Abstract
High-affinity binding of basic fibroblast growth factor (bFGF) to the tyrosine kinase receptor requires cell-surface heparan sulfate proteoglycan or exogenous addition of heparin. The crystal structure of bFGF shows Arg40 and 45 on the surface opposite to the heparin-binding region, suggesting that these charged residues may be involved in the receptor binding. Therefore, these amino acids were mutated to aspartic acid separately or simultaneously, and also a simultaneous mutation to glutamic acid was introduced. These mutants displayed a mitogenic activity decreased greater than tenfold compared to the wild-type protein. Addition of heparin had no effect on the activity, while these mutants showed heparin-binding characteristics resembling those of the native sequence protein. The mutants exhibited decreased stability compared to the native sequence protein. Gradual changes in conformation were observed by circular dichroic and infrared spectroscopy. Heparin chromatography also showed the presence of denatured form for these mutants. However, in the presence of multivalent anions such as citrate, sucrose octasulfate, and heparin, the conformation of the mutants resembled that of the wild-type protein, as revealed by X-ray crystallography and circular dichroism spectra of the mutant with a Arg40 → Asp substitution.
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Abbreviations
- FGF:
-
fibroblast growth factor
- bFGF:
-
basic FGF
- FBS:
-
fetal bovine serum
- DMEM:
-
Dulbecco's Modified Eagles Medium
- LMWH:
-
low-molecular-weight heparin
- PBS:
-
phosphate-buffered saline
- CD:
-
circular dichroism
- FTIR:
-
Fourier transform infrared spectroscopy
- MR:
-
molecular replacement
- SIR:
-
single isomorphous replacement
- EMTS:
-
ethylmercurithiosalicylate
- SOS:
-
sucrose octasulfate
References
Ago, H., Kitagawa, Y., Fujishima, A., Matsuura, Y., and Katsube, Y. (1991).J. Biochem. 110, 360–363.
Arakawa, T., Horan, T. P., Narhi, L. O., Rees, D. C., Schiffer, S. G., Holst, P. L., Prestrelski, S. J., Tsai, L. B., and Fox, G. M. (1993a).Protein Eng. 6, 541–546.
Arakawa, T., Wen, J., and Philo, J. (1993b).J. Protein Chem. 12, 689–693.
Arakawa, T., Wen, J., and Philo, J. S. (1994).Arch. Biochem. Biophys. 308, 267–273.
Baird, A., Schubert, D., Ling, N., and Guillemin, R. (1988).Proc. Natl. Acad. Sci. USA 85, 2324–2328.
Brünger, A. T., Kuriyan, J., and Karplus, M. (1987).Science 235, 458–460.
Covington, A. K., Paalo, M., Robinson, R. A., and Bates, R. G. (1968).Anal. Chem. 40, 700–706.
Crowther, R. A. (1972). InThe Molecular Replacement Method (Rossman, M. G., ed.), Gordon and Breach, New York, pp. 173–178.
Eriksson, E., Cousens, L., Weaver, L., and Matthews, B. (1991).Proc. Natl. Acad. Sci. USA 88, 3441–3445.
Esch, F., Baird, A., Ling, N., Ueno, N., Hill, F., Denoroy, L., Klepper, R., Gospodarowicz, D., Bohlen, P., and Guillemin, R. (1985).Proc. Natl. Acad. Sci. USA 82, 6507–6511.
Folkman, J., and Klagsbrun, M. (1987).Science 235, 442–447.
Fox, G. M., Schiffer, S. G., Tsai, L. B., Banks, A. P., and Arakawa, T. (1988).J. Biol. Chem. 263, 18452–18458.
Gospodarowicz, D., and Cheng, J. (1986).J. Cell. Physiol. 128, 475–488.
Gospodarowicz, D., Cheng, J., Lue, G. M., Baird, A., and Bohlen, P. (1984).Proc. Natl. Acad. Sci. USA 81, 6963–6967.
Jancarik, J., and Kim, S. H. (1991).J. Appl. Crystallogr. 24, 409–411.
Kajio, T., Kawahara, K., and Koto, K. (1992).FEBS Lett. 306, 243–246.
Kraulis, P. J. (1991).J. Appl. Crystallogr. 24, 946–950.
Middaugh, C. R., Mach, H., Burke, C. J., Volkin, D. B., Dabora, J. M., Tsai, P. K., Bruner, M. W., Ryan, J. A., and Marifa, K. E. (1992).Biochemistry 31, 9016–9024.
Moscatelli, D. (1987).J. Cell. Physiol. 131, 123–130.
Neufeld, G., Gospodarowicz, D., Dodge, L., and Fukui, D. K. (1987).J. Cell. Physiol. 131, 131–140.
Norrander, J., Kempe, T., and Messing, J. (1983).Gene 26, 101–106.
Prestrelski, S. J., Fox, G. M., and Arakawa, T. (1992).Arch. Biochem. Biophys. 213, 314–319.
Roghani, M., and Moscatelli, D. (1992).J. Biol. Chem. 267, 22156–22162.
Shing, Y. (1988).J. Biol. Chem. 263, 9059.
Susi, H. and Byler, D. M. (1983).Biochem. Biophys. Res. Commun. 115, 391–397.
Takagi, T. (1990).J. Chromatogr. 280, 409–416.
Tenwilliger, T., and Eisenberg, D. (1983).Acta Crystallogr. A 39, 813–817.
Thomas, K. A., Rios-Candelore, M., Gimenez-Gallego, G., DiSalvo, J., Bennett, C., Rodkey, J., and Fitzpatrick, S. (1985).Proc. Natl. Acad. Sci. USA 82, 6409–6413.
Tronrud, D. E., Ten Eyck, L. F., and Mattews, B. W. (1987).Acta Crystallogr. A 43, 489.
Uhlrich, S., Lagentz, O., Lanfant, M., and Courtois, Y. (1986).Biochem. Biophys. Res. Commun. 137, 1205–1212.
Zhang, J., Cousens, L., Barr, P., and Sprang, S. (1991).Proc. Natl. Acad. Sci. USA 88, 3446–3450.
Zhu, X., Komiya, H., Chirino, A., Faham, S., Fox, G. M., Arakawa, T., Hsu, B. T., and Rees, D. C. (1991).Science 251, 90–93.
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Arakawa, T., Hoist, P., Narhi, L.O. et al. The importance of Arg40 and 45 in the mitogenic activity and structural stability of basic fibroblast growth factor: Effects of acidic amino acid substitutions. J Protein Chem 14, 263–274 (1995). https://doi.org/10.1007/BF01886783
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DOI: https://doi.org/10.1007/BF01886783