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Kevin Campbell is interested in elucidating mechanisms underlying muscular dystrophy. His laboratory is currently focused on why O-glycosylation of dystroglycan is essential for this protein’s function as an extracellular matrix receptor, and how abnormalities in the O-glycosylation of dystroglycan cause muscular dystrophy. The goal of this research is to generate new knowledge regarding the function of dystroglycan, to identify and define disease mechanisms that cause muscular dystrophy and to develop therapeutic strategies for these diseases.

Dystroglycan is a highly glycosylated basement membrane receptor involved in a variety of physiological processes that maintain skeletal muscle membrane integrity as well as the structure and function of the central nervous system. Aberrant posttranslational modification of the α subunit of this protein and concomitant loss of dystroglycan function as an extracellular matrix (ECM) receptor have been observed in several forms of congenital/limb-girdle muscular dystrophies (also called  dystroglycanopathies).  Recent genetic data have shown that mutations in at least sixteen genes encoding known and putative glycosyltransferases disrupt O-glycosylation of dystroglycan and cause muscular dystrophy.  Our current studies focus on the enzymatic function of these proteins, with the aim of understanding the structure and biosynthetic pathway of ECM ligand-binding glycan.

O-mannosyl phosphorylation of α-dystroglycan
Efforts in my laboratory to identify the ECM binding moiety on α-dystroglycan led to the isolation of a novel O-glycan on α-dystroglycan that results from a rare phosphodiester-linked modification. 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 α-dystroglycan-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 LARGE glycosyltransferase-mediated extension to produce the ECM-binding motif.

LARGE is a bifunctional glycosyltransferase
We are also studying post-translational processing of dystroglycan by the novel glycosyltransferase LARGE (the like-acetylglucosaminyltransferase). Previously, my laboratory found that the amino-terminal domain of α-dystroglycan is required for recognition by LARGE. Also, LARGE-mediated modification of α-dystroglycan 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-dystroglycan 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 α-dystroglycan.

Next we discovered that LARGE is a bifunctional glycosyltransferase with both xylosyltransferase and glucuronyltransferase activities that produce repeating units of [-3-Xyl-α1,3-GlcA-β1-].  Using skeletal muscle glycoproteins from the Largemyd mouse (a mutation in the Large gene causes defects in α-dystroglycan glycosylation) as the acceptor substrate, we further demonstrated that LARGE can assemble a polysaccharide with ligand-binding activity onto the immature glycan of the Largemyd α-dystroglycan.  These results and previous studies demonstrate that LARGE synthesizes a (Xyl-GlcA)n  polymer on a phosphorylated O-mannosyl glycan of α-dystroglycan, thereby conferring the ability to bind ECM ligands.

LARGE-Glycan function
Another area of research focuses on the cellular significance of the LARGE-mediated glycosylation of dystroglycan, and on gaining novel insights into how defects in this post-translational modification cause diseases of varying severity. Binding between dystroglycan and its matrix-localized ligands is mediated through the disaccharide repeat added to dystroglycan by LARGE. The amount of this LARGE-glycan that decorates the nearly-ubiquitous dystroglycan is remarkably tissue-specific. In this study we 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 as a result were able to assess the primary cellular impacts of this treatment on the muscle and its disposition to the disease state. Although dystroglycan maintained the ability to bind ligands in the matrix, the dystroglycan that formed in regenerated LargeKD muscles had a significantly reduced ligand-binding capacity. This was a direct consequence of reducing the quantity of LARGE-glycan repeats in each chain, a finding that was confirmed using synthesized LARGE-glycan repeats. Ligand saturation due to insufficiency of the LARGE-glycan in LargeKD-regenerated muscle resulted in reduced basement membrane compaction, defective maturation of the neuromuscular junction, and functionally deficient 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. We propose that ultrastructural organization of the basement membrane can be modified during tissue establishment by extension of the LARGE-glycan. These findings both redefine the cellular significance of dystroglycan and support a new model for the underpinnings of dystroglycan-related disease.

A dystroglycan mutation associated with muscular dystrophy
Despite recent advances in our understanding of the glycosylation defects underlying these  dystroglycanopathies, it remains unclear whether mutated glycosyltransferases are the only causes of these diseases. Recently, we identified—for the first time—a missense mutation (c. 575C->T, T192M) in the dystroglycan gene of a patient with limb-girdle muscular dystrophy and cognitive impairment.  Our in vitro analysis revealed that this mutation does not affect the expression of dystroglycan; instead it impairs the glycosylation of, and thus its ability to bind, laminin.  A mouse model harboring this mutation recapitulates immunohistochemical and neuromuscular abnormalities observed in the patient, and the affected residue selectively impairs the modification of dystroglycan’s post-phosphoryl chains, owing to disruption of the interaction between dystroglycan and LARGE.  These findings led us to propose a novel pathogenic mechanism to account for muscular dystrophy: disruption of the enzyme-substrate complex that is required to initiate maturation of phosphorylated O-mannosyl glycans on dystroglycan.

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

Kevin P. Campbell, Ph.D.
Principal Investigator

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  Kevin P. Campbell
Investigator, Howard Hughes Medical Institute
Chair & Department Executive Officer, Dept. of Molecular Physiology & Biophysics
Roy J. and Lucille A. Carver Biomedical Research Chair
Professor, Neurology and Internal Medicine
Director, Senator Paul D. Wellstone MDCRC
University of Iowa
Carver College of Medicine
4283 Carver Biomedical Research Building
285 Newton Road
Iowa City, Iowa 52242-1101

Phone: (319) 335-7867
or (319) 335-8655
Fax: (319) 335-6957

Email: kevin-campbell@uiowa.edu


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