The basement membrane is a specialized network of extracellular matrix (ECM) macromolecules that surrounds epithelium, endothelium, muscle, fat, and neurons (Rowe and Weiss, 2008). Skeletal muscle cells are bound to the basement membrane through transmembrane receptors, including dystroglycan (DG) and integrins, which help maintain the structural and functional integrity of the muscle cell membrane (Ibraghimov-Beskrovnaya et al., 1992; Han et al., 2009). DG is a central component of the dystrophin-glycoprotein complex (DGC). It is encoded by a single gene, DAG1, and cleaved into α- and β-subunits (α-DG and β-DG, respectively) by post-translational processing (Ibraghimov-Beskrovnaya et al., 1992). Extensive O-glycosylation of α-dystroglycan (α-DG) is required for normal muscle function, and defects in this process result in various forms of muscular dystrophy (Michele et al., 2002; Ohtsubo and Marth, 2006; Yoshida-Moriguchi and Campbell, 2015; Praissman et al., 2016; Sheikh et al., 2017).

α-DG binds to ECM ligands containing laminin-G (LG) domains (e.g., laminin, agrin, perlecan) which are essential components of the basement membrane (Michele et al., 2002). DG, thereby, physically links the cell membrane to the basement membrane. This process requires the synthesis of matriglycan, a heteropolysaccharide [-GlcA-β1,3-Xyl-α1,3-]_n, on α-DG by the bifunctional glycosyltransferase, like-acetylglucosaminyltransferase-1 (LARGE1) (Inamori et al., 2012; Yoshida-Moriguchi and Campbell, 2015; Hohenester, 2019; Ohtsubo and Marth, 2006). Structural studies have shown that a single glucuronic acid-xylose disaccharide (GlcA-Xyl) repeat binds with high-affinity to laminin-α2 LG4,5, and there is a direct correlation between the number of GlcA-Xyl repeats on α-DG and its binding capacity for ECM ligands (Briggs et al., 2016). During skeletal muscle differentiation, LARGE1 elongates matriglycan, thereby increasing the binding capacity of matriglycan for ECM ligands and allowing matriglycan to serve as a matrix scaffold that is required for skeletal muscle function (Goddeeris et al., 2013). Matriglycan synthesis requires the addition of core M3 trisaccharide (GalNAc-β1,3-GlcNAc-β1,4-Man) onto specific threonine residues on α-DG in the endoplasmic reticulum (ER) (Sheikh et al., 2017). Once core M3 is synthesized, the glycosylation-specific kinase, protein O-mannose kinase (POMK) phosphorylates mannose of core M3, and this is required for the subsequent addition of full-length, high-molecular weight forms of matriglycan onto the O-mannose linked modification (Yoshida-Moriguchi and Campbell, 2015; Hohenester, 2019; Jae et al., 2013; Yoshida-Moriguchi et al., 2013; Zhu et al., 2016). In the absence of core M3 phosphorylation by POMK, LARGE1 synthesizes a short, non-elongated form of matriglycan on α-DG (Walimbe et al., 2020). Notably, a loss of function in the post-translational addition of matriglycan leads to a group of disorders known as dystroglycanopathies, congenital and limb-girdle muscular dystrophies with or without brain and eye abnormalities. Disease severity is dependent on the ability of matriglycan to bind ECM ligands, which is dictated by its length and expression (Goddeeris et al., 2013): matriglycan that is of low molecular weight (e.g., short) can cause muscular dystrophy, even if its capacity to bind LG domains is not completely lost (Puckett et al., 2009; Hara et al., 2011a; Hara et al., 2011b; Carss et al., 2013; Cirak et al., 2013; Dong et al., 2015; Walimbe et al., 2020). However, how matriglycan elongation is regulated by factors other than POMK is still unknown.

