Deimination of myelin basic protein. 2. Effect of methylation of MBP on its deimination by peptidylarginine deiminase

Deimination of myelin basic protein (MBP) has been implicated in the chemic al pathogenesis of multiple sclerosis (MS). Degradation of bovine MBP by ca thepsin D, a myelin-associated protease, was increased when 6 arginyl resid ues were deiminated and became very rapid when all 18 arginyl residues were deiminated. Since MBP contains a number of modifications, including methyl ation, phosphorylation, etc., we studied the effect of methylation, an irre versible modification, to determine how this modification affected deiminat ion. Methylation of Arg 106 in bovine MBP (Arg 107 in human), a naturally o ccurring modification of MBP, has been shown to affect the deimination of a rginyl residues in the present study. Since fractionation of MBP into unmet hylated, monomethylated, and dimethylated species cannot be done readily on a preparative scale, mass spectrometry with the Q-TOF instrument resolved these species readily since each differed from the other by 14 atomic mass units (amu). Examination of five different hMBP samples, two from normal br ain and three from MS brain, revealed that increased deimination of arginyl residues correlated with a decreased methylation of Arg 107 (human sequenc e). To study this process in vitro, bovine MBP (bMBP) was used. Component 1 (C-1) is the most cationic of the MBP "charge isomers" and the most unmodi fied, in which all arginyl residues are intact. It was deiminated to variou s extents with purified bovine brain peptidylarginine deiminase, generating a number of species containing 0-13.7 mol of citrulline/mol of bMBP. Mass spectrometry of each of these species permitted us to determine the influen ce of methylation of Arg 106 (bovine sequence) on deimination by this enzym e. We found that bMBP with unmethylated arginine was deiminated at a rate o f 0.081 mol of citrulline/min, with monomethylarginine, 0.068 mol of citrul line/min, and with dimethylarginine, 0.036 mol of citrulline/min. We sugges t that the methylated arginyl residue becomes sequestered in the hydrophobi c beta-sheet structure and disrupts the three-dimensional structure of the protein so that other arginyl residues are less accessible to peptidylargin ine deiminase.