- Email: [email protected]
GLYCOSAMINOGLYCANS ALTER THE CONFORMATION OF INTERFERON-GAMMA
doi:10.1006/cyto.1999.0592, available online at http://www.idealibrary.com on
GLYCOSAMINOGLYCANS ALTER THE CONFORMATION OF INTERFERON-GAMMA Vandana Balasubramanian, Murali Ramanathan Interferon- (IFN-) is a potent immunomodulatory cytokine whose physiological roles are modulated by the extracellular matrix. Here, circular dichroism and fluorescence spectroscopic techniques are used to demonstrate that low molecular weight heparin and chondroitin sulfate cause significant changes in secondary and tertiary structure of IFN-. The results suggest that heparin and chondroitin sulfate modulate IFN- activity by causing structural changes in the IFN- dimer. 2000 Academic Press
A number of cytokines and growth factors, e.g. interleukins 1, 2, 3, 4, 6, fibroblast growth factor, vascular endothelial growth factor, and the chemokines, bind to the extracellular matrix and these cytokine-extracellular matrix interactions may play important roles in the regulation of individual cytokine activities and cytokine networks. The focus of this report is interferon- (IFN-), a potent, pleiotropic, pro-inflammatory cytokine produced by activated T cells and natural killer cells that binds to extracellular matrix components and to cell surface heparan sulfate with high affinity. IFN- is a particularly relevant model for the regulation of cytokine activity by the extracellular matrix because it is disease-promoting in a variety of autoimmune disorders such as multiple sclerosis and septic shock. Heparan sulfate is a potent antagonist of IFN- activity in a variety of cell types including glioblastoma and endothelial cells.1,2 It inhibits the induction of class II MHC molecules, a signature activity of IFN-, and antagonizes the upregulation of ICAM-1 in human umbilical vein endothelial cells (HUVEC)3 and indoleamine 2,3 dioxygenase in glioblastoma cells.1 The extracellular matrix appears to modulate IFN- effects via multiple mechanisms. For example, extracellular matrix components have been hypothFrom the Department of Pharmaceutics, State University of New York at Buffalo, Buffalo, NY 14260-1200, USA Correspondence to: Murali Ramanathan, Department of Pharmaceutics, 543 Cooke Hall, State University of New York at Buffalo, Buffalo, NY 14260-1200, USA; E-mail: [email protected]ffalo.edu Received 10 March 1999; received in revised form 2 June 1999; accepted for publication 27 July 1999 2000 Academic Press 1043–4666/00/050466+06 $35.00/0 KEY WORDS: extracellular matrix/circular dichroism/fluorescence spectroscopy/heparin/chondroitin sulfate/IFN-gamma 466
esized to be involved in storing IFN- thereby providing a pool of cytokine locally.4 The modulation of the cellular response to IFN- by heparin is also a result of the competitive inhibition of receptor binding because the heparin-binding regions of IFN- are also involved in increasing the on-rate for the IFN-–IFN- receptor interaction.5 Pharmacokinetic and pharmacodynamic factors may also be involved. In rats, the half-life of recombinant human IFN- increases from a control value of 1.1 min to 99 min when co-administered with 2000 units/kg heparin.6 The proteolytic degradation pattern of the IFN- carboxyl tail is limited to less than ten amino acid residues in the presence of heparin and this in turn, contributes to a substantial increase in IFN- activity.6 Chondroitin-sulfate proteoglycans also immobilize IFN- in the extracellular matrix and enhance the cellular response to IFN-.7 IFN- binds to basement membrane heparan sulfate with nanomolar dissociation constants4 principally via the KRKR sequence in its carboxyl terminal tail.8 The heparin-like domains of heparan sulfate are involved in binding IFN-9 and the glycosaminoglycan carboxylic groups are necessary for IFN- recognition. However, the N-sulfate groups are dispensable since N-desulfation of heparan sulfate followed by acetylation does not affect binding.9 The IFN- protected region or footprint of heparan sulfate is about 10 Kd long and contains N-acetylated and glucuronic acidrich residues flanked by small N-sulfated oligosaccharides. The data are consistent with a model in which heparin links the two carboxyl tails of the IFN- dimer.10 Although conformational changes in IFN- have been postulated as a potential mechanism for the modulation of IFN- activity by the basement membrane,11 direct evidence for such conformation change CYTOKINE, Vol. 12, No. 5 (May), 2000: pp 466–471
Glycosaminoglycans alter IFN-gamma structure / 467
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is lacking. Here, we have therefore focused on the structural changes that occur in IFN- upon interaction with low molecular weight heparin (LMW heparin) and chondroitin sulfate. We address three specific questions. Do LMW heparin and chondroitin sulfate alter: (i) the secondary structure; (ii) the tertiary structure; (iii) the quaternary structure of IFN-? Our results demonstrate that LMW heparin and chondroitin sulfate cause large structural changes in the secondary structure of IFN- and that these structural changes do not involve aggregation or denaturation of IFN- dimers.
