An Arg/Lys-->Gln mutant of recombinant murine myelin basic protein as a mimic of the deiminated form implicated in multiple sclerosis.
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citrulline, we have constructed a mutant form of recombinant murine MBP (rmMBP) in which 5 Arg and 1 Lys residues have been replaced by Gln as the most reasonable analogue of Cit.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-qCit(6) 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-qCit(6) 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.
Department of Molecular Biology and Genetics, and Biophysics Interdepartmental Group, University of Guelph, 50 Stone Road East, Guelph, Ont., Canada N1G 2W1.
Ian R
IR
Bates
David S
DS
Libich
D Denise
DD
Wood
Mario A
MA
Moscarello
George
G
Harauz
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics; D015153:Blotting, Western; D002402:Cathepsin D / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D005973:Glutamine / Q000235:genetics*; D007700:Kinetics; D008239:Lysine / Q000235:genetics*; D051379:Mice; D008958:Models, Molecular; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009103:Multiple Sclerosis / Q000235:genetics*; D020125:Mutation, Missense / Q000235:genetics*; D004676:Myelin Basic Protein / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization
D004676:Myelin Basic Protein; D020033:Protein Isoforms; D011994:Recombinant Proteins; D005973:Glutamine; D001120:Arginine; D002402:Cathepsin D; D008239:Lysine