Position Of GlcNAc Research Articles

Conformational preferences of heparan sulfate to recognize the CXCL8 dimer in aqueous medium: degree of sulfation and hydrogen bonds.

The sulfation pattern and epimerization of the long-chain sulfated polysaccharide heparan sulfate (HS) cause structural diversity and regulate various physiological and pathological processes when binding with proteins. In this work, we performed a series of molecular dynamics simulations of three variants of the octadecasaccharide HS with varying sulfation positions in aqueous medium in their free forms and in the presence of the chemokine CXCL8 dimer. The free energy of binding depicts the sulfation at the 6-O position of GlcNAc (HS6S), and both 3-O and 6-O positions of GlcNAc (HS3S6S) of HS variants are more likely to bind with the CXCL8 dimer than the triply sulfated HS2S3S6S, which is sulfated at the 2-O position of GlcUA additionally along with 3-O and 6-O positions of GlcNAc. Binding between HS and CXCL8 was driven by electrostatic and van der Waals interactions predominantly regardless of the sulfation pattern; however, unfavorable entropic contribution suppressed the interaction between HS and CXCL8. The contribution of different amino acid residues to the binding energetics suggested that basic amino acids line up the binding site of CXCL8. This study further acknowledges the role of interfacial water that is structured and bound with HS through hydrogen bonds, exhibiting differential hydrogen bond relaxation dynamics compared to when the HS molecules are free. Moreover, this study identifies that with the increase in sulfation, the HS-water hydrogen bond relaxation occurs faster with the complexation, while the reverse trend is followed in their free forms. Significant structural adaptation of the different sulfated HS molecules, as verified from the free energy landscapes generated from various reaction coordinates, root-mean-square-deviations, end-to-end distances, including ring pucker angles, dihedral flexibility, and the high conformational entropy cost arising from the glycosidic bonds, suggests that the different sulfated variants of HS undergo significant structural transformation to bind with CXCL8. The presence of a CXCL8 dimer imposes the bound forms of HS to adopt non-linear structures with skew-boat conformations. The atomistic details of the study would help in understanding the selectivity and conformational diversity, as well as the role of solvents in the recognition of CXCL8 by different sulfated variants of HS molecules.

Glycomics of a novel type-2 N-acetyllactosamine-specific lectin purified from the feather star, Oxycomanthus japonicus (Pelmatozoa: Crinoidea)

A lectin - designated OXYL for the purposes of this study that strongly recognizes complex-type oligosaccharides of serum glycoproteins - was purified from a crinoid, the feather star Oxycomanthus japonicus, the most basal group among extant echinoderms. OXYL was purified through a combination of anion-exchange and affinity chromatography using Q-sepharose and fetuin-sepharose gel, respectively. Lectin was determined to be a 14-kDa polypeptide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions. However, 14-kDa and 28-kDa bands appeared in the same proportion under non-reducing conditions. Gel permeation chromatography showed a 54-kDa peak, suggesting that lectin consists of four 14-kDa subunits. Divalent cations were not indicated, and stable haemagglutination activity was demonstrated at pH 4-12 and temperatures below 60°C. Surface plasmon resonance analysis of OXYL against fetuin showed k(ass) and k(diss) values of 1.4×10(-6)M(-1)s(-1) and 3.1×10(-3)s(-1), respectively, indicating that it has a strong binding affinity to the glycoprotein as lectin. Frontal affinity chromatography using 25 types of prydylamine-conjugated glycans indicated that OXYL specifically recognizes multi-antennary complex-type oligosaccharides containing type-2 N-acetyllactosamines (Galβ1-4GlcNAc) if α2-3-linked sialic acid is linked at the non-reducing terminal. However, type-1 N-acetyllactosamine (Galβ1-3GlcNAc) chains and α2-6-linked sialic acids were never recognized by OXYL. This profiling study showed that OXYL essentially recognizes β1-4-linkage at C-1 position and free OH group at C-6 position of Gal in addition to the conservation of N-acetyl groups at C-2 position and free OH groups at C-3 position of GlcNAc in N-acetyllactosamine. This is the first report on glycomics on a lectin purified from an echinoderm belonging to the subphylum Pelmatozoa.

Enzymes Responsible for Synthesis of Corneal Keratan Sulfate Glycosaminoglycans

Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, beta1,3-N-acetylglucosaminyltansferase-7 (beta3GnT7) and beta1,4-galactosyltransferase-4 (beta4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of beta3GnT7/beta4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of beta3GnT7 or beta4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.

Acquisition of species-specific O-linked carbohydrate chains from oviducal mucins in Rana arvalis. A case study.

The extracellular matrix surrounding amphibian eggs is composed of mucin-type glycoproteins, highly O-glycosylated and plays an important role in the fertilization process. Oligosaccharide-alditols were released from the oviducal mucins of the anuran Rana arvalis by alkali-borohydride treatment in reduced conditions. Neutral and acidic oligosaccharides were fractionated by ion-exchange chromatographies and purified by HPLC. Each compound was identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) spectrometry, NMR spectroscopy, electrospray ionization-tandem mass spectroscopy (ESI-MS/MS) and permethylation analyses. This paper reports on the structures of 19 oligosaccharide-alditols, 12 of which have novel structures. These structures range in size from disaccharide to octasaccharide. Some of them are acidic, containing either a glucuronic acid or, more frequently, a sulfate group, located either at the 6 position of GlcNAc or the 3 or 4 positions of Gal. This latter sulfation is novel and has only been characterized in the species R. arvalis. This structural analysis led to the establishment of several novel carbohydrate structures, demonstrating the structural diversity and species-specificity of amphibian glycoconjugates.

