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, Shinako; Shiba, T.; Ishiguro, Masji; Tagawa, Hideki; Oka, Shogo; Kajihara, Yasuhiro; Kawasaki, Toshisuke; Wakatsuki, Soichi; Kato, Ryuichi

doi:10.1074/jbc.m400622200

Cited by 62 publications

(48 citation statements)

References 46 publications

(38 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recently, we succeeded in the crystallization of the catalytic domain of human GlcAT-P with N-acetyllactosamine, and we revealed the structural basis of the catalytic reaction mechanism at the atomic level (32). Now we are trying to crystallize GlcAT-S and HNK-1ST in a similar way.…”

Section: Discussionmentioning

confidence: 99%

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

Kizuka¹,

Matsui

Takematsu

et al. 2006

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard 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...

show abstract

Section: Discussionmentioning

confidence: 99%

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

Kizuka¹,

Matsui

Takematsu

et al. 2006

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…In other glycosyltransferases also, a few residues determine the sugar donor specificity, such as His308 in glucuronyltransferase-I (GlcAT-I) (His 311 in GlcAT-P) [90,91], Met266 and Ala268 in the blood group B galactosyltransferase, and Leu266 and Gly268 in the blood group A Nacetylgalactosaminyltransferase [92]. Mutation of these residues abrogates their specificities.…”

Section: Sugar Donor Specificity Of the Enzyme Determined By A Few Ammentioning

confidence: 97%

Structure and Function of β -1,4-Galactosyltransferase

Qasba¹,

Ramakrishnan²,

Boeggeman³

2008

CDT

117

112

View full text Add to dashboard Cite

Beta-1,4-galactosylransferase (beta4Gal-T1) participates in the synthesis of Galbeta1-4-GlcNAc-disaccharide unit of glycoconjugates. It is a trans-Golgi glycosyltransferase (Glyco-T) with a type II membrane protein topology, a short N-terminal cytoplasmic domain, a membrane-spanning region, as well as a stem and a C-terminal catalytic domain facing the trans-Golgi-lumen. Its hydrophobic membrane-spanning region, like that of other Glyco-T, has a shorter length compared to plasma membrane proteins, an important feature for its retention in the trans-Golgi. The catalytic domain has two flexible loops, a long and a small one. The primary metal binding site is located at the N-terminal hinge region of the long flexible loop. Upon binding of metal ion and sugar-nucleotide, the flexible loops undergo a marked conformational change, from an open to a closed conformation. Conformational change simultaneously creates at the C-terminal region of the flexible loop an oligosaccharide acceptor binding site that did not exist before. The loop acts as a lid covering the bound donor substrate. After completion of the transfer of the glycosyl unit to the acceptor, the saccharide product is ejected; the loop reverts to its native conformation to release the remaining nucleotide moiety. The conformational change in beta4Gal-T1 also creates the binding site for a mammary gland-specific protein, alpha-lactalbumin (LA), which changes the acceptor specificity of the enzyme toward glucose to synthesize lactose during lactation. The specificity of the sugar donor is generally determined by a few residues in the sugar-nucleotide binding pocket of Glyco-T, conserved among the family members from different species. Mutation of these residues has allowed us to design new and novel glycosyltransferases, with broader or requisite donor and acceptor specificities, and to synthesize specific complex carbohydrates as well as specific inhibitors for these enzymes.

show abstract

“…In the case of wheat XSC, the assembly of the complex would start with the formation of TaGT43-4 homodimers. TaGT43-4 dimerization could possibly involve hydrogen bonds, hydrophobic and electrostatic interactions involving the COOH-terminal end of the globular domain of TaGT43-4, as was observed in the solved crystal structure of human glucuronosyltransferase, GlcAT-P (also belonging to the GT43 family), showing the COOHterminal region forming a long loop that extends to the other molecule in the homodimer (Kakuda et al, 2004). The fact that the monomeric form of TaGT43-4 (;52 kD) is the major species in nonreducing/ nondenaturing gel electrophoresis (Fig.…”

Section: Implications Of Assembly-dependent Trafficking In Xylan Syntmentioning

confidence: 99%

Composition, Assembly, and Trafficking of a Wheat Xylan Synthase Complex

et al. 2016

View full text Add to dashboard Cite

Xylans play an important role in plant cell wall integrity and have many industrial applications. Characterization of xylan synthase (XS) complexes responsible for the synthesis of these polymers is currently lacking. We recently purified XS activity from etiolated wheat (Triticum aestivum) seedlings. To further characterize this purified activity, we analyzed its protein composition and assembly. Proteomic analysis identified six main proteins: two glycosyltransferases (GTs) TaGT43-4 and TaGT47-13; two putative mutases (TaGT75-3 and TaGT75-4) and two non-GTs; a germin-like protein (TaGLP); and a vernalization related protein (TaVER2). Coexpression of TaGT43-4, TaGT47-13, TaGT75-3, and TaGT75-4 in Pichia pastoris confirmed that these proteins form a complex. Confocal microscopy showed that all these proteins interact in the endoplasmic reticulum (ER) but the complexes accumulate in Golgi, and TaGT43-4 acts as a scaffold protein that holds the other proteins. Furthermore, ER export of the complexes is dependent of the interaction between TaGT43-4 and TaGT47-13. Immunogold electron microscopy data support the conclusion that complex assembly occurs at specific areas of the ER before export to the Golgi. A di-Arg motif and a long sequence motif within the transmembrane domains were found conserved at the NH 2 -terminal ends of TaGT43-4 and homologous proteins from diverse taxa. These conserved motifs may control the forward trafficking of the complexes and their accumulation in the Golgi. Our findings indicate that xylan synthesis in grasses may involve a new regulatory mechanism linking complex assembly with forward trafficking and provide new insights that advance our understanding of xylan biosynthesis and regulation in plants.

show abstract

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

Cited by 62 publications

References 46 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

Structure and Function of β -1,4-Galactosyltransferase

Composition, Assembly, and Trafficking of a Wheat Xylan Synthase Complex

Contact Info

Product

Resources

About

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

Cited by 62 publications

References 46 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

Structure and Function of &#946; -1,4-Galactosyltransferase

Composition, Assembly, and Trafficking of a Wheat Xylan Synthase Complex

Contact Info

Product

Resources

About

Structure and Function of β -1,4-Galactosyltransferase