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Evidence that LMW heparin and chondroitin sulfate alter the secondary structure of IFN-
Evidence that LMW heparin and chondroitin sulfate alter the tertiary structure of IFN- To determine the effects of glycosaminoglycans on the tertiary structure of IFN-, we monitored tryptophan fluorescence. There is only one tryptophan, W31, per monomeric subunit of IFN-. The tryptophan is located at the end of B helix and takes part in the formation of a cleft that accommodates the C-terminal F helix of the other monomer.15 Changes in tryptophan fluorescence therefore provide information about the effects of LMW heparin or chondroitin sulfate on dimeric interface of IFN-. The emission spectra were obtained between 300– 400 nm with excitation set at 280 nm. Fluorescence spectra for the IFN- and for IFN- in the presence of
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The effect of glycosaminoglycans on the secondary structure of IFN- was examined using far ultraviolet-CD spectra of the protein in the presence and absence of LMW heparin or chondroitin sulfate. Characteristic bands of an -helix rich protein were noted in the CD spectrum of IFN- (Fig. 1)—a negative band around 220 nm, a shoulder at 208 nm and a positive band at 190 nm.13 These are consistent with the previously reported far ultraviolet-CD data.14 Figure 1 shows IFN- CD spectra in the absence and presence of varying concentrations of LMW heparin. The spectra are corrected for ellipticity of the added LMW heparin. The CD spectrum of IFN- changes significantly upon addition of LMW heparin (Fig. 1). The ellipticity value at 220 nm decreased with the addition of LMW heparin indicating secondary structure changes in the protein. The ellipticity at 220 nm is found to decrease rapidly with increasing LMW heparin concentrations but approaches saturation at higher concentrations (Fig. 1B). A similar decrease in the ellipticity values was observed with chondroitin sulfate (Fig. 2A and B).
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Figure 1. A: Circular dichroism spectra of IFN- in the presence of LMW heparin. The IFN-–concentration was 50 g/ml and the LMW heparin concentrations are indicated. The IFN- spectra shown have been corrected for the ellipticity of the added LMW heparin. B: The dependence of the IFN- ellipticity at 220 nm on LMW heparin concentration.
various concentrations of LMW heparin are shown in Fig. 3A. The protein fluorescence is reduced by the addition of LMW heparin in a concentration dependent manner. A similar reduction in fluorescence was observed with chondroitin sulfate (Fig. 4A). Figure 3B is a plot of percent of fluorescence quenched versus LMW heparin concentration. The quenching of IFN- fluorescence in the presence of LMW heparin shows saturation at higher concentrations of LMW heparin. Figure 4B shows the corresponding results for chondroitin sulfate. These results indicate that LMW heparin and chondroitin sulfate alter the local environment of W31 and suggest that the interactions either cause or are associated with changes in the tertiary structure of IFN-.
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Figure 2. A: Circular dichroism spectra of IFN- in the presence of chondroitin sulfate.
Figure 3. A: Tryptophan fluorescence spectra of IFN- in the presence of LMW heparin.
The IFN--concentration was 50 g/ml and the chondroitin sulfate concentrations are indicated. The IFN- spectra shown have been corrected for the ellipticity of the added chondroitin sulfate. B: Dependence of the IFN- ellipticity at 220 nm on chondroitin sulfate concentration.
The IFN- concentration was 50 g/ml and the LMW heparin concentrations are indicated. The samples were excited at 280 nm and the emission spectra were recorded between 300–400 nm. The IFN- spectra shown have been corrected for the fluorescence background of the added LMW heparin. B: Dependence of the fluorescence intensity at 340 nm on LMW heparin concentration. The solid line is the least squares fit of a quadratic binding equation to the data.
Evidence that LMW heparin and chondroitin sulfate A do not alter the quaternary structure of IFN- Loss of quaternary structure of IFN- can also cause the conformational changes observed using CD and fluorescence. The changes in quarternary structure could also mean formation of larger aggregates or dissociation to individual subunits. To investigate the aggregation or dissociation of the dimeric protein, we probed the quaternary structure by ANS fluorescence. ANS fluorescence is sensitive to the hydrophobicity of proteins and its use for quaternary structural determination and subunit affinity has been well documented.16,17 Thus, ANS fluorescence in the presence of IFN- can be expected to increase several fold if
aggregation occurs, and decrease if dissociation to subunits occurs. The ANS fluorescence intensities for IFN-– LMW heparin and IFN-–chondroitin sulfate mixtures were independent of extracellular matrix component concentrations, as shown in Figures 5 and 6, respectively. Thus, IFN- neither aggregates nor dissociates upon interaction with LMW heparin or chondroitin sulfate.
DISCUSSION Here, we have investigated the effects of glycosaminoglycans on the structure of IFN- and our
Glycosaminoglycans alter IFN-gamma structure / 469
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Figure 5. Normalized % ANS fluorescence at 530 nm in the presence of IFN- plus the indicated concentrations of LMW heparin. The y-axis is the ratio of the ANS fluorescence intensity of the sample to the ANS fluorescence intensity in the presence of IFN-. The IFN- and the ANS concentrations were 10 g/ml and 10 M, respectively. The samples were excited at 380 nm and the emission spectra between 400–600 nm were recorded.