α-DG is composed of three distinct domains: the N-terminal (α-DGN) domain, a central mucin-like domain, and the C-terminal domain (Figure 1; Ibraghimov-Beskrovnaya et al., 1992; Brancaccio et al., 1995; Brancaccio et al., 1997). α-DGN serves as a binding site for LARGE1 in the Golgi and is required for the subsequent functional glycosylation of the mucin-like domain of α-DG (Kanagawa et al., 2004). We, therefore, hypothesized that α-DGN must be involved in regulating the production and elongation of matriglycan. Here, we have used a multidisciplinary approach to show that LARGE1 synthesizes a non-elongated form of matriglycan on DG that lacks α-DGN (i.e., α-DGN-deleted DG) resulting in ~100–125 kDa α-DG. This short form of matriglycan binds laminin and maintains muscle-specific force. However, it fails to prevent lengthening contraction-induced reductions in force, neuromuscular junction (NMJ) abnormalities, or dystrophic changes in muscle. Collectively, our study shows that LARGE1 requires α-DGN to generate full-length matriglycan in skeletal muscle, but the synthesis of a shorter form of matriglycan can proceed independently of this domain.

Figure 1

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Constitutive deletion of DG in mice causes embryonic lethality due to disruption of Reichert’s membrane, an extraembryonic basement membrane required for in utero development (Williamson et al., 1997). Deletion of α-DGN in mice also causes embryonic lethality (de Greef et al., 2019). However, mice that are heterozygous for α-DGN deletion (Dag1^wt/Δα-DGN) are viable and express α-DG of two different sizes (Figure 2—figure supplement 1) corresponding to both wild-type (WT) and the α-DGN-deleted (Δα-DGN) forms of DG. Thus, to successfully ablate α-DGN in skeletal muscle, we used mice expressing Cre recombinase under the control of the paired Box 7 (Pax7) promoter (Pax7^Cre), floxed DG mice (Dag1^flox/flox), and heterozygous α-DGN-deleted mice (Dag1^wt/Δα-DGN) to generate Pax7^Cre; Dag1^{flox/Δα-DGN} (muscle-specific α-DGN knockout [M-α-DGN KO]) mice (Figures 1 and 2).

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To evaluate the gross phenotype of mice harboring a muscle-specific deletion of α-DGN (i.e., M-α-DGN KO mice), we first measured body weight and grip strength. M-α-DGN KO mice were slightly lower in weight than WT littermate (control) mice at 12 weeks of age, and they exhibited decreased forelimb grip strength as well (Figure 2A). To determine whether deletion of α-DGN affects matriglycan expression, we performed histological analysis of quadriceps muscle from control and M-α-DGN KO mice. M-α-DGN KO mice showed characteristic features of muscular dystrophy, including an increase in centrally nucleated fibers (Figure 2B). Immunofluorescence analyses of M-α-DGN KO muscle showed reduced levels of matriglycan relative to controls, but a similar expression of β-DG, the transmembrane subunit of DG (Figure 2B).

Immunoblot analysis demonstrated that skeletal muscle from M-α-DGN KO mice expresses a shorter form of matriglycan which results in an ~100–125 kDa α-DG, a decrease in the molecular weight of the core α-DG, and no change in β-DG (Figure 2C). No matriglycan is seen in Large^myd mice which have a deletion in Large1 (Figure 2C). To investigate how the loss of α-DGN affects ligand binding, we performed a laminin overlay using laminin-111. Skeletal muscle from control mice showed a broad band centered at ~100–250 kDa, indicative of α-DG-laminin binding; in contrast, we observed laminin binding at ~100–125 kDa in M-α-DGN KO skeletal muscle (Figure 2C). To further confirm that the ~100–125 kDa band seen with anti-matriglycan antibodies in M-α-DGN KO muscle is matriglycan-positive α-DG, we digested it overnight with β-glucuronidase (Thermotoga maritima) and α-xylosidase (Sulfolobus solfataricus). Immunoblot analysis with anti-matriglycan antibodies and laminin overlay revealed that ~100–125 kDa was completely lost after enzymatic digestion, indicating that the ~100–125 kDa band is indeed matriglycan-positive α-DG (Figure 2—figure supplement 2). The sensitivity of ~100–125 kDa α-DG to enzymatic digestion also demonstrates that matriglycan is not capped like brain α-DG (Sheikh et al., 2020).

