Acetylglucosamine Residues Research Articles

CIRCULATING METABOLIC BIOMARKERS AS POTENTIAL LEADS FOR GLOBAL PREVENTION OF RENAL DYSFUNCTION – A FLEMISH COHORT STUDY

Objective: Studies of people with normal or slightly impaired renal function offer a unique setting to explore the metabolic profiles associated with the early renal dysfunction. Design and method: In 578 randomly recruited Flemish, we assessed at a 5 year interval the multivariable-adjusted associations of estimated glomerular filtration rate (eGFR, ml/min/1.73 m2) with 43 circulating metabolites quantified by non-targeted nuclear magnetic resonance spectroscopy. Significance of associations, expressed per 1-SD increment in the metabolites, were Bonferroni corrected. Results: At baseline (2005–2010), glycine was associated with a 1.81 lower eGFR (P = 0.011). At follow-up (2009–2013), threonine (-2.14) and glycine (-1.73) were cross-sectionally associated with lower eGFR (P < = 0.02) and aspartate (+2.23), leucine (+2.30) and 2 aminobutyrate (+2.12) with higher eGFR (P < = 0.0033). At follow-up, the odds ratio of having eGFR < 60 was 1.62 for threonine and 1.44 for glycine (P < = 0.039). Partial least squares analyses of baseline and follow-up data were confirmatory. For baseline glycan N acetylglucosamine residues (GlycA), a marker of systemic inflammation, the odds ratio of progressing towards follow-up eGFR < 60 vs. non-progressing (n = 49 vs. 529) was 1.85 (P = 0.0086). Baseline GlycA was a better predictor than 24 h microalbuminuria, increasing (P < = 0.0001) the integrated discrimination (+3.4%) and net reclassification (+40.1%) indexes. Pathway analysis pointed to aminoacyl-tRNA biosynthesis, glycine, serine and threonine metabolism, valine, leucine and isoleucine metabolism, and glycolysis or gluconeogenesis. Conclusions: The glycine to threonine pathway and systemic inflammation as reflected by GlycA are markers of early renal dysfunction, whereas protection against oxidative stress and anaplerotic energy supply from the catabolism of branched amino acids (leucine) might be protective.

Elaboration of stable and antibody functionalized positively charged colloids by polyelectrolyte complexation between chitosan and hyaluronic acid.

In this study, we describe the elaboration of multifunctional positively charged polyelectrolyte complex (PEC) nanoparticles, designed to be stable at physiological salt concentration and pH, for effective targeted delivery. These nanoparticles were obtained by charge neutralization between chitosan (CS) as polycation and hyaluronic acid (HA) as polyanion. We showed that the course of the complexation process and the physico-chemical properties of the resulting colloids were impacted by (i) internal parameters such as the Degree of Acetylation (DA, i.e., the molar ration of acetyl glucosamine residues) and molar mass of CS, the HA molar mass and (ii) external parameters like the charge mixing ratio and the polymer concentrations. As a result, nonstoichiometric colloidal PECs were obtained in water or PBS (pH 7.4) and remained stable over one month. The polymer interactions were characterized by thermal analysis (DSC and TGA) and the morphology was studied by scanning electron microscopy. A model antibody, anti-ovalbumine (OVA) immunoglobulin A (IgA) was sorbed on the particle surface in water and PBS quantitatively in 4 h. The CS-HA/IgA nanoparticles average size was between 425–665 nm with a positive zeta potential. These results pointed out that CS-HA can be effective carriers for use in targeted drug delivery.

Biochemical studies on sphingolipids of Artemia franciscana: novel neutral glycosphingolipids

Neutral glycosphingolipids containing one to six sugars in their oligosaccharide chains have been isolated from cysts of the brine shrimp Artemia franciscana. The structures of these glycolipids were identified by methylation analysis, partial acid hydrolysis, gas-liquid chromatography, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and proton nuclear magnetic resonance spectroscopy to be Glcβ1-Cer, Manβ1-4Glcβ1-Cer, Fucα1-3Manβ1-4Glcβ1-Cer, GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GlcNAcα1-2Fucα1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer (CPS), and GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer (CHS). Two glycosphingolipids, CPS and CHS, were characterized as novel structures. Because Artemia contains a certain series of glycosphingolipids (-Fucα3Manβ4GlcβCer), which differ from the core sugar sequences reported thus far, we tentatively designated the glycosphingolipids characterized as nonarthro-series ones. Furthermore, CHS exhibited a hybrid structure of arthro-series and nonarthro-series sugar chain. Two novel glycosphingolipids were characterized from the brine shrimp Artemia franciscana; one was composed of arthrotetraose and a branching fucose attached to N-acetylglucosamine residue, and the other was composed of CPS with an additional N-acetylglucosamine residue attached to the branching fucose.

