Epitopes in the interacting regions of β-dystroglycan (PPxY motif) and dystrophin (WW domain)
Epitopes in the interacting regions of β-dystroglycan (PPxY motif) and dystrophin (WW domain)
复制标题
DOI：
10.1016/s0304-4165(01)00147-7
复制
发表时间：
2001-07-02
影响因子：
3
通讯作者：
Morris, GE
中科院分区：
文献类型：
--
作者：
Pereboev, AV;Ahmed, N;Morris, GE
肌营养不良蛋白聚糖基因从单个mRNA产生两种产物。细胞外α-肌营养不良聚糖和跨膜β-肌营养不良聚糖。杜氏肌营养不良蛋白，肌营养不良蛋白，通过β-肌营养不良蛋白聚糖与肌膜结合。肌营养不良蛋白的WW结构域与β-肌营养不良蛋白聚糖中的PPxY基序相互作用。使用β-肌营养不良聚糖的最后16个氨基酸作为免疫原产生一组四种单克隆抗体（MANDAGI-4）。mAb识别所有测试细胞和组织的蛋白质印迹上的43 kDa条带，并在广泛动物种属的骨骼肌免疫组织化学中染色肌膜。抗肌营养不良蛋白WW结构域的单克隆抗体（mAb）MANHINGE 4A，使用16-mer合成肽产生，在Western印迹上识别肌营养不良蛋白，并对肌膜进行染色。我们已经确定了精确的序列识别的单克隆抗体使用噬菌体展示的随机15-mer肽库。通过对从文库中选择的17种不同肽进行测序来鉴定参与结合所有四种β-肌营养不良聚糖mAb的7个氨基酸共有序列SPPPYVP。PPY是三种mAb最重要的残基，但PxxVP是第四种mAb的必需残基。MANDAG2.通过对来自文库的五种不同的随机肽进行测序，由mAb MANHINGE 4A识别的肌营养不良蛋白上的表位被鉴定为WW结构域的第一β链中的PWxRA，其中W和R残基总是存在.最近的三维结构证实了这两个表位在肌营养不良蛋白-肌营养不良蛋白聚糖复合物中是相邻的。突出了在原位免疫定位期间两个相互作用基序如何也可接近抗体的问题。(C)2001 Elsevier Science B. V.保留所有权利。
The dystroglycan gene produces two products from a single mRNA. the extracellular alpha -dystroglycan and the transmembrane beta -dystroglycan. The Duchenne muscular dystrophy protein, dystrophin, associates with the muscle membrane via beta -dystroglycan. the WW domain of dystrophin interacting with a PPxY motif in beta -dystroglycan. A panel of four monoclonal antibodies (MANDAGI-4) was produced using the last 16 amino acids of beta -dystroglycan as immunogen. The mAbs recognized a 43 kDa band on Western blots of all cells and tissues tested and stained the sarcolemma in immunohistochemistry of skeletal muscle over a wide range of animal species. A monoclonal antibody (mAb) against the WW domain of dystrophin, MANHINGE4A, produced using a 16-mer synthetic peptide, recognized dystrophin on Western blots and also stained the sarcolemma. We have identified the precise sequences recognized by the mAbs using a phage-displayed random 15-mer peptide library. A 7-amino-acid consensus sequence SPPPYVP involved in binding all four beta -dystroglycan mAbs was identified by sequencing 17 different peptides selected from the library. PPY were the most important residues for three mAbs, but PxxVP were essential residues for a fourth mAb. MANDAG2. By sequencing five different random peptides from the library, the epitope on dystrophin recognized by mAb MANHINGE4A was identified as PWxRA in the first beta -strand of the WW domain, with the W and R residues invariably present. A recent three-dimensional structure confirms that the two epitopes are adjacent in the dystrophin-dystroglycan complex. highlighting the question of how the two interacting motifs can also be accessible to antibodies during immunolocalization in situ. (C) 2001 Elsevier Science B.V. All rights reserved.