Dystroglycanopathies are diseases characterized by progressive muscular degeneration and impairment of patient’s quality of life. They are associated with altered glycosylation of the dystrophin-glycoprotein (DGC) complex components, such as α-dystroglycan (α-DG), fundamental in the structural and functional stability of the muscle fiber. The diagnosis of dystroglycanopathies is currently based on the observation of clinical manifestations, muscle biopsies and enzymatic measures, and the available monoclonal antibodies are not specific for the dystrophic hypoglycosylated muscle condition. Thus, modified α-DG mucins have been considered potential targets for the development of new diagnostic strategies toward these diseases. In this context, this work describes the synthesis of the hypoglycosylated α-DG mimetic glycopeptide NHAc-Gly-Pro-Thr-Val-Thr[αMan]-Ile-Arg-Gly-BSA (1) as a potential tool for the development of novel antibodies applicable to dystroglycanopathies diagnosis. Glycopeptide 1 was used for the development of polyclonal antibodies and recombinant monoclonal antibodies by Phage Display technology. Accordingly, polyclonal antibodies were reactive to glycopeptide 1, which enables the application of anti-glycopeptide 1 antibodies in immune reactive assays targeting hypoglycosylated α-DG. Regarding monoclonal antibodies, for the first time variable heavy (VH) and variable light (VL) immunoglobulin domains were selected by Phage Display, identified by NGS and described by in silico analysis. The best-characterized VH and VL domains were cloned, expressed in E. coli Shuffle T7 cells, and used to construct a single chain fragment variable that recognized the Glycopeptide 1 (GpαDG1 scFv). Molecular modelling of glycopeptide 1 and GpαDG1 scFv suggested that their interaction occurs through hydrogen bonds and hydrophobic contacts involving amino acids from scFv (I51, Y33, S229, Y235, and P233) and R8 and α-mannose from Glycopeptide 1.

中文翻译：

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抗合成α-营养不良糖蛋白粘蛋白糖肽的多方面抗体开发，可用于营养不良性糖病诊断

肌营养不良症是特征在于进行性肌肉变性和患者生活质量受损的疾病。它们与肌营养不良蛋白-糖蛋白（DGC）复杂成分（例如α-肌钙蛋白（α-DG））的糖基化改变有关，这是肌纤维结构和功能稳定性的基础。营养不良性糖病的诊断当前基于临床表现，肌肉活检和酶促措施的观察，并且可用的单克隆抗体对营养不良的低糖基化肌肉状况不具有特异性。因此，修饰的α-DG粘蛋白已被认为是开发针对这些疾病的新诊断策略的潜在靶标。在这种情况下，1）作为开发适用于肌营养不良症诊断的新型抗体的潜在工具。糖肽1通过噬菌体展示技术用于开发多克隆抗体和重组单克隆抗体。因此，多克隆抗体对糖肽1具有反应性，这使得抗糖肽1抗体可以在针对低糖基化α-DG的免疫反应测定中应用。关于单克隆抗体，首次通过噬菌体展示选择可变的重（VH）和可变的轻（VL）免疫球蛋白结构域，通过NGS鉴定并通过计算机分析进行描述。克隆特征最丰富的VH和VL结构域，在大肠杆菌中表达改组T7细胞，并用于构建识别糖肽1（GpαDG1scFv）的单链片段变量。糖肽1和GpαDG1scFv的分子模型表明，它们的相互作用通过氢键和疏水性接触发生，涉及scFv的氨基酸（I51，Y33，S229，Y235和P233）以及R8和糖肽1的α-甘露糖。