Crystal structure of GlcAT-S, a human glucuronyltransferase, involved in the biosynthesis of the HNK-1 carbohydrate epitope

The HNK-1 carbohydrate epitope is found in various neural cell adhesion molecules. Two glucuronyltransferases (GlcAT-P and GlcAT-S) are involved in the biosynthesis of HNK-1 carbohydrate. Our previous study on the crystal structure of GlcAT-P revealed the reaction and substrate recognition mechanisms of this enzyme. Comparative analyses of the enzymatic activities of GlcAT-S and GlcAT-P showed that there are notable differences in the acceptor substrate specificities of these enzymes. To elucidate differences between their specificities, we now solved the crystal structure of GlcAT-S. Residues interacting with UDP molecule, which is a part of the donor substrate, are highly conserved between GlcAT-P and GlcAT-S. On the other hand, there are some differences between these proteins in the manner they recognize their respective acceptor substrates. Phe245, one of the most important GlcAT-P residues for the recognition of acceptors, is a tryptophan in GlcAT-S. In addition, Val320, which is located on the C-terminal long loop of the neighboring molecule in the dimer and critical in the recognition of the acceptor sugar molecule by the GlcAT-P dimer, is an alanine in GlcAT-S. These differences play key roles in establishing the distinct specificity for the acceptor substrate by GlcAT-S, which is further supported by site-directed mutagenesis of GlcAT-S and a computer-aided model building of GlcAT-S/substrate complexes.     

Molecular cloning and expression of a second glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope

A cDNA encoding a novel glucuronyltransferase was cloned from a rat brain cDNA library. The cDNA sequence contained an open reading frame encoding 324 amino acids, with type II transmembrane topology. The amino acid sequence revealed 49% homology to rat GlcAT-P, a glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope of glycoproteins, [Terayama et al. (1997) Proc. Natl. Acad. Sci. USA 94, 6093-6098] and the highest sequence homology was found in the catalytic region. Northern blot analysis indicated that this newly cloned glucuronyltransferase is expressed in the nervous system, consistent with the selective localization of the HNK-1 carbohydrate epitope in the nervous system. Transfection of this cDNA into COS-1 cells induced the expression of the HNK-1 carbohydrate epitope on cell surfaces, and induced the morphological changes in these cells. These results indicated that this newly cloned cDNA is a second glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope.     

A novel glucuronyltransferase in nervous system presumably associated with the biosynthesis of HNK-1 carbohydrate epitope on glycoproteins.

Recently, embryonic chicken brain extract was shown to contain a glucuronyltransferase, which transfers glucuronic acid from UDP-glucuronic acid to glycolipid acceptors (neolactotetraosyl ceramide). The enzyme was also suggested to transfer glucuronic acid to glycoprotein acceptors (asialoorosomucoid) (Das, K. K., Basu, M., Basu, S., Chou, D. K. H., and Jungalwala, F. B. (1991) J. Biol. Chem. 266, 5238-5243). In this study, the glucuronyltransferase activity in rat brain extract was separated into two groups by UDP-glucuronic acid-Sepharose CL-6B column chromatography. The enzyme recovered predominantly in the effluent fraction (GlcAT-L) catalyzed the transfer of glucuronic acid to glycolipid acceptors but not to glycoprotein acceptors, whereas the enzyme recovered in the eluate fraction (GlcAT-P) transferred glucuronic acid most predominantly to glycoprotein acceptors and very little to glycolipid acceptors. GlcAT-P was able to transfer glucuronic acid to oligosaccharide chains on asialoorosomucoid. The enzyme recognized a terminal lactosamine structure, Gal beta 1-4GlcNAc, on glycoproteins. It was localized in the nervous system and was hardly detectable in other tissues, including the thymus, spleen, lung, kidney, and liver. Although GlcAT-L and GlcAT-P shared some properties in common such as tissue distributions and developmental changes, they exhibited marked differences in their phospholipid dependence and in their pH profiles, apart from their respective acceptor preference to glycolipids and glycoproteins. The acceptor specificity and tissue distribution suggest that a novel glucuronyltransferase, GlcAT-P, is involved in the biosynthesis of the sulfoglucuronylgalactose structure in the HNK-1 carbohydrate epitope that is expressed on glycoproteins.     