The NMJs in adult control mice demonstrate a normal pretzel-like morphology whereas NMJs from M-α-DGN KO mice display a variety of abnormalities, including a granular appearance and AChR-rich streaks extending beyond the pre-synaptic terminal (Figure 3A). Post-synaptic morphology in adult M-α-DGN KO mice was predominately irregular in the tibialis anterior (TA), extensor digitorum longus (EDL), and soleus (SOL) muscles (Figure 3B). Although the overall synaptic size did not differ between controls and M-α-DGN KO mice, the dispersion of AChR clusters was greater in the M-α-DGN KOs (Figure 3B), in line with an increased percentage of plaque-like formations and AChR extensions that projected beyond the nerve terminal. Despite the post-synaptic abnormalities, all NMJs from M-α-DGN KO mice were fully innervated.

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To determine what effect the loss of α-DGN has on muscle force production, we characterized the phenotype and function of EDL muscles in 12- to 17-week-old WT (control) and M-α-DGN KO mice. Specifically, we measured muscle mass, muscle cross-sectional area (CSA), production of absolute isometric tetanic force, specific force, and lengthening contraction-induced reduction in force. Muscle mass and CSA were comparable between control and M-α-DGN KO mice (Figure 4A and B). Although the production of absolute isometric tetanic force was significantly lower in M-α-DGN KO mice compared to control mice (Figure 4C), specific forces were comparable between the two groups when normalized to muscle CSA (Figure 4D). Lengthening contraction-induced force reduction in M-α-DGN KO EDL remained greater than those from control EDL for the entire 60 min that muscles were assessed (Figure 4E). These results suggest that the short form of matriglycan on α-DG in M-α-DGN KO EDL enables force production but cannot prevent force reduction caused by lengthening contractions. POMK KO skeletal muscle also expresses a short form of matriglycan, similar to M-α-DGN KO muscle. The short form of matriglycan in POMK KO skeletal muscle maintains force production but similarly cannot prevent lengthening contraction-induced force decline (Walimbe et al., 2020). We, therefore, compared the function of EDL muscles from POMK KO mice with those from M-α-DGN KO mice ex vivo. We did not observe significant differences in lengthening contraction-induced force deficits between the two mouse strains (Figure 4—figure supplement 1). These results suggest that muscle with similar size matriglycan exhibits similar force production and lengthening contraction-induced force decline.

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We next determined if exogenous DG lacking α-DGN (Figure 5) produces the short form of matriglycan. We first produced muscle-specific DG KO mice to achieve a muscle-specific deletion of DG by crossing mice expressing Cre under control of the paired box 7 (Pax7) promoter (Pax7^Cre) to Dag1^flox/flox mice to generate Pax7^Cre; Dag1^flox/flox (M-Dag1 KO) mice. To assess the presence of DG, we performed immunostaining of quadriceps muscles from 12-week-old M-Dag1 KO mice, which showed the absence of β-DG-positive fibers (Figure 5—figure supplement 1A). Immunoblot analysis showed that α-DG derived from skeletal muscle was absent in M-Dag1 KO mice (Figure 5—figure supplement 1B). However, consistent with prior reports, peripheral nerve-derived α-DG of 110 kDa is observed in M-Dag1 KO mice in the presence of EDTA, which improves the extraction of matriglycan-positive α-DG (Saito et al., 2003). To assess muscle function, we evaluated muscle-specific force and lengthening contraction-induced reduction in force ex vivo, which showed that muscle-specific force was significantly decreased and that muscles were more susceptible to lengthening contraction-induced force decline in the absence of DG (Figure 5—figure supplement 1C, D). Collectively, these results show that M-Dag1 KO mice harbor a more complete loss of DG in muscle than the previously generated mouse model that deleted DG with cre driven by the muscle creatine kinase (MCK) promotor (MCK^Cre; Dag1^flox/flox) harboring muscle-specific deletion of DG (Cohn et al., 2002).