Occurrence of reducing terminal N-acetylglucosamine 3-sulfate and fucosylated outer chains in acidic N-glycans of porcine zona pellucida glycoproteins.

Structures of acidic N-glycans released from porcine zona pellucida glycoproteins by hydrazinolysis were studied. The results indicated that the acidic glycans are of mono- to tetraantennary complex-type with and without N-acetyllactosamine repeating units. Sulfated residues are not only located at the C-6 position of GlcNAc included in the N-acetyllactosamine repeating units, but also at the C-6 position of GlcNAc in the non-repeated antennae and at the C-3 position of reducing terminal GlcNAc residue. Analysis of the oligosaccharide fragments released by endo-beta-galactosidase digestion and by hydrazine/nitrous acid treatment also revealed that various sulfated and non-sulfated forms of fucosylated structures such as Fucalpha1 --> 2Galbeta1 --> 4(+/-SO3- --> 6)GlcNAc (type 2H), Galbeta1 --> 4(Fucalpha1 --> 3)(+/-SO3- --> 6)GlcNAc(Lex) and Fucalpha1 --> 3 or 4(+/-SO3- --> 6)GlcNAc, are expressed in the repeated outer chain moieties.

Stable Expression of the Golgi Form and Secretory Variants of Human Fucosyltransferase III from BHK-21 Cells: PURIFICATION AND CHARACTERIZATION OF AN ENGINEERED TRUNCATED FORM FROM THE CULTURE MEDIUM

Stable BHK-21 cell lines were constructed expressing the Golgi membrane-bound form and two secretory forms of the human alpha1, 3/4-fucosyltransferase (amino acids 35-361 and 46-361). It was found that 40% of the enzyme activity synthesized by cells transfected with the Golgi form of the fucosyltransferase was constitutively secreted into the medium. The corresponding enzyme detected by Western blot had an apparent molecular mass similar to those of the truncated secretory forms. The secretory variant (amino acids 46-361) was purified by a single affinity-chromatography step on GDP-Fractogel resin with a 20% final recovery. The purified enzyme had a unique NH2 terminus and contained N-linked endo H sensitive carbohydrate chains at its two glycosylation sites. The fucosyltransferase transferred fucose to the O-4 position of GlcNAc in small oligosaccharides, glycolipids, glycopeptides, and glycoproteins containing the type I Galbeta1-3GlcNAc motif. The acceptor oligosaccharide in bovine asialofetuin was identified as the Man-3 branched triantennary isomer with one Galbeta1-3GlcNAc. The type II motif Galbeta1-4GlcNAc in bi-, tri-, or tetraantennary neutral or alpha2-3/alpha2-6 sialylated oligosaccharides with or without N-acetyllactosamine repeats and in native glycoproteins were not modified. The soluble forms of fucosyltransferase III secreted by stably transfected cells may be used for in vitro synthesis of the Lewisa determinant on carbohydrates and glycoproteins, whereas Lewisx and sialyl-Lewisx structures cannot be synthesized.

A convenient synthesis of β-d-galactosyl disaccharide derivatives using the β-d-galactosidase from Bacillus circulans

β-d-Gal-(1 → 4)-β-d-GlcNAc-OC6H4NO2-p (p-nitrophenyl N-acetyl-β-lactosaminide) and β-d-Gal-(1 → 6)-β-d-GlcNAc-OC6H4NO2-p (p-nitrophenyl N-acetyl-β-isolactosaminide) were regioselectively synthesized from lactose and p-nitrophenyl 2-acetamido-2-deoxy-glucopyranoside, employing transglycosylation by the β-d-galactosidase from Bacillus circulans and by controlling the concentration of organic solvent in the reaction system. The (1 → 4)-linked disaccharide was formed exclusively when the concentration of organic solvent was high, whereas the (1 → 6)-linked isomer was produced with a low concentration. Further utilization of the transglycosylation by the enzyme led to the regioselective formation of β-d-Gal-(1 → 4)-d-GalNAc and β-d-Gal-(1 → 4)-β-d-GalNAc-OC6H4NO2-p. With the enzyme, β-d-galactosyl transfer occurred preferentially at the O-4 position of GlcNAc and GalNAc, regardless of the configuration of the hydroxyl group.

Novel fucolipids of human adenocarcinoma: disialosyl Lea antigen (III4FucIII6NeuAcIV3NeuAcLc4) of human colonic adenocarcinoma and the monoclonal antibody (FH7) defining this structure.

A new fucoganglioside, disialosyl Lea, has been found in the disialoganglioside fraction of human colonic adenocarcinoma. The ganglioside has been isolated from four other disialogangliosides by high-performance liquid chromatography followed by preparative high-performance thin-layer chromatography. The structure of the antigen was characterized by its conversion to lactofucopentaosyl(II)ceramide (Lea-active ceramide pentasaccharide), methylation analysis, and high-mass range electron-impact as well as field-desorption mass spectrometry of the permethylated derivative, as shown below: (Formula; see text) Specific removal of alpha 2----3-linked sialosyl residue by influenza virus A2 sialidase, or preferential hydrolysis of the same residue by Clostridium perfringens sialidase in the absence of detergent, resulted in the formation of an intermediate product, monosialosyl LeaII (III4FucIII6NeuAcLc4), which reacted with anti-Lea antibody and with monoclonal antibody FH7 and may have a sialic acid linked at the 6 position of GlcNAc. The IgG3 monoclonal antibody FH7 was established, which reacts specifically with disialosyl Lea and monosialosyl LeaII as above, but does not react with disialosyllactotetraosylceramide (IV3NeuAcIII6Neu-AcLc4), monosialosyl LeaI (IV3NeuAcIII4FucLc4), and other mono- and disialogangliosides isolated from the same cancer tissue. The antibody FH7 may be useful in the detection of human cancer.