N-acetylglucosamine Groups Research Articles

Differential Expression and Localisation of Acetyl-Glucosamine, Acetyl-Galactosamine, Galactose, Mannose and Glucose Specific Lectins in Lingual Tonsil of Buffalo (Bubalus bubalis)

The present study is the most comprehensive representation of lectin binding sites in various structural components of the lingual tonsil of six adult healthy buffaloes. The study was useful in determining the specific binding affinities of sixteen lectins of the N-acetylglucosamine group, i.e., Triticum vulgaris (WGA), succinylated Triticum vulgaris (s-WGA), Lycopersicon esculentum (LEL), Datura stramonium (DSL), Solanum tuberosum (STL); N-acetylgalactosamine group, i.e., Glycine max (SBA), Dolichos biflorus (DBA), Ricinus communis (RCA), Vicia villosa agglutinin (VVL); galactose group i. e Griffonia simplicifolia isolectin B4 (GS1B4), Arachis hypogaea (PNA), Artocarpus integrifolia (Jacalin), Erythrina crisa-galli (ECL); and glucose/mannose group i.e. Canavalia ensiformis (Con A), Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PSA) at structures of the lingual tonsil. The stratum spinosum of stratified squamous epithelium and modified reticular epithelium was strongly demarcated by lectins of the N-acetylglucosamine group. In addition, VVL, jacalin, and Con A lectins also showed strong responses for the same layer. The RCA, PNA, Con A, and PSA were the best markers for the collagen fibers of the subepithelial connective tissue. The lymphoid cells of the inter- and parafollicular region possessed receptors for ECL, WGA, and Con A. In contrast, the germinal center B cells were labeled only by the lectins of the glucose/mannose group. The mucosal secretions and the endothelium of the blood vessels were predominantly composed of glucosamine, sialic acid, and galactosamine sugars. The characteristic localization of lectins suggests the presence of specific receptor sites that may be useful for studying early disease pathogenesis and developing oral vaccines. In addition, the study will provide a database for comparing histochemical changes in different disease states.

Glycan Stability and Flexibility: Thermodynamic and Kinetic Characterization of Nonconventional Hydrogen Bonding in Lewis Antigens.

We provide evidence for CH-based nonconventional hydrogen bonds (H-bonds) for 10 Lewis antigens and two of their rhamnose analogues. We also characterize the thermodynamics and kinetics of the H-bonds in these molecules and present a plausible explanation for the presence of nonconventional H-bonds in Lewis antigens. Using an alternative method to simultaneously fit a series of temperature-dependent fast exchange nuclear magnetic resonance (NMR) spectra, we determined that the H-bonded conformation is favored by ∼1 kcal/mol over the non-H-bonded conformation. Additionally, a comparison of temperature-dependent 13C linewidths in various Lewis antigens and the two rhamnose analogues reveals H-bonds between the carbonyl oxygen of the N-acetyl group of N-acetylglucosamine and the OH2 group of galactose/fucose. The data presented herein provide insight into the contribution of nonconventional H-bonding to molecular structure and could therefore be used for the rational design of therapeutics.

N-Acetylglucosamine and its dimer ameliorate inflammation in murine colitis by strengthening the gut barrier function.

