Biswas C, Sinha D, Mandal C
Immunobiology Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, 700032, Calcutta, India.
Mol Immunol. 2000 Aug-Sep;37(12-13):745-54. doi: 10.1016/s0161-5890(00)00096-1.
Achatinin, a 9-O-acetyl sialic acid (9-O-AcSA) binding lectin, has been demonstrated to be synthesized in amoebocytes of Achatina fulica snails. This lectin was affinity-purified from Achatina amoebocytes lysate (AAL); it appeared as a single band on native polyacrylamide gel electrophoresis (PAGE) and showed 16 identical subunits of M.W. 15 kDa on sodium dodecyl sulphate (SDS)-PAGE. It was found to be homologous with an earlier reported lectin, Achatinin-H, derived from hemolymph of A. fulica snails (Sen, G., Mandal, C., 1995. The specificity of the binding site of Achatinin-H, a sialic-acid binding lectin from Achantia fulica. Carbohydr. Res., 268, 115-125). Homology between both lectins was confirmed by their similar electrophoretic mobilities, carbohydrate specificity and cross reactivity on immunodiffusion. Achatinin showed in vitro calcium dependent binding to two 9-O-acetylated sialoglyoconjugates (9-O-AcSG) on lipopolysaccharide (LPS) (Escherichia coli 055: B5) of M.W. 40 kDa and 27.5 kDa, which was abolished following de-O-acetylation. Based on the previously defined narrow sugar specificity of Achatinin towards 9-O-AcSAalpha2-->6GalNAc [Sen, G., Mandal, C., 1995. The specificity of the binding site of Achatinin-H, a sialic-acid binding lectin from Achatina fulica. Carbohydr. Res., 268, 115-125], we conclude that LPS contains this lectinogenic epitope at the terminal sugar moiety. The Achatinin-mediated hemagglutination inhibition of rabbit erythrocytes by LPS further confirmed it. The lectin exhibited bacteriostatic effect on Gram-negative bacteria E. coli, DH5alpha and C600. AAL was earlier reported to undergo coagulation in presence of pg level of LPS (Biswas, C., Mandal, C., 1999. The role of amoebocytes in the endotoxin-mediated coagulation in the innate immunity of Achatina fulica snail, Scand. J. Immunol. 49, 131-138). We now demonstrate that Achatinin participates in LPS-mediated coagulation of AAL as indicated by enhanced release of Achatinin from the LPS stimulated amoebocytes and most importantly, by exhibiting a 77% decline in the coagulation of AAL when depleted of Achatinin. Level of Achatinin sharply declined (17-fold) following injection of LPS (20 microg per snail) to the snails, which was reversible by simultaneous injection of LPS and leupeptin implying the presence of LPS-mediated serine protease activity in Achatinin. This was substantiated when purified Achatinin in vitro showed serine protease activity in the presence of LPS followed by its complete blockage in the presence of leupeptin and phenyl methyl sulphonyl fluoride. Therefore, Achatinin, an abundantly available lectin at multiple sites of A. fulica, by virtue of its interaction with LPS, essentially plays a crucial role in the innate immune protection of A. fulica snails.
玛瑙螺凝集素是一种能结合9 - O - 乙酰化唾液酸（9 - O - AcSA）的凝集素，已被证明是由非洲大蜗牛的变形细胞合成的。这种凝集素是从非洲大蜗牛变形细胞裂解物（AAL）中亲和纯化得到的；在非变性聚丙烯酰胺凝胶电泳（PAGE）上它呈现为一条单一的条带，在十二烷基硫酸钠（SDS）-PAGE上显示出16个分子量为15 kDa的相同亚基。发现它与先前报道的源自非洲大蜗牛血淋巴的凝集素玛瑙螺凝集素 - H同源（森，G.，曼达尔，C.，1995年。玛瑙螺凝集素 - H（一种来自非洲大蜗牛的唾液酸结合凝集素）结合位点的特异性。碳水化合物研究，268，115 - 125）。两种凝集素之间的同源性通过它们相似的电泳迁移率、碳水化合物特异性以及免疫扩散中的交叉反应得以证实。玛瑙螺凝集素在体外表现出对脂多糖（LPS）（大肠杆菌055: B5）上两种分子量分别为40 kDa和27.5 kDa的9 - O - 乙酰化唾液酸糖缀合物（9 - O - AcSG）的钙依赖性结合，去O - 乙酰化后这种结合被消除。基于先前确定的玛瑙螺凝集素对9 - O - AcSAα2→6GalNAc的狭窄糖特异性[森，G.，曼达尔，C.，1995年。玛瑙螺凝集素 - H（一种来自非洲大蜗牛的唾液酸结合凝集素）结合位点的特异性。碳水化合物研究，268，115 - 125]，我们得出结论，LPS在末端糖部分含有这种凝集原性表位。LPS对玛瑙螺凝集素介导的兔红细胞血凝抑制作用进一步证实了这一点。该凝集素对革兰氏阴性菌大肠杆菌DH5α和C600表现出抑菌作用。先前报道AAL在皮克水平的LPS存在下会发生凝集（比斯瓦斯，C.，曼达尔，C.，1999年。变形细胞在非洲大蜗牛先天免疫中内毒素介导的凝集作用中的作用，斯堪的纳维亚免疫学杂志。49，131 - 138）。我们现在证明玛瑙螺凝集素参与LPS介导的AAL凝集，这表现为LPS刺激的变形细胞中玛瑙螺凝集素释放增加，最重要的是，当去除玛瑙螺凝集素时，AAL的凝集下降了77%。向蜗牛注射LPS（每只蜗牛20微克）后，玛瑙螺凝集素水平急剧下降（17倍），同时注射LPS和亮肽素可使其恢复，这意味着玛瑙螺凝集素中存在LPS介导的丝氨酸蛋白酶活性。当纯化的玛瑙螺凝集素在体外LPS存在下显示出丝氨酸蛋白酶活性，而在亮肽素和苯甲基磺酰氟存在下完全被阻断时，这一点得到了证实。因此，玛瑙螺凝集素作为非洲大蜗牛多个部位大量存在的一种凝集素，通过与LPS相互作用，在非洲大蜗牛的先天免疫保护中起着至关重要的作用。
