Identification of novel bioactive glycans on dystroglycan

描述（由申请人提供）：肌营养不良聚糖是一种细胞粘附分子，在神经肌肉和皮质发育以及肌营养不良中起重要作用。糖基化对三聚糖醛酸（aDG）的功能至关重要；已经确定了六个基因，当突变时，会导致aDG的糖基化，导致细胞外基质蛋白（包括层粘连蛋白）的结合减少或缺失。这些基因的缺陷也会导致先天性肌营养不良（CMD），其中aDG糖基化不足是导致分子缺陷的原因。已知其中一些基因（POMT1, POMT2, POMGnT1）是参与aDG蛋白上o -连接甘露糖结构合成的糖基转移酶。在哺乳动物中，o链甘露糖只存在于aDG上。因此，这些碳水化合物结构对这种蛋白质具有高度特异性。尽管许多研究人员付出了巨大的努力，但其他三种已知的CMD基因改变aDG糖基化的功能尚不清楚。其中之一是LARGE，在肌营养不良小鼠（LARGEmyd）中缺失的基因。LARGE之所以重要，不仅是因为该基因的缺陷导致人类疾病，还因为它的过表达已被证明可以刺激aDG的糖基化，并挽救CMD患者细胞中的层粘连蛋白结合。因此，了解LARGE的功能可能是开发多种形式CMD治疗方法的关键，也是了解糖营养不良蛋白在神经肌肉发育中的作用的关键。在这里，我们提供的证据表明LARGE合成了一种新的碳水化合物结构。这种结构，如o -甘露糖，在哺乳动物中极为罕见，但在低等生物中很常见。本提案将最终确定这种碳水化合物结构在三聚糖聚糖上的存在，定义其在配体结合中的作用，并定义LARGE作为产生它的酶的功能。因此，这一提议不仅将定义一种治疗上重要的酶的新功能，而且可能确定一种尚未在哺乳动物中描述的聚糖结构。

DESCRIPTION (provided by applicant): Dystroglycan is a cell adhesion molecule that plays important roles in neuromuscular and cortical development and in muscular dystrophy. The glycosylation of a dystroglycan (aDG) is essential for its function; Six genes have been identified that, when mutated, cause the underglycosylation of aDG leading to diminished or absent binding of extracellular matrix proteins, including laminin. Defects in these genes also cause forms of congenital muscular dystrophy (CMD) where underglycosylation of aDG is causative molecular defect. Several of these genes (POMT1, POMT2, POMGnT1) are known to be glycosyltransferases involved in the synthesis of O-linked mannose structures on the aDG protein. In mammals, O-linked mannose has only been shown to exist on aDG. Thus, these carbohydrate structures are highly specific to this one protein. The functions of the three other known CMD genes that alter aDG glycosylation are unknown, despite intense efforts by numerous investigators. One of these is LARGE, the gene deleted in the myodystrophy (LARGEmyd) mouse. LARGE is important not only because defects in this gene cause human disease, but also because its overexpression has been shown to stimulate glycosylation of aDG and rescue laminin binding in cells from CMD patients. Thus, understanding LARGE function may be a key to the development of therapeutics for multiple forms of CMD as well as to understanding the role of dystroglycan in neuromuscular development. Here we provide evidence that suggests that LARGE synthesizes a novel carbohydrate structure. This structure, like O-mannose, appears to be extremely rare in mammals but common in lower organisms. This proposal will conclusively identify the presence of this carbohydrate structure on dystroglycan, define its role in ligand binding, and define the function of LARGE as the enzyme that creates it. This proposal, therefore, will not only define a novel function for a therapeutically important enzyme, but likely identify a glycan structure not yet described in mammals.