Carboxylmethylation affects the proteolysis of myelin basic protein by Staphylococcus aureus V8 proteinase.
Bovine myelin basic protein (MBP), charge isoform 1 (C1) was carboxylmethylated by the enzyme D-aspartyl/L-isoaspartyl protein methyltransferase (EC. 2.1.1.77) and the carboxylmethylated protein was subjected to proteolysis by sequencing grade staphylococcal V8 proteinase at pH 4.0 to identify its carboxylmethylated modified aspartate and/or asparagine residues which are recognized by this methyltransferase. Native MBP, C1 was treated similarly and the proteolysis products were compared, using electrophoretic, chromatographic and amino acid sequencing techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed differences in the kinetics of proteolysis between the native and the carboxylmethylated MBP, C1 which were confirmed using HPLC. Partial sequencing of the native and carboxylmethylated fragments eluting at about 29 min (P29) revealed cleavage of native MBP, C1 at Gly-127-Gly-128 and of the carboxylmethylated MBP, C1 at Phe-124-Gly-125. Additional evidence including tryptic subdigestion of carboxylmethylated P29 disclosed the following partial sequence for this peptide: Gly-Tyr-Gly-Gly-Arg-Ala-Ser-Asp-Tyr-Lys-Ser-Ala-His-Lys-Gly-Leu-Lys- Gly-His-Asp-Ala-Gln-Gly-Thr-Leu-Ser-Lys-Ileu-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 staphylococcal V8 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-118-Gly-119) of MBP, preferring to cleave the protein at a number of hitherto unreported sites.
Sellinger
O Z
OZ
Laboratory of Neurochemistry, University of Michigan Medical Center, Ann Arbor 48109-0720.
Wolfson
M F
MF
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011497:Protein O-Methyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology*
D004676:Myelin Basic Protein; D010446:Peptide Fragments; D012436:S-Adenosylmethionine; D011497:Protein O-Methyltransferase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase