynamic effect rather than a kinetic effect, with the possible exception of the reversal of the ATP effect by glucose. In addition, the Hvh is somewhat greater than the Hcal, suggesting that at pH 4.3 there is some cooperativity of the transition.
Although no Trp residues are found in the immediate vicinity of the putative ATP binding sites, the compactness of folding of the protein can be assessed by measuring the efficiency of quenching of the Trp residues with acrylamide (Fig. 4). There are six Trp residues in GLUT 1. Only one of these is near the center of a transmembrane helix; three are near the membrane-water interface, and the remaining two are in extramembranous loop regions. A lower Stern-Volmer quenching constant would correspond to a more tightly folded molecule, assuming that changes in quenching efficiency are due to a change in the frequency of collisions with the quencher, rather than to a change in fluorophore lifetime. The constants for acrylamide quenching of GLUT 1 at pH 7.4 in a variety of conditions are summarized in Table 2. The quenching studies could not be done at acidic pH because of the presence of high background fluorescence from the protonated form of ATP.
|
|
|
Discussion |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
There is evidence that ATP binds to GLUT 1 at neutral pH and that it prevents the binding of glucose (Heard et al. 2000). However, the binding of ATP at neutral pH does not lead to a change in the parameters of denaturation as monitored by DSC (Table 1). Similarly, ATP at pH 7.4 does not affect cytochalasin B binding (M. Lachaal et al., in prep.). It should be noted that Heard et al. (2000) prepared GLUT 1 in the absence of reducing agent, whereas the preparation used in the present study was made in the presence of mercaptoethanol. It has been suggested that the binding of ATP to GLUT 1 is lost in the presence of reducing agents. However, our acrylamide quenching results (Table 2) indicate that ATP is able to interact with GLUT 1 even at neutral pH, loosening the structure of the protein. This interaction appears without a significant change in the denaturation temperature at pH 7.4. Glucose and ATP appear to have opposite effects on the stability of GLUT 1. While higher concentrations of ATP slightly increase the quenching constant, glucose has the opposite effect of lowering the amount of quenching of the Trp residues of GLUT 1. We have no explanation for the observation that there is slightly lower quenching at 0.5 mM ATP. When D-glucose is added together with ATP, however, the stabilizing effect of D-glucose predominates (Table 2). We also see this for the unfolding of GLUT 1 at low pH, where glucose and ATP have opposing effects, counterbalancing each other (Fig. 3).
Unlike the results at neutral pH, ATP has a marked effect in lowering the denaturation temperature of GLUT 1 at low pH, as measured by DSC. At pH 4.3, 4.8 mM ATP lowers the denaturation temperature by over 10°C (Table 1). This drastic lowering of the transition temperature requires both low pH and the presence of ATP. Neither at pH 4.3 in the absence of ATP nor at pH 7.4 in the presence of ATP is the transition temperature substantially affected. In the presence of 4.8 mM ATP, there is a gradual reduction in the transition temperature of GLUT 1 when the pH is lowered, with the denaturation temperature reaching a value of 53°C at pH 4.3. The pH required for half-maximal reduction of the transition temperature is around 5.
The greater effect of ATP in lowering the denaturation temperature of GLUT 1 at low pH suggests that ATP binds more strongly to the low-pH form of GLUT 1. This would not be unexpected, since the loosening of the protein structure by acidification would provide more access of ATP to the interfacial region where the ATP binding site is thought to exist. However, there are indications that ATP also binds to GLUT 1 at neutral pH. One would expect that also at neutral pH, the ATP would bind more strongly to an at least partly unfolded form of the protein. This is supported by our observation that ATP increases acrylamide quenching at neutral pH. The lack of effect on the denaturation temperature at this pH would indicate that the structure is sufficiently stable that there is no substantial shift in the denaturation temperature.