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We next generated an adeno-associated virus (AAV) construct of DG lacking the α-DGN (AAV-MCK DG-E; Figure 5A), which we injected into M-Dag1 KO mice through the retro-orbital (RO) sinus. A previous report found that matriglycan was not produced when a similar adenoviral construct of DG lacking the α-DGN was used to infect DagI KO ES cells (Kanagawa et al., 2004). However, we found that matriglycan of similar size was produced in M-Dag1 KO mice injected with AAV-MCK DG-E as in M-α-DGN KO mice (Figure 5C). Immunofluorescence analysis of quadriceps muscles from M-Dag1 KO mice injected with AAV-MCK DG-E showed decreased immunoreactivity to matriglycan-positive muscle fibers but restored expression of β-DG (Figure 5B). We previously reported that WT DG is able to restore full-length matriglycan and its function when expressed in DG null cells or muscle (Hara et al., 2011a; Hara et al., 2011b). Immunoblot analysis of skeletal muscle from M-Dag1 KO mice injected with AAV-MCK-DG-E showed expression of α-DG containing matriglycan around ~100–125 kDa (Figure 5C), which was the same size as α-DG with the short form of matriglycan in M-α-DGN KO muscle (Figure 2C). The molecular weight of α-DG was decreased in muscle from M-Dag1 KO mice, similar to that observed in M-α-DGN KO mice, whereas the molecular weight of β-DG was unchanged relative to M-α-DGN KO mice (Figure 2C). We also observed laminin binding at ~100–125 kDa in muscle from M-Dag1 KO + AAV MCK DG-E mice (Figure 5C). In addition, we assessed the physiological effects of expressing DG without the DGN. We observed that the specific force was comparable in M-Dag1 KO + AAV MCK DG-E and M-Dag1 KO mice (Figure 5D) and that the two groups exhibited similar amounts of lengthening contraction-induced force decline (Figure 5E). Therefore, these data demonstrate that AAV-mediated delivery of exogenous DG lacking the α-DGN into M-Dag1 KO mice also produces a short-form matriglycan and such mice exhibit similar muscle function as M-α-DGN KO mice.

Our studies show that DG lacking the α-DGN expresses a short form of matriglycan; this suggests that α-DGN is necessary to produce full-length matriglycan. To test this hypothesis, we determined if matriglycan expression could be restored in mice lacking α-DGN. We injected M-α-DGN KO mice with an AAV expressing α-DGN (AAV-CMV α-DGN) and harvested the skeletal muscles of these mice 8–10 weeks after injection. H&E staining in M-α-DGN KO mice injected with AAV-CMV α-DGN was unchanged from M-α-DGN KO mice (Figure 2B and Figure 6A). Quadriceps muscles from M-α-DGN KO mice injected with AAV-CMV α-DGN showed a reduced intensity of matriglycan relative to littermate controls (Figure 6). Immunoblot analysis of these mice showed that α-DG had a molecular weight of ~100–125 kDa, whereas β-DG remained unchanged (Figure 6B). Laminin binding was observed at ~100–125 kDa in M-α-DGN KO skeletal muscle infected with AAV-CMV α-DGN (Figure 6B). Collectively, this phenotype is similar to that observed in the skeletal muscles of M-α-DGN KO mice. Expressing α-DGN in M-α-DGN KO mice did not alter specific force or improve force deficits induced by lengthening contractions (Figure 6C and D). Thus, supplementing M-α-DGN KO skeletal muscle with α-DGN fails to produce full-length matriglycan. To test if excessive free α-DGN interferes with the binding of endogenous α-DGN to LARGE1, we analyzed immunoblots of skeletal muscle from control mice and those overexpressing free α-DGN (α-DGN transgenic [α-DGN Tg]). Immunoblot analysis of these mice showed that α-DG had a reduced molecular weight of ~100–125 kDa, whereas β-DG remained unchanged (Figure 5—figure supplement 1). This biochemical phenotype is similar to that observed in the skeletal muscles of M-α-DGN KO mice and demonstrates that free α-DGN interferes with the elongation of matriglycan, but that LARGE1 can still synthesize a short form of matriglycan on α-DG.