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Figure 4. A: Tryptophan fluorescence spectra of IFN- in the presence of chondroitin sulfate. The IFN- concentration was 50 g/ml and the chondroitin sulfate concentrations are indicated. The samples were excited at 280 nm and the emission spectra were recorded between 300–400 nm. The IFN- spectra shown have been corrected for the fluorescence background of the added chondroitin sulfate. B: Dependence of the fluorescence intensity at 340 nm on chondroitin sulfate concentration. The solid line is the least squares fit of a quadratic binding equation to the data.
results demonstrate that the interaction with LMW heparin and chondroitin sulfate cause a loss of -helical content and alterations in the tertiary structure of IFN-. The spectroscopic methods used allow the structural changes associated with the interaction to be monitored and do not require separation of the bound and unbound species. The dissociation constant, Kd, for IFN- binding to LMW heparin was estimated to be 29 nM using the fluorescence methodology while the circular dichroism measurements provided a Kd value of 22 nM. The Kd values from the two methods are thus very similar. These values are approximately 15- to 19-fold higher than those previously reported.4 The exact reasons for
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Figure 6. Normalized % ANS fluorescence at 530 nm in the presence of IFN- plus the indicated concentrations of chondroitin sulfate. The y-axis is the ratio of the ANS fluorescence intensity of the sample to the ANS fluorescence intensity in the presence of IFN-. The IFN- and the ANS concentrations were 10 g/ml and 10 M, respectively. The samples were excited at 380 nm and the emission spectra between 400–600 nm were recorded.
this discrepancy are not clear but there are methodological differences that could be contributing factors: e.g. matrigel was used in4 while LMW heparin was used in our studies. The fluorescence studies indicate that the local environment of tryptophan, W31, is altered when IFN- and LMW heparin interact. The binding of LMW heparin to IFN- may directly involve W31 or alternatively, the quenching of fluorescence may be the result of collateral solvent exposure changes associated
470 / Balasubramanian and Ramanathan
with binding. However, while the IFN- peak fluorescence intensity is reduced in the presence of LMW heparin/chondroitin sulfate, the peak wavelength is not red or blue shifted. This finding suggests that LMW heparin binding either does not alter the average hydrophobicity of the tryptophan environments in the dimer or that the changes if any, are offset and not detected. LMW heparin is structurally similar to heparan sulfate and interactions between heparan sulfate and cytokines may contribute to the regulation of immune responses particularly in inflammation and in other pathological states.18,19 Heparan sulfate is released from endothelial cells during inflammation, and T cells and platelets release heparinases upon activation.19–21 Thus, soluble heparan sulfate-cytokine interactions may play important regulatory roles in the crosstalk between endothelial and immune cells in inflammation.
MATERIALS AND METHODS LMW heparin, chondroitin sulfate and IFN- Bovine intestinal mucosa-derived LMW heparin (Catalog number H5027, average molecular weight 3000 daltons) and bovine trachea-derived chondroitin sulfate A (Catalog number C8529, average molecular weight 34 000 daltons) were obtained from Sigma Chemical Company (St Louis, MO, U.S.A.). IFN-, free of macromolecular additives such as human serum albumin, was a gift from Genentech Inc. (South San Francisco, CA, U.S.A.). Slide-a-lyzer dialysis cassettes (Pierce, Rockford, IL, U.S.A.) were used to dialyse IFN- against phosphate buffered saline (PBS). After dialysis, the concentration of the protein was measured using the Biorad protein assay reagent (Biorad, Richmond, CA, U.S.A.). Bovine serum albumin (Fisher Scientific, Springfield, NJ, U.S.A.) solutions were used as calibration standards.
Circular dichroism spectroscopy The CD studies were carried out using a Jasco J500 spectropolarimeter calibrated with d 10-camphorsulphonic acid (Sigma Chemical, St Louis, MO, U.S.A.). The measurements were made using 1 mm path length quartz cuvets. The instrument time constant and sensitivity were 4 s and 1 m, respectively. The samples were scanned over the wavelength range of 260–190 nm for secondary structure analysis. For titration, two solutions were prepared in phosphate buffered saline (pH 7.4): solution A contained 50 g/ml IFN- and solution B contained 25 M LMW heparin or chondroitin sulfate plus 50 g/ml IFN-. The spectra of solution A was recorded and solution B was added in aliquots to solution A. After each addition, the solutions were mixed by inversion and spectra recorded. The percent reduction in ellipticity was plotted against total LMW heparin or chondroitin sulfate concentration, and the data were fitted to the quadratic binding equation described in Data analysis.
