Spiroplasma citri spiralin acts in vitro as a lectin binding to glycoproteins from its insect vector Circulifer haematoceps
Spiroplasma citri spiralin acts in vitro as a lectin binding to glycoproteins from its insect vector Circulifer haematoceps
复制标题
DOI：
10.1094/phyto-95-0541
复制
发表时间：
2005-05-01
期刊：
影响因子：
3.2
通讯作者：
Saillard, C
中科院分区：
文献类型：
--
作者：
Killiny, N;Castroviejo, M;Saillard, C
为了了解叶蝉Circulifer hoematoceps传播柑橘螺原体的分子机制，我们使用蛋白质印迹的螺原体重叠测定（Far-western测定）筛选了作为假定的柑橘螺原体结合分子的叶蝉蛋白。通过一维或二维十二烷基硫酸钠聚丙烯酰胺凝胶电泳分离昆虫蛋白，进行印迹，并用柑橘链霉菌蛋白进行探测。在这项体外测定中，我们发现螺原体蛋白对七种叶蝉蛋白表现出亲和力。柑橘链霉菌蛋白与分子量为 50 和 60 kDa 的昆虫蛋白之间的相互作用被发现对糖敏感。这些昆虫蛋白被鉴定为高甘露糖 N-糖蛋白，支持糖蛋白-凝集素类型与柑橘链霉菌蛋白的相互作用。柑橘链球菌中的凝集素检测仅显示一种 24 kDa 的蛋白质。通过对所有柑橘链霉菌蛋白印迹进行叶蝉蛋白重叠测定，一种具有相似分子量 24 kDa 的螺原体蛋白显示出昆虫蛋白结合能力。该蛋白被鉴定为螺旋蛋白，它是柑橘链球菌最丰富的膜蛋白。远西方用纯化的螺旋蛋白和昆虫糖蛋白进行的实验证实了螺旋蛋白与 50 和 60 kDa 的昆虫糖蛋白的结合。因此，螺旋蛋白可以通过介导螺原体粘附到昆虫载体肠道或唾液腺的上皮细胞，在柑橘链球菌的传播中发挥关键作用。
In order to understand the molecular mechanisms underlying transmission of Spiroplasma citri by the leafhopper Circulifer hoematoceps, we screened leafhopper proteins as putative S. citri-binding molecules using a spiroplasma overlay assay of protein blots (Far-western assay). Insect proteins were separated by one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, blotted, and probed with S. citri proteins. In this in vitro assay, we found that spiroplasma proteins exhibited affinity for seven leafhopper proteins. The interactions between S. citri proteins and insect proteins with molecular masses of 50 and 60 kDa were found to be sugar sensitive. These insect proteins were identified as high mannose N-glycoproteins, which support an interaction of glycoprotein-lectin type with S. citri proteins. Lectin detection in S. citri has revealed only one protein of 24 kDa. Using a leafhopper protein overlay assay on all S. citri protein blot, one spiroplasma protein with a similar molecular mass of 24 kDa was shown to display an insect protein-binding capacity. This protein was identified as the spiralin, which is the most abundant membrane protein of S. citri. Far-western experiments performed with purified spiralin and insect glycoproteins confirmed the binding of spiralin to the insect glycoproteins of 50 and 60 kDa. Thus, the spiralin could play a key role in the transmission of S. citri by mediating spiroplasma adherence to epithelial cells of insect vector gut or salivary gland.