Abstract
Recombinant human glial cell line-derived neurotrophic factor has been implicated to have therapeutic potential in the treatment of neurodegenerative diseases. The mature protein is a single polypeptide of 134 amino acid residues and functions as a disulfide-linked dimer. Reduction of the protein with dithiothreitol at pH 7.0 and in the absence of denaturant showed that the single intermolecular cystine bridge was reduced preferentially. Direct alkylation of the generated free sulfhydryl group using iodoacetamide or iodoacetate without denaturant was incomplete. Unfolding the protein in 6 M guanidine hydrochloride prior to the modification showed rapid disulfide scrambling. However, the sulfhydryl-modifying reagent N-ethylmaleimide was able to label quantitatively the free cysteinyl residue in the absence of any added chaotropic agent. By a combination of peptide mapping, Edman degradation, and mass spectrometric analysis, the labeled residue was identified to be Cys101, hence verifying the location of the intermolecular disulfide bond. The modified protein behaved as a noncovalent dimer when chromatographed through a Superdex 75 column under nondenaturing conditions and was comparable in biological activity to an unmodified control sample. The results therefore indicate that the intermolecular disulfide bridge of the protein is not essential for its biological function.
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REFERENCES
Baloh, R. H., Tansey, M. G., Lampe, P. A., Fahrner, T. J., Enomoto, H., Simburger, K. S., Leitner, M. L., Araki, T., Johnson, E. M., and Milbrandt, J. (1998). Neuron 21, 1291–1302.
Bohn, M. C. (1999). Biochem. Pharmacol. 57, 135–142.
Daopin, S., Li, M., and Davies, D. R. (1993). Proteins Struct. Funct. Genet. 17, 176–192.
Durbec, P., Marcos-Gutierrez, C. V., Kilkenny, C., Grigoriou, M., Wartiowaara, K., Suvanto, P., Smith, D., Ponder, B., Costantini, F., Saarma, M., Sariola, H., and Pachnis, V. (1996). Nature 381, 789–793.
Eigenbrot, C., and Gerber, N. (1997). Nature Struct. Biol. 4, 435–438.
Fullmer, C. S. (1984). Anal. Biochem. 142, 336–339.
Gash, D. M., Zhang, Z., and Gerhardt, G. (1998). Ann. Neurol. 44(Suppl. 1), S121-S125.
Gregory, J. D. (1955). J. Am. Chem. Soc. 77, 3922–3923.
Grondin, R., and Gash, D. M. (1998). J. Neurol. 245(Suppl.), 35–42.
Haniu, M., Hui, J., Young, Y., Le, J., Katta, V., Lee, R., Shimamoto, G., and Rohde, M. F. (1996). Biochemistry 35, 16799–16805.
Henderson, C. E. (1996). Curr. Opin. Neurobiol. 6, 64–70.
Hui, J. O., Le, J., Katta, V., Rohde, M. F., and Haniu, M. (1997). In Techniques in Protein Chemistry, Vol. VIII (D. Marshak, D., ed.), Academic Press, San Diego, California, pp. 277–287.
Jing, S. Q., Wen, D., Yu, Y., Holst, P., Luo, Y., Fang, M., Tamir, R., Antonio, L., Hu, Z., Cupples, R., Louis, J.-C., Hu, S., Altrock, B. W., and Fox, G. M. (1996). Cell 85, 1113–1124.
Kotzbauer, P. T., Lampe, P. A., Heuckeroth, R. O., Golden, J. P., Creedon, D. J., Johnson, E. M., and Milbrandt, J. D. (1996). Nature 384, 467–470.
Kress, L. F., and Laskowski, Sr., M. (1967). J. Biol. Chem. 242, 4925–4929.
Laemmli, U. K. (1970). Nature 227, 680–685.
Li, T., Bender, M., Yamane, H., Arakawa, T., Narhi, L. O., and Philo, J. (1999). Biochemistry (submitted).
Light, A., Hardwick, B. C., Hatfield, L. M., and Sondack, D. L. (1969). J. Biol. Chem. 244, 6289.
Lin, L.-F. H., Doherty, D. H., Lile, J. D., Bektesh, S., and Collins, F. (1993). Science 260, 1130–1132.
Lin, L.-F. H., Zhang, T. J., Collins, F., and Armes, L. G. (1994). J. Neurochem. 63, 758–768.
Milbrandt, J., de Sauvage, F., Fahrner, T. J., Baloh, R. H., Leitner, M. L., Tansey, M. G., Lampe, P. A., Heuckeroth, R. O., Kotzbauer, P. T., Simburger, K. S., et al. (1998). Neuron 20, 245–253.
Reeve, J. R., Cheng, K.-W., and Pierce, J. G. (1975). Biochem. Biophys. Res. Commun. 67, 149–155.
Reeve, Jr., J. R., and Pierce, J. G. (1981). Int. J. Peptide Protein Res. 18, 79–87.
Sondack, D. L., and Light, A. (1971). J. Biol. Chem. 246, 1630–1637.
Sperling, R., Burstein, Y., and Steinberg, I. Z. (1969). Biochemistry 9, 3810–3820.
Thoenen, H. (1991). Trends Neurosci. 14, 165–170.
Trupp, M., Arenas, E., Fainzilber, M., Nilsson, A.-S., Sieber, B.-A., Grigoriou, M., Kilkenny, C., Salazar-Grueso, E., Pachnis, V., Arumae, U., Sariola, H., Saarma, M., and Ibàñez, C. F. (1996). Nature 381, 785–789.
Voytik-Harbin, S. L., Brightman, A. O., Waisner, B., Lamar, C. H., and Badylak, S. F. (1998). In-Vitro Cell Dev. Biol. Anim. 34, 239–246.
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Hui, J.O., Woo, G., Chow, D.T. et al. The Intermolecular Disulfide Bridge of Human Glial Cell Line-Derived Neurotrophic Factor: Its Selective Reduction and Biological Activity of the Modified Protein. J Protein Chem 18, 585–593 (1999). https://doi.org/10.1023/A:1020607501910
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DOI: https://doi.org/10.1023/A:1020607501910