CYTOKINE, Vol. 12, No. 5 (May, 2000: 466–471)
Fluorescence spectroscopy Fluorescence studies were carried out at room temperature using an SLM Aminco 8000 fluorometer with 4 mm excitation and emission slits. Tryptophan fluorescence emission spectra were recorded over the wavelength range of 300–400 nm with the excitation wavelength set at 280 nm. A 295 nm long pass filter was used during the measurements to minimize the effect of Raman bands on the emission maxima. Spectra were recorded in I-shaped, 2 mm/10 mm dual path length cuvets so that corrections for the inner filter effect could be made. Titrations were carried out as described for the circular dichroism studies. The percent reduction in fluorescence was plotted against total LMW heparin or chondroitin sulfate concentration and the data were fitted to a quadratic binding equation. The fluorescence of 1, 8 anilinonaphthalene sulfonate (ANS; Sigma Chemical, St Louis, MO, U.S.A.) is sensitive to quaternary structure and was used to determine the effects of LMW heparin or chondroitin sulfate on the homodimeric quaternary structure of IFN-. The final IFN- and the ANS concentrations were 10 g/ml and 10 M, respectively. Emission spectra for ANS were obtained over a range of 400 and 600 nm with excitation set at 380 nm.
Data analysis The quadratic binding equation was used for fitting instead of the Michaelis–Menten type simple binding hyperbola because under the experimental conditions used, the free LMW heparin or chondroitin sulfate concentration are not measured.12 The percent reductions in fluorescence or in ellipticity were plotted against total LMW heparin (or chondroitin sulfate) concentration, DT. If Db is the concentration of the LMW heparin (or chondroitin sulfate)-IFN- complex, PT is the total IFN- concentration, Kd is the dissociation constant and S=DT +PT +Kd, then according to the quadratic binding equation, bound LMW heparin (or chondroitin sulfate) concentration Db, is given by:
Since, the bound state is associated with loss of ellipticity or intrinsic fluorescence without loss of generality, the percent reduction in fluorescence or ellipticity is given by:
This quadratic binding equation has two fitted parameters, Maximal % reduction and Kd. The least squares curve fitting routine in Kaleidagraph 3.08 (Synergy Software, Reading PA) was used on a Power Macintosh (Apple Computer, Cupertino, CA) to determine the unknown parameters from plots of % reduction vs DT.
Glycosaminoglycans alter IFN-gamma structure / 471
Acknowledgements This work was supported by a grant 1R29GM54087-01 from the National Institute of General Medical Sciences. Funding for the Ramanathan laboratory from the National Multiple Sclerosis Society is also gratefully acknowledged. We thank Dr R. M. Straubinger for use of the circular dichroism and fluorimetry instrumentation. We gratefully acknowledge Genentech Inc., South San Francisco, U.S.A. for generously providing us the IFN- used in these studies.
REFERENCES 1. Daubener W, Nockemann S, Gutsche M, Hadding U (1995) Heparin inhibits the antiparasitic and immune modulatory effects of human recombinant interferon-gamma. Eur J Immunol 25:688– 692. 2. Korlipara LV, Pleass HC, Taylor RM, Proud G, Forsythe JL, Kirby JA (1994) Cultured endothelium. Heparin antagonizes the induction of class II MHC molecules by interferon-gamma. Transplantation 58:1426–1429. 3. Rix DA, Douglas MS, Talbot D, Dark JH, Kirby JA (1996) Role of glycosaminoglycans (GAGs) in regulation of the immunogenicity of human vascular endothelial cells. Clin Exp Immunol 104:60–65. 4. Lortat-Jacob H, Kleinman HK, Grimaud JA (1991) Highaffinity binding of interferon-gamma to a basement membrane complex (matrigel). J Clin Invest 87:878–883. 5. Sadir R, Forest E, Lortat-Jacob H (1998) The heparan sulfate binding sequence of interferon-gamma increased the on rate of the interferon-gamma-interferon-gamma receptor complex formation. J Biol Chem 273:10919–10925. 6. Lortat-Jacob H, Baltzer F, Grimaud JA (1996) Heparin decreases the blood clearance of interferon-gamma and increases its activity by limiting the processing of its carboxyl-terminal sequence. J Biol Chem 271:16139–16143. 7. Camejo EH, Rosengren B, Camejo G, Sartipy P, Fager G, Bondjers G (1995) Interferon gamma binds to extracellular matrix chondroitin-sulfate proteoglycans, thus enhancing its cellular response. Arteriosclerosis, Thrombosis Vascular Biol 15:1456–1465.
