Identification of a 521-kilodalton protein (Gli521) involved in force generation or force transmission for Mycoplasma mobile gliding
Identification of a 521-kilodalton protein (Gli521) involved in force generation or force transmission for Mycoplasma mobile gliding
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DOI：
10.1128/jb.187.10.3502-3510.2005
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发表时间：
2005-05-01
影响因子：
3.2
通讯作者：
Miyata, M
中科院分区：
文献类型：
--
作者：
Seto, S;Uenoyama, A;Miyata, M
已知几种支原体物种可以在固体表面（例如玻璃）上沿膜突出的方向滑动，但这种运动的机制尚不清楚。为了鉴定一种参与滑行的新型蛋白质，我们针对最快滑翔机支原体的不溶性蛋白部分制备了单克隆抗体，并筛选了抗体对滑行的抑制作用。五种单克隆抗体阻止了滑行支原体的运动，将它们保留在玻璃表面上，并且它们都在免疫印迹中识别出大蛋白质。这种名为 Gli521 的蛋白质由 4,738 个氨基酸组成，预计分子量为 520,559 Da，编码在另一种滑动蛋白 Gli349 的基因下游，已知 Gli349 负责滑动过程中的玻璃结合。 Edman 降解分析表明，N 端区域在第 43 和第 44 位氨基酸残基之间的肽键处进行加工。对先前分离的滑动突变体的分析表明，Gli521 蛋白在非结合突变体 m9 中缺失，其中 gli521 基因被第 1170 位氨基酸密码子处的无义突变截断。免疫荧光和免疫电子显微镜表明，Gli521 定位在膜突出的底部，在“颈部”，如之前在 Gli349 中观察到的。抗Gli521抗体对滑行运动的抑制作用的分析表明，该蛋白负责力的产生或力的传递，其作用与Gli349不同，并且还表明Gli349和Gli521在滑行过程中的构象变化。
Several mycoplasma species are known to glide on solid surfaces such as glass in the direction of the membrane protrusion, but the mechanism underlying this movement is unknown. To identify a novel protein involved in gliding, we raised monoclonal antibodies against a detergent-insoluble protein fraction of Mycoplasma mobile, the fastest glider, and screened the antibodies for inhibitory effects on gliding. Five monoclonal antibodies stopped the movement of gliding mycoplasmas, keeping them on the glass surface, and all of them recognized a large protein in immunoblotting. This protein, named Gli521, is composed of 4,738 amino acids, has a predicted molecular mass of 520,559 Da, and is coded downstream of a gene for another gliding protein, Gli349, which is known to be responsible for glass binding during gliding. Edman degradation analysis indicated that the N-terminal region is processed at the peptide bond between the amino acid residues at positions 43 and 44. Analysis of gliding mutants isolated previously revealed that the Gli521 protein is missing in a nonbinding mutant, m9, where the gli521 gene is truncated by a nonsense mutation at the codon for the amino acid at position 1170. Immunofluorescence and immunoelectron microscopy indicated that Gli521 localizes all around the base of the membrane protrusion, at the "neck," as previously observed for Gli349. Analysis of the inhibitory effects of the anti-Gli521 antibody on gliding motility revealed that this protein is responsible for force generation or force transmission, a role distinct from that of Gli349, and also suggested conformational changes of Gli349 and Gli521 during gliding.