LARGE Enzyme Activity Deciphered: a New Therapeutic Target for Muscular Dystrophies Jane E Hewitt*

Hewitt Genome Medicine 2012, 4:23 http://genomemedicine.com/content/4/3/23 RESEARCH HIGHLIGHT LARGE enzyme activity deciphered: a new therapeutic target for muscular dystrophies Jane E Hewitt* because of its association with congenital muscular Abstract dystrophy (CMD). CMDs are a group of clinically and A signicant proportion of severe, inherited congenital genetically heterogeneous disorders that are currently muscular dystrophies are due to aberrant glycosylation untreatable. ese diseases are usually severe and often of the extracellular matrix receptor α-dystroglycan involve other organs, particularly the central nervous and a consequent lack of ligand-binding activity. system [1]. e genetic basis of a significant proportion A key member of this glycosylation pathway is of CMDs is still unknown; further understanding of the the LARGE protein, which was originally identied molecular pathways already identified may help to find through genome sequencing and genetic studies. these missing gene loci and aid both diagnosis and the Until recently, the biochemical activity of this enzyme development of therapeutic strategies. proved frustratingly elusive, but a recent study shows Around 10 years ago, through positional cloning of the that LARGE encodes a bifunctional glycosyltransferase mouse myodystrophy (myd) mutation, the LARGE gene that synthesizes a novel polysaccharide structure, was shown to be necessary for the functional glyco sy- which is required for functional dystroglycan. lation of the extracellular matrix receptor α-dystroglycan Identication of this structure should lead to [2] and to be required for ligand binding by this receptor development of new diagnostic tools and therapeutic [3]. Subsequently, from their association with human strategies for these dystrophies. muscular dystrophies, a number of additional genes were Keywords Muscular dystrophy, dystroglycan, identified within the same glycosylation pathway [1]. glycosylation, glycosaminoglycan, glycosyltransferase, ese disorders are all associated with hypoglycosylation extracellular matrix of α-dystroglycan and thus are collectively termed dystroglycanopathies. e medical relevance of dystroglycan glycosylation is not restricted to the muscular dystrophy field; arenaviruses, which can cause severe The mysterious role of LARGE in muscular disease in humans, use the same glycan structures to bind dystrophies α-dystroglycan as a co-receptor [4], and many epithelial Advances in genome sequencing and bioinformatics have cancers lose α-dystroglycan glycosylation, in some cases led to the identification of hundreds of genes of unknown because of silencing of LARGE [5]. or only inferred function. Often, the first insights into the e role of LARGE in the dystroglycan glycosylation function of a gene come from identification of mutations pathway has been of particular interest; circumstantial associated with a human disease and the task is then to evidence suggested that it synthesizes a novel glycan determine the underlying molecular pathways in which structure that is directly responsible for ligand inter- the gene is involved. is may be challenging and could action. Uniquely, overexpression of LARGE rescues func- require a combination of in vivo, cellular and biochemical tional glycosylation of α-dystroglycan in cells defective experiments to provide the answer. In the fields of glyco- for other enzyme components of this pathway [6]. How- biology and medicine, determination of the enzymatic ever, although it became clear that unusual O-mannosyl activity of LARGE, a putative bifunctional glycosyltrans- glycans on dystroglycan are required for ligand binding ferase, has been the focus of a great deal of recent interest [6], the biochemical activity of LARGE remained frustratingly elusive until a recent publication in Science [7]. *Correspondence: [email protected] School of Biology, University of Nottingham, Queen’s Medical Centre, Nottingham, Defective glycosylation of dystroglycan and NG7 2UH, UK disease Dystroglycan is a widely expressed protein with roles in © 2010 BioMed Central Ltd © 2012 BioMed Central Ltd muscle stabilization, cell migration, basement membrane Hewitt Genome Medicine 2012, 4:23 Page 2 of 3 http://genomemedicine.com/content/4/3/23 assembly and signaling [6]. In skeletal muscle, dystro- (a) glycan links dystrophin and the actin cytoskeleton inside the cell to the extracellular matrix (ECM) through laminin. Sia e protein is synthesized as a precursor molecule that is post-translationally cleaved into α and β subunits. e 2-3 extracellular α subunit is extensively glycosylated with Gal GalNAc O-linked glycans that include unusual O-mannose and 1-4 1-3 O-phosphomannose structures [6,8]. Although these GlcNAc GlcNAc