Purification and Characterization of a Glucuronyltransferase Involved in the Biosynthesis of the HNK-1 Epitope on Glycoproteins from Rat Brain

Terayama, Koji; Seiki, Takashi; Nakamura, Akemi; Matsumori, Kanae; Ohta, Satoru; Oka, Shogo; Sugita, Mutsumi; Kawasaki, Toshisuke

doi:10.1074/jbc.273.46.30295

Cited by 36 publications

(17 citation statements)

References 40 publications

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“…Under these assay conditions, the bacterially expressed FLAG-Pcat and FLAG-Scat exhibit the specific activities of about 1,100 and 450 nmol/min/mg protein, respectively (17). The specific activities were comparable with that of GlcAT-P purified from rat brain (9). As shown in Fig.…”

Section: Effect Of the Interaction On The Glucuronyltransferase And Ssupporting

confidence: 55%

Physical and Functional Association of Glucuronyltransferases and Sulfotransferase Involved in HNK-1 Biosynthesis

Kizuka¹,

Matsui

Takematsu

et al. 2006

Journal of Biological Chemistry

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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 (HSO 3 -3GlcA␤1-3Gal␤1-4GlcNAc-) 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 coimmunoprecipitated 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.Glycosylation is one of the major post-translational protein modifications, especially for cell surface proteins, that play important roles in a variety of cellular functions, including recognition and adhesion. Among them, human natural killer-1 (HNK-1) 2 carbohydrate, which is recognized by HNK-1 monoclonal antibody, is predominantly expressed in the nervous system (1, 2). HNK-1 carbohydrate is expressed on several glycoproteins and glycolipids and is considered to be involved in cell migration, recognition, and adhesion. The unique structural feature of this carbohydrate is a sulfated trisaccharide (HSO 3 -3GlcA␤1-3Gal␤1-4GlcNAc) (3, 4). Recently, we cloned two glucuronyltransferases (GlcAT-P and GlcAT-S) (5-9) and one sulfotransferase (HNK-1ST) (10) that are involved in the biosynthesis of HNK-1 carbohydrate. To investigate the biological function of HNK-1 carbohydrate in vivo, we generated GlcAT-P gene-deficient mice and revealed that HNK-1 carbohydrate is involved in synaptic plasticity (11). More recently, we found a nonsulfated form of HNK-1 carbohydrate in mouse kidney (12), where only GlcAT-S was expressed and not HNK-1ST. In the nervous system, however, all the HNK-1 carbohydrate structures identified so far are sulfated forms because of the presence of glucuronyltransferases (GlcAT-P and GlcAT-S) and HNK-1ST (13, 14). The fact that only the existence of HNK-1ST is sufficient for the sulfation of HNK-1 carbo...

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Section: Effect Of the Interaction On The Glucuronyltransferase And Ssupporting

confidence: 55%

Physical and Functional Association of Glucuronyltransferases and Sulfotransferase Involved in HNK-1 Biosynthesis

Kizuka¹,

Matsui

Takematsu

et al. 2006

Journal of Biological Chemistry

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“…The GlcAT-D expression pattern was slightly different from that of GlcAT-P. GlcAT-D transcripts were positive in the pallidum and retina where GlcAT-P expression was not specifically observed, suggesting different in vivo functions between the two GlcATs. GlcAT-D had catalytic activity on both glycoprotein and glycolipid acceptors in our in vitro assay, whereas GlcAT-P was reported to be active on only glycoprotein acceptors (20). This may reflect different localization patterns of the GlcATs, where the GlcAT-D is expressed in a wider region than GlcAT-P.…”

Section: Fig 10 Hplc Analysis Of Smith Degradation Productsmentioning

confidence: 48%

“…The present study showed that GlcAT-D acts on both glycoprotein and glycolipid acceptors in vitro, whereas GlcAT-P has been reported to be specific to glycoprotein acceptors (20,35). It remains to be determined what these glucuronyltransferases use as acceptor substrates in the nervous system.…”