Structural basis for acceptor substrate recognition of a human glucuronyltransferase, GlcAT-P, an enzyme critical in the biosynthesis of the carbohydrate epitope HNK-1

The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules. Its structure is characterized by a terminal sulfated glucuronyl acid. The glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of the HNK-1 epitope, GlcAT-P as the major enzyme. We overexpressed and purified the recombinant human GlcAT-P from Escherichia coli. Analysis of its enzymatic activity showed that it catalyzed the transfer reaction for N-acetyllactosamine (Galbeta1-4GlcNAc) but not lacto-N-biose (Galbeta1-3GlcNAc) as an acceptor substrate. Subsequently, we determined the first x-ray crystal structures of human GlcAT-P, in the absence and presence of a donor substrate product UDP, catalytic Mn(2+), and an acceptor substrate analogue N-acetyllactosamine (Galbeta1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The asymmetric unit contains two independent molecules. Each molecule is an alpha/beta protein with two regions that constitute the donor and acceptor substrate binding sites. The UDP moiety of donor nucleotide sugar is recognized by conserved amino acid residues including a DXD motif (Asp(195)-Asp(196)-Asp(197)). Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate. In addition, Val(320) and Asn(321), which are located on the C-terminal long loop from a neighboring molecule, and Phe(245) contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity.     

The N-glycan acceptor specificity of a glucuronyltransferase, GlcAT-P, associated with biosynthesis of the HNK-1 epitope

The acceptor specificity of a rat brain glucuronyltransferase, GlcAT-P, associated with biosynthesis of the HNK-1 epitope on glycoproteins, was investigated using asialoorosomucoid as a model acceptor substrate. Structural analysis of N-linked oligosaccharides, to which glucuronic acid was transferred by GlcAT-P, by means of two-dimensional mapping of pyridylamino-oligosaccharides and MS spectrometry, demonstrated that the enzyme transferred glucuronic acid to bi-, tri-, and tetra-antennary complex type sugar chains, with almost equal efficiency, indicating that the enzyme has no preference as to the number of acceptor sugar branches. Next, we studied the branch specificity of this enzyme by means of the selective branch scission method involving two step exoglycosidase digestion using authentic pyridylamino-oligosaccharides. The GlcAT-P is highly specific for the terminal N-acetyllactosamine structure and no glucuronic acid was incorporated into a Gal1-3GlcNAc moiety. The GlcAT-P transferred glucuronic acid to the galactose residues in the N-acetyllactosamine branches of bi-, tri-, and tetra-antennary oligosaccharide chains, with different efficiencies and most preferentially to those in the Gal1-4GlcNAc1-4Man1-3 branch.     

Different acceptor specificities of two glucuronyltransferases involved in the biosynthesis of HNK-1 carbohydrate

The biosynthesis of HNK-1 carbohydrate is mainly regulated by two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase (HNK-1 ST). To determine how the two glucuronyltransferases are involved in the biosynthesis of the HNK-1 carbohydrate, we prepared soluble forms of GlcAT-P and GlcAT-S fused with the IgG-binding domain of protein A and then compared the enzymatic properties of the two enzymes. Both GlcAT-P and GlcAT-S transferred glucuronic acid (GlcA) not only to a glycoprotein acceptor, asialoorosomucoid (ASOR), but also to a glycolipid acceptor, paragloboside. The activity of GlcAT-P toward ASOR was enhanced fivefold in the presence of sphingomyelin, but there were no effects on that of GlcAT-S. The activities of the two enzymes toward paragloboside were only detected in the presence of phospholipids such as phosphatidylinositol. Kinetic analysis revealed that the K(m) value of GlcAT-P for ASOR was 10 times lower than that for paragloboside. Furthermore, acceptor specificity analysis involving various oligosaccarides revealed that GlcAT-P specifically recognized N-acetyllactosamine (Galbeta1-4GlcNAc) at the nonreducing terminals of acceptor substrates. In contrast, GlcAT-S recognized not only the terminal Galbeta1-4GlcNAc structure but also the Galbeta1-3GlcNAc structure and showed the highest activity toward triantennary N-linked oligosaccharides. GlcAT-P transferred GlcA to NCAM about twice as much as to ASOR, whereas GlcAT-S did not show any activity toward NCAM. These lines of evidence indicate that these two enzymes have significantly different acceptor specificities, suggesting that they may synthesize functionally and structurally different HNK-1 carbohydrates in the nervous system.     