8. Lortat-Jacob H, Grimaud JA (1991) Interferon-gamma binds to heparan sulfate by a cluster of amino acids located in the C-terminal part of the molecule. FEBS Letters 280:152–154. 9. Lortat-Jacob H, Grimaud JA (1992) Binding of interferongamma to heparan sulfate is restricted to the heparin-like domains and involves carboxylic—but not N-sulfated—groups. Biochim Biophys Acta 1117:126–130. 10. Lortat-Jacob H, Turnbull JE, Grimaud JA (1995) Molecular organization of the interferon gamma-binding domain in heparan sulphate. Biochem J 310:497–505. 11. Lortat-Jacob H, Grimaud JA (1991) Interferon-gamma C-terminal function: new working hypothesis. Heparan sulfate and heparin, new targets for IFN-gamma, protect, relax the cytokine and regulate its activity. Cell Mol Biol 37:253–260. 12. Epps DE, Raub TJ, Kezdy FJ (1995) A general, wide-rage spectrofluorometric method for measuring the site-specific affinities of drugs toward human serum albumin. Analyt Biochem 227:342– 350. 13. Fasman GD (1993) Distinguishing transmembrane helices from peripheral helices by circular dichrosim. Biotech Appl Biochem 18:111–138. 14. Hogrefe HH, McPhie P, Bekisz JB, Enterline JC, Dyer D, Webb DS, Gerrard TL, Zoon KC (1989) Amino terminus is essential to the structural integrity of recombinant human interferon-gamma. J Biol Chem 264:12179–12186. 15. Ealick SE, Cook WJ, Vijay-Kumar S, Carson M, Nagabhushan TL, Trotta PP, Bugg CE (1991) Three-dimensional structure of recombinant human interferon-gamma. Science 252:698–702. 16. Merz WE (1988) Evidence for impaired subunit interaction in chemically deglycosylated human choriogonadotropin. Biochem Biophys Res Commun 156:1271–1278. 17. Aloj SM, Edelhoch H, Ingham KC, Morgan FJ, Canfield RE, Ross GT (1973) The rates of dissociation and reassociation of the subunits of human chorionic gonadotropin. Arch Biochem Biophys 159:497–504. 18. Grimaud JA, Lortat-Jacob H (1994) Matrix receptors to cytokines: from concept to control of tissue fibrosis dynamics. Pathol Res Pract 190:883–890. 19. Ihrcke NS, Wrenshall LE, Lindman BJ, Platt JL (1993) Role of heparan sulfate in immune system-blood vessel interactions. Immunol Today 14:500–505. 20. Geller RL, Ihrcke NS, Platt JL (1994) Release of endothelial cell-associated heparan sulfate proteoglycan by activated T cells. Transplantation 57:770–774. 21. Ihrcke NS, Platt JL (1996) Shedding of heparan sulfate proteoglycan by stimulated endothelial cells: evidence for proteolysis of cell-surface molecules. J Cell Physiol 168:625–637.
GLYCOSAMINOGLYCANS ALTER THE CONFORMATION OF INTERFERON-GAMMA Vandana Balasubramanian, Murali Ramanathan Interferon- (IFN-) is a potent immunomodulatory cytokine whose physiological roles are modulated by the extracellular matrix. Here, circular dichroism and fluorescence spectroscopic techniques are used to demonstrate that low molecular weight heparin and chondroitin sulfate cause significant changes in secondary and tertiary structure of IFN-. The results suggest that heparin and chondroitin sulfate modulate IFN- activity by causing structural changes in the IFN- dimer. 2000 Academic Press
A number of cytokines and growth factors, e.g. interleukins 1, 2, 3, 4, 6, fibroblast growth factor, vascular endothelial growth factor, and the chemokines, bind to the extracellular matrix and these cytokine-extracellular matrix interactions may play important roles in the regulation of individual cytokine activities and cytokine networks. The focus of this report is interferon- (IFN-), a potent, pleiotropic, pro-inflammatory cytokine produced by activated T cells and natural killer cells that binds to extracellular matrix components and to cell surface heparan sulfate with high affinity. IFN- is a particularly relevant model for the regulation of cytokine activity by the extracellular matrix because it is disease-promoting in a variety of autoimmune disorders such as multiple sclerosis and septic shock. Heparan sulfate is a potent antagonist of IFN- activity in a variety of cell types including glioblastoma and endothelial cells.1,2 It inhibits the induction of class II MHC molecules, a signature activity of IFN-, and antagonizes the upregulation of ICAM-1 in human umbilical vein endothelial cells (HUVEC)3 and indoleamine 2,3 dioxygenase in glioblastoma cells.1 The extracellular matrix appears to modulate IFN- effects via multiple mechanisms. For example, extracellular matrix components have been hypothFrom the Department of Pharmaceutics, State University of New York at Buffalo, Buffalo, NY 14260-1200, USA Correspondence to: Murali Ramanathan, Department of Pharmaceutics, 543 Cooke Hall, State University of New York at Buffalo, Buffalo, NY 14260-1200, USA; E-mail: [email protected]ffalo.edu Received 10 March 1999; received in revised form 2 June 1999; accepted for publication 27 July 1999 2000 Academic Press 1043–4666/00/050466+06 $35.00/0 KEY WORDS: extracellular matrix/circular dichroism/fluorescence spectroscopy/heparin/chondroitin sulfate/IFN-gamma 466