Section: Discussionmentioning

confidence: 94%

“…The glucuronyltransferase activity involved in the biosynthesis of HNK-1 epitope has been studied using brain extracts from chick embryo (33), rat embryo (34), and postnatal rat (35,36) as well as purified enzyme from postnatal rat forebrain (20). The present study showed that GlcAT-D acts on both glycoprotein and glycolipid acceptors in vitro, whereas GlcAT-P has been reported to be specific to glycoprotein acceptors (20,35).…”

Section: Discussionmentioning

confidence: 95%

“…The key enzymes in the biosynthesis of HNK-1 are a ␤1,3-glucuronyltransferase, which transfers a GlcA to a terminal galactose, and a sulfotransferase, which adds a sulfate group to the GlcA. Recently, GlcAT-P, a glucuronyltransferase involved in the biosynthesis of HNK-1 on glycoprotein, was purified, and its cDNA was cloned (20,21). Subsequently, a sulfotransferase that directs a final step of the biosynthesis of HNK-1 was cloned by an expression cloning strategy that involved cotransfection of GlcAT-P cDNA (22,23).…”

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confidence: 99%

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Cloning and Expression of a Novel Galactoside β1,3-Glucuronyltransferase Involved in the Biosynthesis of HNK-1 Epitope

Shimoda

Tajima

Nagase

et al. 1999

Journal of Biological Chemistry

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We isolated a cDNA encoding a novel glucuronyltransferase, designated GlcAT-D, involved in the biosynthesis of the HNK-1 carbohydrate epitope from rat embryo cDNA by the degenerate polymerase chain reaction method. The new cDNA sequence revealed an open reading frame coding for a protein of 324 amino acids with type II transmembrane protein topology. The amino acid sequence of GlcAT-D displayed 50.0% identity to rat GlcAT-P, which is involved in the biosynthesis of the HNK-1 epitope on glycoproteins. Expression of GlcAT-D in COS-7 cells resulted in the formation of the HNK-1 epitope on the cell surface. The enzyme expressed in COS-7 cells transferred a glucuronic acid (GlcA) not only to asialo-orosomucoid, a glycoprotein bearing terminal N-acetyllactosamine structure, but also to paragloboside (lacto-N-neotetraosylceramide), a precursor of the HNK-1 epitope on glycolipids. Furthermore, substrate specificity analysis using a soluble chimeric form of GlcAT-D revealed that GlcAT-D transfers a GlcA not only to Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc-pyridylamine but also to Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc-pyridylamine. Enzymatic hydrolysis and Smith degradation of the reaction product indicated that GlcAT-D transfers a GlcA through a ␤1,3-linkage to a terminal galactose. The GlcAT-D transcripts were detected in embryonic, postnatal, and adult rat brain. In situ hybridization analysis revealed that the expression pattern of GlcAT-D transcript in embryo is similar to that of GlcAT-P, but distinct expression of GlcAT-D was observed in the embryonic pallidum and retina. Regions that expressed GlcAT-D and/or GlcAT-P were always HNK-1-positive, indicating that both GlcATs are involved in the synthesis of the HNK-1 epitope in vivo.HNK-1, also known as CD57, is recognized as an important epitope involved in neurogenesis. Expression of HNK-1 epitope is spatiotemporally regulated, and the epitope is found on migrating neural crest cells (1), odd-numbered rhombomeres (2), and myelinating Schwann cells in motor neurons (3). The HNK-1 epitope is a glycan expressed on glycoproteins, glycolipids, and proteoglycans (reviewed in Refs. 4 and 5). In particular, the epitope has been discovered on a series of cell adhesion molecules, such as myelin-associated glycoproteins (6), L1 (7), neural cell adhesion molecule (7), P0 (8), and transiently expressed axonal glycoprotein-1 (9). It is interesting that only a subpopulation of these molecules expresses HNK-1 epitope. The epitope has also been identified as a ligand for P0 (10), laminin (11), and L-and P-selectins (12). Moreover, it was demonstrated that HNK-1 antibody or isolated HNK-1 glycan interferes with cell-cell or cell-substrate interaction (13-15). These observations indicate that HNK-1 epitope plays a significant role in cell-cell and cell-matrix interaction.Structures of HNK-1 epitope determined to date almost invariably carry HSO 3 -3GlcA␤1-3Gal␤1-4GlcNAc at nonreducing termini (16 -19). The precursor of the epitope, Gal␤1-4GlcNAc sequence, is commonly found on glycoproteins and glycolipids, ...

