Isolation of the murine S100 protein MRP14 (14 kDa migration-inhibitory-factor-related protein) from activated spleen cells: Characterization of post-translational modifications and zinc binding

MRP14 (macrophage migration-inhibitory factor-related protein of molecular mass 14 kDa) is an S100 calcium binding protein constitutively expressed in human neutrophils which may be associated with cellular activation/inflammation. Murine MRP14 expression was up-regulated following concanavalin A activation of spleen cells, and the protein was isolated from conditioned medium in high yield (approx. 500 ng/ml). MRP14 had a mass of 12,972 ± 2 Da by electrospray ionization MS, whereas the theoretical mass derived from the cDNA sequence, after removal of the initiator Met, was 12,918 Da, suggesting that the protein was post-translationally modified. We identified four post-translational modifications of MRP14: removal of the N-terminal Met, N-terminal acetylation, disulphide bond formation between Cys⁷⁹ and Cys⁹⁰, and 1-methylation of His¹⁰⁶; the calculated mass was then 12,971.8 Da. Methylation of His¹⁰⁶ was further characterized after incubation of spleen cells with L-[methyl-³H]Met during concanavalin A stimulation. Sequential analysis of a peptide (obtained by digestion with Lys C) containing methylated His indicated that > 80% of the label in the cycle corresponded to His¹⁰⁶, suggesting that the methyl residue was transferred from S-adenosyl-L-methionine. Comparison of the C18 reverse-phase HPLC retention times of phenylthiocarbamoyl derivatives of a hydrolysed digest peptide of MRP14 with those of standards confirmed methyl substitution on the 1-position of the imidazole ring. MRP14 bound more ⁶⁵Zn²⁺ than the same amounts of the 10 kDa chemotactic protein (CP10) or S100β. Ca²⁺ decreased Zn²⁺ binding in S100β but it did not influence binding to MRP14, suggesting that the Zn²⁺ binding site was distinct from and independent of the two Ca²⁺ binding domains.