Tachylectin-2: crystal structure of a specific GlcNAc/GalNAc-binding lectin involved in the innate immunity host defense of the Japanese horseshoe crab Tachypleus tridentatus.
(9/450)
Tachylectin-2, isolated from large granules of the hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), is a 236 amino acid protein belonging to the lectins. It binds specifically to N-acetylglucosamine and N-acetylgalactosamine and is a part of the innate immunity host defense system of the horseshoe crab. The X-ray structure of tachylectin-2 was solved at 2.0 A resolution by the multiple isomorphous replacement method and this molecular model was employed to solve the X-ray structure of the complex with N-acetylglucosamine. Tachylectin-2 is the first protein displaying a five-bladed beta-propeller structure. Five four-stranded antiparallel beta-sheets of W-like topology are arranged around a central water-filled tunnel, with the water molecules arranged as a pentagonal dodecahedron. Tachylectin-2 exhibits five virtually identical binding sites, one in each beta-sheet. The binding sites are located between adjacent beta-sheets and are made by a large loop between the outermost strands of the beta-sheets and the connecting segment from the previous beta-sheet. The high number of five binding sites within the single polypeptide chain strongly suggests the recognition of carbohydrate surface structures of pathogens with a fairly high ligand density. Thus, tachylectin-2 employs strict specificity for certain N-acetyl sugars as well as the surface ligand density for self/non-self recognition. (+info)
The tumor suppressor EXT-like gene EXTL2 encodes an alpha1, 4-N-acetylhexosaminyltransferase that transfers N-acetylgalactosamine and N-acetylglucosamine to the common glycosaminoglycan-protein linkage region. The key enzyme for the chain initiation of heparan sulfate.
(10/450)
We previously demonstrated a unique alpha-N-acetylgalactosaminyltransferase that transferred N-acetylgalactosamine (GalNAc) to the tetrasaccharide-serine, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser (GlcA represents glucuronic acid), derived from the common glycosaminoglycan-protein linkage region, through an alpha1,4-linkage. In this study, we purified the enzyme from the serum-free culture medium of a human sarcoma cell line. Peptide sequence analysis of the purified enzyme revealed 100% identity to the multiple exostoses-like gene EXTL2/EXTR2, a member of the hereditary multiple exostoses (EXT) gene family of tumor suppressors. The expression of a soluble recombinant form of the protein produced an active enzyme, which transferred alpha-GalNAc from UDP-[3H]GalNAc to various acceptor substrates including GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser. Interestingly, the enzyme also catalyzed the transfer of N-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc to GlcAbeta1-3Galbeta1-O-naphthalenemethanol, which was the acceptor substrate for the previously described GlcNAc transferase I involved in the biosynthetic initiation of heparan sulfate. The GlcNAc transferase reaction product was sensitive to the action of heparitinase I, establishing the identity of the enzyme to be alpha1, 4-GlcNAc transferase. These results altogether indicate that EXTL2/EXTR2 encodes the alpha1,4-N-acetylhexosaminyltransferase that transfers GalNAc/GlcNAc to the tetrasaccharide representing the common glycosaminoglycan-protein linkage region and that is most likely the critical enzyme that determines and initiates the heparin/heparan sulfate synthesis, separating it from the chondroitin sulfate/dermatan sulfate synthesis. (+info)
Involvement of the core protein in the first beta-N-acetylgalactosamine transfer to the glycosaminoglycan-protein linkage-region tetrasaccharide and in the subsequent polymerization: the critical determining step for chondroitin sulphate biosynthesis.
(11/450)
alpha-Thrombomodulin (alpha-TM) with a truncated glycosaminoglycan-protein linkage tetrasaccharide, GlcAbeta1-3Galbeta1-3Galbeta1-4Xyl, was tested as an acceptor together with a sugar donor, UDP-N-[3H]acetylgalactosamine, using a cell-free enzyme system prepared from the serum-free culture medium of a human melanoma cell line. The truncated tetrasaccharide on alpha-TM served as an acceptor, whereas the linkage tetrasaccharide-serine did not. Our characterization of the radioactively labelled product by enzymic digestion revealed that the N-[3H]acetylgalactosamine residue was transferred to alpha-TM through a beta1,4-linkage. The substrate competition experiments with the chondro-hexasaccharide and alpha-TM reinforced our speculation that a common N-acetylgalactosaminyltransferase catalysed the transfer of N-acetylgalactosamine to both the linkage tetrasaccharide and the longer chondroitin oligosaccharides. Moreover, chondroitin polymerization was demonstrated on the tetrasaccharide of alpha-TM using both UDP-glucuronic acid and UDP-N-acetylgalactosamine as sugar donors. Much longer chains were synthesized on alpha-TM than on the linkage penta- and hexa-saccharide-serines. Together, these results indicated that the core protein is required for the transfer of the first N-acetylgalactosamine residue through a beta1,4-linkage and also for subsequent efficient chain polymerization reactions, and that the critical determining step for chondroitin sulphate biosynthesis is the transfer of the first N-acetylgalactosamine residue. (+info)
Incorporation of N-acetylgalactosamine into consecutive threonine residues in MUC2 tandem repeat by recombinant human N-acetyl-D-galactosamine transferase-T1, T2 and T3.
