Bifunctional Glycosyltransferase Research Articles

Structural basis for matriglycan synthesis by the LARGE1 dual glycosyltransferase.

LARGE1 is a bifunctional glycosyltransferase responsible for generating a long linear polysaccharide termed matriglycan that links the cytoskeleton and the extracellular matrix and is required for proper muscle function. This matriglycan polymer is made with an alternating pattern of xylose and glucuronic acid monomers. Mutations in the LARGE1 gene have been shown to cause life-threatening dystroglycanopathies through the inhibition of matriglycan synthesis. Despite its major role in muscle maintenance, the structure of the LARGE1 enzyme and how it assembles in the Golgi are unknown. Here we present the structure of LARGE1, obtained by a combination of X-ray crystallography and single-particle cryo-EM. We found that LARGE1 homo-dimerizes in a configuration that is dictated by its coiled-coil stem domain. The structure shows that this enzyme has two canonical GT-A folds within each of its catalytic domains. In the context of its dimeric structure, the two types of catalytic domains are brought into close proximity from opposing monomers to allow efficient shuttling of the substrates between the two domains. Together, with putative retention of matriglycan by electrostatic interactions, this dimeric organization offers a possible mechanism for the ability of LARGE1 to synthesize long matriglycan chains. The structural information further reveals the mechanisms in which disease-causing mutations disrupt the activity of LARGE1. Collectively, these data shed light on how matriglycan is synthesized alongside the functional significance of glycosyltransferase oligomerization.

Structure of the human heparan sulfate polymerase complex EXT1-EXT2

Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating N-acetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Here, we present the cryo-electron microscopy structure of human EXT1-EXT2, which reveals the formation of a tightly packed hetero-dimeric complex harboring four glycosyltransferase domains. A combination of in vitro and in cellulo mutational studies is used to dissect the functional role of the four catalytic sites. While EXT1 can catalyze both glycosyltransferase reactions, our results indicate that EXT2 might only have N-acetylglucosamine transferase activity. Our findings provide mechanistic insight into heparan sulfate chain elongation as a nonprocessive process and lay the foundation for future studies on EXT1-EXT2 function in health and disease.

Biochemical Characterization of GacI, a Bifunctional Glycosyltransferase-Phosphatase Enzyme Involved in Acarbose Biosynthesis in Streptomyces glaucescens GLA.O.

Acarbose, a pseudotetrasaccharide produced by several strains of Actinoplanes and Streptomyces, is an α-glucosidase inhibitor clinically used to control type II diabetes. Bioinformatic analysis of the biosynthetic gene clusters of acarbose in Actinoplanes sp. SE50/110 (the acb cluster) and Streptomyces glaucescens GLA.O (the gac cluster) revealed their distinct genetic organizations and presumably biosynthetic pathways. However, to date, only the acarbose pathway in the SE50/110 strain has been extensively studied. Here, we report that GacI, one of the proteins that appear to be different between the two pathways, is a bifunctional glycosyltransferase family 5 (GT5)-phosphatase (PP) enzyme that functions at two different steps in acarbose biosynthesis in S. glaucescens GLA.O. In the acb pathway, the GT and the PP reactions are performed by two different enzymes. Truncated GacI proteins having only the GT or the PP domain showed comparable catalytic activity with the full-length GacI, indicating that domain separation does not significantly affect their respective catalytic activity. GacI, which is widely distributed in many Streptomyces, represents the first example of naturally occurring GT5-PP bifunctional enzymes biochemically characterized.

Variation at the common polysaccharide antigen locus drives lipopolysaccharide diversity within the Pseudomonas syringae species complex.