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To determine if excess LARGE1 produces extended matriglycan in M-α-DGN KO muscle, we analyzed immunoblots of skeletal muscle from littermate controls, M-α-DGN KO and M-α-DGN KO mice injected with AAV-MCK-Large1. M-α-DGN KO mice injected with AAV-MCK-Large1 demonstrated no change in the molecular weight of α-DG and β-DG relative to M-α-DGN KO. A laminin overlay using laminin-111 also showed no change (Figure 6—figure supplement 1). These results indicate that even if LARGE1 is overexpressed, full-length matriglycan cannot be produced without α-DGN.

The ability of matriglycan to bind ECM ligands correlates with its length (Goddeeris et al., 2013). We hypothesized that the susceptibility to force decline after lengthening contractions would differ depending on the length of matriglycan. To test this hypothesis, we performed physiological muscle tests in three mouse models with different size α-DG to determine the difference in susceptibility to lengthening contraction-induced force reduction. Specifically, we used: (1) M-α-DGN KO mice, which express a short form of matriglycan, (2) Dag1^T190M mice, which harbor a knock-in mutation (T190M) in Dag1 that inhibits the DG-LARGE1 interaction and leads to incomplete post-translational modification of α-DG (Hara et al., 2011a), and (3) C57BL/6J WT (C57) mice, which express full-length matriglycan. The percent deficit value of the eight EC shows the largest difference in the EC protocol; we, therefore, compared these values between our three different mouse models (Figure 7A). M-α-DGN KO mice showed a significantly higher percent deficit (70.2%±5.7) compared to C57 (41.7%±8.0) and Dag1^T190M (41.6%±6.7) mice, with no difference observed between the latter groups. Laminin overlay assay in skeletal muscle showed α-DG laminin binding at ~150–250 kDa in skeletal muscle from C57 mice, ~100–150 kDa in skeletal muscle from Dag1^T190M mice, and ~100–125 kDa in skeletal muscle from M-α-DGN KO mice (Figure 7C). Moreover, the percentage of centrally nucleated fibers differed significantly in Dag1^T190M (1.73%±0.31) and M-α-DGN KO (9.28%±2.41) mice compared to C57 mice (1.22%±0.15) (Figure 7B). The reduction of laminin-binding activity of α-DG is thought to be the main cause of dystroglycanopathy (Kanagawa et al., 2009; Goddeeris et al., 2013). Indeed, we observed a reduced binding capacity (relative B_max) for laminin-111 in solid-phase binding analyses in skeletal muscle from M-α-DGN KO and Dag1^T190M mice compared to skeletal muscle from C57 mice (10.7-fold and 2.3-fold difference relative to WT, respectively) (Figure 7D). However, the binding capacity of skeletal muscle from Dag1^T190M and M-α-DGN KO mice was higher than that of Large1^myd muscle (Figure 7E). M-α-DGN KO and Dag1^T190M also displayed a greater dissociation constant (Figure 7E). Collectively, these results suggest that matriglycan-positive α-DG of at least ~150 kDa is sufficient to prevent force decline from lengthening contractions and significant dystrophic changes, despite a 45% reduction in laminin-binding activity.

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Matriglycan is a scaffold for LG domain-containing ECM proteins (e.g., laminin, agrin, and perlecan). During skeletal muscle differentiation, matriglycan is elongated by LARGE1, increasing its binding capacity for ECM ligands, which allows it to serve as a matrix scaffold that is required for skeletal muscle function. Consequently, defects in matriglycan synthesis led to dystroglycanopathies, such as congenital and limb-girdle muscular dystrophies that can be accompanied by structural brain and eye abnormalities. Notably, there is a direct correlation between the number of GlcA-Xyl repeats on α-DG and its binding capacity for ECM ligands. Indeed, patients with increased clinical severity of dystroglycanopathy have few GlcA-Xyl repeats and a shorter form of matriglycan. Despite the importance of mature, full-length matriglycan for proper muscle structure and function, little is known about mechanisms that control its elongation.

