Department of Preclinical/Clinical Pharmacology, ML Bio Solutions, a BridgeBio company, Palo Alto, USA.
Department of Clinical Development, ML Bio Solutions, a BridgeBio company, Palo Alto, USA.
J Muscle Res Cell Motil. 2024 Sep;45(3):123-138. doi: 10.1007/s10974-024-09670-y. Epub 2024 Apr 18.
The cell membrane protein, dystroglycan, plays a crucial role in connecting the cytoskeleton of a variety of mammalian cells to the extracellular matrix. The α-subunit of dystroglycan (αDG) is characterized by a high level of glycosylation, including a unique O-mannosyl matriglycan. This specific glycosylation is essential for binding of αDG to extracellular matrix ligands effectively. A subset of muscular dystrophies, called dystroglycanopathies, are associated with aberrant, dysfunctional glycosylation of αDG. This defect prevents myocytes from attaching to the basal membrane, leading to contraction-induced injury. Here, we describe a novel Western blot (WB) assay for determining levels of αDG glycosylation in skeletal muscle tissue. The assay described involves extracting proteins from fine needle tibialis anterior (TA) biopsies and separation using SDS-PAGE followed by WB. Glycosylated and core αDG are then detected in a multiplexed format using fluorescent antibodies. A practical application of this assay is demonstrated with samples from normal donors and patients diagnosed with LGMD2I/R9. Quantitative analysis of the WB, which employed the use of a normal TA derived calibration curve, revealed significantly reduced levels of αDG in patient biopsies relative to unaffected TA. Importantly, the assay was able to distinguish between the L276I homozygous patients and a more severe form of clinical disease observed with other FKRP variants. Data demonstrating the accuracy and reliability of the assay are also presented, which further supports the potential utility of this novel assay to monitor changes in ⍺DG of TA muscle biopsies in the evaluation of potential therapeutics.
细胞膜蛋白，dystroglycan，在将各种哺乳动物细胞的细胞骨架与细胞外基质连接方面起着至关重要的作用。dystroglycan 的α-亚基（αDG）的特点是高度糖基化，包括独特的 O-甘露糖基 matriglycan。这种特定的糖基化对于 αDG 与细胞外基质配体的有效结合是必不可少的。一组称为 dystroglycanopathy 的肌肉疾病与 αDG 的异常、功能失调的糖基化有关。这种缺陷阻止肌细胞附着在基底膜上，导致收缩诱导的损伤。在这里，我们描述了一种用于确定骨骼肌组织中αDG 糖基化水平的新型 Western blot（WB）测定法。该测定法涉及从细针胫骨前肌（TA）活检中提取蛋白质，并使用 SDS-PAGE 进行分离，然后进行 WB。然后使用荧光抗体以多重格式检测糖基化和核心 αDG。该测定法的实际应用是用来自正常供体和诊断为 LGMD2I/R9 的患者的样本进行的。使用来自正常 TA 的校准曲线进行的 WB 的定量分析表明，与未受影响的 TA 相比，患者活检中的αDG 水平显著降低。重要的是，该测定法能够区分 L276I 纯合子患者和与其他 FKRP 变体观察到的更严重形式的临床疾病。还提供了证明该测定法准确性和可靠性的数据，这进一步支持了该新型测定法在评估潜在治疗方法时监测 TA 肌肉活检中αDG 变化的潜在效用。