Ulcerative colitis (UC) is a chronic gastrointestinal disease whose incidence is increasing rapidly worldwide. Anti-inflammatory medications, including 5-aminosalicylic acid (5-ASA), corticosteroids, and immunosuppressants, are used for its treatment; however, new alternatives would be required due to the serious side effects of some of these medications. N-Acetylglucosamine (NAG) is an amino sugar composed of mucin that is secreted by intestinal epithelial cells. It is also used to promote the growth of intestinal bacteria. The current study aimed to determine the efficacy of NAG against dextran sulfate sodium (DSS)-induced chronic colitis and elucidate its mechanism of action. Mice were randomly divided into control, DSS, 0.1% sulfasalazine, 0.1% NAG, 0.3% NAG, and 0.3% NAG-dimer (NAG-D) groups, and results showed that colitis-induced body weight loss, disease activity, colonic tissue damage, colon length shortening, and the loss of mucin-secreting area were significantly improved in the NAG-D group. The intestinal permeability indicator, serum CD 14 level, and expression of the tight junction protein, occludin, were both improved in the 0.3% NAG group. Inflammatory biomarkers, including GATA3, IFN-γ, p-IκBα, COX2, TGF-β1, and Smad7, were significantly lower in the 0.3% NAG and NAG-D groups than in the DSS group. The intestinal microbial composition was most significantly altered in the 0.3% NAG group, showing decreased ratios of pathogenic bacteria, such as Betaproteobacteria, especially Burkholderiales. The results overall suggested that NAG or NAG-D supplementation can alleviate inflammation by strengthening the intestinal barrier function and maintaining gut microbiota homeostasis in a DSS-induced colitis mouse model.

Numerous severely twisted N-acetylglucosamine conformations found in the protein databank.

Taking advantage of the known planarity of the N-acetyl group of N-acetylglucosamine, an analysis of the quality of carbohydrate structures found in the protein databank was performed. Few obvious defects of the local geometry of the carbonyl group were observed. However, the N-acetyl group was often found in the less favorable cis conformation (12% of the cases). It was also found severely twisted in numerous instances, especially in structures with a resolution poorer than 1.9 Å determined between 2000 and 2015. Though the automated PDB-REDO procedure has proved able to improve nearly 85% of the structural models deposited to the PDB, and does prove able to cure most severely twisted conformations of the N-acetyl group, it fails to correct its high rate of cis conformations. More generally, for structures with a resolution poorer than 1.6 Å, it produces N-acetylglucosamine models in slightly poorer agreement with experimental data, as measured using real-space correlation coefficients. Significant improvements are thus still needed, at least as far as this carbohydrate structure is concerned.

Conversion of N-Acetylglucosamine to Protected Amino Acid over Ru/C Catalyst

Chitin is the most abundant marine biomass, containing nitrogen atoms in its monomer units, N-acetylglucosamine (NAG). Thus, NAG is a potential feedstock for the production of renewable organonitrogen chemicals. Here, we report a synthetic pathway of a widely used standard amino acid in a protected form, acetylglycine (AcGly), from NAG via two steps: conversion of NAG to N-acetylmonoethanolamine (AMEA) and its transformation to AcGly. In the first step, a Ru/C catalyst converts NAG to AMEA in an NaHCO3 aqueous solution under H2 pressure through a retro-aldol reaction and hydrogenation reaction. Model reactions and density functional theory calculations for this reaction have revealed that resonance stabilization of the acetamido group of NAG avoids its isomerization proceeding to side reactions, which is different from glucose regardless of their similar structures. For the second step of the synthesis of AcGly from AMEA under O2 pressure, the same combination of Ru/C and NaHCO3 is applicable. A mechanist...

Construction of a high-mannose-type glycan library by a renewed top-down chemo-enzymatic approach.

A comprehensive method for the construction of a high-mannose-type glycan library by systematic chemo-enzymatic trimming of a single Man9-based precursor was developed. It consists of the chemical synthesis of a non-natural tridecasaccharide precursor, the orthogonal demasking of the non-reducing ends, and trimming by glycosidases, which enabled a comprehensive synthesis of high-mannose-type glycans in their mono- or non-glucosylated forms. It employed glucose, isopropylidene, and N-acetylglucosamine groups for blocking the A-, B-, and C-arms, respectively. After systematic trimming of the precursor, thirty-seven high-mannose-type glycans were obtained. The power of the methodology was demonstrated by the enzymatic activity of human recombinant N-acetylglucosaminyltransferase-I toward M7-M3 glycans, clarifying the substrate specificity in the context of high-mannose-type glycans.

