Total chemical synthesis and chemotactic activity of human S100A12 (EN-RAGE)

• Published in: FEBS letters 2001-01, Vol.488 (1), p.85-90
• Main Authors: Miranda, Les P., Tao, Tony, Jones, Alun, Chernushevich, Igor, Standing, Kenneth G., Geczy, Carolyn L., Alewood, Paul F.
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Binding Sites; CaBP, calcium-binding protein; Calcium; Calcium - metabolism; Calcium - pharmacology; Calcium-Binding Proteins - chemical synthesis; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - pharmacology; CD, circular dichroism; Cell Line; Chemotactic; Chemotactic Factors - chemical synthesis; Chemotactic Factors - chemistry; Chemotactic Factors - pharmacology; Chemotaxis - drug effects; Chromatography, High Pressure Liquid; Circular Dichroism; Dimerization; ESI-MS, electrospray ionisation mass spectrometry; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HL-60 Cells; Humans; Macrophages - cytology; Macrophages - drug effects; Mass Spectrometry; Molecular Sequence Data; MRP, migration inhibitory factor related protein; Neutrophils - cytology; Neutrophils - drug effects; NMR, nuclear magnetic resonance; Non-covalent interaction; Protein Structure, Secondary - drug effects; Q, quadrupole; RAGE, receptor for advanced glycosylation end products; RP-HPLC, reversed phase-high performance liquid chromatography; S100; S100 Proteins; S100A12 Protein; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; Sequence Alignment; Solid-phase peptide synthesis; Standard IUPAC single and triple letter codes for amino acids are used throughout; TOF, time-of-flight; Zinc; Zinc - metabolism; Zinc - pharmacology
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(00)02392-9

Human S100A12 (extracellular newly identified RAGE (receptor for advanced glycosylation end products)-binding protein), a new member of the S100 family of EF-hand calcium-binding proteins, was chemically synthesised using highly optimised 2-(1 H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/ tert-butoxycarbonyl in situ neutralisation solid-phase chemistry. Circular dichroism studies indicated that CaCl 2 decreased the helical content by 27% whereas helicity was marginally increased by ZnCl 2. The propensity of S100A12 to dimerise was examined by electrospray ionisation time-of-flight mass spectrometry which clearly demonstrated the prevalence of the non-covalent homodimer (20 890 Da). Importantly, synthetic human S100A12 in the nanomolar range was chemotactic for neutrophils and macrophages in vitro.