esized to be involved in storing IFN- thereby providing a pool of cytokine locally.4 The modulation of the cellular response to IFN- by heparin is also a result of the competitive inhibition of receptor binding because the heparin-binding regions of IFN- are also involved in increasing the on-rate for the IFN-–IFN- receptor interaction.5 Pharmacokinetic and pharmacodynamic factors may also be involved. In rats, the half-life of recombinant human IFN- increases from a control value of 1.1 min to 99 min when co-administered with 2000 units/kg heparin.6 The proteolytic degradation pattern of the IFN- carboxyl tail is limited to less than ten amino acid residues in the presence of heparin and this in turn, contributes to a substantial increase in IFN- activity.6 Chondroitin-sulfate proteoglycans also immobilize IFN- in the extracellular matrix and enhance the cellular response to IFN-.7 IFN- binds to basement membrane heparan sulfate with nanomolar dissociation constants4 principally via the KRKR sequence in its carboxyl terminal tail.8 The heparin-like domains of heparan sulfate are involved in binding IFN-9 and the glycosaminoglycan carboxylic groups are necessary for IFN- recognition. However, the N-sulfate groups are dispensable since N-desulfation of heparan sulfate followed by acetylation does not affect binding.9 The IFN- protected region or footprint of heparan sulfate is about 10 Kd long and contains N-acetylated and glucuronic acidrich residues flanked by small N-sulfated oligosaccharides. The data are consistent with a model in which heparin links the two carboxyl tails of the IFN- dimer.10 Although conformational changes in IFN- have been postulated as a potential mechanism for the modulation of IFN- activity by the basement membrane,11 direct evidence for such conformation change CYTOKINE, Vol. 12, No. 5 (May), 2000: pp 466–471
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is lacking. Here, we have therefore focused on the structural changes that occur in IFN- upon interaction with low molecular weight heparin (LMW heparin) and chondroitin sulfate. We address three specific questions. Do LMW heparin and chondroitin sulfate alter: (i) the secondary structure; (ii) the tertiary structure; (iii) the quaternary structure of IFN-? Our results demonstrate that LMW heparin and chondroitin sulfate cause large structural changes in the secondary structure of IFN- and that these structural changes do not involve aggregation or denaturation of IFN- dimers.
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Evidence that LMW heparin and chondroitin sulfate alter the secondary structure of IFN-
Evidence that LMW heparin and chondroitin sulfate alter the tertiary structure of IFN- To determine the effects of glycosaminoglycans on the tertiary structure of IFN-, we monitored tryptophan fluorescence. There is only one tryptophan, W31, per monomeric subunit of IFN-. The tryptophan is located at the end of B helix and takes part in the formation of a cleft that accommodates the C-terminal F helix of the other monomer.15 Changes in tryptophan fluorescence therefore provide information about the effects of LMW heparin or chondroitin sulfate on dimeric interface of IFN-. The emission spectra were obtained between 300– 400 nm with excitation set at 280 nm. Fluorescence spectra for the IFN- and for IFN- in the presence of
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The effect of glycosaminoglycans on the secondary structure of IFN- was examined using far ultraviolet-CD spectra of the protein in the presence and absence of LMW heparin or chondroitin sulfate. Characteristic bands of an -helix rich protein were noted in the CD spectrum of IFN- (Fig. 1)—a negative band around 220 nm, a shoulder at 208 nm and a positive band at 190 nm.13 These are consistent with the previously reported far ultraviolet-CD data.14 Figure 1 shows IFN- CD spectra in the absence and presence of varying concentrations of LMW heparin. The spectra are corrected for ellipticity of the added LMW heparin. The CD spectrum of IFN- changes significantly upon addition of LMW heparin (Fig. 1). The ellipticity value at 220 nm decreased with the addition of LMW heparin indicating secondary structure changes in the protein. The ellipticity at 220 nm is found to decrease rapidly with increasing LMW heparin concentrations but approaches saturation at higher concentrations (Fig. 1B). A similar decrease in the ellipticity values was observed with chondroitin sulfate (Fig. 2A and B).
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Figure 1. A: Circular dichroism spectra of IFN- in the presence of LMW heparin. The IFN-–concentration was 50 g/ml and the LMW heparin concentrations are indicated. The IFN- spectra shown have been corrected for the ellipticity of the added LMW heparin. B: The dependence of the IFN- ellipticity at 220 nm on LMW heparin concentration.
various concentrations of LMW heparin are shown in Fig. 3A. The protein fluorescence is reduced by the addition of LMW heparin in a concentration dependent manner. A similar reduction in fluorescence was observed with chondroitin sulfate (Fig. 4A). Figure 3B is a plot of percent of fluorescence quenched versus LMW heparin concentration. The quenching of IFN- fluorescence in the presence of LMW heparin shows saturation at higher concentrations of LMW heparin. Figure 4B shows the corresponding results for chondroitin sulfate. These results indicate that LMW heparin and chondroitin sulfate alter the local environment of W31 and suggest that the interactions either cause or are associated with changes in the tertiary structure of IFN-.
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Figure 2. A: Circular dichroism spectra of IFN- in the presence of chondroitin sulfate.
Figure 3. A: Tryptophan fluorescence spectra of IFN- in the presence of LMW heparin.
