Tamura H, Tanaka S, Oda T, Uemura Y, Aketagawa J, Hashimoto Y
Department of Biochemistry, Faculty of Science, Saitama University, Urawa, Japan.
Carbohydr Res. 1996 Dec 13;295:103-16. doi: 10.1016/s0008-6215(96)90128-7.
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.
一种新型的（1→3）-β-D-葡聚糖结合蛋白（T-GBP）已从日本鲎（Tachypleus tridentatus）的血细胞裂解物中纯化出来。它是一种碱性蛋白（pI 9.2），似乎是由亚基组成的同四聚体，亚基的表观分子量为168000，其氨基末端序列（20个残基）为KSGFILTAPKSLTLGRNNRL。T-GBP对用纯化的G因子制剂和裂解物中的促凝血酶重建的（1→3）-β-D-葡聚糖引发的凝血级联反应具有抑制作用。使用Boc-Leu-Gly-Arg-4-硝基苯胺作为显色底物，通过测量凝血级联反应产物凝血酶的残留酰胺水解活性来评估（1→3）-β-D-葡聚糖与T-GBP的结合。一系列（1→3）-β-D-葡聚糖和其他多糖对T-GBP的结合特异性通过该途径中第一个蛋白酶原G因子的激活相对抑制率（%）来表示，G因子通过与（1→3）-β-D-葡聚糖结合而被激活。发现T-GBP对线性（1→3）-β-D-葡聚糖具有高亲和力，例如茯苓聚糖、可德兰多糖和异淀粉酶多糖。它能够与具有侧链分支和混合连接的（1→3）-β-D-葡聚糖结合，如裂褶菌多糖、香菇多糖、海带多糖、酵母β-D-葡聚糖，以及（1→3），（1→4）-β-D-葡聚糖，如地衣多糖和大麦β-D-葡聚糖。使用针对T-GBP产生的特异性抗体（兔IgG）通过酶联免疫吸附测定法证明了茯苓聚糖与T-GBP的结合。