A Novel Glucosyltransferase Is Required for Glycosylation of a Serine-rich Adhesin and Biofilm Formation by Streptococcus parasanguinis

Fap1-like serine-rich glycoproteins are conserved in streptococci, staphylococci, and lactobacilli, and are required for bacterial biofilm formation and pathogenesis. Glycosylation of Fap1 is mediated by a gene cluster flanking the fap1 locus. The key enzymes responsible for the first step of Fap1 glycosylation are glycosyltransferases Gtf1 and Gtf2. They form a functional enzyme complex that catalyzes the transfer of N-acetylglucosamine (GlcNAc) residues to the Fap1 polypeptide. However, until now nothing was known about the subsequent step in Fap1 glycosylation. Here, we show that the second step in Fap1 glycosylation is catalyzed by nucleotide-sugar synthetase-like (Nss) protein. The nss gene located upstream of fap1 is also highly conserved in streptococci and lactobacilli. Nss-deficient mutants failed to catalyze the second step of Fap1 glycosylation in vivo in Streptococcus parasanguinis and in a recombinant Fap1 glycosylation system. Nss catalyzed the direct transfer of the glucosyl residue to the GlcNAc-modified Fap1 substrate in vitro, demonstrating that Nss is a glucosyltransferase. Thus we renamed Nss as glucosyltransferase 3 (Gtf3). A gtf3 mutant exhibited a biofilm defect. Taken together, we conclude that this new glucosyltransferase mediates the second step of Fap1 glycosylation and is required for biofilm formation.

Adhesins acquired in the aquatic environment andVibrio choleraecolonization of intestinal cells

Recent results for Vibrio cholerae interactions with bivalves and chitin-containing substrates are reviewed. Chitin, composed of b-1,4-linked N -acetylglucosamine residues, is one of the most abundant biopolymers in nature and the most abundant in the marine environment. V. cholerae connection to chitin is a well known phenomenon and one of the best documented examples of a successful bacteriasubstrate interaction, affecting both the lifestyle of the microorganisms and natural system functioning. In sea water, vibrios can be entrapped by filter-feeding invertebrates that sieve suspended food particles from the aquatic environment. Persistence of bacteria in bivalves depends on different factors including their sensitivity to the bactericidal activity of hemolymph. Both V. cholerae survival inside bivalves and chitin colonization depend on cell surface properties, with contribution of different ligands (e.g. chitin-binding proteins, MSHA). The role of these ligands in interactions with human surfaces is discussed and evidence is presented supporting (i) the link between vibrio persistence in the environment (both inside bivalves and adhering to chitin-containing surfaces) and infection of the human host, and (ii) the hypothesis that virulence mechanisms of bacteria that have environmental reservoirs represent a consequence of adaptive mechanisms to the environment. Key words: Vibrio cholerae, chitin, virulence, adaptive mechanisms, environmental reservoir

Growing Through a Wall

CELL BIOLOGY In bacteria, the cell wall must be firm enough to define cell shape and allow a high internal osmotic pressure, while at the same time sufficiently dynamic to allow cell growth and division. Hayhurst et al. provide insight into how the cell wall in the rod-shaped organism , Bacillus subtilis , is structurally organized to achieve these functions. The main structural component of the cell wall is peptidoglycan, comprising glycan strands cross-linked by peptides. Atomic force microscopy (AFM) on purified glycan revealed individual strands up to 5 μm long (5000 disaccharides). Fluorescence microscopy in whole cells showed that B. subtilis displays very few terminal N -acetyl glucosamine (GlcNAc) residues and that internal peptidoglycan-associated GlcNAc residues exhibit a pattern suggestive of a helical structure. AFM imaging of B. subtilis peptidoglycan sacculi revealed little indication of structural features on the outer surface, probably because surface layers are hydrolyzed during cell wall turnover. However, the inner surface exhibited 50-nm-wide cables running across the short axis of the cell with cross striations consistent with a helical structure. The authors suggest that during biosynthesis, glycan strands are polymerized and cross-linked and then coiled to form the inner-surface cables. New helices are likely inserted into the cell wall by being cross-linked between two existing cables, while the external surface is cleaved to allow cell growth. — VV Proc. Natl. Acad. Sci. U.S.A. 105 , 14600 (2008).