The IFN--concentration was 50 g/ml and the chondroitin sulfate concentrations are indicated. The IFN- spectra shown have been corrected for the ellipticity of the added chondroitin sulfate. B: Dependence of the IFN- ellipticity at 220 nm on chondroitin sulfate concentration.
The IFN- concentration was 50 g/ml and the LMW heparin concentrations are indicated. The samples were excited at 280 nm and the emission spectra were recorded between 300–400 nm. The IFN- spectra shown have been corrected for the fluorescence background of the added LMW heparin. B: Dependence of the fluorescence intensity at 340 nm on LMW heparin concentration. The solid line is the least squares fit of a quadratic binding equation to the data.
Evidence that LMW heparin and chondroitin sulfate A do not alter the quaternary structure of IFN- Loss of quaternary structure of IFN- can also cause the conformational changes observed using CD and fluorescence. The changes in quarternary structure could also mean formation of larger aggregates or dissociation to individual subunits. To investigate the aggregation or dissociation of the dimeric protein, we probed the quaternary structure by ANS fluorescence. ANS fluorescence is sensitive to the hydrophobicity of proteins and its use for quaternary structural determination and subunit affinity has been well documented.16,17 Thus, ANS fluorescence in the presence of IFN- can be expected to increase several fold if
aggregation occurs, and decrease if dissociation to subunits occurs. The ANS fluorescence intensities for IFN-– LMW heparin and IFN-–chondroitin sulfate mixtures were independent of extracellular matrix component concentrations, as shown in Figures 5 and 6, respectively. Thus, IFN- neither aggregates nor dissociates upon interaction with LMW heparin or chondroitin sulfate.
DISCUSSION Here, we have investigated the effects of glycosaminoglycans on the structure of IFN- and our
Glycosaminoglycans alter IFN-gamma structure / 469
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8
0.6 µM 10 µM
120
100
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4 60 0 0 300
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340 360 Wavelength (nm)
380
400
B
4 6 8 Heparin concentration (µM)
10
Figure 5. Normalized % ANS fluorescence at 530 nm in the presence of IFN- plus the indicated concentrations of LMW heparin. The y-axis is the ratio of the ANS fluorescence intensity of the sample to the ANS fluorescence intensity in the presence of IFN-. The IFN- and the ANS concentrations were 10 g/ml and 10 M, respectively. The samples were excited at 380 nm and the emission spectra between 400–600 nm were recorded.
60
40 160
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0 0
10 2 4 6 8 Chondroitin sulfate concentration (µM)
Figure 4. A: Tryptophan fluorescence spectra of IFN- in the presence of chondroitin sulfate. The IFN- concentration was 50 g/ml and the chondroitin sulfate concentrations are indicated. The samples were excited at 280 nm and the emission spectra were recorded between 300–400 nm. The IFN- spectra shown have been corrected for the fluorescence background of the added chondroitin sulfate. B: Dependence of the fluorescence intensity at 340 nm on chondroitin sulfate concentration. The solid line is the least squares fit of a quadratic binding equation to the data.
results demonstrate that the interaction with LMW heparin and chondroitin sulfate cause a loss of -helical content and alterations in the tertiary structure of IFN-. The spectroscopic methods used allow the structural changes associated with the interaction to be monitored and do not require separation of the bound and unbound species. The dissociation constant, Kd, for IFN- binding to LMW heparin was estimated to be 29 nM using the fluorescence methodology while the circular dichroism measurements provided a Kd value of 22 nM. The Kd values from the two methods are thus very similar. These values are approximately 15- to 19-fold higher than those previously reported.4 The exact reasons for
Normalised intensity (%)
% Quenching
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140 120 100 80 60 0
2 4 6 8 Chondroitin sulfate concentration (µM)
10
Figure 6. Normalized % ANS fluorescence at 530 nm in the presence of IFN- plus the indicated concentrations of chondroitin sulfate. The y-axis is the ratio of the ANS fluorescence intensity of the sample to the ANS fluorescence intensity in the presence of IFN-. The IFN- and the ANS concentrations were 10 g/ml and 10 M, respectively. The samples were excited at 380 nm and the emission spectra between 400–600 nm were recorded.
this discrepancy are not clear but there are methodological differences that could be contributing factors: e.g. matrigel was used in4 while LMW heparin was used in our studies. The fluorescence studies indicate that the local environment of tryptophan, W31, is altered when IFN- and LMW heparin interact. The binding of LMW heparin to IFN- may directly involve W31 or alternatively, the quenching of fluorescence may be the result of collateral solvent exposure changes associated
470 / Balasubramanian and Ramanathan
with binding. However, while the IFN- peak fluorescence intensity is reduced in the presence of LMW heparin/chondroitin sulfate, the peak wavelength is not red or blue shifted. This finding suggests that LMW heparin binding either does not alter the average hydrophobicity of the tryptophan environments in the dimer or that the changes if any, are offset and not detected. LMW heparin is structurally similar to heparan sulfate and interactions between heparan sulfate and cytokines may contribute to the regulation of immune responses particularly in inflammation and in other pathological states.18,19 Heparan sulfate is released from endothelial cells during inflammation, and T cells and platelets release heparinases upon activation.19–21 Thus, soluble heparan sulfate-cytokine interactions may play important regulatory roles in the crosstalk between endothelial and immune cells in inflammation.
