title: Factor G utilizes a carbohydrate-binding cleft that is conserved between horseshoe crab and bacteria for the recognition of beta-1,3-D-glucans.
abstract: In the horseshoe crab, the recognition of beta-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 beta-1,3-D-glucans. To gain an insight into the recognition of beta-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 approximately 30 and 3 microM, respectively. Z1-Z2 and Z2A-Z2A bound more strongly to an insoluble beta-1,3-D-glucan (curdlan) than Z2A. The affinity of Z2A for a soluble beta-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 beta-sheet in a predicted beta-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 beta-1,3-D-glucans by factor G is accomplished via a carbohydrate-binding cleft that is evolutionally conserved between horseshoe crab and bacteria.
authors.affiliation: Graduate School of Systems Life Sciences, Faculty of Sciences, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ueda
authors.forename: Shuhei
authors.initials: S
authors.lastname: Ohwada
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Abe
authors.forename: Toshio
authors.initials: T
authors.lastname: Shibata
authors.forename: Manabu
authors.initials: M
authors.lastname: Iijima
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Yoshimitsu
authors.forename: Takumi
authors.initials: T
authors.lastname: Koshiba
authors.forename: Munehiro
authors.initials: M
authors.lastname: Nakata
authors.forename: Tadashi
authors.initials: T
authors.lastname: Ueda
authors.forename: Shun-ichiro
authors.initials: S
authors.lastname: Kawabata
mesh_terms: D000818:Animals; D001665:Binding Sites; D002480:Cellulase / Q000737:chemistry; D020633:Cellvibrio / Q000737:chemistry* / Q000201:enzymology; D017124:Conserved Sequence; D043364:Endo-1,4-beta Xylanases; D019143:Evolution, Molecular; D005936:Glucans; D006737:Horseshoe Crabs / Q000737:chemistry*; D037102:Lectins / Q000235:genetics*; D011134:Polysaccharides / Q000378:metabolism; D011509:Proteoglycans; D047071:beta-Glucans / Q000378:metabolism*
chemical_list: D005936:Glucans; D037102:Lectins; D011134:Polysaccharides; D011509:Proteoglycans; D047071:beta-Glucans; C010770:polysaccharide-K; C038459:curdlan; C008247:laminaran; D002480:Cellulase; D043364:Endo-1,4-beta Xylanases