mannose structures were shown to be necessary for 1-2 POMGnT1 1-4 ligand binding in vivo, the precise structure of the glycans 6 Man involved in the ECM interaction remained unclear. Man P X e severity of the dystroglycanopathies varies from POMT1/2 Walker-Warburg syndrome, a severe form of CMD that is Ser/Thr Ser/Thr also associated with ocular abnormalities and CNS defects, to milder forms of limb girdle muscular dystrophy with no CNS involvement [1]. e severity of the clinical phenotype does not correlate with the particular (b) gene mutated but probably relates to the pathogenicity of 1-3 1-3 1-3 the specific mutations and the extent of the loss of α- n dystroglycan glycosylation. LARGE is one of at least six dystroglycanopathy genes, along with POMT1, POMT2, LARGE ? POMGnT1, Fukutin and FKRP [1]; several of these genes ? Fukutin encode enzymes that are known to be directly involved in FKRP synthesis of O-linked mannose structures (Figure 1). Figure 1. Key glycans on α-dystroglycan. Dystroglycan contains Initially, biochemical and genetic studies pointed towards unusual O-mannosyl glycans that are necessary for ligand binding an O-mannose tetrasaccharide as a likely candidate for activity. (a) Two structures that have been implicated in ligand the laminin interaction (Figure 1). However, enzymatic binding by genetic and biochemical studies. Proteins mutated in removal of most of this structure does not eliminate dystroglycanopathies are shown in red boxes, and their enzyme laminin binding [9]. Further biochemical and mass spec- activities, where known, are indicated by arrows. POMT1 and POMT2 encode protein O-mannosyltransferases. Although this tro s copy studies identified a phosphomannose structure glycan was originally considered a candidate for the laminin- on dystroglycan [8] and showed that LARGE and Fukutin binding moiety (and mutation of POMT1, POMT2 or POMGnT1 (and probably FKRP) act downstream of formation of results in dystroglycanopathy), enzymatic removal does not destroy this structure (Figure 1). us, the ligand-binding struc- the laminin-binding activity in vitro [9]. The recently described ture is thought to be synthesized, at least in part, by phosphomannose structure [8] contains an unknown structure (X) thought to be the glycan responsible for binding. LARGE and LARGE and is presumed to be attached to this phosphate. Fukutin (and probably FKRP) act downstream of the formation of this structure. Gal, galactose; GlcNAc, N-acetylglucosamine; Man, LARGE enzyme activity determined mannose; P, phosphorylation; Sia, sialic acid.(b) LARGE synthesizes a LARGE was first identified by bioinformatic analysis of polysaccharide with repeating units of [Xyl(α1→3)GlcA(β1→3)] that the human genome sequence. e name LARGE derives is required for ligand-binding activity (star, xylose; diamond, GlcA). This structure is a good candidate for the X moiety attached to the from the fact that this gene, at 650 kb, is one of the phosphomannose in (a). The functions of Fukutin and FRKP and their biggest in the human genome, even though the coding relationship to the synthesis of this structure are currently unknown. region covers only 2 kb. e protein contains two predicted catalytic domains, each showing homology to a different family of glycosyltransferases, suggesting that addition to the expected components of the known O- LARGE is a bifunctional enzyme [2]. However, the enzyme glycans identified by previous studies, they identified activity of LARGE proved difficult to elucidate from significant amounts of two unexpected sugars - glucuronic sequence homologies alone. acid (GlcA) and xylose. Furthermore, a Chinese hamster Kevin Campbell and colleagues [7] have now shown ovary (CHO) cell line defective in synthesis of that LARGE indeed encodes a bifunctional glycosyl trans- UDP-xylose did not show increased immunoreactivity ferase that has both xylosyltransferase and glucuronyl- with the monoclonal antibody IIH6, which recognizes transferase activities. In this study, the authors [7] fully glycosylated dystroglycan, when transfected with performed compositional analysis of the sugars present LARGE, in contrast to UDP-xylose producing cell lines on α-dystroglycan expressed as a secreted, recombinant [7]. Although both GlcA and xylose are components of protein by human embryonic kidney (HEK293) cells. In some glycosaminoglycan (GAG) chains [10], previous Hewitt Genome Medicine 2012, 4:23 Page 3 of 3 http://genomemedicine.com/content/4/3/23 studies had indicated that GAGs were not necessary for should help to generate more reliable or specific anti αdystroglycan binding to laminin. bodies, for example. Little is known about the dystro Hypothesizing that LARGE has both xylosyltransferase glycanligand interaction, but now it will be possible

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