A serine protease zymogen functions as a pattern-recognition receptor for lipopolysaccharides Shigeru Ariki, Kumiko Koori, Tsukasa Osaki, Kiyohito Motoyama, Kei-ichiro Inamori, and Shun-ichiro Kawabata* Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan Edited by John H. Law, University of Arizona, Tucson, AZ, and approved November 20, 2003 (received for review October 24, 2003) Bacterial lipopolysaccharide (LPS)-induced exocytosis of granular the Imd pathway (13). Overexpression of peptidoglycan- hemocytes is a key component of the horseshoe crab’s innate recognition protein-LE, a receptor for the diaminopimelic-acid- immunity to infectious microorganisms; stimulation by LPS induces type peptidoglycan, leads to the activation of prophenoloxidase the secretion of various defense molecules from the granular cascade in Drosophila larvae (14). hemocytes. Using a previously uncharacterized assay for exocyto- The presence of circulating hemocytes is essential to inverte- sis, we clearly show that hemocytes respond only to LPS and not brates’ innate immunity, such as self-͞non-self recognition, to other pathogen-associated molecular patterns, such as ␤-1,3- phagocytosis, encapsulation, and melanization (15). In horse- glucans and peptidoglycans. Furthermore, we show that a granular shoe crabs, granular hemocytes comprise 99% of all hemocytes protein called factor C, an LPS-recognizing serine protease zymo- and are involved in the storage and release of defense molecules, gen that initiates the hemolymph coagulation cascade, also exists including serine protease zymogens, a clottable protein coagu- on the hemocyte surface as a biosensor for LPS. Our data demon- logen, protease inhibitors, antimicrobial peptides, and lectins strate that the proteolytic activity of factor C is both necessary and (16–18). In response to stimulation by LPS, the defense mole- sufficient to trigger exocytosis through a heterotrimeric GTP- cules stored in granules are immediately secreted by exocytosis binding protein-mediating signaling pathway. Exocytosis of he- (16, 19). This response is important for the host defense’s ability mocytes was not induced by thrombin, but it was induced by to engulf and kill invading microbes. The granular components hexapeptides corresponding to the tethered ligands of protease- include two PAMP-sensitive zymogens, factor C (20) and factor activated G protein-coupled receptors (PARs). This finding sug- G (21, 22). These serine protease zymogens are autocatalytically gested the presence of a PAR-like receptor on the hemocyte activated by LPS and ␤-1,3-glucans, respectively. The activated surface. We conclude that the serine protease zymogen on factor C activates factor B, which in turn converts the proclotting the hemocyte surface functions as a pattern-recognition protein enzyme into the clotting enzyme, which converts coagulogen to for LPS. an insoluble coagulin gel. On the other hand, the activated factor G activates the proclotting enzyme directly. he innate immune system is a sensitive non-self-recognizing In horseshoe crabs, however, no specific LPS-recognizing Tcascade triggered by microbial cell wall constituents called protein has been identified as a pattern-recognition protein pathogen-associated molecular patterns (PAMP), such as lipo- involved in signal transduction to trigger exocytosis. Microscopic BIOCHEMISTRY polysaccharides (LPS) of Gram-negative bacteria, ␤-1,3-glucans observation and microfluorometric analysis of intracellular 2ϩ 2ϩ of fungi, and peptidoglycans of Gram-positive bacteria (1–4). Mg and Ca on LPS-induced exocytosis suggest that the These PAMP are recognized via a set of pattern-recognition exocytosis is mediated by a heterotrimeric GTP-binding protein receptors and proteins that are germ-line-encoded receptors of (G protein) that stimulates the inositol-1,4,5-triphosphate- signaling pathway leading to the increase of intracellular Mg2ϩ the innate immune system. Recent studies have revealed that 2ϩ insects and mammals conserve a signaling pathway of the innate and Ca (23). The exocytosis is not induced by a phorbol ester immune system through cell-surface receptors called Tolls and that mimics diacylglycerol to activate protein kinase C. Likewise, through Toll-like receptors (TLRs) (5, 6). In mammals, TLRs on exocytosis is not affected by ethanol or chelerythrine, both of specialized antigen-presenting cells function as signal transduc- which inhibit phospholipase D, or by herbimycin, a tyrosine ers by way of NF-␬B, leading to the production of proinflam- kinase inhibitor (23). In the present study, we have established matory cytokines and the expression of costimulatory molecules a quantitative assay for LPS-induced exocytosis. Furthermore, on