Jun Aketagawa

Characterization of β-Glucan Recognition Site on C-Type Lectin, Dectin 1

ABSTRACT Dectin 1 is a mammalian cell surface receptor for (1→3)-β-d-glucans. Since (1→3)-β-d-glucans are commonly present on fungal cell walls, it has been suggested that dectin 1 is important for recognizing fungal invasion. In this study we tried to deduce the amino acid residues in dectin 1 responsible for β-glucan recognition. HEK293 cells transfected with mouse dectin 1 cDNA could bind to a gel-forming (1→3)-β-d-glucan, schizophyllan (SPG). The binding of SPG to a dectin 1 transfectant was inhibited by pretreatment with other β-glucans having a (1→3)-β-d-glucosyl linkage but not by pretreatment with α-glucans. Dectin 1 has a carbohydrate recognition domain (CRD) consisting of six cysteine residues that are highly conserved in C-type lectins. We prepared 32 point mutants with mutations in the CRD and analyzed their binding to SPG. Mutations at Trp221 and His223 resulted in decreased binding to β-glucan. Monoclonal antibody 4B2, a dectin- 1 monoclonal antibody which had a blocking effect on the β-glucan interaction, completely failed to bind the dectin-1 mutant W221A. A mutant with mutations in Trp221 and His223 did not have a collaborative effect on Toll-like receptor 2-mediated cellular activation in response to zymosan. These amino acid residues are distinct from residues in other sugar-recognizing peptide sequences of typical C-type lectins. These results suggest that the amino acid sequence W221-I222-H223 is critical for formation of a β-glucan binding site in the CRD of dectin 1.

Relationship between conformation and biological response for (1→3)-β-d-glucans in the activation of coagulation Factor G from limulus amebocyte lysate and host-mediated antitumor activity. Demonstration of single-helix conformation as a stimulant.

The relationship between the conformation of (1----3)-beta-D-glucans in gel or hydrated form and the stimulation of two types of biological responses, namely, activation of coagulation Factor G from limulus amebocyte lysate (LAL) and host-mediated antitumor activity was examined. Both types were activated by the single-helical conformation, as revealed by high-resolution, solid-state 13C-n.m.r. spectroscopy. The potency of activation of Factor G was increased over 100-fold by treatment with a NaOH solution which leads to a complete or partial conversion from the triple to the single helix. Such a single-helix specific response was also demonstrated for the antitumor activity of curdlan, although the distinction was less pronounced for branched (1----3)-beta-D-glucans. The presence of the single-helix conformation was observed in schizophyllan gel, even though the triple helix is the most stable form of branched glucans in aqueous media.

Activation of a limulus coagulation factor G by (1 → 3)-g⊝d-glucans

Abstract Various oligo- and poly-saccharides differing in sugar compositions and types of linkage were examined for their ability to activate a limulus coagulation factor G, the first protease in an alternative coagulation cascade of the horseshoe crab, Tachypleus tridentatus , whose amebocytes have originally been known to contain a lipopolysacchaide(LPS)-driven pathway leading to the formationof coagulin gel. Linear and branched (1 → 3)-β- d -glucans and mixed linkage (1 → 3),(1 → 4)-β- d -glucans were found to exhibit the ability to activate factor G at concentrations of 10 −8 –10 −10 g/mL as assayed by amidolytic activity of the clotting enzyme. Laminaran oligosaccharides, laminaran dextrins (number-average mol. wt. ≦ 5800), and other polysaccharides including carboxymethylcellulose, amylose, starch, d -fructans, α- l -arabinan, β- d -xylans, (1 → 3)-β- d -galactan, water-soluble chitin derivatives, chondroitin, chondroitin sulfates, hyaluronan, keratan sulfate, heparins, heparan sulfate, and LPSs were virtually inactive as activators even at a concentration of 10 −7 g/mL. The activating ability increased with increasing number-average mol. wt. (6800–216 000) of linear (1 → 3)-β- d -glucans. The apparent activating ability of gyrophoran, nigeran, and yeast α- d -mannan was largely abolished by digestion with a highly purified Arthrobacter luteus endo-(1 → 3)-β- d -glucanase, which provided supportive evidence for the activation to be ascribed to contaminating (1 → 3)-β- d -glucan(s). Possible participation of ordered structures of (1 → 3)-β- d -glucans in the activation of factor G is discussed.

Inhibition of high-molecular-weight-(1 → 3)-β-d-glucan-dependent activation of a limulus coagulation factor G by laminaran oligosaccharides and curdlan degradation products

Extensive surveys for the effects of various beta-D-glucans on the coagulation cascade in horseshoe crab amebocyte lysates showed that low-mol-wt-(1-->3)-beta-D-glucans and laminaran oligosaccharides inhibit the activation of a limulus coagulation factor G by high-mol-wt-(1-->3)-beta-D-glucans. The inhibitory properties are exclusively dependent upon their number-average mol wt (Mn) in a range of 342-58,100, which correspond to a degree of polymerization (dp) range of 2-359. The most effective is a laminaran dextrin of Mn 5800 (dp of 35-36), which causes 50% inhibition of factor G activation at a concentration of 3.16 ng/mL. The inhibition of the activation of factor G proportional to the concentration of the inhibitor, and the adsorption of factor G by inhibitory beta-D-glucan-conjugated cellulose suggested a high affinity of the inhibitory saccharides for the activator-recognition site of factor G. Branched (1-->6), (1-->3)-beta-D-glucans, laminarans, mixed linkage (1-->3), (1-->4)-beta-D-glucans, and partially substituted curdlan and laminaran were found to be inhibitory, possibly owing to clusters of consecutive (1-->3)-beta-D-glucopyranosyl residues as intrachain units. The inhibition appears to be related to the inability of the inhibitory (1-->3)-beta-D-glucans to form ordered conformations and to their tendency to take a random-coil structure in aqueous solution.