Purification and characterization of two recombinant human glucuronyltransferases involved in the biosynthesis of HNK-1 carbohydrate in Escherichia coli

Two glucuronyltransferases (GlcAT-P and GlcAT-S) are involved in the biosynthesis of HNK-1 carbohydrate, which is spatially and temporally regulated in the nervous system. To clarify the enzymatic properties of the respective glucuronyltransferases, we established an expression system for producing large amounts of soluble forms of flag-tagged human GlcAT-P and GlcAT-S in Escherichia coli. Approximately 15 and 6 mg of enzymatically active flag-GlcAT-P and flag-GlcAT-S were purified from E. coli cells in 5 liters of culture medium, respectively. These recombinant enzymes transferred GlcA to a glycoprotein acceptor, asialo-orosomucoid (ASOR), as well as a glycolipid acceptor, paragloboside. The specific activity of the recombinant GlcAT-P (1100 nmol/min/mg) toward a glycoprotein acceptor, ASOR, was comparable to that of the enzyme (4300 nmol/min/mg) purified from rat brain. Phosphatidylinositol (PI) is specifically required for expression of the activity of the recombinant enzymes toward a glycolipid acceptor, paragloboside. The recombinant GlcAT-P was highly specific for the terminal type II structure, Galbeta1-4GlcNAc, while the recombinant GlcAT-S recognized not only the type II structure, Galbeta1-4GlcNAc, but also the type I structure, Galbeta1-3GlcNAc. These acceptor specificities were similar to those of the native enzymes.     

Molecular basis for acceptor substrate specificity of the human beta1,3-glucuronosyltransferases GlcAT-I and GlcAT-P involved in glycosaminoglycan and HNK-1 carbohydrate epitope biosynthesis, respectively

The human beta1,3-glucuronosyltransferases galactose-beta1,3-glucuronosyltransferase I (GlcAT-I) and galactose-beta1,3-glucuronosyltransferase P (GlcAT-P) are key enzymes involved in proteoglycan and HNK-1 carbohydrate epitope synthesis, respectively. Analysis of their acceptor specificity revealed that GlcAT-I was selective toward Galbeta1,3Gal (referred to as Gal2-Gal1), whereas GlcAT-P presented a broader profile. To understand the molecular basis of acceptor substrate recognition, we constructed mutants and chimeric enzymes based on multiple sequence alignment and structural information. The drastic effect of mutations of Glu227, Arg247, Asp252, and Glu281 on GlcAT-I activity indicated a key role for the hydrogen bond network formed by these four conserved residues in dictating Gal2 binding. Investigation of GlcAT-I determinants governing Gal1 recognition showed that Trp243 could not be replaced by its counterpart Phe in GlcAT-P. This result combined with molecular modeling provided evidence for the importance of stacking interactions with Trp at position 243 in the selectivity of GlcAT-I toward Galbeta1,3Gal. Mutation of Gln318 predicted to be hydrogen-bonded to 6-hydroxyl of Gal1 had little effect on GlcAT-I activity, reinforcing the role of Trp243 in Gal1 binding. Substitution of Phe245 in GlcAT-P by Ala selectively abolished Galbeta1,3Gal activity, also highlighting the importance of an aromatic residue at this position in defining the specificity of GlcAT-P. Finally, substituting Phe245, Val320, or Asn321 in GlcAT-P predicted to interact with N-acetylglucosamine (GlcNAc), by their counterpart in GlcAT-I, moderately affected the activity toward the reference substrate of GlcAT-P, N-acetyllactosamine, indicating that its active site tolerates amino acid substitutions, an observation that parallels its promiscuous substrate profile. Taken together, the data clearly define key residues governing the specificity of beta1,3-glucuronosyltransferases.     

