Carboxylmcthylation affects the . . . . ' - - " piOtcoty ~ lS o f myelin basic protein by Staphylococcus aureus Vs proteinase

Bovine myelin basic protein (MBP), charge isoform I (CI) was carboxylmethylated by the enzyme a-aspar ty l /Lisoaspartyl protein methyltransferase (EC. 2.1.1.77) and the carboxylmethylated protein was subjected to proteolysis by sequencing grade staphylococcal V 8 proteinase at pH 4.0 to identify its carboxylmethylated modified aspartate a n d / o r asparagine residues which are recognized by this methyltransferase. Native MBP, CI was treated similarly and the proteolysis products were compared, using electrophoretic, chromatographic and amino acid sequencing techniques. Sodium dodecyl sulfate polyacrylamide gel eleetrophoresis (SDS-PAGE) revealed differences in the kinetics of proteolysis between the native and the carboxylmethylated MBP, CI which were confirmed using HPLC. Partial sequencing of the native and carboxylmethylated fragments eluting at about 29 min (P29) revealed cleavage of native MBP, CI at Gly-127-Gly-128 and of the carboxylmethylated MBP, CI at Phe-124-Gly-125. Additional evidence including tryptic subdigestion of carboxylmethylated P29 disclosed the following partial sequence for this peptide: G~y-Tyr~G~y~G~y~Arg~A~a-Ser-Asp-Tyr~Lys~Ser~A~a~His-Lys~G~y-Leu-Lys-G~y~His-Asp~A~a~G in-Gly-Thr-LeuSer-Lys-lleu-Phe-Lys-. This sequence matches MBP residues 125-154. As a result of these findings, Asp-132 and Asp-144 were identified as two of the modified (isomerized or racemized) methyl-accepting L-aspartates in MBP. The results of the proteolysis experiments wherein the sequencing grade staph~iococcal V s proteinase was used at the rarely tested pH of 4.0, rather than at its commonly tested pH of 7.8, also disclose that the proteinase totally failed to recognize and hence cleave the two Glu-X bonds (Glu-82-Asn-83 and Glu l l 8 -Gly l l 9 ) of MBP, preferring to cleave the protein at a number of hitherto unreported sites.