Sulfated hyaluronan/collagen I matrices enhance the osteogenic differentiation of human mesenchymal stromal cells in vitro even in the absence of dexamethasone

Glycosaminoglycans (GAG) are multifunctional components of the extracellular matrix (ECM) involved in different steps of the regulation of cellular differentiation. In this study artificial extracellular matrices (aECM) consisting of collagen (Col) I and different GAG derivatives were used as a substrate for human mesenchymal stromal cells (hMSC) to study osteogenic differentiation in vitro. hMSC were cultured on aECM containing col and hyaluronan sulfates (HyaS) with increasing degrees of sulfation (DSS) and were compared with aECM containing col and the natural GAG hyaluronan or chondroitin 4-sulfate. hMSC were analyzed for osteogenic differentiation markers such as calcium phosphate deposition, tissue non-specific alkaline phosphatase (TNAP) and expression of runt-related transcription factor 2 (runx2), osteocalcin (ocn) and bone sialoprotein II (bspII). Compared with aECM containing Col and natural GAG all Col/HyaS-containing aECM induced an increase in calcium phosphate deposition, TNAP activity and tnap expression. These effects were also seen in the absence of dexamethasone (an established osteogenic supplement). The expression of runx2 and ocn was not altered and the expression of bspII was diminished on the col/HyaS-containing aECM. The impact of the Col/HyaS-containing aECM on hMSC differentiation was independent of the DSS of the HyaS derivatives, indicating the importance of the primary (C-6) hydroxyl group of N-acetylglucosamine. These results suggest that Col/HyaS-containing aECM are able to stimulate hMSC to undergo osteogenic differentiation even in the absence of dexamethasone, which makes these matrices an interesting tool for hMSC-based tissue engineering applications and biomaterial functionalizations to enhance bone formation.

Hybrid Quantum Mechanical/Molecular Mechanical Investigation of the β-1,4-Galactosyltransferase-I Mechanism

The enzyme beta-1,4-galactosyltransferase-1 (beta4Gal-T1) catalyzes the transfer of a galactose residue from UDP-Gal to the C4-hydroxyl group of N-acetylglucosamine. The catalytic mechanism of beta4Gal-T1 was investigated using the hybrid quantum mechanical/molecular mechanical (QM/MM) method, with the QM portion containing 253 atoms treated with density functional theory (DFT) at the BP/DZP and BP/TZ2P levels. The remaining parts of the beta4Gal-T1 complex, 4527 atoms in all, were modeled using the AMBER molecular force field. A theoretical model of the Michaelis complex was built using the X-ray structure of beta4Gal-T1 in a complex with the donor or acceptor substrate, respectively. The hybrid QM(DFT)/MM calculations identified an S(N)2-type transition state for the nucleophilic attack of the O4(a) oxygen on the anomeric carbon C1 and the breaking of the C1-O1 glycosidic linkage. The activation barrier found for this process is 15 kcal/mol. In the transition state (TS) model, the sugar donor is partially cleaved from pyrophosphate, while nucleophilic oxygen O4(a) remains protonated with a low barrier hydrogen bond to the catalytic base D318. The structure of TS is characterized by the O4(a)-C1 and C1-O1 distances of 2.703 and 2.092 A, respectively. When the obtained reaction sequence was used, the nature of the captured intermediate resembling the transition state structure (PDB/2FYD) was elucidated. This modeling QM/MM study has provided detailed insight into the mechanism of the Gal transfer catalyzed by beta4Gal-T1 and has supplied further evidence for a concerted S(N)2-type displacement mechanism employed by inverting glycosyltransferases.

Crystal structure and association behaviour of the GluR2 amino-terminal domain

Fast excitatory neurotransmission is mediated largely by ionotropic glutamate receptors (iGluRs), tetrameric, ligand-gated ion channel proteins comprised of three subfamilies, AMPA, kainate and NMDA receptors, with each subfamily sharing a common, modular-domain architecture. For all receptor subfamilies, active channels are exclusively formed by assemblages of subunits within the same subfamily, a molecular process principally encoded by the amino-terminal domain (ATD). However, the molecular basis by which the ATD guides subfamily-specific receptor assembly is not known. Here we show that AMPA receptor GluR1- and GluR2-ATDs form tightly associated dimers and, by the analysis of crystal structures of the GluR2-ATD, propose mechanisms by which the ATD guides subfamily-specific receptor assembly.