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On the Origin of Oligosaccharide Species—Glycosyltransferases in Action

Eijnden

Ernst

Hart

et al. 2000

Carbohydrates in Chemistry and Biology

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Glycoproteins and glycolipids, jointly called glycoconjugates, are macromolecules essential to eukaryotic life. The oligosaccharide chains carried by glycoconjugates often contain biological intelligence which is encoded by the oligosaccharide structure. These chains thus confer specific biological functions on the proteins and lipids carrying them. When embedded in the cellular plasma membrane they decorate the cell surface with specific oligosaccharide structures which can be crucial to cell function. The synthesis of protein-and lipid-linked oligosaccharides proceeds by the sequential addition of monosaccharide building blocks to a growing chain that is covalently linked to the protein or lipid aglycone. In a similar way oligosaccharides present in some secretions (e.g. milk) are built up from a single sugar (glucose).Unlike the processes of protein and nucleic acid synthesis, in which the order of attachment of amino acids and nucleotides is read from a nucleic acid matrix, glycosylation is a non-template-directed process. This enables the formation of branches which are common to oligosaccharides but are not found in nucleic acids and proteins. The enzymes that are responsible for the assembly of the oligosaccharides on glycoconjugates are known as glycosyltransferases (in the early stages of N-glycosylation glycosidases also play a role-see later in this chapter). Being proteins, glycosyltransferases are primary products of their respective genes and, therefore, the oligosaccharide chains formed by their action can be considered as secondary gene products. Glycosyltransferases typically catalyze the following general reaction: nucleoside-P-Psugar + acceptor + sugar-acceptor + nucleoside-P-P In this reaction nucleotideesugars are the donor substrates (for some glycosyltransferases involved in the early stages of protein N-glycosylation a sugarphosphate-dolichol is the donor molecule; see later). In sequential reactions mono- Carbohydrates in Chemistrysaccharides are attached, one at a time, through glycosidic linkages at nonreducing ends and at branching points in a specific order; the sugar-acceptor product of one reaction is the acceptor substrate in the next. In vitro glycosyltransferases generally have optimum activity at a pH of ca 7.0. In most instances Mn2+ ions are required for enzymatic activity (except for sialyltransferases and some p6-N-acetylglucosaminyltransferases which are cation-independent). Because glycosyltransferases are generally membrane-associated proteins, determination of their activity in vitro requires the solubilization of the enzymes with detergents. Some glycosyltransferases, however, occur in a soluble form in body fluids, probably as a result of proteolytic release of the catalytically active part of the enzyme.Glycosyltransferases of eukaryotic organisms have high specificity for the sugar they transfer and the linkage they establish. This has enabled their classification in groups, such as the sialyltransferases which all exclusively utilize CMP-sialic acid as a donor subst...

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Purification and Characterization of a Glucuronyltransferase Involved in the Biosynthesis of the HNK-1 Epitope on Glycoproteins from Rat Brain

Cited by 36 publications

References 40 publications

Physical and Functional Association of Glucuronyltransferases and Sulfotransferase Involved in HNK-1 Biosynthesis

Physical and Functional Association of Glucuronyltransferases and Sulfotransferase Involved in HNK-1 Biosynthesis

Cloning and Expression of a Novel Galactoside β1,3-Glucuronyltransferase Involved in the Biosynthesis of HNK-1 Epitope

On the Origin of Oligosaccharide Species—Glycosyltransferases in Action

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