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

Abstract

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 rules of variation: Amino acid exchange according to the rotating circular genetic code

“…The hydrophobic and essential amino acids can be considered here as the “engines of variation” constituting the collective “clusters of hydrophobicity” (Margoliash and Smith, 1965). Examples of such exchanges are Ala to Val (Lecomte et al, 1999), Val to Ala (Worn, 1998), Ile to Val (Wang et al, 2009), Val to Ile (Hayes et al, 2000), Val to Leu (Shemirani and Muszbek, 2004), Leu to Val (Junca et al, 2002), Leu to Ile (Elahi et al, 2003), Ile to Leu (Ieiri et al, 2000), Val to Phe (Koene et al, 1997), Leu to Met (Lu and Elzinga, 1977), Phe to Ala or to Ile (Lutsenko et al, 2007), Trp to Ala (Ueda et al, 2009), Phe to Leu (Hoffmeyer et al, 2000), Leu to Phe (Jann et al, 2008), etc. Notice that this rule does not say that all hydrophobic codons are located in the same quadrants' position.…”

Protein Crosslinking by Transglutaminase Controls Cuticle Morphogenesis in Drosophila

“…The incorporation of Bi-PA was detected with biotinylated streptavidin-HRP (upper panel). Z2A (C) is a recombinant version of horseshoe crab β-1,3-D-glucan-binding protein [43], which was used as negative control for the TG-dependent incorporation. Loaded recombinant proteins were detected by Western blotting with an anti-6×His tag antibody or anti-Z2A antibody (lower panels).…”

“…Chitin-binding assays were performed as previously reported [43]. Briefly, proteins were mixed with chitin in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and incubated at 4°C for 10 min.…”

Presence of Candida cell wall derived polysaccharides in the sera of intensive care unit patients: relation with candidaemia and Candida colonisation

“…In a pilot study evaluating plasma BDG measurement for the diagnosis of deep-seated mycoses and fungal febrile episodes, Obayashi and colleagues reported a sensitivity of 90% for a cutoff point of 20 pg/ml [15]. However, subsequent studies gave disparate results from different colorimetric and turbidimetric methods [16] using glucans as standards, in the absence of knowledge regarding the nature of the molecule(s) detected [17]. With the Fungitell test, the multicentre evaluation by Ostrosky-Zeichner and colleagues, including 107 cases of proven candidosis, reported a sensitivity/specificity ratio of 0.60/0.92 for a cutoff point of 80 pg/ml, although their use of control sera from healthy subjects could have led to an overestimation [18].…”