N-Acetylglucosamine Reduces Inflammatory Response during Acute Peritonitis in Uremic Rats

Background: Peritoneal dialysis (PD) induces intraperitoneal inflammation and that process may be uremia. The goal of this study is to evaluate the effect of ure- mia on the kinetics of peritonitis and furthermore test the anti-inflammatory potential of N-acetylglucosamine (NAG) in a uremic environment. Methods: Experiments were performed on healthy Wistar rats and on animals with impaired renal function. Acute PD was performed in all animals with dialysis fluid containing either glucose (GLU) or NAG as osmotic solutes. Peritonitis was induced by addition of lipopolysaccharide from Escherichia coli (LPS) to the dialysis fluid. Transperitoneal transport of water and solutes as well as intraperitoneal and systemic inflammation were evaluated. Results: Uremia reduces peritoneal permeability to total protein during peritonitis (–33% vs. control, p < 0.001) and increases net ultrafiltration (+2.5 ± 2.2 vs. –2.7 ± 3.2 ml in control, p < 0.001). In uremic rats with peritonitis, reduced dialysate levels of the following inflammatory mediators were detected as compared to healthy animals: MCP-1 (–15%, p < 0.01); IL-1β (–53%, p < 0.001), and elastase (–28%, p < 0.02). In the serum of uremic rats, the increase in TNFα and MCP-1 concentrations was smaller than in control rats: –44% (p < 0.02) and –39% (p < 0.001), respectively. NAG used as an osmotic solute in rats with preserved renal function decreases intraperitoneal and systemic inflammation during acute peritonitis. Drained dialysate volume was increased in the NAG group by 32% (p < 0.001) and transperitoneal loss of protein was reduced by 21% (p < 0.002). When NAG was used as the osmotic solute instead of GLU, intraperitoneal inflammation in uremic animals was further reduced: TNFα (–40%, p < 0.05); IL-1β (–49%, p < 0.005); MCP-1 (–21%, p < 0.005). The presence of NAG also reduced the increased blood level of IL-1β (–47%,p < 0.02) and MCP-1 (–36%, p < 0.02). Conclusions: Intensity of acute peritonitis is reduced during uremia. NAG exerts a systemic and peritoneal anti-inflammatory action under conditions of uremia that confirms the potential use of this compound as an osmotic agent in the PD fluids that also decreases inflammation.

N-Acetylglucosamine – An Osmotic Solute for Peritoneal Dialysis without Inducing Hyperinsulinemia

Methods: N-Acetylglucosamine (NAG) was compared to glucose as an osmotic solute during peritoneal dialysis in rats. The effect of the tested solutes on blood glucose and insulin levels during dialysis was evaluated. Results: During 6-hour exchange with NAG (220 mmol/l) solution, the dialysate volume was higher than in rats dialyzed with fluid containing glucose (220 mmol/l; GLU: 34.5 ± 1.7 vs. 32.8 ± 1.1 ml, respectively; p < 0.05). The peritoneal permeability to protein (D/S × 1,000) was lower in the NAG group (9.7 ± 2.5 vs. 16.3 ± 5.6 in GLU; p < 0.02). Dialysis with GLU-based solution resulted in hyperglycemia up to 180 ± 39 mg/dl; in the NAG group the increase in the blood glucose level was moderate (up to 91 ± 9 mg/dl; p < 0.001). Dialysis with GLU fluid caused an increase in blood insulin level by 53.2 ± 62.4 pmol/l, whereas the insulin blood concentration in NAG-treated animals was increased by 5.0 ± 5.4 pmol/l (p < 0.001). Conclusions: NAG is more effective than GLU osmotic solute during peritoneal dialysis and it reduces peritoneal permeability to protein. Dialysis with NAG results in lower hyperglycemia and hyperinsulinemia, both effects are favorable in diabetic peritoneal dialysis patients.