(12/450)
An oligopeptide containing three consecutive Thr residues mimicking the tandem repeat portion of MUC2 (PTTTPLK) was investigated for the acceptor specificity to UDP-N-acetyl-D-galactosamine:peptide N-acetylgalactosaminyltransferase isozymes, UDP-N-acetyl-D-galactosamine:peptide N-acetylgalactosaminyltransferase-T1, T2 and T3. The enzymatic reaction products were fractionated by the reversed-phase high performance liquid chromatography, then characterized by matrix-assisted laser desorption ionization time of flight mass spectrometry and by a peptide sequencing analysis. A maximum of two, one or three N-acetyl-D-galactosamine residues was transferred by UDP-N-acetyl-D-galactosamine:peptide N-acetylgalactosaminyltransferase-T1, T2 or T3, respectively. The preferential orders of N-acetyl-D-galactosamine incorporation were Thr-2, then Thr-4 for UDP-N-acetyl-D-galactosamine:peptide N-acetylgalactosaminyltransferase-T1, Thr-2 for UDP-N-acetyl-D-galactosamine:peptide N-acetylgalactosaminyltransferase-T2 and Thr4, Thr-3, then Thr-2 for UDP-N-acetyl-D-galactosamine:peptide N-acetylgalactosaminyltransferase-T3. (+info)
O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts.
(13/450)
The plasma membrane of polarised epithelial cells is characterised by two structurally and functionally different domains, the apical and basolateral domains. These domains contain distinct protein and lipid constituents that are sorted by specific signals to the correct surface domain [1]. The best characterised apical sorting signal is that of glycophosphatidylinositol (GPI) membrane anchors [2], although N-linked glycans on some secreted proteins [3] and O-linked glycans [4] also function as apical sorting signals. In the latter cases, however, the underlying sorting mechanisms remain obscure. Here, we have analysed the role of O-glycosylation in the apical sorting of sucrase-isomaltase (SI), a highly polarised N- and O-glycosylated intestinal enzyme, and the mechanisms underlying this process. Inhibition of O-glycosylation by benzyl-N-acetyl-alpha-D-galactosaminide (benzyl-GalNAc) was accompanied by a dramatic shift in the sorting of SI from the apical membrane to both membranes. The sorting mechanism of SI involves its association with sphingolipid- and cholesterol-rich membrane rafts because this association was eliminated when O-glycosylation was inhibited by benzyl-GaINAc. The results demonstrate for the first time that O-linked glycans mediate apical sorting through association with lipid rafts. (+info)
Demonstration of a novel sulfotransferase in fetal bovine serum, which transfers sulfate to the C6 position of the GalNAc residue in the sequence iduronic acid alpha1-3GalNAc beta1-4iduronic acid in dermatan sulfate.
(14/450)
A novel sulfotransferase activity was discovered in fetal bovine serum using pig skin dermatan sulfate as an acceptor and [35S]3'-phosphoadenosine 5'-phosphosulfate as a sulfate donor. The enzyme was separated from chondroitin:GalNAc 6-O-sulfotransferase by chromatographic techniques. Enzymatic analysis of the reaction products demonstrated that the enzyme transferred sulfate to the C6 position of the GalNAc residue in the sequence -iduronic acid alpha1-3GalNAc beta1-4iduronic acid-. Thus, the enzyme has been identified as a hitherto unreported dermatan sulfate:GalNAc 6-O-sulfotransferase. The finding is in sharp contrast to the current concept that in dermatan sulfate biosynthesis GalNAc 4-O-sulfation is a prerequisite for iduronic acid formation by C5 epimerase. (+info)
Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies.
(15/450)
Circulating immune complexes (CICs) isolated from sera of patients with IgA nephropathy (IgAN) consist of undergalactosylated, mostly polymeric, and J chain-containing IgA1 and IgG antibodies specific for N-acetylgalactosamine (GalNAc) residues in O-linked glycans of the hinge region of IgA1 heavy chains. Antibodies with such specificity occur in sera of IgAN patients, and in smaller quantities in patients with non-IgA proliferative glomerulonephritis and in healthy controls; they are present mainly in the IgG (predominantly IgG2 subclass), and less frequently in the IgA1 isotype. Their specificity for GalNAc was determined by reactivity with IgA1 myeloma proteins with enzymatically removed N-acetylneuraminic acid (NeuNAc) and galactose (Gal); removal of the O-linked glycans of IgA1 resulted in significantly decreased reactivity. Furthermore, IgA2 proteins that lack the hinge region with O-linked glycans but are otherwise structurally similar to IgA1 did not react with IgG or IgA1 antibodies. The re-formation of isolated and acid-dissociated CICs was inhibited more effectively by IgA1 lacking NeuNAc and Gal than by intact IgA1. Immobilized GalNAc and asialo-ovine submaxillary mucin (rich in O-linked glycans) were also effective inhibitors. Our results suggest that the deficiency of Gal in the hinge region of IgA1 molecules results in the generation of antigenic determinants containing GalNAc residues that are recognized by naturally occurring IgG and IgA1 antibodies. (+info)
Characterization of olfactory receptor organs in Xenopus laevis Daudin.
(16/450)
Xenopus laevis is highly suitable for studying the mechanisms of olfactory reception for water-soluble odorants and for airborne odorants. However, the functional differences of cells and component protein molecules in the olfactory receptors of Xenopus have remained obscure. In recent studies, the patterns of sugar residues expressed on the cell surface have been utilized to analyze the characteristics of neurons, because the sugar chains in neurons play very important roles in targeting and cell-to-cell communication. In this study, we have determined the distribution of sugar residues and glycoproteins in the olfactory receptor organs of Xenopus using lectins as labeling agents, and characterized the receptors of water-soluble odorants and of airborne odorants. The results of lectin histochemical analysis show distributional differences of GlcNAc, GalNAc and mannose between the middle chamber and the lateral chamber of the main nasal cavity. Furthermore, a 65 kDa glycoprotein containing mannose, GlcNAc and GalNAc was specifically detected in the medial chamber of the main cavity epithelium in receptor organs of airborne odorants by SDS-PAGE and lectin blotting. The characteristics of the epithelia demonstrated in this study should further our understanding of the functional differences between the receptors of water-soluble odorants and of airborne odorants at the molecular level. (+info)