SummaryThe common polysaccharide antigen (CPA) of the lipopolysaccharide (LPS) from Pseudomonas syringae is highly variable, but the genetic basis for this is poorly understood. We have characterized the CPA locus from P. syringae pv. actinidiae (Psa). This locus has genes for l‐ and d‐rhamnose biosynthesis and an operon coding for ABC transporter subunits, a bifunctional glycosyltransferase and an o‐methyltransferase. This operon is predicted to have a role in the transport, elongation and termination of the CPA oligosaccharide and is referred to as the TET operon. Two alleles of the TET operon were present in different biovars (BV) of Psa and lineages of the closely related pathovar P. syringae pv. actinidifoliorum. This allelic variation was reflected in the electrophoretic properties of purified LPS from the different isolates. Gene knockout of the TET operon allele from BV1 and replacement with that from BV3, demonstrated the link between the genetic locus and the biochemical properties of the LPS molecules in Psa. Sequence analysis of the TET operon from a range of P. syringae and P. viridiflava isolates displayed a phylogenetic history incongruent with core gene phylogeny but correlates with previously reported tailocin sensitivity, suggesting a functional relationship between LPS structure and tailocin susceptibility.

Leloir glycosyltransferases of natural product C-glycosylation: structure, mechanism and specificity.

A prominent attribute of chemical structure in microbial and plant natural products is aromatic C-glycosylation. In plants, various flavonoid natural products have a β-C-d-glucosyl moiety attached to their core structure. Natural product C-glycosides have attracted significant attention for their own unique bioactivity as well as for representing non-hydrolysable analogs of the canonical O-glycosides. The biosynthesis of natural product C-glycosides is accomplished by sugar nucleotide-dependent (Leloir) glycosyltransferases. Here, we provide an overview on the C-glycosyltransferases of microbial, plant and insect origin that have been biochemically characterized. Despite sharing basic evolutionary relationships, as evidenced by their common membership to glycosyltransferase family GT-1 and conserved GT-B structural fold, the known C-glycosyltransferases are diverse in the structural features that govern their reactivity, selectivity and specificity. Bifunctional glycosyltransferases can form C- and O-glycosides dependent on the structure of the aglycon acceptor. Recent crystal structures of plant C-glycosyltransferases and di-C-glycosyltransferases complement earlier structural studies of bacterial enzymes and provide important molecular insight into the enzymatic discrimination between C- and O-glycosylation. Studies of enzyme structure and mechanism converge on the view of a single displacement (SN2)-like mechanism of enzymatic C-glycosyl transfer, largely analogous to O-glycosyl transfer. The distinction between reactions at the O- or C-acceptor atom is achieved through the precise positioning of the acceptor relative to the donor substrate in the binding pocket. Nonetheless, C-glycosyltransferases may differ in the catalytic strategy applied to induce nucleophilic reactivity at the acceptor carbon. Evidence from the mutagenesis of C-glycosyltransferases may become useful in engineering these enzymes for tailored reactivity.

Bifunctional glycosyltransferases catalyze both extension and termination of pectic galactan oligosaccharides.

Pectins are the most complex polysaccharides of the plant cell wall. Based on the number of methylations, acetylations and glycosidic linkages present in their structures, it is estimated that up to 67 transferase activities are involved in pectin biosynthesis. Pectic galactans constitute a major part of pectin in the form of side-chains of rhamnogalacturonan-I. In Arabidopsis, galactan synthase 1 (GALS1) catalyzes the addition of galactose units from UDP-Gal to growing β-1,4-galactan chains. However, the mechanisms for obtaining varying degrees of polymerization remain poorly understood. In this study, we show that AtGALS1 is bifunctional, catalyzing both the transfer of galactose from UDP-α-d-Gal and the transfer of an arabinopyranose from UDP-β-l-Arap to galactan chains. The two substrates share a similar structure, but UDP-α-d-Gal is the preferred substrate, with a 10-fold higher affinity. Transfer of Arap to galactan prevents further addition of galactose residues, resulting in a lower degree of polymerization. We show that this dual activity occurs both invitro and invivo. The herein described bifunctionality of AtGALS1 may suggest that plants can produce the incredible structural diversity of polysaccharides without a dedicated glycosyltransferase for each glycosidic linkage.

Structural flexibility of human α-dystroglycan.