Solution structure of hyaluronic acid oligomers by experimental and theoretical NMR, and molecular dynamics simulation.

The conformational properties of hyaluronic acid (HA) oligomers in aqueous solution were investigated by combining high-resolution NMR experimental results, theoretical simulation of NMR two-dimensional (2D) spectra by Complete Relaxation Matrix Analysis (CORMA), and molecular dynamics calculations. New experimental findings recorded for the tetra- and hexasaccharides enabled the stiffness of the HA and its viscoelastic properties to be interpreted. In particular, rotating frame nuclear Overhauser effect spectroscopy spectra provided new information about the arrangement of the glycosidic linkage. From (13)C NMR relaxation the rotational correlation time (tau(c)) were determined. The tau(c) were employed in the calculation of geometrical constraints, by using the MARDIGRAS algorithm. Restrained simulated annealing and 1 ns of unrestrained molecular dynamic simulations were performed on the hexasaccharide in a box of 1215 water molecules. The beta(1 --> 3) and beta(1 --> 4) glycosidic links were found to be rigid. The lack of rotational degree of freedom is due to direct and/or water-mediated interresidue hydrogen bonding. Both single or tandem water bridges were found between carboxylate group and N-acetil group. The carboxylate group of glucuronic acid is not involved in a direct link with the amide group of N-acetyl glucosamine and this facilitated bonding between the residue and the water molecules.

Affinity Labelling at the 4-Hydroxyl Group of N-Acetylglucosamine and N-Acetylgalactosamine

Derivatives of N-acetylgalactosamine and N-acetylglucosamine in which the 4-OH group could be selectively labelled have been prepared from a common precursor.

Germ-cell warfare in ascidians: sperm from one species can interfere with the fertilization of a second species.

Ascidians (invertebrate chordates) are very abundant in many marine subtidal areas. They often live in dense multispecies clumps; thus, interspecific competition for space may be intense. Although most noncolonial species are broadcast spawners, their eggs can be fertilized only by sperm of the same species (1). Multiple fertilization is lethal and all animals have evolved blocks to polyspermy. Ascidian eggs block polyspermy by enzymatic (2) and electrical mechanisms (3). Sperm bind to N-acetylglucosamine groups on the vitelline coat (4, 5, 6, 7). Follice cells surrounding the vitelline coat release N-acetylglucosaminidase during egg activation (8), preventing the binding of all sperm but a few (2). I show here that this interaction is not species-specific; sperm from one species can cause glycosidase release from follicle cells of a second species. Furthermore, once glycosidase release has been induced, the subsequent addition of sperm from the egg-producing species fails to fertilize a substantial proportion of these eggs. This leads to the hypothesis that sperm from one species of ascidian can interfere with fertilization of a second species. While intraspecific sperm competition has been well documented in several taxa (9, 10), this is the first record of sperm competition between species, or interspecific sperm competition.

Predicted Influence of N-Acetyl Group Content on the Conformational Extension of Chitin and Chitosan Chains

ABSTRACT Conformational analysis of chitosan molecules has been performed using the MM3(92) force field to investigate the role played by the acetamido groups on the stiffness of these chains. A high dielectric constant value was needed to model an aqueous environment and to reproduce the distribution of the N-acetyl glucosamine group orientation that is observed by NMR. Disaccharidic fragments, differently substituted at C2, were selected as models for chitin and chitosan chains. Their conformational space has been explored by means of adiabatic mapping of the glycosidic Φ,Ψ torsion angles. Although the overall features of all the potential energy surfaces created appear similar, the accessible conformational space of a glycosidic bond is affected by the nature of the substituent at C2 on the non-reducing residue of the disaccharide unit. This is illustrated by the differences in the calculated partition functions together with the predicted average homonuclear and heteronuclear coupling constants. Computed maps were used to predict polymeric unperturbed dimensions, characteristic ratio and persistence length of idealized chitin and chitosan chains, by Monte Carlo methods. Pure chitosan is predicted to be more coiled than pure chitin chains. At low N-acetyl group contents, chain extension appears to be dependent on the degree of substitution. Average chain dimensions increase monotonically for increases in content up to 60% of N-acetyl groups, but show no significant variation at higher contents. For molecules consisting of 50% amino and 50% N-acetylated residues, random, alternate and block patterns of substitution have been investigated. It has also been shown that the spatial extension of the polymer chains is dependent on the primary structure. Comparison with the literature experimental data is difficult because of the extreme diversity of the reported conformationally dependent values. However, such study provides a unique insight into the dependence of these two factors (degree of acetylation and distribution of acetyl groups) on the stiffness and flexibility of different chitin and chitosan chains.