Purification and characterization of a (1 → 3)-β-d-glucan-binding protein from horseshoe crab (Tachypleus tridentatus) amoebocytes

A novel (1-->3)-beta-D-glucan-binding protein (T-GBP) has been purified from the amoebocyte lysate of the Japanese horseshoe crab, Tachypleus tridentatus. It is a basic protein (pI 9.2) which appears to be a homotetramer composed of subunits with an apparent mol wt of 168000 and with an amino-terminal sequence (20 residues) KSGFILTAPKSLTLGRNNRL. T-GBP exerted an inhibitory effect on the (1-->3)-beta-D-glucan-initiated coagulation cascade reconstituted with purified preparations of factor G and the proclotting enzyme from the lysate. The binding of (1-->3)-beta-D-glucans to T-GBP was evaluated by measuring the residual amidolytic activity of the clotting enzyme, the product of the coagulation cascade, using Boc-Leu-Gly-Arg-4-nitroanilide as the chromogenic substrate. The binding specificity of a wide range of (1-->3)-beta-D-glucans and other polysaccharides towards T-GBP was expressed by the relative inhibition (%) of the activation of factor G, the first protease zymogen in the pathway, which is activated by binding to (1-->3)-beta-D-glucans. T-GBP was found to have a high affinity for linear (1-->3)-beta-D-glucans, e.g. pachyman, curdlan, and paramylon. It was able to bind to (1-->3)-beta-D-glucans with side-chain branches and mixed linkage such as schizophyllan, lentinan, laminarins, yeast beta-D-glucan, and (1-->3),(1-->4)-beta-D-glucans such as lichenin and barley beta-D-glucan. Binding of pachyman to T-GBP was demonstrated by an enzyme-linked immunosorbent assay using a specific antibody (rabbit IgG) raised against T-GBP.

Development of a new endotoxin sensor with intermittent injection of limulus reagent for continuous monitoring of dialysate fluid

This report describes a method of continuously, stably, and inexpensively measuring endotoxin (ET) concentrations in dialysate fluid using an ET sensor with intermittent injection of limulus reagent. An ET solution simulating dialysate fluid was sampled in a single tube at a flow rate of 260 microliters/min and mixed with 30 microliters of limulus reagent intermittently injected into the tube. The absorbance of the solution was measured after the limulus reaction at 313 or 318 degrees K at 26 min. A good linear relationship (r = 0.98) between peak area of absorbance and ET concentration at ET concentrations ranging from 0 to 0.12 endotoxin unit (EU)/ml was obtained, using a spectrophotometer with a cell volume of 8 microliter. The baseline rose after the measurements were taken because the cell volume was so small that the cell was stuffed with gel. A good linear relationship (r = 1.00) at ET concentration of 0.1-0.25 EU/ml was also obtained, and the baseline was unchanged after measurements, using a metal free spectrophotometer with a cell volume of 420 microliters. In conclusion, to measure ET concentrations below 0.1 EU/ml, the cell volume of a metal free spectrophotometer should be minimal.

Development of a stopped flow reactor for the rapid, high sensitivity measurement of endotoxin concentration in dialysate

Continuous measurement of endotoxin concentration in dialysate, using a separated endotoxin-specific limulus reagent, promises rapid measurement without the complex operating procedures of the limulus reagent. To achieve high sensitivity measurements in a short period of time, an improved system featuring stopped-flow operation was developed. To prevent dispersion of the limulus reagent and residence of reacting solution containing the limulus reagent in the system reactor, the circuit in the reactor was changed from a coil configuration to a straight line, and its length was reduced. An endotoxin test solution was supplied at 760 microl/min, into which 40 microl of limulus reagent was pulse-injected. Flow was stopped at the point where the test solution entered the reactor. After the completion of the reaction, the solution was passed through a spectrophotometer and the relationship between reaction time and absorbance was determined. Peak tailing was less than that obtained by the conventional technique, good correlation was obtained from the peak height, and a decrease in sensitivity caused by broadening of the peak was suppressed. The lower detection limit of dialysate was 100 endotoxin units (EU)/L at a reaction time of 20 minutes, and 60 EU/L at 30 minutes. Change from the monitoring system to stop-flow operation made high sensitivity monitoring of endotoxin concentration with a short reaction time possible.

A new procedure for stable quantification of endotoxin in dialysate fluid using limulus reagent

For reliable determination of endotoxin, the activation of enzymes in lysate before measurement should be prevented, and the authors have designed a new procedure to effect this by dissolving the enzymes in lysate in a buffer solution of low pH. A given amount of the enzymes in lysate was dissolved in a lower pH buffer solution (pH 6.1-6.3) and the substrate was dissolved in a higher pH buffer solution (pH 8.0). After standing for 0-24 hr, both solutions were mixed with the sample solution. Data on blank absorbance and calibration line slope obtained by the new procedure were compared with those obtained by the conventional procedure. In the conventional procedure, blank absorbance increased with standing time, reaching approximately seven times the initial value in 24 hr, whereas in the improved procedure, it increased by 1.5 times at a standing time of 3 hr, after which it was independent of standing time. The change in slope of the calibration line with standing time was more gradual in the improved procedure than in the conventional procedure. The authors conclude that the activation of enzymes in lysate can be prevented by dissolving the enzymes in a buffer solution of low pH, and that this procedure is effective for long-term monitoring of endotoxin concentration.