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

Abstract

A novel (1 → 3)- β-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 168 000 and with an amino-terminal sequence (20 residues) KSGFILTAPKSLTLGRNNRL. T-GBP exerted an inhibitory effect on the (1 → 3)- β-d-glucan-initiated coagulation cascade reconstituted with purified preparations of factor G and the proclotting enzyme from the lysate. The binding of (1 → 3)- β-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)- β-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)- β-d-glucans. T-GBP was found to have a high affinity for linear (1 → 3)- β-d-glucans, e.g. pachyman, curdlan, and paramylon. It was able to bind to (1 → 3)- β-d-glucans with side-chain branches and mixed linkage such as schizophyllan, lentinan, laminarins, yeast β-d-glucan, and (1 → 3),(1 → 4)- β-d-glucans such as lichenin and barley β-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.