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

被引:50
作者
Bates, IR
Libich, DS
Wood, DD
Moscarello, MA
Harauz, G
机构
[1] Univ Guelph, Dept Mol Biol & Genet, Guelph, ON N1G 2W1, Canada
[2] Univ Guelph, Biophys Interdept Grp, Guelph, ON N1G 2W1, Canada
[3] Hosp Sick Children, Dept Struct Biol & Biochem, Toronto, ON M5G 1X8, Canada
基金
加拿大健康研究院; 加拿大自然科学与工程研究理事会;
关键词
myelin basic protein; multiple sclerosis; deimination; citrulline; lipid vesicle aggregation; cathepsin D; circular dichroism;
D O I
10.1016/S1046-5928(02)00017-7
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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citrulline, we have constructed a mutant form of recombinant murine MBP (rmMBP) in which 5 Arg and 1 Lys residues have been replaced by Gln as the most reasonable analogue of Cit.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-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 rrnMBP. 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. (C) 2002 Elsevier Science (USA). All rights reserved.
引用
收藏
页码:330 / 341
页数:12
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