Retardation of the unfolding process by single N-glycosylation of ribonuclease A based on molecular dynamics simulations

The conformational characteristics of glycosylated- and unglycosylated bovine pancreatic ribonuclease A (RNaseA) were traced with unfolding molecular dynamics simulations using CHARMM program at 470 K. The glycosylated RNase (Glc_RNase) possesses nearly identical protein structure with RNaseA, differing only by presence of a single acetylglucosamine residue N-linked to Asn34 in the RNaseA. Attaching of monomeric N-acetylglucosamine residue to the Asn34 in RNaseA resulted in a change of denaturing process of Glc_RNase. Simulations showed that the unfolding of RNaseA involved significant weakening of nonlocal interactions whereas the glycosylation led Glc_RNase to preserve the nonlocal interactions even in its denatured form. Even in simulations over 8 ns at 470 K, Glc_RNase remained relatively stable as a less denatured conformation. However, conformation of RNaseA was changed to a fully unfolded state before 3 ns of the simulations at 470 K. This difference was due to fact that formation of hydrogen bond bridges and nonlocal contacts induced by the attached N-acetylglucosamine of Glc_RNase showing in the unfolding simulations. These high-temperature unfolding MD simulations provided a theoretical basis for the previous experimental work in which Glc_RNase showed slower unfolding kinetics compared with unglycosylated RNaseA, suggesting that single N-glycosylation induced retardation of unfolding process of the ribonuclease protein.

Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines

In the past decades, a large number of studies in mammalian cells have revealed that processing of glycoproteins is compartmentalized into several subcellular organelles that process N-glycans to generate complex-type oligosaccharides with terminal N -acetlyneuraminic acid. Recent studies also suggested that processing of N-glycans in insect cells appear to follow a similar initial pathway but diverge at subsequent processing steps. N-glycans from insect cell lines are not usually processed to terminally sialylated complex-type structures but are instead modified to paucimannosidic or oligomannose structures. These differences in processing between insect cells and mammalian cells are due to insufficient expression of multiple processing enzymes including glycosyltransferases responsible for generating complex-type structures and metabolic enzymes involved in generating appropriate sugar nucleotides. Recent genomics studies suggest that insects themselves may include many of these complex transferases and metabolic enzymes at certain developmental stages but expression is lost or limited in most lines derived for cell culture. In addition, insect cells include an N -acetylglucosaminidase that removes a terminal N -acetylglucosamine from the N-glycan. The innermost N -acetylglucosamine residue attached to asparagine residue is also modified with alpha(1,3)-linked fucose, a potential allergenic epitope, in some insect cells. In spite of these limitations in N-glycosylation, insect cells have been widely used to express various recombinant proteins with the baculovirus expression vector system, taking advantage of their safety, ease of use, and high productivity. Recently, genetic engineering techniques have been applied successfully to insect cells in order to enable them to produce glycoproteins which include complex-type N-glycans. Modifications to insect N-glycan processing include the expression of missing glycosyltransferases and inclusion of the metabolic enzymes responsible for generating the essential donor sugar nucleotide, CMP- N -acetylneuraminic acid, required for sialylation. Inhibition of N -acetylglucosaminidase has also been applied to alter N-glycan processing in insect cells. This review summarizes current knowledge on N-glycan processing in lepidopteran insect cell lines, and recent progress in glycoengineering lepidopteran insect cells to produce glycoproteins containing complex N-glycans.

Evaluation of β1,4-galactosyltransferase as a potential biomarker for the detection of subclinical disease after the completion of primary therapy for ovarian cancer