The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan‐polymerizing penicillin‐binding protein 1b of Escherichia coli

The penicillin-binding protein (PBP) 1b of Escherichia coli catalyses the assembly of lipid-transported N-acetyl glucosaminyl-beta-1, 4-N-acetylmuramoyl-L-alanyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl+ ++- (L)-D-alanyl-D-alanine disaccharide pentapeptide units into polymeric peptidoglycan. These units are phosphodiester linked, at C1 of muramic acid, to a C55 undecaprenyl carrier. PBP1b has been purified in the form of His tag (M46-N844) PBP1bgamma. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46-N844) PBP1bgamma possesses an amino-terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an congruent with 100-amino-acid insert, to a D198-G435 glycosyl transferase module that possesses the five motifs characteristic of the PBPs of class A. In in vitro assays, the glycosyl transferase of the PBP catalyses the synthesis of linear glycan chains from the lipid carrier with an efficiency of congruent with 39 000 M-1 s-1. Glu-233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu-233 gamma-COOH donates its proton to the oxygen atom of the scissile phosphoester bond of the lipid carrier, leading to the formation of an oxocarbonium cation, which then undergoes attack by the 4-OH group of a nucleophile N-acetylglucosamine. Asp-234 of motif 1 or Glu-290 of motif 3 could be involved in the stabilization of the oxocarbonium cation and the activation of the 4-OH group of the N-acetylglucosamine. In turn, Tyr-310 of motif 4 is an important component of the amino acid sequence-folding information. The glycosyl transferase module of PBP1b, the lysozymes and the lytic transglycosylase Slt70 have much the same catalytic machinery. They might be members of the same superfamily. The glycosyl transferase module is linked, via a short junction site, to the amino end of a Q447-N844 acyl transferase module, which possesses the catalytic centre-defining motifs of the penicilloyl serine transferases superfamily. In in vitro assays with the lipid precursor and in the presence of penicillin at concentrations sufficient to derivatize the active-site serine 510 of the acyl transferase, the rate of glycan chain synthesis is unmodified, showing that the functioning of the glycosyl transferase is acyl transferase independent. In the absence of penicillin, the products of the Ser-510-assisted double-proton shuttle are glycan strands substituted by cross-linked tetrapeptide-pentapeptide and tetrapeptide-tetrapeptide dimers and uncross-linked pentapeptide and tetrapeptide monomers. The acyl transferase of the PBP also catalyses aminolysis and hydrolysis of properly structured thiolesters, but it lacks activity on D-alanyl-D-alanine-terminated peptides. This substrate specificity suggests that carbonyl donor activity requires the attachment of the pentapeptides to the glycan chains made by the glycosyl transferase, and it implies that one and the same PBP molecule catalyses transglycosylation and peptide cross-linking in a sequential manner. Attempts to produce truncated forms of the PBP lead to the conclusion that the multimodular polypeptide chain behaves as an integrated folding entity during PBP1b biogenesis.

The Role of Ligand Density in the Enzymatic Glycosylation of Carbohydrates Presented on Self-Assembled Monolayers of Alkanethiolates on Gold.

The biological activity of immobilized carbohydrates can show a dramatic dependence on the density of carbohydrate. This is the result of investigations with self-assembled monolayers that present N-acetylglucosamine groups as a model substrate for glycosylation by bovine β-1,4-galactosyltransferase (GalTase; see picture). Surface plasmon resonance spectroscopy and carbohydrate-binding lectins were used to characterize the reaction at the interface. UDP-Gal=uridine diphosphogalactose.