While the DSC results suggest that both low pH and the presence of ATP are required to lower the stability of GLUT 1, the acrylamide quenching results indicate the ATP is capable of loosening the structure of GLUT 1 at neutral pH. Most of the Trp residues are in the loop regions or in the membrane interface. It is possible that at neutral pH, ATP binds to GLUT 1 at the membrane interface, locally destabilizing the protein, resulting in the Trp residues being more easily quenched by acrylamide, without causing a gross destabilization of the molecule which would result in a changed denaturation temperature. At lower pH, changes in electrostatic interactions caused by changes in the state of protonation of several groups lead to a loss in the overall thermal stability of the protein when ATP is bound. A high NaCl concentration of 0.4 M has no effect on the transition temperature of GLUT 1. However, this salt concentration partially reverses the effect of ATP in lowering the denaturation temperature of GLUT 1, suggesting that the interaction between GLUT 1 and ATP must, at least in part, be electrostatic.
As we have previously noted (Epand et al. 1999), the enthalpy of denaturation of GLUT 1 is lower than that for globular proteins on a weight basis. This observation is in agreement with the behavior of other integral membrane proteins and is likely a consequence of the transmembrane helices not completely unfolding during the thermal denaturation. Thus, an effect of ligands on the denaturation temperature is probably a consequence of changes in the conformational stability of the loop regions. There are several charged residues in the loop regions, including two cationic Lys residues in the exofacial ATP binding motif and three Arg residues in the cytoplasmic motif. It is likely that a component of the binding of ATP to this site involves electrostatic interaction between these cationic residues and the negative charges on the phosphates of ATP. The changes in the electrostatic interactions among groups in the loop region appear to lead to a destabilization of the protein and a loosening of its structure. The binding of glucose, probably to sites that facilitate its diffusion across the membrane, requires that the protein be folded in its native conformation. The binding of glucose thus stabilizes the native structure and raises the denaturation temperature. In contrast, ATP may bind less specifically. Part of the force for the binding of ATP is the electrostatic interaction between the cationic amino acid residues and the negative charge on ATP. If these cationic residues become more accessible in the denatured state, it would explain the observation that ATP binds more strongly to the denatured form of the protein, with the resulting lowering of the transition temperature. Hence both low pH and ATP are required for the destabilization of GLUT 1.
|
Materials and methods |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Differential scanning calorimetry (DSC)
A Nanocal instrument from Calorimetry Sciences was used for all runs. The reference cell of the calorimeter contained an identical buffer with all other components except for GLUT 1, at the same pH. All runs contained 0.8 mg/mL GLUT 1 in 10 mM Tris buffer, adjusted to the desired pH with small amounts of dilute HCl. Three cycles of freezing and thawing were done on all samples before the samples were loaded into the calorimeter. In our reconstituted preparation, both cytoplasmic and extracellular putative binding sites are accessible after freeze-thawing. ATP is supplied as the disodium salt and will thus acidify solutions that are not sufficiently buffered. Carefully controlled pH conditions are required with ATP binding studies.
DSC data analysis
All scans were analyzed with the software program Origin version 5.0 to obtain the transition temperature (Tm) and the calorimetric enthalpy (Hcal). A modified version of Origin v2.9, developed for irreversible transitions (Lepock et al. 1992), was used to obtain the Arrhenius energy of activation of thermal unfolding, E, calculated from the shape of the DSC denaturation transition. Estimates for E and HvH were also obtained, when applicable, for the variation of Tm with scan rate, as described previously (Sanchez-Ruiz et al. 1988; Morin et al. 1990; Epand et al. 1999).
Fluorescence measurements
Fluorescence was carried out in an SLM Aminco-Bowman Series 2 Luminescence Spectrometer, equipped with stirring and temperature control. All spectra were corrected for instrumental factors. Excitation and emission monochromator slits were set for a spectral bandwidth of 4 nm.
A suspension of GLUT 1-containing membranes (12.6 µL of a 2 mg protein/mL solution) was placed in 2 mL of 10 mM Tris pH 7.4 (final concentration of protein, 0.23 µM). The pH was adjusted to the desired value with small aliquots of dilute HCl, and the samples were subjected to two cycles of freezing and thawing. The sample was then transferred to a quartz cuvette and placed in the fluorimeter with magnetic stirring and temperature control. Measurements were made at 25°C, with excitation at 295 nm. Emission spectra were recorded between 305 nm and 450 nm. This procedure was repeated after the addition of specific concentrations of D-glucose and/or ATP. All runs were done in duplicate and averaged. Spectra were corrected for instrumental factors. Corrections for inner filter effects were made where required, based on the absorption spectra.
To a suspension of GLUT 1, made as described above, aliquots of 4 µL each of a 5 mM acrylamide solution were added successively. The mixture was subjected to two cycles of freezing and thawing after each addition of acrylamide solution, so as to assure penetration of the quencher into the entire sample. The fluorescence spectrum was subsequently recorded. Fluorescence emission spectra were measured at 25°C using a quartz cuvette with stirring and temperature control. The excitation monochromator was set at 295 nm. The procedure was repeated in the presence of specific concentrations of D-glucose or ATP. Instrument corrections were applied before constructing Stern-Volmer plots (Lehrer 1971), following the equation:
 |
All runs were done in duplicate and averaged. Linear regression values were obtained with the program Graphpad Prism version 3.0.
|
Acknowledgments |
|---|
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
|
References |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Diamond, D.L. and Carruthers, A. 1993. Metabolic control of sugar transport by derepression of cell surface glucose transporters. An insulin-independent recruitment-independent mechanism of regulation. J. Biol. Chem. 268: 6437–6444.
Epand, R.F., Epand, R.M., and Jung, C.Y. 1999. Glucose-induced thermal stabilization of the native conformation of GLUT 1. Biochemistry 38: 454–458.[Medline]
Haber, R.S., Rathan, A., Weiser, K.R., Pritsker, A., Itzkowitz, S.H., Bodian, C., Slater, G., Weiss, A., and Burstein, D.E. 1998. GLUT1 glucose transporter expression in colorectal carcinoma: A marker for poor prognosis. Cancer 83: 34–40.[CrossRef][Medline]
Haber, R.S., Weiser, K.R., Pritsker, A., Reder, I., and Burstein, D.E. 1997. GLUT1 glucose transporter expression in benign and malignant thyroid nodules. Thyroid 7: 363–367.[Medline]
Heard, K.S., Fidyk, N., and Carruthers, A. 2000. ATP-dependent substrate occlusion by the human erythrocyte sugar transporter. Biochemistry 39: 3005–3014.[CrossRef][Medline]
Hruz, P.W. and Mueckler, M.M. 1999. Cysteine-scanning mutagenesis of transmembrane segment 7 of the GLUT1 glucose transporter. J. Biol. Chem. 274: 36176–36180.
Hruz, P.W. and Mueckler, M.M. 2000. Cysteine-scanning mutagenesis of transmembrane segment 11 of the GLUT1 facilitative glucose transporter. Biochemistry 39: 9367–9372.[CrossRef][Medline]
Hu, X.J., Peng, F., Zhou, H.Q., Zhang, Z.H., Cheng, W.Y., and Feng, H.F. 2000. The abnormality of glucose transporter in the erythrocyte membrane of Chinese type 2 diabetic patients. Biochim. Biophys. Acta 1466: 306–314.[Medline]
Ismail-Beigi, F. 1993. Metabolic regulation of glucose transport. J. Membr. Biol. 135: 1–10.[Medline]
Kasahara, T. and Kasahara, M. 1998. Tryptophan 388 in putative transmembrane segment 10 of the rat glucose transporter Glut1 is essential for glucose transport. J. Biol. Chem. 273: 29113–29117.
Khayat, Z.A., Tsakiridis, T., Ueyama, A., Somwar, R., Ebina, Y., and Klip, A. 1998. Rapid stimulation of glucose transport by mitochondrial uncoupling depends in part on cytosolic Ca2+ and cPKC. Am. J. Physiol 275: C1487–C1497.
Lachaal, M., Rampal, A.L., Lee, W., Shi,Y., and Jung, C.Y. 1996. GLUT1 transmembrane glucose pathway. Affinity labeling with a transportable D-glucose diazirine. J. Biol. Chem. 271: 5225–5230.
Lachaal, M., Spangler, R.A., and Jung, C.Y. 2001. Adenosine and adenosine triphosphate modulate the substrate binding affinity of glucose transporter GLUT 1 in vitro. Biochim. Biophys. Acta 1511: 123–133.[Medline]
Lehrer, S.S. 1971. Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry 10: 3254–3263.[CrossRef][Medline]
Lepock, J.R., Ritchie, K.P., Kolios, M.C., Rodahl, A.M., Heinz, K.A., and Kruuv, J. 1992. Influence of transition rates and scan rate on kinetic simulations of differential scanning calorimetry profiles of reversible and irreversible protein denaturation. Biochemistry 31: 12706–12712.[CrossRef][Medline]
Levine, K.B., Cloherty, E.K., Fidyk, N.J., and Carruthers, A. 1998. Structural and physiologic determinants of human erythrocyte sugar transport regulation by adenosine triphosphate. Biochemistry 37: 12221–12232.[CrossRef][Medline]
Morin, P.E., Diggs, D., and Freire, E. 1990. Thermal stability of membrane-reconstituted yeast cytochrome c oxidase. Biochemistry 29: 781–788.[CrossRef][Medline]
Mueckler, M. 1994. Facilitative glucose transporters. Eur. J. Biochem. 219: 713–725.[Medline]
Mueckler, M. and Makepeace, C. 1999. Transmembrane segment 5 of the Glut1 glucose transporter is an amphipathic helix that forms part of the sugar permeation pathway. J. Biol. Chem. 274: 10923–10926.
Nelson, D.L. and Cox, M.M. 2000. Lehninger principles of biochemistry. Worth Publishers, New York.
Pinkofsky, H.B., Dwyer,D.S., and Bradley, R.J,. 2000. The inhibition of GLUT1 glucose transport and cytochalasin B binding activity by tricyclic antidepressants. Life Sci. 66: 271–278.[CrossRef][Medline]
Sanchez-Ruiz, J.M., Lopez-Lacomba, J.L., Cortijo, M, and Mateo, P.L. 1988. Differential scanning calorimetry of the irreversible thermal denaturation of thermolysin. Biochemistry 27: 1648–1652.[CrossRef][Medline]
Sofue, M., Yoshimura, Y., Nishida, M., and Kawada, J. 1993. ADP modifies the function of the glucose transporter: Studies with reconstituted liposomes. Biochem. J. 292 (Pt 3): 877–881.
Wang, B.Y., Kalir, T., Sabo, E., Sherman, S.E., Cohen, C., and Burstein, DE. 2000. Immunohistochemical staining of GLUT1 in benign, hyperplastic, and malignant endometrial epithelia. Cancer 88: 2774–2781.[Medline]
Wood, R.E. and Morgan, H.E. 1969. Regulation of sugar transport in avian erythrocytes. J. Biol. Chem. 244: 1451–1460.
Younes, M., Lechago, L.V., Somoano, J.R., Mosharaf, M., and Lechago, L. 1996. Wide expression of the human erythrocyte glucose transporter Glut1 in human cancers. Cancer Res. 56: 1164–1167.
Zeng, H., Parthasarathy, R., Rampal, A.L., and Jung, C.Y. 1996. Proposed structure of putative glucose channel in GLUT1 facilitative glucose transporter. Biophys. J. 70: 14–21.
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