We now show that LARGE1 synthesizes a non-elongated form of matriglycan on α-DG lacking α-DGN (i.e., α-DGN-deleted DG). This short form of matriglycan binds laminin and maintains specific force. However, it fails to prevent reduced force production after ECs, NMJ abnormalities, or dystrophic changes in muscle. Collectively, our study shows that LARGE1 requires α-DGN to generate full-length matriglycan in skeletal muscle and thus prevents muscle pathophysiology. However, synthesis of a shorter form of matriglycan can proceed independently of the α-DGN domain.

Our results suggest that the interaction between LARGE1 and α-DGN holds the enzyme-substrate complex together over multiple cycles of sugar addition. These findings build on our previous study demonstrating that phosphorylation of the core M3 trisaccharide by POMK is also necessary for matriglycan elongation (Walimbe et al., 2020). Thus, the generation of full-length matriglycan on α-DG (~150–250 kDa) by LARGE1 requires both POMK and α-DGN; in the absence of either, LARGE1 synthesizes a shorter form of matriglycan. Thus, the interaction between LARGE1 and α-DGN is essential for efficient matriglycan polymerization. Although deep-learning tools could be used to predict protein-protein interactions, it is unlikely that these tools will be able to predict the interaction between LARGE1 and DG as this requires post-translational modifications (Walimbe et al., 2020).

In our study, muscle-specific deletion of α-DGN resulted in the production of short forms of matriglycan on α-DG (~100–125 kDa). Mice lacking α-DGN exhibited low body weight and grip strength, and histological characterization of quadriceps muscles revealed mild muscular dystrophy and a lack of homogeneous matriglycan expression. Physiological examination revealed that M-α-DGN KO muscle was susceptible to lengthening contraction-induced force decline, although specific force was maintained. These results are consistent with those obtained when α-DGN-deleted DG was expressed in muscle-specific DG null mice and confirm that α-DG lacking α-DGN produces short forms of matriglycan, which do not prevent dystrophic muscle changes in this mouse model. Furthermore, DG in the post-synaptic membrane is known to play a key role in synaptic maturation (Nishimune et al., 2008). However, the NMJs in M-α-DGN KO mice in our study were abnormal and irregularly shaped. This indicates both that DG and full-length matriglycan are required for synaptic maturation.

If LARGE1 binding to α-DGN enables its ability to elongate matriglycan, then we would expect that rescuing M-α-DGN KO skeletal muscle with α-DGN would restore the expression of full-length matriglycan. However, this failed to occur and suggests that restoring α-DGN expression alone is not sufficient for LARGE1 to elongate matriglycan. These results indicate that the ability of LARGE1 to elongate matriglycan requires α-DG with α-DGN. This finding is consistent with data showing that matriglycan is not elongated when α-DGN is deleted, even when LARGE1 is overexpressed. Therefore, α-DGN acts as a recognition site for the glycosyltransferase LARGE1 and establishes a model where α-DGN, together with phosphorylated core M3, anchors LARGE1 to the matriglycan production site to enable its synthesis and elongation. Notably, although the molecular recognition of α-DGN by LARGE1 is considered essential for the expression of functional α-DG (Kanagawa et al., 2004), our results show that LARGE1 can synthesize a short non-elongated form of matriglycan in the absence of α-DGN, indicating that LARGE1 does not need to interact with α-DGN to function.

To determine how much matriglycan is needed to prevent lengthening contraction-induced reductions in force, we studied mice that express different sizes of matriglycan. Muscle from M-α-DGN KO mice showed an increased force deficit and a 7.6-fold increase in centrally nucleated fibers compared to muscle from C57 mice, indicating that the short form of matriglycan does not prevent dystrophic changes. However, despite the lower amount of matriglycan in muscle from Dag1^T190M mice compared to that from C57 mice, the force deficit was not different between the two groups, and centrally nucleated fibers were increased in α-DGN mutant (Dag1^T190M) mice by only 1.4-fold. This indicates that a matriglycan-positive α-DG over 150 kDa can prevent muscular dystrophy.