the cell surface. These cytokines and costimulatory molecules we report that the LPS-sensitive protease zymogen factor C also are necessary to activate naive T cells; it may be that TLRs are exists on the hemocyte surface, and that the proteolytic activity assembled at the cell membrane as signaling receptor complexes triggers a G protein-mediating exocytosis for innate immune (4, 7, 8). responses. We have made an identification and characterization Although Drosophila Toll controls the host defense to fungal of a pattern-recognition protein that leads to LPS-induced and Gram-positive bacterial infection, it does not function as a exocytosis. pattern-recognition receptor (9). Initially, Drosophila Toll was Materials and Methods identified as a transmembrane protein that controls dorsoventral Materials. ␣ patterning in the Drosophila embryo (10). In the embryo, a Factor C and human -thrombin were purified as proteolytic cascade that includes three proteases (Gastrulation described (20, 24). LPS (Salmonella minnesota R595) and lipid Defective, Snake, and Easter) cleaves a cytokine-like protein, A(Escherichia coli K12) were from List Biological Laboratories (Campbell, CA). Laminarin, thapsigargin, pertussis toxin, Spaetzle, as a ligand for Drosophila Toll. These three proteases, ␣ however, are dispensable in the induction of the Drosophila A23187, L- -phosphatidylcholine, 1,2-diacyl-sn-glycero-3- Toll-mediated immune response (5). During infection, the cleaved form of Spaetzle is produced through another proteo- This paper was submitted directly (Track II) to the PNAS office. lytic cascade, one that includes Persephone, a newly identified Abbreviations: LPS, lipopolysaccharide; PAR, protease-activated G protein-coupled recep- serine protease (11, 12). However, no pattern-recognition pro- tor; PAMP, pathogen-associated molecular patterns; TLR, Toll-like receptor; PPACK, D-Phe- teins initiating the proteolytic cascade have been identified. The Pro-Arg-chloromethylketone; TL-2, tachylectin-2. Drosophila immune system also detects bacteria through pepti- *To whom correspondence should be addressed. E-mail: [email protected] doglycan-recognition proteins, and Gram-negative diamin- u.ac.jp. opimelic-acid-type peptidoglycan is the most potent inducer of © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0306904101 PNAS ͉ January 27, 2004 ͉ vol. 101 ͉ no. 4 ͉ 953–958 Downloaded by guest on October 2, 2021 phospho-L-serine, L-␣-phosphatidylinositol, 1,2-diacyl-sn- glycero-3-phosphoethanolamine, sphingomyelin, and choles- terol were from Sigma. Peptidoglycan (Staphylococcus aureus) was from Fluka. Curdlan was from Wako Pure Chemical (Osaka). U-73122 and U-73343 were from Calbiochem. Masto- paran, D-Phe-Pro-Arg-chloromethylketone (PPACK), and Ala- Ala-Phe-chloromethylketone were from Bachem. Bovine pan- creatic ␣-chymotrypsin treated with N-tosyl-L-lysyl chloromethylketone and trypsin treated with N-tosyl-L- phenylalanyl chloromethylketone were from Worthington. Hexapeptides SFLLRN, SLIGRL, and GYPGKF were synthe- sized at Genenet (Oita, Japan). The COOH-terminal amino acids of these peptides were amidated. Assay of Exocytosis. Hemolymph (1 ml) was collected into 50 ml of pyrogen-free 10 mM Hepes-NaOH, pH 7.0, containing 0.5 M NaCl. The diluted hemolymph (200 ␮l each) was applied to Fig. 1. Effects of PAMP on exocytosis. Hemocytes collected into a sterilized pyrogen-free 24-well plates filled with 800 ␮l of the same buffer, plate were treated with various concentrations of PAMP at 23°Cfor1h.The and then incubated at 23°C for 10 min for the attachment of amount of TL-2 secreted in the supernatant was determined by ELISA. Ⅺ, LPS; ⅜ ‚ ᭛ hemocytes. PAMP, proteases, and activators were added in assay , curdlan; , laminarin; , peptidoglycan. buffer (10 mM Hepes-NaOH, pH 7.0, containing 0.5 M NaCl, 50 mM MgCl2, and 10 mM CaCl2). For inhibition studies, hemo- The resulting immunocomplex bound to protein A-Sepharose cytes were pretreated with inhibitors at 23°C for 20 min for was collected by centrifugation and washed with the same buffer. U-73122 or 1 h for pertussis toxin. Hemocytes were then The pellet was subjected to SDS͞PAGE (27) and transferred to stimulated with activators or LPS. After incubation at 23°C for nitrocellulose membranes overnight at 20 V by using an elec- 1 h, each exocytosed fluid was collected by centrifugation troblot apparatus (Bio-Rad). After blocking, the antigens were at 2,000 ϫ g for 5 min and quantitated by ELISA by using visualized by streptavidin-biotinylated horseradish-peroxidase anti-coagulogen or anti-tachylectin-2 (TL-2) antibody. complex (Amersham Pharmacia). ELISA. The amount of coagulogen in the exocytosed fluid was Preparation of Activated Factor C