Chemical modification and site-directed mutagenesis of methionine residues in recombinant human granulocyte colony-stimulating factor: Effect on stability and biological activity

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 Met(1)>Met(138)>Met(127)>>>Met(122). H2O2 oxidation of Met(122) is relatively slow and is biphasic with a faster second reaction phase being affected by the oxidation of Met(127). 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 Met(127) and Met(122) is unstable and exhibits decreased ability to dimerize the receptor after exposure to acid or elevated temperature. All modified forms, except Met(1)-oxidized G-CSF, also show significantly lower biological activity. Our data suggest that Met(138) is solvent accessible and its surrounding microenvironment may be critical for G-CSF function, whereas Met(127) is less accessible to solvent and Met(122) is near the hydrophobic core. Oxidation at both Met(127) and Met(122) 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 Met(127) or at Met(138) 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. (C) 1999 Academic Press.