Objective: Approximately 50% of patients with ovarian cancer who have normal CA 125 levels at the completion of therapy have persistent disease. In an effort to improve the ability to detect small volume disease, we have evaluated the usefulness of N -acetylglucosamine:β1,4-galactosyltransferase as a potential biomarker for the detection of subclinical disease after the completion of primary therapy for ovarian cancer. Study Design: The sera of 33 patients with stage IIIC epithelial ovarian cancer in complete clinical remission after chemotherapy (CA 125 <35 units/mL and negative computed tomography scan) who underwent second-look surgery were examined for N -acetylglucosamine:β1,4-galactosyltransferase activity. The values were determined from sera that had been obtained before primary cytoreductive operation and before second-look surgery after the completion of platinum-based chemotherapy. Determinations of the levels of CA 125 were performed with the Bayer Immuno ITM CA-125 II assay. N -acetylglucosamine:β1,4-galactosyltransferase activity was determined by measuring the transfer of galactose from uridine diphosphate– carbon 14–labeled galactose to the terminal N -acetylglucosamine residue of a very well-defined synthetic acceptor, N -acetylglucosamine:β1,6GalNAcα-o-benzyl, which is a portion of the core structure of mucin glycoproteins. The cutoff value of N -acetylglucosamine:β1,4-galactosyltransferase was determined to be 22,000 counts/min, based on the analysis of 25 healthy control subjects. Correlation between serum CA 125 and N -acetylglucosamine:β1,4-galactosyltransferase levels was determined with the use of the Pearson correlation coefficient. The ability of galactosyltransferase to identify small volume disease correctly was also evaluated. Results: There was a significant correlation between serum CA 125 and N -acetylglucosamine:β1,4-galactosyltransferase levels before the operation (r = 0.57; P =.03) but not before second-look surgery (r = 0.10; P =.57). Thirteen patients (39.4%) had residual disease at second-look surgery. Elevated N -acetylglucosamine:β-1,4galactosyltransferase activity >22,000 cpm correctly identified 10 of these patients (76.9%). The sensitivity, specificity, and positive and negative predictive values of N -acetylglucosamine:β1,4-galactosyltransferase activity (>22,000 counts/min) for the prediction of residual disease at second-look surgery were 77%, 45%, 48%, and 77%, respectively. Conclusion: Our comparative study of serum CA 125 and N -acetylglucosamine:β1,4-galactosyltransferase levels showed a significant correlation between the two tumor markers before the beginning of ovarian cancer therapy. This correlation disappeared before second-look surgery because 60% of patients with normal serum CA 125 and N -acetylglucosamine:β1,4-galactosyltransferase levels. CA 125 antigen appears to be inferior to N -acetylglucosamine:β1,4-galactosyltransferase in the detection of small-volume residual disease. N -acetylglucosamine:β1,4-galactosyltransferase may be useful as a biomarker in the monitoring of patients with ovarian cancer when the serum CA 125 level is normal. These findings require confirmation in larger studies. (Am J Obstet Gynecol 2002;187:575-80.)

Human N-Acetylglucosaminyltransferase I. Expression in Escherichia coli as a Soluble Enzyme, and Application as an Immobilized Enzyme for the Chemoenzymatic Synthesis of N-Linked Oligosaccharides

N-Acetylglucosaminyltransferase I (GnT-I), which catalyzes the transfer of an N-acetylglucosamine residue from UDP-N-acetylglucosamine to the alpha1,3-linked mannose on Man5GlcNAc2 (M5), is a critical enzyme for the synthesis of high-mannose-type to complex-type glycan structures in N-linked glycan processing. We developed a large-scale preparation system for recombinant human GnT-I (hGnT-I) using the maltose binding protein (MBP) fusion system to facilitate the chemoenzymatic route for complex-type N-linked glycan synthesis. MBP-fused GnT-I was purified by affinity chromatography on an amylose resin column. The relative activity of MBP-fused GnT-I toward high-mannose-type N-linked oligosaccharides was 100% for Man5GlcNAc2, 52% for Man3GlcNAc2, 17% for Man6GlcNAc2. MBP-fused GnT-I exhibited optimal activity at pH 6.5-9.5 and was more active between pH 6.5-9.0. The optimum temperature for MBP-fused GnT-I activity was 40 degrees C, but the enzyme was active between 0-70 degrees C. Mn2+ and Co2+ were critical for the enzyme activity, while Zn2+ and Ca2+ inhibited the activity. Kinetic analysis of the purified enzyme showed an apparent K(m) value of 0.483 mM and a V(max) of 101 nmol/mg/min for M5. Immobilization of MBP-fused GnT-I on the amylose resin led to an 80% yield of the high mannose-type-of oligosaccharide.

