Structure-function analysis of Pattem-recognition Proteins

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In the horseshoe crab, LPS induces hemocyte exocytotic degranulation, resulting in the secretion of various defense molecules, such as coagulation factors, antimicrobial peptides, and lectins. Recent studies have demonstrated that the zymogen form of the serine protease factor C, a major granular component of hemocyte, also exists on the hemocyte surface and functions as a biosensor for LPS. The proteolytic activity of activated factor C initiates hemocyte exocytosis via a G protein mediated signal transduction pathway. Furthermore, it has become clear that an endogenous mechanism for the feedback amplification of the innate immune response exists and is dependent upon a granular component of the horseshoe crab hemocyte. We report that Factor C associates with LPS on the hemocyte surface and directly recognizes Gram-negative bacteria. Structure-function analyses reveal that the LPS-binding site is present in the amino-terminal cysteine-rich (Cys-rich) region of the molecule, and that it contains a tri-peptide sequence consisting of an aromatic residue flanked by two basic residues, which is conserved in other mammalian LPS-recognizing proteins. Moreover, we demonstrate that the Cys-rich region specifically binds to LPS on Gram-negative bacteria, and that mutations in the tri-peptide motif abrogate its association with both LPS and Gram-negative bacteria, underscoring the importance of the tri-peptide in LPS interaction. Although the innate immune response to LPS in the horseshoe crab is distinct from that of mammals, it appears to rely on structural features that are conserved among LPS-recognizing proteins from diverse species.