Activation of the fifth component of human complement, C5, without cleavage, by methionine oxidizing agents.
Purified human C5 was incubated with chloramine T (Cl-T) or N-chloro-succinimide (N-Cl-S) in barbital buffer, pH 7.2. The treatment led to C5 activation: Cl-T- and N-Cl-S-treated C5 acquired a binding site for C6; upon incubation with C6 and subsequent addition of C7, C8 and C9 a membrane attack complex formed which lysed non-sensitized guinea pig red cells (reactive lysis). While the physiological activation of C5 follows its specific cleavage, the resulting fragment C5b representing the activated C5 and expressing the C6 binding site, the treatment with the mentioned chemicals does not lead to fragmentation of the C5 protein. So, functionally, the product of the chemical treatment is C5b-like, but chemically, it comprises the whole protein; no C5a is released. Cl-T and N-Cl-S are known to more or less selectively oxidize methionine residues in proteins, dependent on the conditions. Other sensitive amino acid residues are tryptophan and cysteine. Conditions were chosen for treatment of C5 with Cl-T which exclude attack on tryptophan, and we have ensured that human C5 does not contain free cysteine residues. Further, oxidation of about 60% of the methionine residues of C5 by Cl-T was demonstrated by amino acid analysis. So, all evidence points to methionine residue(s) as the site of attack of Cl-T and probably also of N-Cl-S. The oxidation product of methionine, its sulphoxide, may cause a change in structural conformation of C5 which involves expression of the C6 binding site. Earlier it was found that oxidation of C5 by hydroxyl radicals leads to its activation without cleavage. Since the properties of this C5b-like product resemble those of the product of treatment with Cl-T and N-Cl-S, it is suggested that the formerly found activation of human C5 by hydroxyl radicals is also mediated by oxidation of methionine residue(s) in the C5 protein.
Vogt
W
W
Max Planck Institute for Experimental Medicine, Göttingen, Germany.
Zimmermann
B
B
Hesse
D
D
Nolte
R
R
D002700:Chloramines / Q000737:chemistry; D003167:Complement Activation; D003182:Complement C5 / Q000737:chemistry*; D015938:Complement Membrane Attack Complex; D006801:Humans; D008715:Methionine / Q000737:chemistry*; D010084:Oxidation-Reduction; D012680:Sensitivity and Specificity; D013388:Succinimides / Q000737:chemistry; D014105:Tosyl Compounds
D002700:Chloramines; D003182:Complement C5; D015938:Complement Membrane Attack Complex; D013388:Succinimides; D014105:Tosyl Compounds; C016300:chloramine-T; C015752:N-chlorosuccinimide; D008715:Methionine