The preparation of antibodies reactive against citrulline-containing charge isomers of myelin basic protein but not against the arginine-containing charge isomers.
Human myelin basic protein (MBP) is composed of several charge isomers, the result of various post-translational modifications. One of the charge isomers C-8, has been shown in our laboratory to contain six citrullinyl residues which replace arginyl residues at selected sites in the MBP. In order to determine the disposition of the citrulline-containing charge isomers in the myelin stack, we prepared specific antisera against the citrullinyl group. Since 9-fluorenylmethoxycarbonyl (Fmoc)-citrulline, required for the preparation of the synthetic peptides to be used for antibody production, was not commercially available, synthesis of the Fmoc-citrulline was a necessary prerequisite. The synthesis and purification of the N-9-fluorenylmethyloxycarbonyl derivative of citrulline is described. It was characterized by thin layer chromatography, 1H and 13C NMR spectroscopy, fast-atom bombardment mass spectroscopy, and thermal analyses. It was used in the automated peptide synthesis of a peptide Ala-Cit-His-Gly-Phe-Leu-Pro-Cit-His-Arg corresponding to residues 24-33 and Gly-Cit-Asp-Ser-Arg-Ser-Gly-Ser-Pro-Met-Ala-Cit-Arg, corresponding to residues 158-170 of the C-8 sequence, a naturally occurring charge isomer of human myelin basic protein, and a tetracitrulline peptide, Cit-Cit-Cit-Cit-Gly. The tetracitrulline peptide was used for the production of an antibody shown to react only with synthetic peptides and proteins containing citrulline. This antibody was used to distinguish between a citrulline-containing protein, C-8, a naturally occurring charge isomer of MBP, and a non-citrulline-containing charge isomer of MBP, C-1.
McLaurin
J
J
Research Institute, Hospital for Sick Children, Toronto, Canada.
Moscarello
M A
MA
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001120:Arginine / Q000032:analysis*; D002152:Calorimetry, Differential Scanning; D002855:Chromatography, Thin Layer; D002956:Citrulline / Q000032:analysis / Q000276:immunology*; D006801:Humans; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000032:analysis / Q000276:immunology*; D016339:Spectrometry, Mass, Fast Atom Bombardment
D000906:Antibodies; D004676:Myelin Basic Protein; D002956:Citrulline; D001120:Arginine