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Carboxylmethylation affects the proteolysis of myelin basic protein by Staphylococcus aureus V8 proteinase

O Z Sellinger1, M F Wolfson

  • 1Laboratory of Neurochemistry, University of Michigan Medical Center, Ann Arbor 48109-0720.

Biochimica Et Biophysica Acta
|October 25, 1991
PubMed

Insights

This study identifies specific aspartate residues in bovine myelin basic protein (MBP) that are modified by D-aspartyl/L-isoaspartyl protein methyltransferase. These findings reveal novel cleavage sites for staphylococcal V8 proteinase at pH 4.0.

Area of Science:

  • Biochemistry
  • Proteomics
  • Enzymology

Background:

  • Myelin basic protein (MBP) is crucial for myelin sheath integrity.
  • Protein methylation, particularly at aspartate residues, plays a role in protein function and aging.
  • D-aspartyl/L-isoaspartyl protein methyltransferase (EC. 2.1.1.77) catalyzes the repair of isomerized aspartate residues.

Purpose of the Study:

  • To identify the specific aspartate and/or asparagine residues in bovine MBP (charge isoform 1, C1) that are recognized and carboxylmethylated by D-aspartyl/L-isoaspartyl protein methyltransferase.
  • To investigate the effect of this methylation on the proteolysis of MBP by staphylococcal V8 proteinase.
  • To characterize novel cleavage sites of staphylococcal V8 proteinase at pH 4.0.

Main Methods:

  • Carboxymethylation of native bovine MBP, C1 using D-aspartyl/L-isoaspartyl protein methyltransferase.
  • Proteolysis of native and carboxylmethylated MBP, C1 using sequencing grade staphylococcal V8 proteinase at pH 4.0.
  • Analysis of proteolysis products using SDS-PAGE, HPLC, and amino acid sequencing.

Main Results:

  • Carboxymethylation altered the proteolysis kinetics of MBP, C1, confirmed by SDS-PAGE and HPLC.
  • Partial sequencing identified cleavage of native MBP, C1 at Gly-127-Gly-128 and carboxylmethylated MBP, C1 at Phe-124-Gly-125.
  • Asp-132 and Asp-144 were identified as methyl-accepting L-aspartate residues in MBP.
  • Staphylococcal V8 proteinase at pH 4.0 failed to cleave Glu-X bonds (Glu-82-Asn-83 and Glu-118-Gly-119) but cleaved at new sites.

Conclusions:

  • Asp-132 and Asp-144 are key methyl-accepting sites for D-aspartyl/L-isoaspartyl protein methyltransferase in bovine MBP.
  • Carboxymethylation of MBP influences its susceptibility to V8 proteinase digestion.
  • The study reveals previously unreported cleavage specificities of staphylococcal V8 proteinase at pH 4.0.

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