Identification of a peptide mimic of the L2/HNK-1 carbohydrate epitope

The L2/HNK-1 carbohydrate is carried by many neural recognition molecules and is involved in neural cell interactions during development, regeneration in the peripheral nervous system, synaptic plasticity, and autoimmune-based neuropathies. Its key structure consists of a sulfated glucuronic acid linked to lactosaminyl residues. Because of its biological importance but limited availability, the phage display method was used to isolate a collection of peptide mimics that bind specifically to an L2/HNK-1 antibody. The phages isolated from a 15-mer peptide library by adsorption to this antibody share a consensus sequence of amino acids. The peptide mimicked several important functions of the L2/HNK-1 carbohydrate, such as binding to motor neurons in vitro, and preferential promotion of in vitro neurite outgrowth from motor axons compared with sensory neurons. A scrambled version of the peptide had no activity. The combined observations indicate that we have isolated a mimic of the L2/HNK-1 carbohydrate that is able to act as its functional substitute.     

Physical and functional association of glucuronyltransferases and sulfotransferase involved in HNK-1 biosynthesis

HNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human natural killer-1 (HNK-1) carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO3-3GlcAbeta1-3Galbeta1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). Considering that almost all the HNK-1 carbohydrate structures so far determined in the nervous system are sulfated, we hypothesized that GlcAT-P or GlcAT-S functionally associates with HNK-1ST, which results in efficient sequential biosynthesis of HNK-1 carbohydrate. In this study, we demonstrated that both GlcAT-P and GlcAT-S were co-immunoprecipitated with HNK-1ST with a transient expression system in Chinese hamster ovary cells. Immunofluorescence staining revealed that these enzymes are mainly co-localized in the Golgi apparatus. To determine which domain is involved in this interaction, we prepared the C-terminal catalytic domains of GlcAT-P, GlcAT-S, and HNK-1ST, and we then performed pulldown assays with the purified enzymes. As a result, we obtained evidence that mutual catalytic domains of GlcAT-P or GlcAT-S and HNK-1ST are important and sufficient for formation of an enzyme complex. With an in vitro assay system, the activity of HNK-1ST increased about 2-fold in the presence of GlcAT-P or GlcAT-S compared with that in its absence. These results suggest that the function of this enzyme complex is relevant to the efficient sequential biosynthesis of the HNK-1 carbohydrate.     

Expression and function of the HNK-1 carbohydrate

Glycosylation is a major post-translational protein modification, especially for cell surface proteins, which play important roles in a variety of cellular functions, including recognition and adhesion. Among them, we have been interested in HNK-1 (human natural killer-1) carbohydrate, which is characteristically expressed on a series of cell adhesion molecules in the nervous system. The HNK-1 carbohydrate has a unique structural feature, i.e. a sulfated glucuronic acid is attached to the non-reducing terminal of an N-acetyllactosamine residue (HSO(3)-3GlcAbeta1-3Galbeta1-4GlcNAc-). We have cloned and characterized the biosynthetic enzymes (two glucuronyltransferases and a sulfotransferase), and also obtained evidence that the HNK-1 carbohydrate is involved in synaptic plasticity and memory formation. In this review, we describe recent findings regarding the expression mechanism and functional roles of this carbohydrate.     

Changes in the expression of the carbohydrate epitope HNK-1 associated with mesoderm induction in the chick embryo

We report that a monoclonal antibody, HNK-1, identifies specific regions and cell types during primitive streak formation in the chick blastoderm. Immunohistochemical studies show that the cells of the forming hypoblast are HNK-1 positive from the earliest time at which they can be identified. Some cells of the margin of the blastoderm are also positive. The mesoderm cells of the primitive streak stain strongly with the antibody from the time of their initial appearance. In the epiblast, some cells are positive and some negative at pre-primitive-streak stages, but as the primitive streak develops a gradient of staining intensity is seen within the upper layer, increasing towards the primitive streak. At later stages of development, the notochord and the mesenchyme of the headfold are positive, while the rest of the mesoderm (lateral plate) no longer expresses HNK-1 immunoreactivity. This antibody therefore reveals changes associated with mesodermal induction: before induction, it recognizes the 'inducing' tissue (the hypoblast) and reveals a mosaic pattern in the responding tissue (the epiblast); after primitive streak formation, the mesoderm of the primitive streak that results from the inductive interactions expresses the epitope strongly. Affinity purification of HNK-1-related proteins in various tissues was carried out, followed by SDS-PAGE to identify them. The hypoblast, mesoderm and epiblast of gastrulating chick embryos have some HNK-1-related proteins in common, while others are unique to specific tissues. Attempts have been made to identify these proteins using Western blots and antibodies known to recognize HNK-1-related molecules, but none of the antibodies used identify the bands unique to any of the tissues studied. We conclude that these proteins may be novel members of the HNK-1/L2 family, and that they may have a role in cell interactions during early development.     

