Kevin Campbell is interested in elucidating the mechanisms that underlie muscular dystrophy. His laboratory currently focuses on understanding why O-glycosylation of the dystroglycan protein is essential for its function as an extracellular matrix receptor and how abnormalities in this modification cause muscular dystrophy. The goal of this research is to understand how dystroglycan functions, to identify and define the mechanisms that lead to muscular dystrophy, and to develop therapeutic strategies for these diseases.
Dystroglycan is a widely expressed transmembrane glycoprotein that requires complex post-translational processing to function as an extracellular matrix (ECM) receptor. This protein is involved in a variety of physiological and developmental processes, including maintenance of the function of skeletal muscle, as well as formation and function of the central nervous system. The extensively glycosylated α dystroglycan (α-DG) subunit acts as a receptor for laminin G (LG) domain-containing ECM proteins that are major constituents of the ECM of skeletal muscle and other tissues, namely: laminin, agrin, and perlecan. Abnormalities in α-DG post-translational processing that disrupt interactions with the ECM result in various congenital and limb-girdle muscular dystrophies, referred to collectively as secondary dystroglycanopathies. The ECM-binding glycan of α-DG also mediates interactions with the LG domains of neurexins, pikachurin and Slit, thereby contributing to synapse formation and axon guidance. Finally, α-DG serves as a cellular receptor for Old World arenaviruses, including the highly pathogenic Lassa fever virus. The requirements for virus entry mirror those for LG domain binding.
Biosynthesis of the glycan that confers functionality to α-DG involves at least 18 gene products, many of them glycosyltransferases. Our current studies focus on the enzymatic functions of these proteins with the aim of understanding the structure and biosynthetic pathway of the ECM ligand-binding glycan.
O-Mannosyl Phosphorylation of α-Dystroglycan
Efforts in my laboratory to identify the ECM-binding moiety on α-DG led to the isolation of a novel O-glycan that results from a rare phosphodiester linkage. Nuclear magnetic resonance (NMR)-based analysis identified this O-glycan as a phosphorylated O-mannosyl trisaccharide (N-acetylgalactosamine-β3-N-acetylglucosamine-β4-mannose, designated as core M3). We further demonstrated that a hydroxyl residue of the phosphate at the C6 position of O-mannose is linked to the ECM ligand-binding motif. Recently, we identified the enzymes that synthesize this novel glycan.
First, we found that glycosyltransferase-like domain-containing 2 (GTDC2) is an endoplasmic reticulum (ER)-localized O-linked mannose β1,4-N-acetylglucosaminyltransferase (designated as POMGNT2). Second, we confirmed that GTDC2 and β1,3-N-acetylgalactosaminyltransferase2 (B3GALNT2) act coordinately on O-mannose to synthesize the core M3 glycan structure. Finally, we identified SGK196, which was previously thought to be an inactive protein kinase, as an active enzyme that phosphorylates the C6 position of O-mannose at the ER, specifically after the mannose is modified by both POMGNT2 and B3GALNT2. This strict specificity of SGK196 for the α-DG-linked core M3 glycan explains why mutations in GTDC2 and B3GALNT2 cause muscular dystrophy although their products are not directly involved in recognition of the ECM ligand. Collectively, these findings demonstrate that the core M3 glycan is phosphorylated on mannose before extension by the LARGE glycosyltransferase produces the ECM-binding motif.
Campbell Research Abstract Slideshow
Figure 1: Model depicting the role of LARGE-glycan in organizing the basement membrane in wild-type muscle.
From Goddeeris, M.M. et al. 2013 Nature 503:136-40.
Figure 2: Model depicting the abnormal structure basement membrane in dystroglycanopathy muscle.
From Goddeeris, M.M. et al. 2013 Nature 503:136-40.
