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This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: jimmunol.0900430v1 183/6/3810    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Google Scholar Articles by Ueda, Y. Articles by Kawabata, S.-i. PubMed PubMed Citation Articles by Ueda, Y. Articles by Kawabata, S.-i.

Factor G Utilizes a Carbohydrate-Binding Cleft That Is Conserved between Horseshoe Crab and Bacteria for the Recognition of β-1,3-D-Glucans¹

Yuki Ueda^2,*, Shuhei Ohwada^2,, Yoshito Abe, Toshio Shibata^*, Manabu Iijima, Yukiko Yoshimitsu, Takumi Koshiba^*,, Munehiro Nakata, Tadashi Ueda and Shun-ichiro Kawabata^3,*,

^* Graduate School of Systems Life Sciences, Department of Biology, Faculty of Sciences, and Department of Immunology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan; and Department of Applied Biochemistry, Tokai University, Kanagawa, Japan

In the horseshoe crab, the recognition of β-1,3-D-glucans by factor G triggers hemolymph coagulation. Factor G contains a domain of two tandem xylanase Z-like modules (Z1-Z2), each of which recognizes β-1,3-D-glucans. To gain an insight into the recognition of β-1,3-D-glucans from a structural view point, recombinants of Z1-Z2, the C-terminal module Z2, Z2 with a Cys to Ala substitution (Z2A), and its tandem repeat Z2A-Z2A were characterized. Z2 and Z1-Z2, but not Z2A and Z2A-Z2A, formed insoluble aggregates at higher concentrations more than 30 and 3 µM, respectively. Z1-Z2 and Z2A-Z2A bound more strongly to an insoluble β-1,3-D-glucan (curdlan) than Z2A. The affinity of Z2A for a soluble β-1,3-D-glucan (laminarin) was equivalent to those of Z1-Z2, Z2A-Z2A, and native factor G, suggesting that the binding of a single xylanase Z-like module prevents the subsequent binding of another module to laminarin. Interestingly, Z2A as well as intact factor G exhibited fungal agglutinating activity, and fungi were specifically detected with fluorescently tagged Z2A by microscopy. The chemical shift perturbation of Z2A induced by the interaction with laminaripentaose was analyzed by nuclear magnetic resonance spectroscopy. The ligand-binding site of Z2A was located in a cleft on a β-sheet in a predicted β-sandwich structure, which was superimposed onto cleft B in a cellulose-binding module of endoglucanase 5A from the soil bacterium Cellvibrio mixtus. We conclude that the pattern recognition for β-1,3-D-glucans by factor G is accomplished via a carbohydrate-binding cleft that is evolutionally conserved between horseshoe crab and bacteria.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (priority area 839 to S.K. and no. 18370045 to S.K.) and the Naito Foundation (to S.K. and M.I.).

² Y.U. and S.O. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Shun-ichiro Kawabata, Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-Ku, Fukuoka 812-8581, Japan. E-mail address: skawascb{at}kyudai.jp

⁴ The online version of this article contains supplemental material.

⁵ Abbreviations used in this paper: NMR, nuclear magnetic resonance; SPR, surface plasmon resonance; QCM, quartz crystal microbalance; ITC, isothermal titration calorimetry; EGFP, enhanced GFP; DP, degree of polymerization; CBM6, family 6 carbohydrate-binding modules; CmCBM6-2, the C-terminal noncatalytic cellulose-binding module of endoglucanase 5A from Cellvibrio mixtus; HSQC, heteronuclear signal quantum correlation.