S100A9 Structural Determinants Affecting Complex Formation with S100A8 (Calprotectin)

Calprotectin, an antimicrobial protein complex, consists of two heterodimeric subunits, S100A8 and S100A9. Unlike S100A8 and other members of the S100 family, the domain structure of S100A9 includes an extended C-terminal tail with amino acids 89-114 which is believed to be crucial for antimicrobial function. In addition, reducing calcium binding by S100A9 may alter complex-dependent functions. Objective: To determine structural features of S100A9, which mediate dimerization of calprotectin. Methods: We used in vitro site-directed mutagenesis to ablate calcium binding sites (E36Q, E78Q and E36Q + E78Q) and deletion mutagenesis to truncate the S100A9 subunit at the C-terminal tail (deletion of 89-114, 113-114). Using the pIRES and pIRES-EGFP expression system, we then transfected S100A8 and mutated S100A9 encoded plasmids into KB cells. Stable calprotectin expressing KB cells were selected in medium containing G418 sulfate. The transfected cells were then selected for expression of green fluorescent protein using a FACSorter. The expression of S100A8 and S100A9 in transfected cells was determined by RT-PCR. The dimerization of these two subunits was determined by reaction with the calprotectin complex-specific murine monoclonal antibody 27E10 in ELISA. Results: Deletion of the C-terminal tail region of S100A9, amino acids 89-114, or 113-114 (the phosphorylation site at threonine-113) did not appear to affect the dimerization of calprotectin. Amino acid substitutions in S100A9 ablating calcium-binding sites destroyed the epitope that binds 27E10. Conclusion: Intact EF-hand calcium-binding domains are required for recognition by the 27E10 antibody. The C-terminal domain of S100A9 is not necessary for recognition by 27E10 and complex formation. This system will enable studies of the structural features of S100A9 essential of the cell autonomous antibacterial properties of calprotectin and model complexing and heterodimer formation of calprotectin as an alternative to the yeast two-hybrid system. Supported by NIH/NIDCR grant R01DE11831 and a scholarship from the Royal Thai Government.