Yeast-derived Recombinant DG42 Protein of Xenopus Can Synthesize Hyaluronan in Vitro

We demonstrate in this report that the Xenopus DG42 gene product made in the yeast Saccharomyces cerevisiae can synthesize authentic high molecular weight hyaluronan (hyaluronic acid; HA) in vitro. Saccharomyces are eukaryotes that do not naturally produce HA or any other molecules known to contain glucuronic acid. Therefore bakers' yeast is a good model system to determine the enzymatic activity of the DG42 protein, which is the topic of recent debate. Membrane extracts prepared from cells expressing DG42 encoded on a plasmid incorporated [14C]glucuronic acid and N-[3H]acetylglucosamine from exogenously supplied UDP-sugar nucleotides into a high molecular mass (10(6) to 10(7) Da) polymer in the presence of magnesium ions. Both sugar precursors were simultaneously required for elongation. Control extracts prepared from cells with the vector plasmid alone or the DG42 cDNA in the antisense orientation did not display this activity. The double-labeled polysaccharide product synthesized in vitro was deemed to be HA by enzymatic analyses; specific HA lyase could degrade the polymer, but it was unaffected by protease or chitinase treatments. The fragments generated by HA lyase were identical to fragments derived from authentic vertebrate HA as compared by high performance liquid chromatography. We conclude that DG42 is a membrane-associated HA synthase capable of transferring both glucuronic acid and N-acetylglucosamine groups.

Attachment of the ascidian sperm surface egg receptor N‐acetylglucosaminidase to the cell membrane

Ascidian sperm bind to vitelline coat N-acetylglucosamine groups of the egg via sperm surface N-acetylglucosaminidase. This sperm surface egg receptor remains anchored throughout penetration. Localization to the sperm surface was verified by biotinylation of intact sperm followed by solubilization in Triton X-100 and binding to streptavidin agarose. The enzyme was determined to be an integral membrane protein as judged by resistance to release by Kl and high pH. Linkage of the enzyme to the sperm surface was probed through differential solubilization followed by measuring released enzymatic activity with a fluorogenic substrate. Nonionic detergents released 90% of the activity. Proteases released about 40%. No activity was released by a phosphatidylinositol specific phospholipase C. This finding, combined with the similarity of release level by all the detergents, including Triton X-100 and octylglucoside, argues against a phosphatidyl-inositol linkage. The release form enters the hydrophilic phase of a Triton X-114 phase separation experiment. This observation, coupled with the findings of release by nonionic detergents, suggests that the protein is hydrophilic once released from the membrane. Thus, although clearly an integral membrane protein, the enzyme has limited hydrophobicity such as would be present in a single transmembrane sequence or extensive glycosylation.

Synthesis ofN-Acetylglucosamine derivatives as probes for specificity of chicken hepatic lectin

Syntheses of the following compounds are described: 6-(Trifluoroacetylamino)hexyl 2-acetamido-2,6-dideoxy-beta-D-glucopyranoside and 2-acetamido-2-deoxy-alpha-D-xylopyranoside, two allyl 2-acetamido-2-deoxy-alpha-D-glucopyranosiduronic acid derivatives, and several allyl 2-acylamido-2-deoxy-beta-D-glucopyranosides having different acyl groups. These and other compounds were used as inhibitors in the binding assay for the chicken hepatic lectin specific for N-acetylglucosamine. We found that: 1) The inhibitory potency of N-acylglucosamine derivatives decreased progressively with increase in the size of acyl group, 2) absence of either 3- or 4-OH group of N-acetylglucosamine lowered the binding affinity more than 100-fold, and 3) the presence of a negatively charged group (carboxylic acid) at the C-6 position did not lower the affinity. The first two items are similar to the mammalian hepatic galactose/N-acetylgalactosamine lectins, but the last item is in a strong contrast to the mammalian lectins.