The Most Abundant Glycoprotein of Amebic Cyst Walls (Jacob) Is a Lectin with Five Cys-Rich, Chitin-Binding Domains

The infectious stage of amebae is the chitin-walled cyst, which is resistant to stomach acids. In this study an extraordinarily abundant, encystation-specific glycoprotein (Jacob) was identified on two-dimensional protein gels of cyst walls purified from Entamoeba invadens. Jacob, which was acidic and had an apparent molecular mass of ∼100 kDa, contained sugars that bound to concanavalin A and ricin. The jacob gene encoded a 45-kDa protein with a ladder-like series of five Cys-rich domains. These Cys-rich domains were reminiscent of but not homologous to the Cys-rich chitin-binding domains of insect chitinases and peritrophic matrix proteins that surround the food bolus in the insect gut. Jacob bound purified chitin and chitin remaining in sodium dodecyl sulfate-treated cyst walls. Conversely, the E. histolytica plasma membrane Gal/GalNAc lectin bound sugars of intact cyst walls and purified Jacob. In the presence of galactose, E. invadens formed wall-less cysts, which were quadranucleate and contained Jacob and chitinase (another encystation-specific protein) in secretory vesicles. A galactose lectin was found to be present on the surface of wall-less cysts, which phagocytosed bacteria and mucin-coated beads. These results suggest that the E. invadens cyst wall forms when the plasma membrane galactose lectin binds sugars on Jacob, which in turn binds chitin via its five chitin-binding domains.

阿米巴的感染阶段为几丁质壁包囊（chitin-walled cyst），其可抵御胃酸。本研究从侵袭内阿米巴（Entamoeba invadens）纯化的包囊壁的二维蛋白质凝胶（two-dimensional protein gels）中，鉴定出一种丰度极高的成囊特异性糖蛋白（encystation-specific glycoprotein，Jacob）。该蛋白呈酸性，表观分子量约为100 kDa，其所含糖基可与刀豆蛋白A（concanavalin A）及蓖麻毒素（ricin）结合。Jacob基因编码一个45 kDa的蛋白质，包含5个呈梯状分布的半胱氨酸富集结构域（Cys-rich domains）。这类半胱氨酸富集结构域与昆虫几丁质酶（insect chitinases）及包裹昆虫肠道食糜的围食膜蛋白（peritrophic matrix proteins）所含的半胱氨酸富集几丁质结合结构域具有相似性，但并无同源性。Jacob可结合纯化的几丁质，以及经十二烷基硫酸钠（sodium dodecyl sulfate，SDS）处理的包囊壁中残留的几丁质。与之相反，溶组织内阿米巴（E. histolytica）质膜上的半乳糖/N-乙酰半乳糖胺凝集素（Gal/GalNAc lectin）可结合完整包囊壁的糖基及纯化的Jacob蛋白。在半乳糖存在的条件下，侵袭内阿米巴可形成无壁包囊，此类包囊为四核结构，其分泌囊泡（secretory vesicles）中含有Jacob及几丁质酶（chitinase，另一种成囊特异性蛋白）。研究人员在无壁包囊的表面发现了半乳糖凝集素，这类包囊可吞噬细菌及黏蛋白包被微球（mucin-coated beads）。上述结果表明，当质膜半乳糖凝集素结合Jacob蛋白的糖基后，Jacob可通过其5个几丁质结合结构域与几丁质结合，进而完成侵袭内阿米巴包囊壁的形成。