Characterisation of sugar residues in glycoconjugates of pig mandibular gland by traditional and lectin histochemistry

Sugar residues are important components of salivary gland secretion. Traditional histochemical methods and lectin histochemistry were used to characterise glycoconjugates present in the mandibular gland of normal adult pigs. Acinar cells contained abundant quantities of glycoconjugates with the terminal trisaccharide sialic acid – (α2→3, 6) galactosyl (β1→3) N -acetylgalactosamine. Mandibular acinar cells also contained α and βN -acetylgalactosamine and N -acetylglucosamine residues, whereas the demilunar cells contained glycoconjugates with fucose, mannose and N -acetylglucosamine residues. In the duct system a range of sugar residues were localised throughout the cell cytoplasm or limited to the apical surface. These results provide new knowledge concerning the structure of salivary glycoconjugates in normal adult pig and a basis for future pathological studies.

Bovine mammary gland UDP-GalNAc:GlcNAcbeta-R beta1-->4-N-acetylgalactosaminyltransferase is glycoprotein hormone nonspecific and shows interaction with alpha-lactalbumin.

We have identified a novel N -acetylgalactosaminyltransferase activity in lactating bovine mammary gland membranes. Acceptor specificity studies and analysis of products obtained in vitro by 400 MHz1H-NMR spectroscopy revealed that the enzyme catalyses the transfer of N -acetylgalactosamine (GalNAc) from UDP-GalNAc to acceptor substrates carrying a terminal, beta-linked N -acetylglucosamine (GlcNAc) residue and establishes a beta1-->4-linkage forming a GalNAcbeta1-->4GlcNAc ( N, N '-diacetyllactosediamine, lacdiNAc) unit. Therefore, the enzyme can be identified as a UDP-GalNAc:GlcNAcbeta-R beta1-->4-N-acetylgalactosaminyltransferase (beta4-GalNAcT). This enzyme resembles invertebrate beta4-GalNAcT as well as mammalian beta4-galactosyltransferase (beta4-GalT) in acceptor specificity. It can, however, be clearly distinguished from the pituitary hormone-specific beta4-GalNAcT by its incapability of acting with an elevated activity on a glycoprotein substrate carrying a hormone-specific peptide motif. Furthermore, the GalNAcT activity appeared not to be due to a promiscuous action of a beta4-GalT as could be demonstrated by comparing the beta4-GalNAcT and beta4-GalT activities of the mammary gland, bovine colostrum, and purified beta4-GalT, by competition studies with UDP-GalNAc and UDP-Gal, and by use of an anti-beta4-GalT polyclonal inhibiting antibody. Interestingly, under conditions where mammalian beta4-GalT forms with alpha-lactalbumin (alpha-LA) the lactose synthase complex, the mammary gland beta4-GalNAcT was similarly induced by alpha-LA to act on Glc with an increased efficiency yielding the lactose analog GalNAcbeta1-->4Glc. This enzyme thus forms the second example of a mammalian glycosyltransferase the specificity of which can be modified by this milk protein. It is proposed that the mammary gland beta4-GalNAcT functions in the synthesis of lacdiNAc-based, complex-type glycans frequently occurring on bovine milk glycoproteins. The action of this enzyme is to be considered when aiming at the production of properly glycosylated protein biopharmaceuticals in the milk of transgenic dairy animals.

Glycosylasparaginase as a Marker Enzyme in the Detection of I-Cell Disease

Glycosylasparaginase (GA; EC 3.5.1.26) is a lysosomal enzyme that cleaves the N-glycosidic bond between asparagine and N -acetylglucosamine residues in the degradation of glycoproteins (1). Deficient enzyme activity leads to the lysosomal storage disease aspartylglycosaminuria (McKusick 208400). Human GA is synthesized as a single, enzymatically inactive precursor polypeptide. The single-chain precursor is thought to be activated on its way to lysosomes (2)(3) by cleavage into two N-glycosylated subunits of 24 kDA (α) and 18 kDA (β) and association of these subunits to form enzymatically active heterodimer or heterotetramer structures (4). GA is actively transported into human lysosomes via mannose-6-phosphate receptor-mediated endocytosis (5). I-Cell disease (mucolipidosis II, McKusick 252500) and a clinically milder, form pseudo-Hurler polydystrophy (mucolipidosis III, McKusick 252600), are autosomal, recessively inherited lysosomal storage diseases in which the transport of newly synthesized lysosomal enzymes into lysosomes is affected (6). The defect is in the enzyme, UDP- N -acetylglucosamine:lysosomal enzyme N -acetylglucosamine-1-phosphotransferase (EC 2.7.8.17), which adds the phosphate trafficking signal to lysosomal enzymes. The activity of phosphotransferase can be measured using radioactive UDP- N -acetylglucosamine as the donor substrate and endogenous lysosomal enzymes or artificial carbohydrate structures as the assay acceptors (7). The defective synthesis of mannose-6-phosphate recognition markers on the carbohydrate moieties …

The Pneumocystis carinii major surface glycoprotein (MSG): its potential involvement in the pathophysiology of pneumocystosis.