LARGE: a Bifunctional Glycosyltransferase
We are also studying posttranslational processing of α-DG by the novel glycosyltransferase LARGE (the like-acetylglucosaminyltransferase). Previously, my laboratory found that the amino-terminal domain of α-DG is required for recognition by LARGE, and that LARGE-mediated modification of this protein is essential for its binding to various ECM-localized ligands such as laminin, agrin, and neurexin. In 2012, we found that xylose (Xyl) and glucuronic acid (GlcA) are component sugars of α-DG produced in LARGE-overexpressing cells, and that mutant cells deficient for UDP-xylose synthase (and thus lacking cellular xylosylation) are defective for functional modification of this protein.
We subsequently discovered that LARGE is a bifunctional glycosyltransferase, possessing xylosyltransferase and glucuronyltransferase activities that produce repeating units of [-3-Xyl-α1, 3-GlcA-β1-]. Using skeletal muscle glycoproteins from the Largemyd mouse (contains a mutation that causes defects in α-DG glycosylation) as the acceptor substrate, we discovered that LARGE can assemble a polysaccharide with ligand-binding activity on the immature glycan of the Largemyd α-DG. These results and those of previous studies demonstrate that LARGE synthesizes a (Xyl-GlcA)n polymer on a phosphorylated O-mannosyl glycan of α-DG, thereby conferring the ability to bind ECM ligands.
A Novel Glucuronyl-Xylosyl Acceptor for Initiation of LARGE-Mediated Glycosylation
Once LARGE activity was characterized, we shifted our focus to the biosynthetic pathway that produces the acceptor polysaccharide extended by LARGE. Using a multidisciplinary approach, we demonstrated that LARGE-mediated addition of the Xyl-GlcA polymer requires B4GAT1-dependent synthesis of a novel glucuronyl-xylosyl acceptor. We showed that B4GAT1 is a novel xylosyl glucuronyltransferase and contributes to the glycan to which the ligand binding LARGE glycan is attached. These findings reveal the molecular mechanism responsible for neuromuscular pathologies in patients with B4GAT1 defects and open new avenues for diagnostic, therapeutic, and anti-viral strategies. Moreover, they demonstrate that LARGE acts on a novel glucuronyl-xylosyl acceptor to initiate synthesis of the laminin binding polysaccharide.
Function of the LARGE-Glycan
Another focus of our research is understanding the cellular significance of the LARGE-mediated glycosylation of α-DG and how defects in this posttranslational modification lead to diseases of varying severity. Binding of α-DG to its matrix-localized ligands is mediated through the disaccharide repeat that is added by LARGE, and the amount of this LARGE-glycan that decorates the nearly ubiquitous α-DG is remarkably tissue-specific. We have demonstrated that the levels of LARGE-glycan in muscle are established during myogenesis.
Using a novel Large knockdown mouse (LargeKD) we interrupted extension of the LARGE-glycan during muscle regeneration in vivo, and assessed the primary cellular impacts of this treatment on the muscle and its disposition to the disease state. Although α-DG initially maintained the ability to bind ligands in the matrix, when formed in regenerated LargeKD muscles it had a significantly reduced ligand-binding capacity. This effect was a direct consequence of a reduction in the number of LARGE-glycan repeats in each chain, as confirmed using synthesized LARGE-glycan repeats. Ligand saturation due to insufficiency of the LARGE-glycan in LargeKD-regenerated muscle led to reduced compaction of the basement membrane, defective maturation of the neuromuscular junction, and functional deficiency in muscle predisposed to dystrophy. Consistent with these findings, disease severity in patients correlates directly with the degree to which extension of the LARGE-glycan is reduced. These findings led us to propose that the ultrastructural organization of the basement membrane can be modified by extension of the LARGE-glycan. Our findings both redefine the cellular significance of dystroglycan and support a new model for the underpinnings of dystroglycan-related disease.
This work was supported in part by grants from the National Institutes of Health and the Muscular Dystrophy Association, and was facilitated by the Iowa Wellstone Muscular Dystrophy Cooperative Research Center.
As of March 7, 2016