N-terminal domain on dystroglycan enables LARGE1 to extend matriglycan on α-dystroglycan and prevents muscular dystrophy.

Dystroglycan (DG) requires extensive post-translational processing and O-glycosylation to function as a receptor for extracellular matrix (ECM) proteins containing laminin-G (LG) domains. Matriglycan is an elongated polysaccharide of alternating xylose (Xyl) and glucuronic acid (GlcA) that binds with high affinity to ECM proteins with LG domains and is uniquely synthesized on α-dystroglycan (α-DG) by like-acetylglucosaminyltransferase-1 (LARGE1). Defects in the post-translational processing or O-glycosylation of α-DG that result in a shorter form of matriglycan reduce the size of α-DG and decrease laminin binding, leading to various forms of muscular dystrophy. Previously, we demonstrated that protein O-mannose kinase (POMK) is required for LARGE1 to generate full-length matriglycan on α-DG (~150-250 kDa) (Walimbe et al., 2020). Here, we show that LARGE1 can only synthesize a short, non-elongated form of matriglycan in mouse skeletal muscle that lacks the DG N-terminus (α-DGN), resulting in an ~100-125 kDa α-DG. This smaller form of α-DG binds laminin and maintains specific force but does not prevent muscle pathophysiology, including reduced force production after eccentric contractions (ECs) or abnormalities in the neuromuscular junctions. Collectively, our study demonstrates that α-DGN, like POMK, is required for LARGE1 to extend matriglycan to its full mature length on α-DG and thus prevent muscle pathophysiology.

Identification of a short, single site matriglycan that maintains neuromuscular function in the mouse.

Matriglycan (-1,3-ß-glucuronic acid-1,3-α-xylose-) is a polysaccharide that is synthesized on α-dystroglycan, where it functions as a high-affinity glycan receptor for extracellular proteins, such as laminin, perlecan and agrin, thus anchoring the plasma membrane to the extracellular matrix. This biological activity is closely associated with the size of matriglycan. Using high-resolution mass spectrometry and site-specific mutant mice, we show for the first time that matriglycan on the T317/T319 and T379 sites of α-dystroglycan are not identical. T379-linked matriglycan is shorter than the previously characterized T317/T319-linked matriglycan, although it maintains its laminin binding capacity. Transgenic mice with only the shorter T379-linked matriglycan exhibited mild embryonic lethality, but those that survived were healthy. The shorter T379-linked matriglycan exists in multiple tissues and maintains neuromuscular function in adult mice. In addition, the genetic transfer of α-dystroglycan carrying just the short matriglycan restored grip strength and protected skeletal muscle from eccentric contraction-induced damage in muscle-specific dystroglycan knock-out mice. Due to the effects that matriglycan imparts on the extracellular proteome and its ability to modulate cell-matrix interactions, our work suggests that differential regulation of matriglycan length in various tissues optimizes the extracellular environment for unique cell types.

Large1 gene transfer in older myd mice with severe muscular dystrophy restores muscle function and greatly improves survival.

Muscular dystrophy is a progressive and ultimately lethal neuromuscular disease. Although gene editing and gene transfer hold great promise as therapies when administered before the onset of severe clinical symptoms, it is unclear whether these strategies can restore muscle function and improve survival in the late stages of muscular dystrophy. Largemyd/Largemyd (myd) mice lack expression of like-acetylglucosaminyltransferase-1 (Large1) and exhibit severe muscle pathophysiology, impaired mobility, and a markedly reduced life span. Here, we show that systemic delivery of AAV2/9 CMV Large1 (AAVLarge1) in >34-week-old myd mice with advanced disease restores matriglycan expression on dystroglycan, attenuates skeletal muscle pathophysiology, improves motor and respiratory function, and normalizes systemic metabolism, which collectively and markedly extends survival. Our results in a mouse model of muscular dystrophy demonstrate that skeletal muscle function can be restored, illustrating its remarkable plasticity, and that survival can be greatly improved even after the onset of severe muscle pathophysiology.

POMK regulates dystroglycan function via LARGE1-mediated elongation of matriglycan.

Matriglycan [-GlcA-ß1,3-Xyl-α1,3-]n serves as a scaffold in many tissues for extracellular matrix proteins containing laminin-G domains including laminin, agrin, and perlecan. Like-acetyl-glucosaminyltransferase 1 (LARGE1) synthesizes and extends matriglycan on α-dystroglycan (α-DG) during skeletal muscle differentiation and regeneration; however, the mechanisms which regulate matriglycan elongation are unknown. Here, we show that Protein O-Mannose Kinase (POMK), which phosphorylates mannose of core M3 (GalNAc-ß1,3-GlcNAc-ß1,4-Man) preceding matriglycan synthesis, is required for LARGE1-mediated generation of full-length matriglycan on α-DG (~150 kDa). In the absence of Pomk gene expression in mouse skeletal muscle, LARGE1 synthesizes a very short matriglycan resulting in a ~ 90 kDa α-DG which binds laminin but cannot prevent eccentric contraction-induced force loss or muscle pathology. Solution NMR spectroscopy studies demonstrate that LARGE1 directly interacts with core M3 and binds preferentially to the phosphorylated form. Collectively, our study demonstrates that phosphorylation of core M3 by POMK enables LARGE1 to elongate matriglycan on α-DG, thereby preventing muscular dystrophy.

Tubular aggregate myopathy caused by a novel mutation in the cytoplasmic domain of STIM1.

OBJECTIVE: To identify the gene mutation of tubular aggregate myopathy (TAM) and gain mechanistic insight into the pathogenesis of the disorder. METHODS: We described a family affected by autosomal dominant TAM and performed exome and Sanger sequencing to identify mutations. We further analyzed the functional significance of the identified mutation by expression studies and intracellular Ca(2+) measurements. RESULTS: A 42-year-old man presented with slowly progressive muscle weakness and atrophy in all 4 limbs and the trunk. Muscle biopsy and microscopic examination revealed tubular aggregates in his skeletal muscle. Genetic analysis of this family identified a novel heterozygous mutation, c.1450_1451insGA (p.Ile484ArgfsX21), in stromal interaction molecule 1 (STIM1), a Ca(2+) sensor in sarcoplasmic reticulum. We transfected cultured cells with STIM1 and demonstrated that the mutant STIM1 exhibited aggregation-like appearance in shrunk cytoplasm. Furthermore, we revealed that the intracellular Ca(2+) influx is decreased by the mutant STIM1. CONCLUSIONS: The novel mutation p.Ile484ArgfsX21 is located in the cytoplasmic C-terminal inhibitory domain (CTID) of STIM1. However, all mutations reported so far in TAM reside in the luminal N-terminal EF hand region. The aggregation-like appearance of STIM1 and the decreased intracellular Ca(2+) influx in cells transfected with CTID mutant are in sharp contrast to these previous reports. Taken together, these findings indicate that mutations of STIM1 cause TAM through the dysregulation of Ca(2+) homeostasis.