Dystroglycan (DG), composed of α and β subunits, belongs to the dystrophin‐associated glycoprotein complex. α‐DG is an extracellular matrix protein that undergoes a complex post‐translational glycosylation process. The bifunctional glycosyltransferase like‐acetylglucosaminyltransferase (LARGE) plays a crucial role in the maturation of α‐DG, enabling its binding to laminin. We have already structurally analyzed the N‐terminal region of murine α‐DG (α‐DG‐Nt) and of a pathological single point mutant that may affect recognition of LARGE, although the structural features of the potential interaction between LARGE and DG remain elusive. We now report on the crystal structure of the wild‐type human α‐DG‐Nt that has allowed us to assess the reliability of our murine crystallographic structure as a α‐DG‐Nt general model. Moreover, we address for the first time both structures in solution. Interestingly, small‐angle X‐ray scattering (SAXS) reveals the existence of two main protein conformations ensembles. The predominant species is reminiscent of the crystal structure, while the less populated one assumes a more extended fold. A comparative analysis of the human and murine α‐DG‐Nt solution structures reveals that the two proteins share a common interdomain flexibility and population distribution of the two conformers. This is confirmed by the very similar stability displayed by the two orthologs as assessed by biochemical and biophysical experiments. These results highlight the need to take into account the molecular plasticity of α‐DG‐Nt in solution, as it can play an important role in the functional interactions with other binding partners.

LARGE2-dependent glycosylation confers laminin-binding ability on proteoglycans.

Both LARGE1 (formerly LARGE) and its paralog LARGE2 are bifunctional glycosyltransferases with xylosy- and glucuronyltransferase activities, and are capable of synthesizing polymers composed of a repeating disaccharide [-3Xylα1,3GlcAβ1-]. Post-translational modification of the O-mannosyl glycan of α-dystroglycan (α-DG) with the polysaccharide is essential for it to act as a receptor for ligands in the extracellular matrix (ECM), and both LARGE paralogs contribute to the modification in vivo. LARGE1 and LARGE2 have different tissue distribution profiles and enzymatic properties; however, the functional difference of the homologs remains to be determined, and α-DG is the only known substrate for the modification by LARGE1 or LARGE2. Here we show that LARGE2 can modify proteoglycans (PGs) with the laminin-binding glycan. We found that overexpression of LARGE2, but not LARGE1, mediates the functional modification on the surface of DG−/−, Pomt1−/− and Fktn−/− embryonic stem cells. We identified a heparan sulfate-PG glypican-4 as a substrate for the LARGE2-dependent modification by affinity purification and subsequent mass spectrometric analysis. Furthermore, we showed that LARGE2 could modify several additional PGs with the laminin-binding glycan, most likely within the glycosaminoglycan (GAG)-protein linkage region. Our results indicate that LARGE2 can modify PGs with the GAG-like polysaccharide composed of xylose and glucuronic acid to confer laminin binding. Thus, LARGE2 may play a differential role in stabilizing the basement membrane and modifying its functions by augmenting the interactions between laminin globular domain-containing ECM proteins and PGs.

Endogenous Glucuronyltransferase Activity of LARGE or LARGE2 Required for Functional Modification of α-Dystroglycan in Cells and Tissues

Mutations in the LARGE gene have been identified in congenital muscular dystrophy (CMD) patients with brain abnormalities. Both LARGE and its paralog, LARGE2 (also referred to as GYLTL1B) are bifunctional glycosyltransferases with xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, and are capable of forming polymers consisting of [-3Xyl-α1,3GlcAβ1-] repeats. LARGE-dependent modification of α-dystroglycan (α-DG) with these polysaccharides is essential for the ability of α-DG to act as a receptor for ligands in the extracellular matrix. Here we report on the endogenous enzymatic activities of LARGE and LARGE2 in mice and humans, using a newly developed assay for GlcA-T activity. We show that normal mouse and human cultured cells have endogenous LARGE GlcA-T, and that this activity is absent in cells from the Large(myd) (Large-deficient) mouse model of muscular dystrophy, as well as in cells from CMD patients with mutations in the LARGE gene. We also demonstrate that GlcA-T activity is significant in the brain, heart, and skeletal muscle of wild-type and Large2(-/-) mice, but negligible in the corresponding tissues of the Large(myd) mice. Notably, GlcA-T activity is substantial, though reduced, in the kidneys of both the Large(myd) and Large2(-/-) mice, consistent with the observation of α-DG/laminin binding in these contexts. This study is the first to test LARGE activity in samples as small as cryosections and, moreover, provides the first direct evidence that not only LARGE, but also LARGE2, is vital to effective functional modification of α-DG in vivo.

A Bifunctional Glycosyltransferase from Agrobacterium tumefaciens Synthesizes Monoglucosyl and Glucuronosyl Diacylglycerol under Phosphate Deprivation

Glycolipids are mainly found in phototrophic organisms (like plants and cyanobacteria), in Gram-positive bacteria, and a few other bacterial phyla. Besides the function as bulk membrane lipids, they often play a role under phosphate deprivation as surrogates for phospholipids. The Gram-negative Agrobacterium tumefaciens accumulates four different glycolipids under phosphate deficiency, including digalactosyl diacylglycerol and glucosylgalactosyl diacylglycerol synthesized by a processive glycosyltransferase. The other two glycolipids have now been identified by mass spectrometry and nuclear magnetic resonance spectroscopy as monoglucosyl diacylglycerol and glucuronosyl diacylglycerol. These two lipids are synthesized by a single promiscuous glycosyltransferase encoded by the ORF atu2297, with UDP-glucose or UDP-glucuronic acid as sugar donors. The transfer of sugars differing in their chemistry is a novel feature not observed before for lipid glycosyltransferases. Furthermore, this enzyme is the first glucuronosyl diacylglycerol synthase isolated. Deletion mutants of Agrobacterium lacking monoglucosyl diacylglycerol and glucuronosyl diacylglycerol or all glycolipids are not impaired in growth or virulence during infection of tobacco leaf discs. Our data suggest that the four glycolipids and the nonphospholipid diacylglyceryl trimethylhomoserine can mutually replace each other during phosphate deprivation. This redundancy of different nonphospholipids may represent an adaptation mechanism to enhance the competitiveness in nature.

TbGT8 is a bifunctional glycosyltransferase that elaborates N-linked glycans on a protein phosphatase AcP115 and a GPI-anchor modifying glycan in Trypanosoma brucei

The procyclic form of Trypanosoma brucei expresses procyclin surface glycoproteins with unusual glycosylphosphatidylinositol-anchor side chain structures that contain branched N-acetyllactosamine and lacto-N-biose units. The glycosyltransferase TbGT8 is involved in the synthesis of the branched side chain through its UDP-GlcNAc: βGal β1-3N-acetylglucosaminyltransferase activity. Here, we explored the role of TbGT8 in the mammalian bloodstream form of the parasite with a tetracycline-inducible conditional null T. brucei mutant for TbGT8. Under non-permissive conditions, the mutant showed significantly reduced binding to tomato lectin, which recognizes poly-N-acetyllactosamine-containing glycans. Lectin pull-down assays revealed differences between the wild type and TbGT8 null-mutant T. brucei, notably the absence of a broad protein band with an approximate molecular weight of 110kDa in the mutant lysate. Proteomic analysis revealed that the band contained several glycoproteins, including the acidic ecto-protein phosphatase AcP115, a stage-specific glycoprotein in the bloodstream form of T. brucei. Western blotting with an anti-AcP115 antibody revealed that AcP115 was approximately 10kDa smaller in the mutant. Enzymatic de-N-glycosylation demonstrated that the underlying protein cores were the same, suggesting that the 10-kDa difference was due to differences in N-linked glycans. Immunofluorescence microscopy revealed the colocalization of hemagglutinin epitope-tagged TbGT8 and the Golgi-associated protein GRASP. These data suggest that TbGT8 is involved in the construction of complex poly-N-acetyllactosamine-containing type N-linked and GPI-linked glycans in the Golgi of the bloodstream and procyclic parasite forms, respectively.

The transgenic expression of LARGE exacerbates the muscle phenotype of dystroglycanopathy mice

Mutations in fukutin-related protein (FKRP) underlie a group of muscular dystrophies associated with the hypoglycosylation of α-dystroglycan (α-DG), a proportion of which show central nervous system involvement. Our original FKRP knock-down mouse (FKRP(KD)) replicated many of the characteristics seen in patients at the severe end of the dystroglycanopathy spectrum but died perinatally precluding its full phenotyping and use in testing potential therapies. We have now overcome this by crossing FKRP(KD) mice with those expressing Cre recombinase under the Sox1 promoter. Owing to our original targeting strategy, this has resulted in the restoration of Fkrp levels in the central nervous system but not the muscle, thereby generating a new model (FKRP(MD)) which develops a progressive muscular dystrophy resembling what is observed in limb girdle muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) is a bifunctional glycosyltransferase previously shown to hyperglycosylate α-DG. To investigate the therapeutic potential of LARGE up-regulation, we have now crossed the FKRP(MD) line with one overexpressing LARGE and show that, contrary to expectation, this results in a worsening of the muscle pathology implying that any future strategies based upon LARGE up-regulation require careful management.

Identification of a Bifunctional Maize C- and O-Glucosyltransferase

Flavonoids accumulate in plant vacuoles usually as O-glycosylated derivatives, but several species can also synthesize flavonoid C-glycosides. Recently, we demonstrated that a flavanone 2-hydroxylase (ZmF2H1, CYP93G5) converts flavanones to the corresponding 2-hydroxy derivatives, which are expected to serve as substrates for C-glycosylation. Here, we isolated a cDNA encoding a UDP-dependent glycosyltransferase (UGT708A6), and its activity was characterized by in vitro and in vivo bioconversion assays. In vitro assays using 2-hydroxyflavanones as substrates and in vivo activity assays in yeast co-expressing ZmF2H1 and UGT708A6 show the formation of the flavones C-glycosides. UGT708A6 can also O-glycosylate flavanones in bioconversion assays in Escherichia coli as well as by in vitro assays with the purified recombinant protein. Thus, UGT708A6 is a bifunctional glycosyltransferase that can produce both C- and O-glycosidated flavonoids, a property not previously described for any other glycosyltransferase.

P.8.2 Reduction of LARGE expression in different types of muscular dystrophies other than dystroglycanopathy

Mutations in the genes coding for putative or demonstrated glycosyltransferases or other proteins involved in alpha-dystroglycan (ADG) glycosylation pathway result in the failure of alpha-dystroglycan to be properly glycosylated and lead to genetic forms of muscular dystrophy, collectively termed as dystroglycanopathies. An important enzyme which is involved in maintaining muscle cell viability, known as LARGE, has been shown to participate in O-mannosyl phosphorylation of ADG. It could also act as a bifunctional glycosyltransferase and allow ADG to bind laminin-G domain containing ligands. Additionally, transient overexpression of LARGE enzyme demonstrated a marked increase in hyperglycosylation of ADG and a corresponding increase in high affinity binding to several extracellular matrix ligands. To date, it has not been investigated whether LARGE enzyme affects the basic pathogenic mechanisms of muscular dystrophies, except for dystroglycanopathies and the influence of LARGE gene expression in different types of muscular dystrophies is not known. In this study, the expression level of LARGE and ADG immunofluorescence were examined in skeletal muscle biopsies from 26 patients with different forms of muscular dystrophy (i.e. DMD, BMD, calpainopathy, sarcoglycanopathy, dysferlinopathy, and merosin and collagen VI deficient CMDs) and correlation with different histopathological findings was investigated. We detected reduced expression level of LARGE gene in different types of muscular dystrophies, partly correlating with the severity of dystrophic changes, but we did not find any significant relationship between reduction of LARGE expression and ADG hypoglycosylation. Our results suggest that LARGE enzyme might have another function in skeletal muscle fibers that is probably distinct from adding a critical sugar chain onto ADG.

Characterization of a trifunctional glucosyltransferase essential for Moraxella catarrhalis lipooligosaccharide assembly

The human respiratory tract pathogen Moraxella catarrhalis expresses lipooligosaccharides (LOS), glycolipid surface moieties that are associated with enhanced colonization and virulence. Recent studies have delineated the major steps required for the biosynthesis and assembly of the M. catarrhalis LOS molecule. We previously demonstrated that the glucosyltransferase enzyme Lgt3 is responsible for the addition of at least one glucose (Glc) molecule, at the β-(1-4) position, to the inner core of the LOS molecule. Our data further suggested a potential multifunctional role for Lgt3 in LOS biosynthesis. The studies reported here demonstrate that the Lgt3 enzyme possesses two glycosyltransferase domains (A1 and A2) similar to that of other bifunctional glycosyltransferase enzymes involved in surface polysaccharide biosynthesis in Escherichia coli, Pasteurella multocida and Streptococcus pyogenes. Each Lgt3 domain contains a conserved DXD motif, shown to be involved in the catalytic activity of other glycosyltransferases. To determine the function of each domain, A1 (N-terminal), A2 (C-terminal) and double A1A2 site-directed DAD to AAA mutants were constructed and the resulting LOS phenotypes of these modified strains were analyzed. Our studies indicate that the Lgt3 N-terminal A1 catalytic domain is responsible for the addition of the first β-(1-3) Glc to the first Glc on the inner core. The C-terminal catalytic domain A2 then adds the β-(1-4) Glc and the β-(1-6) Glc, confirming the bifunctional nature of this domain. The results from these experiments demonstrate that Lgt3 is a novel, multifunctional transferase responsible for the addition of three Glcs with differing linkages onto the inner core of M. catarrhalis LOS.

Identification of Antithrombin-Modulating Genes. Role of LARGE, a Gene Encoding a Bifunctional Glycosyltransferase, in the Secretion of Proteins?

The haemostatic relevance of antithrombin together with the low genetic variability of SERPINC1, and the high heritability of plasma levels encourage the search for modulating genes. We used a hypothesis-free approach to identify these genes, evaluating associations between plasma antithrombin and 307,984 polymorphisms in the GAIT study (352 individuals from 21 Spanish families). Despite no SNP reaching the genome wide significance threshold, we verified milder positive associations in 307 blood donors from a different cohort. This validation study suggested LARGE, a gene encoding a protein with xylosyltransferase and glucuronyltransferase activities that forms heparin-like linear polysaccharides, as a potential modulator of antithrombin based on the significant association of one SNPs, rs762057, with anti-FXa activity, particularly after adjustment for age, sex and SERPINC1 rs2227589 genotype, all factors influencing antithrombin levels (p = 0.02). Additional results sustained this association. LARGE silencing inHepG2 and HEK-EBNA cells did not affect SERPINC1 mRNA levels but significantly reduced the secretion of antithrombin with moderate intracellular retention. Milder effects were observed on α1-antitrypsin, prothrombin and transferrin. Our study suggests LARGE as the first known modifier of plasma antithrombin, and proposes a new role for LARGE in modulating extracellular secretion of certain glycoproteins.

Molecular Basis for Dystroglycan Binding to Laminin‐G Domain‐Containing Ligands

Dystroglycan (DG) is a highly glycosylated extracellular matrix (ECM) receptor. The like‐acetylglucosaminyltransferase (LARGE) is responsible for posttranslational modification of alpha‐dystroglycan (α‐DG) which is required for its function. Reduced ligand binding activity of α‐DG due to perturbed glycosylation is a common pathologic feature among several different forms of muscular dystrophy (called dystroglycanopathies). We now report that LARGE is a bifunctional glycosyltransferase, with both xylosyltransferase (Xyl‐T) and glucuronyltransferase (GlcA‐T) activities. We examined the enzymatic activity of LARGE with respect to Xyl‐α‐pNP as well as GlcA‐β‐MU as the acceptors and UDP‐Xyl and UDP‐GlcA as donors, using a secreted form of LARGE that lacks its transmembrane domain (LARGEdTM). We have identified the enzymatic function of LARGE, which produces a polysaccharide with repeating units of [‐3Xyl‐α1,3GlcA‐β1‐] onto these acceptors. We further show that this modification confers binding ability on α‐DG to laminin‐G domain‐containing ECM ligands: laminin, agrin, and neurexin. α‐DG binds the LG4 and 5 domains of laminin alpha chains, and it is suggested that three basic patches contribute to these interactions. The LARGE‐synthesized, negatively charged glycan on α‐DG likely binds to laminin through electrostatic associations with these basic patches.

Structural and functional evidence for two separate oligosaccharide binding sites of Pasteurella multocida hyaluronan synthase

Pasteurella multocida hyaluronan synthase (PmHAS) is a bi-functional glycosyltransferase, containing a β1,3-glucuronyltransferase and β1,4-N-acetylglucosaminetransferase domain. PmHAS catalyzes the elongation of hyaluronan (HA) through the sequential addition of single monosaccharides to the non-reducing end of the hyaluronan chain. Research is focused on the relation between the length of the HA oligosaccharide and the single-step elongation kinetics from HA4 up to HA9. It was found that the turnover number kcat increased with length to maximum values of 11 and 14 s-1 for NAc- and UA-transfer, respectively. Interestingly, the specificity constant kcat/KM increased with polymer length from HA5 to HA7 to a value of 44 mM-1s-1, indicating an oligosaccharide binding site with increasing specificity towards a heptasaccharide at the UA domain. The value of kcat/KM remained moderately constant around 8 mM-1s-1 for HA4, HA6, and HA8, indicating a binding site with significantly lower binding specificity at the NAc domain than at the UA domain. These findings are further corroborated by a structural homology model of PmHAS, revealing two distinct sites for binding of oligosaccharides of different sizes, one in each transferase domain. Structural alignment studies between PmHAS and glycosyltransferases of the GT-A fold showed significant similarity in the binding of the UDP-sugars and the orientation of the acceptor substrate. These similarities in substrate orientation in the active site and in essential amino acid residues involved in substrate binding were utilized to localize the two HA oligosaccharide binding sites.

LARGE2 generates the same xylose- and glucuronic acid-containing glycan structures as LARGE

LARGE (like-glycosyltransferase) and LARGE2 (glycosyltransferase-like 1B (GYLTL1B)) are homologous Golgi glycosyltransferases possessing two catalytic domains with homology to members of glycosyltransferase families GT8 and GT49. Mutations in human and mouse Large result in muscular dystrophy due to underglycosylation of dystroglycan. The systemic function of LARGE2 is unknown, but at a cellular level the enzyme can substitute for LARGE in glycosylating dystroglycan. Here, we show that LARGE2 catalyzes the same glycosylation reaction as LARGE. It is a bifunctional glycosyltransferase using uridine diphosphate (UDP)-xylose (Xyl) and UDP-glucuronic acid (GlcA) as donor sugars to produce a xyloglucuronan with alternating Xyl and GlcA residues.

Xylosyl- and glucuronyltransferase functions of LARGE in α-dystroglycan modification are conserved in LARGE2

LARGE-dependent modification enables α-dystroglycan (α-DG) to bind to its extracellular matrix ligands. Mutations in the LARGE gene and several others involved in O-mannosyl glycan synthesis have been identified in congenital and limb-girdle muscular dystrophies that are characterized by perturbed glycosylation and reduced ligand-binding affinity of α-DG. LARGE is a bifunctional glycosyltransferase that alternately transfers xylose and glucuronic acid, thereby generating the heteropolysaccharides on α-DG that confer its ligand binding. Although the LARGE paralog LARGE2 (also referred to as GYLTL1B) has likewise been shown to enhance the functional modification of α-DG in cultured cells, its enzymatic activities have not been identified. Here, we report that LARGE2 is also a bifunctional glycosyltransferase and compare its properties with those of LARGE. By means of a high-performance liquid chromatography-based enzymatic assay, we demonstrate that like LARGE, LARGE2 has xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, as well as polymerizing activity. Notably, however, the pH optima of the Xyl-T and GlcA-T of LARGE2 are distinct from one another and also from those of LARGE. Our results suggest that LARGE and LARGE2 catalyze the same glycosylation reactions for the functional modification of α-DG, but that they have different biochemical properties.