Deimination of Human Myelin Basic Protein by a Peptidylarginine Deiminase from Bovine Brain

Abstract

A peptidylarginine deiminase (PAD; EC 3.5.3.15) has been isolated from bovine brain and some of its characteristics have been studied. The enzyme showed an absolute requirement for Ca²⁺, a temperature optimum at ∼50°C, and two K_mvalues when benzoylarginine ethyl ester was used as substrate, 0.78 mMand 11.2 mM.The higher K_mhas not been reported previously. Protein substrates for the enzyme included polyarginine and myelin basic protein but not histones. Because one of the components of MBP contains six citrullinyl residues per mole, enzymic deimination appeared to be a likely mechanism. When the most cationic component (C-1) was subjected to PAD in solution, 17 of the 19 arginyl residues were modified. From sequence analyses we concluded that the nature of the amino acid residues adjacent to the deiminated arginine were not modifiers of the reaction as arginyl residues in a variety of environments were deiminated. This deimination was reflected in a large increase in random structure, as measured by [θ]₂₀₀. At 5°C, the [θ]₂₀₀of the deiminated protein was -70 × 10³ compared with -30 × 10³ deg cm²/dmol for the native protein. When the temperature was increased to 70°C, the [θ]₂₀₀ was -44 × 10³ for the deiminated protein and -20 × 10⁷ deg cm²/ dmol for the native C-1. When plotted as a function of temperature, [θ]₂₀₀ decreased linearly from 5°C to 50°C for both proteins and did not change from 50°C to 70°C. PAD provides a mechanism for deimination of arginyl residues of myelin basic protein. The selective deimination of the six arginyl residues that are consistently found deiminated in C-8 may be determined by the orientation of the protein in the membrane and/or the more complex lipid composition of myelin may affect the selectivity of the deimination.