TY - JOUR
T1 - An Arg/Lys → Gln mutant of recombinant murine myelin basic protein as a mimic of the deiminated form implicated in multiple sclerosis
AU - Bates, Ian R.
AU - Libich, David S.
AU - Wood, D. Denise
AU - Moscarello, Mario A.
AU - Harauz, George
PY - 2002/11/23
Y1 - 2002/11/23
N2 - 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citrulline, we have constructed a mutant form of recombinant murine MBP (rmMBP) in which 5 Arg and 1 Lys residues have been replaced by Gln as the most reasonable analogue of Cit.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.
AB - 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citrulline, we have constructed a mutant form of recombinant murine MBP (rmMBP) in which 5 Arg and 1 Lys residues have been replaced by Gln as the most reasonable analogue of Cit.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.
KW - Cathepsin D
KW - Circular dichroism
KW - Citrulline
KW - Deimination
KW - Lipid vesicle aggregation
KW - Multiple sclerosis
KW - Myelin basic protein
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U2 - 10.1016/S1046-5928(02)00017-7
DO - 10.1016/S1046-5928(02)00017-7
M3 - Article
C2 - 12135568
AN - SCOPUS:0036025305
VL - 25
SP - 330
EP - 341
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
IS - 2
ER -