The major antigens of rat Pneumocystis carinii are 45, 60 and 116 kDa, and those of human P. carinii 20, 40, 66, and 95 kDa [1]. The predominant band on acrylamide gels is a surface antigen, which varies in size (95–140 kDa) depending on the host and method of preparation. This antigen is referred to by a variety of names, including gpA, gp95, gp116, gp120 and MSG. In the following the latter term will be used for this antigen. MSG is encoded by a family of approximately 100 genes, each of which is responsible for the expression of a different isoform of MSG [2, 3]. Human MSG is 95 kDa, slightly smaller than the corresponding rat antigen [4,5]. Polyclonal and monoclonal antibodies directed against rat and human P. carinii MSG show that species-specific as well as antigenic determinants are shared and that individual isolates of human and rat P. carinii are antigenically different [4–8]. Biochemical studies indicate that MSG is an acidic glycoprotein composed of a protein core and a carbohydrate portion. N -Linked carbohydrates rich in mannose, glucose, and N -acetylglucosamine residues represent one tenth of the MSG mass [4,9]. Expression of recombinant MSG has largely been unsuccessful. Rat P. carinii MSG has been recombinantly expressed using the Escherichia coli or baculovirus expression system, but differs in glycosylation from native MSG [10]. The difficulty of expressing human recombinant MSG is believed to rely on critical upstream conserved sequences not yet understood. Native MSG preparations from all species are obtained by solubilization of P. carinii organisms with zymolyase and purification utilizing high performance liquid chromatography [11]. Zymolyase hydrolyzes linear glucose polymers with β-1,3-linkages [12]. Studies of the cyst wall have demonstrated that zymolyase treatment removes …

Female gamete membrane glycoproteins potentially involved in gamete recognition in Ectocarpus siliculosus (Phaeophyceae)

In the plasma membrane-enriched fraction of female gametes of Ectocarpus siliculosus (Phaeophyceae) four sex-specific glycoproteins (P2–3, P5, P16) exposing N- acetylglucosamine (GlcNAc) residues were detected in vitro. In immunoblots these proteins were labelled with the GlcNAc complementary lectin wheat germ agglutinin (WGA) conjugated to digoxigenin. P2 also cross-reacted with the mannose-specific Galanthus nivalis agglutinin (GNA). Previously, a key and lock mechanism of gamete recognition was demonstrated in vivo: fertilization can be selectively inhibited by a WGA pretreatment of female gametes, or by GlcNAc pretreated male gametes. Lectins with different carbohydrate requirements such as GNA do not affect fertilization in vivo. Thus, the remaining glycoproteins P3, P5 and P16 seem to be involved in cell-cell recognition during fertilization, and possibly one of them functions as the male receptor in the plasma membrane of female gametes. Membrane proteins of both gamete types showed higher differences than cytosolic proteins or proteins extracted from whole cells when examined by high resolution two-dimensional gel electrophoresis.

Comparative studies of asparagine-linked sugar chains of immunoglobulin G from eleven mammalian species

1. 1. Asparagine-linked sugar chains released by hydrazinolysis from IgGs of porcine, equine, bovine, goat, ovine, canine, rabbit, guinea-pig and rat were comparatively analyzed by microsequencing and lectin affinity chromatography. 2. 2. Sugar chains of all IgGs basically consisted of biantennary complex-type oligosaccharides containing 0–2 sialic acid residue(s). More than 70% of the oligosaccharides were neutral, except for guinea-pig IgG, and fucosylated trimannosyl core structures were dominant except for rabbit IgG. Bisecting N- acetylglucosamine residue was absent in porcine and equine IgGs. 3. 3. A large quantity of galactose-less oligosaccharides were present in IgGs of porcine, equine, canine and rat.

Campylobacter jejuni strains from patients with Guillain-Barré syndrome belong mostly to Penner serogroup 19 and contain beta-N-acetylglucosamine residues.