MATERIALS AND METHODS LMW heparin, chondroitin sulfate and IFN- Bovine intestinal mucosa-derived LMW heparin (Catalog number H5027, average molecular weight 3000 daltons) and bovine trachea-derived chondroitin sulfate A (Catalog number C8529, average molecular weight 34 000 daltons) were obtained from Sigma Chemical Company (St Louis, MO, U.S.A.). IFN-, free of macromolecular additives such as human serum albumin, was a gift from Genentech Inc. (South San Francisco, CA, U.S.A.). Slide-a-lyzer dialysis cassettes (Pierce, Rockford, IL, U.S.A.) were used to dialyse IFN- against phosphate buffered saline (PBS). After dialysis, the concentration of the protein was measured using the Biorad protein assay reagent (Biorad, Richmond, CA, U.S.A.). Bovine serum albumin (Fisher Scientific, Springfield, NJ, U.S.A.) solutions were used as calibration standards.
Circular dichroism spectroscopy The CD studies were carried out using a Jasco J500 spectropolarimeter calibrated with d 10-camphorsulphonic acid (Sigma Chemical, St Louis, MO, U.S.A.). The measurements were made using 1 mm path length quartz cuvets. The instrument time constant and sensitivity were 4 s and 1 m, respectively. The samples were scanned over the wavelength range of 260–190 nm for secondary structure analysis. For titration, two solutions were prepared in phosphate buffered saline (pH 7.4): solution A contained 50 g/ml IFN- and solution B contained 25 M LMW heparin or chondroitin sulfate plus 50 g/ml IFN-. The spectra of solution A was recorded and solution B was added in aliquots to solution A. After each addition, the solutions were mixed by inversion and spectra recorded. The percent reduction in ellipticity was plotted against total LMW heparin or chondroitin sulfate concentration, and the data were fitted to the quadratic binding equation described in Data analysis.
CYTOKINE, Vol. 12, No. 5 (May, 2000: 466–471)
Fluorescence spectroscopy Fluorescence studies were carried out at room temperature using an SLM Aminco 8000 fluorometer with 4 mm excitation and emission slits. Tryptophan fluorescence emission spectra were recorded over the wavelength range of 300–400 nm with the excitation wavelength set at 280 nm. A 295 nm long pass filter was used during the measurements to minimize the effect of Raman bands on the emission maxima. Spectra were recorded in I-shaped, 2 mm/10 mm dual path length cuvets so that corrections for the inner filter effect could be made. Titrations were carried out as described for the circular dichroism studies. The percent reduction in fluorescence was plotted against total LMW heparin or chondroitin sulfate concentration and the data were fitted to a quadratic binding equation. The fluorescence of 1, 8 anilinonaphthalene sulfonate (ANS; Sigma Chemical, St Louis, MO, U.S.A.) is sensitive to quaternary structure and was used to determine the effects of LMW heparin or chondroitin sulfate on the homodimeric quaternary structure of IFN-. The final IFN- and the ANS concentrations were 10 g/ml and 10 M, respectively. Emission spectra for ANS were obtained over a range of 400 and 600 nm with excitation set at 380 nm.
Data analysis The quadratic binding equation was used for fitting instead of the Michaelis–Menten type simple binding hyperbola because under the experimental conditions used, the free LMW heparin or chondroitin sulfate concentration are not measured.12 The percent reductions in fluorescence or in ellipticity were plotted against total LMW heparin (or chondroitin sulfate) concentration, DT. If Db is the concentration of the LMW heparin (or chondroitin sulfate)-IFN- complex, PT is the total IFN- concentration, Kd is the dissociation constant and S=DT +PT +Kd, then according to the quadratic binding equation, bound LMW heparin (or chondroitin sulfate) concentration Db, is given by:
Since, the bound state is associated with loss of ellipticity or intrinsic fluorescence without loss of generality, the percent reduction in fluorescence or ellipticity is given by:
This quadratic binding equation has two fitted parameters, Maximal % reduction and Kd. The least squares curve fitting routine in Kaleidagraph 3.08 (Synergy Software, Reading PA) was used on a Power Macintosh (Apple Computer, Cupertino, CA) to determine the unknown parameters from plots of % reduction vs DT.
Glycosaminoglycans alter IFN-gamma structure / 471
Acknowledgements This work was supported by a grant 1R29GM54087-01 from the National Institute of General Medical Sciences. Funding for the Ramanathan laboratory from the National Multiple Sclerosis Society is also gratefully acknowledged. We thank Dr R. M. Straubinger for use of the circular dichroism and fluorimetry instrumentation. We gratefully acknowledge Genentech Inc., South San Francisco, U.S.A. for generously providing us the IFN- used in these studies.
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