Mice deficient in nervous system-specific carbohydrate epitope HNK-1 exhibit impaired synaptic plasticity and spatial learning

The HNK-1 carbohydrate epitope, a sulfated glucuronic acid at the non-reducing terminus of glycans, is expressed characteristically on a series of cell adhesion molecules and is synthesized through a key enzyme, glucuronyltransferase (GlcAT-P). We generated mice with a targeted deletion of the GlcAT-P gene. The GlcAT-P -/- mice exhibited normal development of gross anatomical features, but the adult mutant mice exhibited reduced long term potentiation at the Schaffer collateral-CA1 synapses and a defect in spatial memory formation. This is the first evidence that the loss of a single non-reducing terminal carbohydrate residue attenuates brain higher functions.     

Differential expression of two glucuronyltransferases synthesizing HNK-1 carbohydrate epitope in the sublineages of the rat myogenic progenitors

HNK-1 epitope is a cell-surface carbohydrate mediating various cell-cell or cell-substrate interactions. We found HNK-1 epitope in longitudinally arrayed fibers in the subpopulation of the epaxial myotome, and hypaxial myoblasts migrating into the limb bud in the rat embryo. We next investigated the expression patterns of genes encoding two glucuronyltransferases (GlcAT-P, GlcAT-D) and sulfotransferase (Sul-T), which are required for biosynthesis of HNK-1 epitope. GlcAT-P gene was expressed in the non-migrating longitudinal fibers, whereas GlcAT-D gene was expressed in the migrating myoblasts in the limb bud. Sul-T gene expression was ubiquitously observed in all these myogenic populations. Thus, differential expression of GlcAT genes may relate to the epaxial/hypaxial or migrating/non-migrating myoblast lineages.     

Determination of structural elements of the L2/HNK-1 carbohydrate epitope required for its function

The L2/HNK-1 carbohydrate epitope has been shown to carry an unusual 3-sulfoglucuronic acid linkedO-glycosidically through a neolactosyl-type back bone to a ceramide residue. Using monoclonal antibodies, the same or a closely related epitope has also been detectedN-glycosidically linked to glycoproteins, amongst them several neural cell adhesion molecules. We used synthetic glycolipids carrying sulfated or non-sulfated glucuronic acid attached to ceramide through glycans of different length to show that not only the sulfated glucuronic acid but also the neolactosyl-type backbone is essential for the recognition of the L2/HNK-1 carbohydrate by a monoclonal antibody, its binding to laminin and its role in neural cell migration and outgrowth of processes from neurons and astrocytes.Abbreviations mab monoclonal antibody - TLC thin layer chromatography - HRP horseradish peroxidase - glcA glucuronic acid - gal galactose - glcNAc N-acetyl-glucosamine - man mannose     

Characterization of recombinant human glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans.

We characterized the recombinant glucuronyltransferase I (GlcAT-I) involved in the glycosaminoglycan-protein linkage region biosynthesis. The enzyme showed strict specificity for Galbeta1-3Galbeta1-4Xyl, exhibiting negligible incorporation into other galactoside substrates including Galbeta1-3Galbeta1-O-benzyl, Galbeta1-4GlcNAc and Galbeta1-4Glc. A comparison of the GlcAT-I with another beta1,3-glucuronyltransferase involved in the HNK-1 epitope biosynthesis revealed that the two beta1,3-glucuronyltransferases exhibited distinct and no overlapping acceptor substrate specificities in vitro. Nevertheless, the transfection of the GlcAT-I cDNA into COS-1 cells induced the significant expression of the HNK-1 epitope. These results suggested that the high expression of the GlcAT-I gene rendered the cells capable of synthesizing the HNK-1 epitope.