In Vitro Methionine Oxidation of Recombinant Human Leptin
Purpose. To investigate the role and importance of the four methionines in recombinant human leptin, and the effect of methionine oxidation in leptin structural stability and biological activity.
Methods. Oxidized leptin derivatives were prepared in the presence of H2O2 and t-butylhydroperoxide, separated by RP-HPLC, and characterized by peptide mapping and LC/MS. Their biophysical and biological properties were studied.
Results. Six major species of oxidized leptins were detected: two mono-oxidized, one di-oxidized, two tri-oxidized, and one tetra-oxidized. Further oxidation at cystine disulfide was also detected. Kinetic analysis indicated that oxidation at Met1 and Met69 proceeded first and independently. In 48 mM t-butylhydroperoxide, the pseudo first-order rate constants, k1 and k69, were 1.5 × 10−3 and 2.3 × 10−4 min−1. No change in the secondary or tertiary structure was detected for Met1 mono-oxidized and Met1, Met69 di-oxidized leptins. The Met1 mono-oxidized leptin retained full potency as compared to native leptin. A slight decrease of thermostability and a significant loss of the in vitro bioactivity were observed for Met1, Met69 di-oxidized leptin. Both Met55 and Met137 were not oxidized in t-butylhydroperoxide but only in H2O2. They appeared to be much less accessible to oxidation and might interact with the hydrophobic core structure of the leptin molecule.
Conclusions. The oxidation of leptin occurred in the order of Met1 > Met69 >> Met55 ≈ Met137, and the importance for maintaining leptin structural integrity was Met55 ≈ Met137 >> Met69≈ Met1. Met69, but not Met1, plays a critical role in the protein stability and activity.
Unable to display preview. Download preview PDF.
- 1.Y. Zhang, R. Proenca, M. Maffei, M. Barone, L. Leopold, and J. M. Friedman. Positional cloning of the mouse obese gene and its human homologue. Nature 372:425–432 (1994).Google Scholar
- 2.L. A. Tartaglia, M. Dembrski, X. Weng, N. Deng, J. Culpepper, R. Devos, G. J. Richards, L. A. Campfield, F. T. Clark, J. Deeds, C. Muir, S. Sanker, A. Moriarty, K. J. Moore, J. S. Smutko, G. G. Mays, E. A. Woolf, C. A. Monroe, and R. I. Tepper. Identification and expression cloning of a leptin receptor, OB-R. Cell 83:1263–1271 (1995).Google Scholar
- 3.R. Devos, J. G. Richards, L. A. Campfield, L. A. Tartaglia, Y. Guisez, J. V. Heyden, J. Travernier, G. Plaetinck, and P. Burn. OB protein binds specifically to the choroid plexus of mice and rats. Proc. Natl. Acad. Sci. USA 93:5668–5673 (1996).Google Scholar
- 4.S. L. Cohen, J. L. Halaas, J. M. Friedman, B. T. Chait, L. Bannett, D. Chang, R. Hecht, and F. Collins. Human leptin characterization. Nature 382:589 (1996).Google Scholar
- 5.M. A. Pellymounter, M. J. Cullen, M. B. Baker, R. Hecht, D. Winters, T. Boone, and F. Collins. Effects of the obese gene product on body weight regulation in oblob mice. Science 269:540–543 (1995).Google Scholar
- 6.J. L. Halaas, K. S. Gajiwala, M. Maffei, S. L. Cohen, B. T. Chait, D. Rabinowitz, R. L. Lallone. S. K. Burley, and J. M. Friedman. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269:543–546 (1995).Google Scholar
- 7.L. A. Campfield, F. J. Smith, Y. Guisez, R. Devos, and P. Burn. Recombinant mouse OB protein: Evidence for a peripheral signal linking adiposity and central neural networks. Science 269:546–548 (1995).Google Scholar
- 8.F. Sherman, J. W. Stewart, and S. Tsunasawa. Methionine or not methionine at the beginning of a protein. BioEssays 3:27–31 (1985).Google Scholar
- 9.H. S. Lu, C. L. Clogston, L. A. Merewether, L. O. Narhi, and T. C. Boone. Protein Folding: In Vivo and In Vitro (J. Cleland, Ed.) ACS symposium series 526, chap. 15, American Chemical Society, Washington, DC, 1993.Google Scholar
- 10.J. L. Liu, T. Eris, S. L. Lauren, G. W. Stearns, K. R. Westcott, and H. S. Lu. Techniques in Protein Chemistry (Marshak, D. R., ed) Vol. VIII, pp. 155–163, Academic Press, San Diego, 1997.Google Scholar
- 11.M. C. Manning, K. Patel, and R. T. Borchardt. Stability of protein pharmaceuticals. Pharm. Res. 6:903–918 (1989).Google Scholar
- 12.Y.-R. Hsu, L. O. Narhi, C. Spahr, K. E. Langley, and H. S. Lu. In vitro methionine oxidation of Escherichia coli-derived human stem cell factor: effects on the molecular structure, biological activity, and dimerization. Protein Sci. 5:1165–1173 (1996).Google Scholar
- 13.R. G. Keck. The use of t-butyl hydroperoxide as a probe for methionine oxidation in proteins. Anal. Biochem. 236:56–62 (1996).Google Scholar
- 14.M. Greenfield, and G. D. Fasman. Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry 8:4108–4116 (1969).Google Scholar
- 15.R. E. Pacifici, and A. R. Thomason. Hybrid tyrosine kinase/cytokine receptors transmit mitogenic signals in response to ligand. J. Biol. Chem. 269:1571–1574 (1994).Google Scholar
- 16.D. W. White, K. K. Kuropatwinski, R. Devos, H. Baumann, and L. A. Tartaglia. Leptin receptor (OB-R) signaling. J. Biol. Chem. 272:4065–4071 (1997).Google Scholar
- 17.T. H. Nguyen, J. Burnier, and W. Meng. The kinetics of relaxin oxidation by hydrogen peroxide. Pharm. Res. 10:1563–1571 (1993).Google Scholar
- 18.F. Zhang, M. B. Basinski, J. M. Beals, S. L. Briggs, L. M. Churgay, D. K. Clawson, R. D. DiMarchi, T. C. Furman, J. E. Hale, H. M. Hsiung, B. E. Schoner, D. P. Smith, X. Y. Zhang, J.-P. Wery, and R. W. Schevitz. Crystal structure of the obese protein leptin-E100. Nature 387:206–209 (1997).Google Scholar