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 12972 +/- 2 Da by electrospray ionization MS, whereas the theoretical mass derived from the cDNA sequence, after removal of the initiator Met, was 12918 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 12971.8 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 85Zn2+ than the same amounts of the 10 kDa chemotactic protein (CP10) or S100 beta. Ca2+ decreased Zn2+ binding in S100 beta 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.
Raftery
M J
MJ
Immunology Group, Heart Research Institute, Camperdown, New South Wales, Australia.
Harrison
C A
CA
Alewood
P
P
Jones
A
A
Geczy
C L
CL
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D040502:Calgranulin B; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A / Q000494:pharmacology; D003545:Cysteine; D004220:Disulfides / Q000032:analysis; D005786:Gene Expression Regulation / Q000187:drug effects*; D006639:Histidine; D006801:Humans; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D013058:Mass Spectrometry; D008715:Methionine / Q000378:metabolism; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000276:immunology / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology
D000943:Antigens, Differentiation; D002135:Calcium-Binding Proteins; D040502:Calgranulin B; D004220:Disulfides; D010446:Peptide Fragments; D003208:Concanavalin A; D006639:Histidine; D008715:Methionine; D015032:Zinc; D003545:Cysteine