Macrophage migration inhibition related protein 14 kDa (MRP14) is an S100 calcium binding protein, possibly involved in macrophage differentiation/maturation, but the exact function of the protein is not known. The protein sequence of murine MRP14 was first deduced from the cDNA sequence by screening a murine spleen cell cDNA expression library with human MRP14 cDNA under low stringency conditions1. The cDNA sequence indicated that MRP14 was comprised of 113 amino acids with a calculated mass of 13049 Da.
We have isolated MRP14 in high yield from supernatants of Concanavalin A stimulated mouse spleen cells after DEAE sepharose, heparin sepharose and C4 RP chromatography. The experimentally determined mass using electrospray mass spectrometry was 12972±2 Da which was 77 Da less than the theoretical mass derived from the cDNA sequence.
We have identified several post translational modifications, using a combination of mass spectrometry and N-terminal sequencing of peptides derived from endoprotease digestion of MRP14, including N-acetylation, an internal disulphide bond between Cys79 and Cys90 and a particularly unusual modification comprising specific methylation of Hisl06. The calculated mass of MRP14 including all the PT modification is 12971.8 Da which now compares well with the experimentally determined mass (12972±2 Da).
The methylation of Hisl06 was characterised further because it is a very unusual modif1cation. To identify the source of the methyl group of Hisl06 the cell culture medium was supplemented wilh L-[methyl-3H] Met and MRP14 isolated. After Lys C treatment a C-terminal peptide was isolated and N-terminally sequenced, except after each cycle of Edman degradation the PTH amino acids were collected and analysed for incorporated radioactivity. Approximately 80% of the radioactivity was found in the cycle which contained the methylated His indicating that the methyl residue was derived from Met, via S-adenosyl Met.
His can be methylated at either the 1 or 3 position of the imidazole ring. The PTC derivatives of the synthetic isomers, 1-methyl His and 3-methyl His, were readily separated by using C18 RP HPLC. Comparison of the retention times of the PTC derivatives of a hydrolysed digest peptide of MRP14 indicated the substitution to be at the 1 position of the imidazole ring of His.