Muscular dystrophy is not observed in a mouse model of Fukuyama congenital muscular dystrophy, which occurs due to a retrotransposition insertion in the mouse fukutin ortholog and leads to laminin binding at 50% of normal levels (Kanagawa et al., 2009). In Dag1^T190M mice, the laminin-binding level is about 45% of normal, which likely explains the mild increase in centrally nucleated fibers compared to muscle from C57 mice. However, in muscle from M-α-DGN KO mice, the laminin-binding level is only 9% relative to that of C57 mice and leads to a marked increase in centrally nucleated fibers and force deficit induced by lengthening contractions. This indicates that matriglycan length is critical for regulating damage induced by lengthening contractions and that the production of ~120–150 kDa α-DG significantly prevents dystrophic change, suggesting that this pathologic effect can be prevented without the expression of full-length matriglycan. Thus, our results describe a relationship between matriglycan size, damage induced by lengthening contractions, and the degree of dystrophic change.

α-DGN is cleaved by the proprotein convertase furin at the sequence RVRR (amino acids 309–312) (Singh et al., 2004). It has a globular structure that is organized into two subdomains (Brancaccio et al., 1997). The first subdomain is a typical Ig-like domain; the second subdomain resembles ribosomal RNA-binding proteins (Bozic et al., 2004). α-DGN is secreted by cells in culture (Saito et al., 2011) and has been detected in a wide variety of human bodily fluids, including serum and plasma (Saito et al., 2008; Saito et al., 2011). Decreased levels of α-DGN have been reported in the serum of patients with Duchenne muscular dystrophy (DMD) and in the serum of utrophin-deficient mdx mice, a mouse model for DMD (Crowe et al., 2016). Finally, we have also studied the protective role of secreted α-DGN (de Greef et al., 2019) on influenza A virus proliferation.

Collectively our study demonstrates that synthesis of full-length matriglycan on α-DG (~150–250 kDa) requires α-DG containing α-DGN. In the absence of α-DGN, LARGE1 can synthesize a short non-elongated form of matriglycan on α-DG (~100–125 kDa) in skeletal muscle in a process that does not require that LARGE1 interacts with α-DGN. Taken together, these findings demonstrate that matriglycan length regulates the severity of muscular dystrophy. Interestingly, α-DG of approximately 100–125 kDa was seen in muscle from mild FKRP patients (Brockington et al., 2001). Our work, thereby, enhances our understanding of the mechanisms underlying matriglycan synthesis and further identifies this pathway as a possible therapeutic target for the treatment of α-dystroglycanopathy.

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 2C, 5C, 6B, 7B, Figure 2-figure supplement 1, Figure 2-figure supplement 2, Figure 5-figure supplement 1B, and Figure 6-figure supplement 1.

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Author details

1. Hidehiko Okuma

   Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, Department of Molecular Physiology and Biophysics and Department of Neurology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2749-9855

2. Jeffrey M Hord

   Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, Department of Molecular Physiology and Biophysics and Department of Neurology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

3. Ishita Chandel

   Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, Department of Molecular Physiology and Biophysics and Department of Neurology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, United States

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

4. David Venzke

   Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, Department of Molecular Physiology and Biophysics and Department of Neurology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, United States

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8180-9562

5. Mary E Anderson

   Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, Department of Molecular Physiology and Biophysics and Department of Neurology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, United States

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

6. Ameya S Walimbe

   Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, Department of Molecular Physiology and Biophysics and Department of Neurology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, United States

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

7. Soumya Joseph

   Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, Department of Molecular Physiology and Biophysics and Department of Neurology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, United States

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

8. Zeita Gastel

   Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, Department of Molecular Physiology and Biophysics and Department of Neurology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, United States

   Contribution

   Methodology

   Competing interests

   No competing interests declared

9. Yuji Hara

   Department Pharmaceutical Sciences, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

10. Fumiaki Saito

    Department of Neurology, School of Medicine, Teikyo University, Tokyo, Japan

    Contribution

    Investigation, Methodology

    Competing interests

    No competing interests declared

11. Kiichiro Matsumura

    Department of Neurology, School of Medicine, Teikyo University, Tokyo, Japan

    Contribution

    Investigation, Methodology

    Competing interests

    No competing interests declared

12. Kevin P Campbell

    Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, Department of Molecular Physiology and Biophysics and Department of Neurology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, United States

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    kevin-campbell@uiowa.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2066-5889

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