An Arg/Lys → Gln mutant of recombinant murine myelin basic protein as a mimic of the deiminated form implicated in multiple sclerosis

Ian R. Bates, David S. Libich, D. Denise Wood, Mario A. Moscarello, George Harauz

Research output: Contribution to journalArticle

50 Scopus citations

Abstract

The degree of post-translational enzymatic deimination (conversion of arginyl to citrullinyl residues) of myelin basic protein (MBP) is correlated with the severity of the human autoimmune disease multiple sclerosis (MS). It is difficult to obtain large quantities of deiminated MBP from natural sources (autopsy material), and in vitro deimination using peptidylarginine deiminase (EC 3.5.3.15) is both non-specific and irreproducible. Since there is no known codon for citrulline, we have constructed a mutant form of recombinant murine MBP (rmMBP) in which 5 Arg and 1 Lys residues have been replaced by Gln as the most reasonable analogue of Cit. The residues were chosen to correspond to the 6 Arg residues in human MBP which are most commonly deiminated in chronic MS. The mutant species, rmMBP-qCit6 where the "q" represents "quasi-," was probed by numerous biochemical and biophysical techniques. Highly homogeneous protein preparations were obtained using a modified expression system which minimised spurious misincorporation of Lys for Arg, as ascertained by electrospray ionisation mass spectrometry. The mutant form rmMBP-qCit6 had a reduced ability to aggregate lipid vesicles, a slightly greater susceptibility to digestion by cathepsin D, a greater proportion of random secondary structure, and different conformational responses to lipids, compared with the unmodified rmMBP. Overall, the mutant protein's properties were consistent with the effects of deimination and support its use as a model for evaluating the effects of this modification.

Original languageEnglish (US)
Pages (from-to)330-341
Number of pages12
JournalProtein Expression and Purification
Volume25
Issue number2
DOIs
StatePublished - Nov 23 2002
Externally publishedYes

Keywords

  • Cathepsin D
  • Circular dichroism
  • Citrulline
  • Deimination
  • Lipid vesicle aggregation
  • Multiple sclerosis
  • Myelin basic protein

ASJC Scopus subject areas

  • Biotechnology

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