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  • Title: Chemical modification and site-directed mutagenesis of methionine residues in recombinant human granulocyte colony-stimulating factor: effect on stability and biological activity.
    Author: Lu HS, Fausset PR, Narhi LO, Horan T, Shinagawa K, Shimamoto G, Boone TC.
    Journal: Arch Biochem Biophys; 1999 Feb 01; 362(1):1-11. PubMed ID: 9917323.
    Abstract:
    Chemical modification and mutagenesis of methionines in recombinant human granulocyte colony-stimulating factor (G-CSF) were investigated. Selective oxidation of G-CSF by H2O2 and t-butyl hydroperoxide leads to generation of different oxidized forms. Four modified forms were isolated and shown to contain 1 to 4 oxidized methionyl residues. All methionines in G-CSF are reactive, with reaction kinetics following the order of Met1>Met138>Met127>>>Met122. H2O2 oxidation of Met122 is relatively slow and is biphasic with a faster second reaction phase being affected by the oxidation of Met127. All oxidized forms retain gross G-CSF conformation similar to that of the native molecule and are able to bind the soluble G-CSF receptor. However, G-CSF form oxidized at both Met127 and Met122 is unstable and exhibits decreased ability to dimerize the receptor after exposure to acid or elevated temperature. All modified forms, except Met1-oxidized G-CSF, also show significantly lower biological activity. Our data suggest that Met138 is solvent accessible and its surrounding microenvironment may be critical for G-CSF function, whereas Met127 is less accessible to solvent and Met122 is near the hydrophobic core. Oxidation at both Met127 and Met122 results in alterations of G-CSF structure that affect the apparent molecular size, polarity, and stability and lead to the loss of G-CSF biological function. G-CSF variants with Leu replacement at Met127 or at Met138 are not completely resistant to oxidation-induced inactivation, while the variant with Leu replacement at both sites is more stable and can retain in vitro biological activity following oxidation.
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