Campylobacter jejuni was isolated from stool cultures from 14 (30%) of 46 patients with Guillain-Barré syndrome and from 6 (1.2%) of 503 healthy persons, and the difference was highly significant (p < 0.0001). In addition, serological evidence of recent C. jejuni infection was found in 5 of 29 patients with negative stool cultures. Therefore, 41% of patients were associated with C. jejuni infection. Ten of 12 (83%) isolates from patients with Guillain-Barré syndrome belonged to Penner serogroup 19, which is a rare serogroup in sporadic patients with C. jejuni enteritis. In the lectin typing study, all serogroup 19 strains from patients with Guillain-Barré syndrome were shown to contain terminal beta-N-acetylglucosamine residues on their cell surface, but serogroup 19 strains from patients with enteritis were not.

Production of Polyclonal Antibody Specific for Mannose-Containing Triglycosylceramide (Amino-CTH) and Immunochemical Detection of Its Hapten in Crustacean

An antibody against Amino-CTH containing mannose, GlcNAcβ1-3 Manβ1-4 Glcβ1-Ceramide (ArOse3 Cer) isolated from the larvae of the green-bottle fly, Lucilia caesar, was purified by affinity chromatography from rabbit antiserum. The specificity of the antibody was verified by ELISA and TLC-immunostaining. It was found to bind to ArOse3Cer and ArOse3-sphingosine, and to a lesser extent to certain other compounds containing an N -acetylglucosamine residue at their termini, such as GlcNAcβ 1-3 Gal β1-4 Glcβ 1-Ceramide (Amino-CTH) and GlcNAcβ 1-2 Man α 1-3 Manβ1-4 Glcβ 1-Ceramide (MlOse4 Cer). This antibody was used to detect the natural hapten in crustacean glycolipids. The purified antibody reacted with a neutral glycosphingolipid in Euphausia superba (antarctic krill) and Macrobrachiurn nipponense (fresh-water shrimp), as indicated by TLC-immunostaining. The crustacean glycolipid antigen was isolated and characterized as GlcNAcβ1-3 Manβ 1-4 Glc-Ceramide. This is the first paper to demonstrate the presence of an Amino-CTH containing mannose in the crustacean. Cryostat sections of M. nipponense were examined to determine the distribution of ArOse3Cer with the purified antibody. Clear staining was observed in the green gland, esophagus and gills.

Crystal Structure of Glucose Oxidase from Aspergillus niger Refined at 2·3 Å Reslution

Glucose oxidase (β- d-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) is an FAD-dependent enzyme that catalyzes the oxidation of β- d-glucose by molecular oxygen. The crystal structure of the partially deglycosylated enzyme from Aspergillus niger has been determined by isomorphous replacement and refined to 2·3 Å resolution. The final crystallographic R-value is 18·1% for reflections between 10·0 and 2·3 Å resolution. The refined model includes 580 amino acid residues, the FAD cofactor, six N -acetylglucosamine residues, three mannose residues and 152 solvent molecules. The FAD-binding domain is topologically very similar to other FAD-binding proteins. The substrate-binding domain is formed from non-continuous segments of sequence and is characterized by a deep pocket. One side of this pocket is formed by a six-stranded antiparallel β-sheet with the flavin ring system of FAD located at the bottom of the pocket on the opposite side. Part of the entrance to the active site pocket is at the interface to the second subunit of the dimeric enzyme and is formed by a 20-residue lid, which in addition covers parts of the FAD-binding site. The carbohydrate moiety attached to Asn89 at the tip of this lid forms a link between the subunits of the dimer.

Peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F cannot release glycans with fucose attached α1 → 3 to the asparagine‐linked N‐acetylglucosamine residue

The ability of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N-glycopeptides and N-glycoproteins from plants was compared. Bromelain glycopeptide and horseradish peroxidase-C glycoprotein, which contain xylose linked beta 1----2 to beta-mannose and fucose linked alpha 1----3 to the innermost N-acetylglucosamine, were used as substrates. In contrast to PNGase A, the enzyme from F. meningosepticum did not act upon these substrates even at concentrations 100-fold higher than required for complete deglycosylation of commonly used standard substrates. After removal of alpha 1----3-linked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations. Hence we conclude that alpha 1----3 fucosylation of the inner N-acetylglucosamine impedes the enzymatic action of PNGase F. Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool.