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

Self Cite

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...

Section: Discussionmentioning

confidence: 93%

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

Kizuka¹,

Matsui

Takematsu

et al. 2006

Self Cite

“…Calf intestine alkaline phosphatase and bovine liver ␤-glucuronidase (EC 3.2.1.31) were purchased from Roche Molecular Biochemicals (Tokyo, Japan) and Sigma, respectively. The linkage trisaccharide serines, Gal␤1-3Gal␤1 Expression of the Soluble Form of GlcAT-I and Enzyme AssayThe construction of a soluble form of GlcAT-I fused with the cleavable insulin signal sequence and the protein A IgG-binding domain was carried out as described (35,36), except for the replacement of the expression vector pSVL with pEF-BOS (40). Each expression plasmid (10 g) was transfected into COS-1 cells on 100-mm plates using Lipofectamine (Invitrogen) according to the instructions provided by the manufacturer.…”

Section: Materials-udp-[u-mentioning

confidence: 99%

“…We have cloned and characterized the substrate specificity of GlcAT-I (35,36). A crystal structure of the ternary complex of the enzyme with the donor substrate product UDP and the acceptor substrate analogue, Gal␤1-3Gal␤1-4Xyl, revealed the key amino acid residues required for the recognition of the donor and acceptor substrates (37).…”

mentioning

confidence: 99%

2-O-Phosphorylation of Xylose and 6-O-Sulfation of Galactose in the Protein Linkage Region of Glycosaminoglycans Influence the Glucuronyltransferase-I Activity Involved in the Linkage Region Synthesis

Tone

Pedersen

Yamamoto

et al. 2008

Self Cite

Sulfated glycosaminoglycans (GAGs), including heparan sulfate and chondroitin sulfate, are synthesized on the so-called common GAG-protein linkage region (GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser) of core proteins, which is formed by the stepwise addition of monosaccharide residues by the respective specific glycosyltransferases. Glucuronyltransferase-I (GlcAT-I) is the key enzyme that completes the synthesis of this linkage region, which is a prerequisite for the conversion of core proteins to functional proteoglycans bearing GAGs. The Xyl and Gal residues in the linkage region can be modified by phosphorylation and sulfation, respectively, although the biological significance of these modifications remains to be clarified. Here we present evidence that these modifications can significantly influence the catalytic activity of GlcAT-I. Enzyme assays showed that the synthetic substrates, Gal-Gal-Xyl

“…These enzymes catalyze the transfer of GlcA from a donor substrate, uridine diphosphoglucuronic acid (UDP-GlcA), to a reducing terminal residue of oligosaccharide chain in the pres-ence of manganese. GlcAT-I transfers GlcA to Gal␤1-3Gal␤1-4Xyl␤1-O-Ser in a biosynthesis pathway of proteoglycan (19,26). The substrate binding and the reaction mechanisms of GlcAT-I have been discussed at an atomic level based on its crystal structure in complex with donor and acceptor substrate (31)(32)(33).…”

mentioning

confidence: 99%

Structural Basis for Acceptor Substrate Recognition of a Human Glucuronyltransferase, GlcAT-P, an Enzyme Critical in the Biosynthesis of the Carbohydrate Epitope HNK-1

Kakuda

Shiba

Ishiguro

et al. 2004

Self Cite

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 (Gal␤1-4GlcNAc) but not lacto-N-biose (Gal␤1-3GlcNAc) 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 (Gal␤1-4GlcNAc) contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity.