1H-NMR study of GM2 ganglioside: evidence that an interresidue amide-carboxyl hydrogen bond contributes to stabilization of a preferred conformation

Several properties of the exchangeable amide protons of the ganglioside G_M2 were studied in detail by¹H-NMR spectroscopy in fully deuterated dimethylsulfoxide [²H₆]DMSO/2% H₂O, and compared with data obtained for the simpler constituent glycosphingolipids G_A2 and G_M3. In addition to chemical shifts,³ J _2,HN coupling constants, and temperature shift coefficients, the kinetics of NH/²H chemical exchange were examined by following the disappearance of the amide resonances in [²H₆]DMSO/2%²H₂O. The results included observation of an increase in half-life of theN-acetylgalactosamine acetamido HN by more than an order of magnitude in G_M2 compared to G_A2, attributable to the presence of the additionalN-acetylneuraminic acid residue. Additional one-dimensional dipolar cross relaxation experiments were also performed on nonexchangeable protons of G_M2. The results of all of these experiments support a three-dimensional model for the terminal trisaccharide in which a hydrogen bond is formed between theN-acetylgalactosamine acetamido NH and theN-acetylneuraminic acid carboxyl group. The interaction is proposed to be of the -acceptor type, a possibility which has not yet been explored in the literature on carbohydrates. The proposed model is discussed in comparison with that of Sabesanet al. (1984,Can J Chem 62: 1034–45), and the models of G_M1 proposed more recently by Acquottiet al. (1990,J Am Chem Soc 112:7772–8) and Scarsdaleet al. (1990,Biochemistry 29:9843–55).     

N-Acetylneuraminic acid biosynthesis in rat liver and kidney

Rat liver and kidney tissue slices incubated withN-acetyl [³H]mannosamine incorporated radioactivity into free and boundN-acetylneuraminic acid and CMP-N-acetylneuraminic acid (CMP-NeuAc). Liver and kidney also incorporated radioactivity from intravenously injected [³H]ManNAc intoN-acetylneuraminic acid and CMP-NeuAc. From the decrease in the specific radioactivity of CMP-NeuAc after a single injection ofN-acetyl[³H]mannosamine the half-life of CMP-NeuAc was determined. From this half-life and the pool size of CMP-NeuAc a synthesis rate of CMP-NeuAc was calculated, being 1.2 nmol/min/g wet weight of kidney. In previous experiments a value of 1.0 nmol/min/g wet weight was determined for liver [Ferwerdaet al. (1983) Biochem J 216: 87–92]. The synthesis rate of CMP-NeuAcin vivo was in the same range as the synthesis rate calculated from the turnover of boundN-acetylneuraminic acid, which was 2.7 and 0.4 nmol/min/g wet weight for liver and kidney respectively.The assay conditions for UDP-N-acetylglucosamine 2-epimerase andN-acetylmannosamine kinase were adapted to measure low activitiesin vitro. It appeared that the kinase activity detected in kidney can synthesizeN-acetylmannosamine6-phosphate at a rate sufficient for the observed production ofN-acetylneuraminic acidin vivo. Also a low, but measurable activity of UDP-N-acetylglucosamine 2-epimerase was detected in kidneyin vitro, suggesting that the biosynthetic pathway ofN-acetylneuraminic acid in kidney is the same as in liver. The synthesis rate ofN-acetylneuraminic acid in liver determinedin vivo is approximately 12 times slower than the maximal potential rate calculated from the activities of theN-acetylneuraminic acid (precursor-) forming enzymes as detectedin vitro. This indicates that in liverin vivo the enzymes are working far below their maximal capacity.     

Structures of the α(1-3)-galactose-containing asparagine-linked glycans of a Lewis lung carcinoma cell subline resistant toAleuria aurantia agglutinin: elucidation by1H-NMR spectroscopy

Four bi-antennary glycan fractions of theN-acetyllactosamine-type, derived from a Lewis lung carcinoma (LL₂) cell subline resistant to theAleuria aurantia agglutinin were studied by 400 MHz¹H-NMR spectroscopy. By this method, their antennae were found to be terminated either by (2-3 or 6)-linkedN-acetylneuraminic acid or (1-3)-linked galactose residues. The primary structure of glycans of these four glycopeptide or derived oligosaccharide-alditols has been determined in full detail.Abbreviations NAc N-acetyl group - NGc N-glycolyl group - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - Man mannose - Gal galactose - Fuc fucose - Con A concanavalin A - LCA Lens culinaris agglutinin - AAA Aleuria aurantia agglutinin - WGA Wheat germ agglutinin - RCA II Ricinus communis agglutinin II - PBS phosphate buffered saline, 0.01m Na₂HPO₄/0.14m NaCl, pH 7.2 - HPLC high performance liquid chromatography - EMEM Eagle's Minimal Essential Medium - Lec^R lectin resistant - MG -methylglycoside     

Primary structure of the xylose-containingN-linked carbohydrate moiety from ascorbic acid oxidase ofCucurbita pepo medullosa

Ascorbic acid oxidase (E.C.1.10.3.3) from the green zucchini squash (Cucurbita pepo medullosa) is a copper-containing glycoprotein which catalyzes the reaction:l-ascorbic acid +1/2 O₂l-dehydroascorbic acid + H₂O. The carbohydrate content of the purified plant glycoprotein amounted to 3% (w/w), and monosaccharide analysis revealed the carbohydrate moiety to be of theN-glycosidic type. The carbohydrate chains were released from the apoenzyme by digestion with PNGase-F immobilized on Sepharose 4B. After fractionation on Bio-Gel P-2 and purification on Mono-Q, the neutral oligosaccharide was investigated by 500-MHz¹H-NMR spectroscopy. The primary structure of theN-linked carbohydrate chain was established to be: Abbreviations AAO ascorbic acid oxidase - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - Man mannose - Xyl xylose - GLC gas-liquid chromatography - FPLC fast protein liquid chromatography - NMR nuclear magnetic resonance - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

1H NMR Ganglioside Ceramide Resonance Region on the Differential Diagnosis of Low and High Malignancy of Brain Gliomas

1. The high-resolution ¹H NMR (MRS) spectra of human brain tumor homogenates revealed a broad resonance at 5.3–5.4 ppm in glioblastoma multiforme (N = 16) and brain metastases (N = 2). The broad resonance was identified as ceramide, a sphingosine–fatty acid combination portion of ganglioside, indicating an elevated abundance of monounsaturated fatty acids. GLC analysis of gangliosides in the highly malignant glioblastoma multiforme revealed that the elevated monounsaturated fatty acid is oleic acid (C18:1). The resonance at 5.3–5.4 ppm region was not detectable in normal human brain (N = 2), in meningiomas (N = 2), or in low-grade astrocytomas (N = 12). In normal human brain the abundance of monounsaturated fatty acid is minimal.2. This investigation was made possible because the method of producing homogenate resulted in (i) no loss of lipids during the process and (ii) a well-homogenised sample, with (iii) no loss in chemical integrity.3. The properties of tumor gangliosides include antigenic specificity and immunosuppresive activity and the ceramide, a sphingosine–fatty acid combination, noticeably influences the ganglioside immunosuppressive activity.4. The observation of ¹H NMR ceramide resonance in high-malignant brain tumors emphasizes the dramatic role of aberrant gangliosides and ceramide precursors on the grade of malignancy and invasiveness.5. Further insight into the specific nature of the ceramide portion of gangliosides in grading the malignancy of brain tumors should be investigated further.     

Differential effect of various inhibitors on four types of rat sialidase

The inhibitory effect of various compounds on the activities of four types of rat sialidase was investigated. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid andN-acetylneuraminic acid were competitive inhibitors for the sialidases. The former was effective against cytosolic sialidase and intralysosomal sialidase more than two membrane-associated sialidases I and II, the latter being a much weaker inhibitor. A heavy metal ion such as Cu²⁺ (1mm) and thiol-modifying 4-hydroxymercuribenzoate (50 µm) caused complete inhibition of the activities of cytosolic sialidase and membrane sialidase I, while no decrease in the activities of intralysosomal sialidase and membrane sialidase II was observed. When 4-nitrophenyloxamic acid and siastatin B, inhibitors of bacterial sialidases, and synthetic thioglycoside GM3 analogue Neu5Ac-s-(2-6)Gal(1-4)Glc(1-1) ceramide, an inhibitor of influenza virus sialidase, were tested, they did not affect any activity of the rat sialidases. By the differential effect of these inhibitors, the four types of rat sialidase could be discriminated from one another and furthermore from viral and bacterial sialidases.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-dehydro-N-acetylneuraminic acid - 4MU-Neu5Ac 4-methylumbelliferyl--N-acetyl-d-neuraminic acid     

Complete 1H, 13C and 15N NMR assignments and secondary structure of the 269-residue serine protease PB92 from Bacillus alcalophilus

Summary The ¹H, ¹³C and ¹⁵N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CH_n carbons, all amide (NH and NH₂) nitrogens and 99.2% of the amide and CH_n protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D two-/three-/four-dimensional - HSQC heteronuclear single-quantum coherence - HMQC heteronuclear multiple-quantum coherence - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - NOE nuclear Overhauser enhancement (connectivity) - NOESY 2D NOE spectroscopy Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667].     

Isolation and properties of a sialidase fromTrypanosoma rangeli

In the culture supernatant ofTrypanosoma rangeli, strain El Salvador, a sialidase was present with an activity of 0.1 U/mg protein as determined with the 4-methylumbelliferyl glycoside of -N-acetylneuraminic acid as substrate. This enzyme was purified about 700-fold almost to homogeneity by gel chromatography on Sephadex G-100 and Blue Sepharose, and affinity chromatographies on 2-deoxy-2,3-didehydroneuraminic acid and horse submandibular gland mucin, both immobilized on Sepharose. The pH optimum is at 5.4–5.6, and the molecular weight was determined by gel chromatography, high performance liquid chromatography and sodium dodecyl sulphate gel electrophoresis to be 70 000. The substrate specificity of the enzyme is comparable to bacterial, viral and mammalian sialidases with cleavage rates for the following substrates in decreasing order: N-acetylneuraminyl-(2–3)-lactose> N-glycoloylneuraminy-(2–3)-lactose> N-acetylneuraminyl-(2–6)-lactose >sialoglycoproteins>gangliosides>9-O-acetylated sialoglycoproteins.4-O-Acetylated derivatives are resistant towards the action of this sialidase. The enzyme activity can be inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, Hg²⁺ ions, andp-nitrophenyloxamic acid; it is not dependent on the presence of Ca²⁺ Mn²⁺ or Mg²⁺ ions.Abbreviations BSA bovine serum albumin - BSM bovine submandibular gland mucin - CMP cytidine monophosphate - EDIA ethylenediaminetetraacetic acid - ESM equine submandibular gland mucin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HPLC high performance liquid chromatography - Lac lactose - MU-Neu5Ac 4-methylumbelliferyl glycoside of -N-acetylneuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - Neu4Ac5Gc N-glycoloyl-4-O-acetylneuraminic acid - Neu2en 2-deoxy-2,3-didehydroneuraminic acid - Neu5Gc N-glycoloylneuraminic acid - PMSF phenylmethylsulfonyl fluoride - PSM pig submandibular gland mucin - SDS sodium dodecyl sulfate - Tris tris-(hydroxymethyl)aminomethane Dedicated to Professor Dr. Heinz Mühlpfordt on the occasion of his 65th birthday.     

Characterization of novel mono-O-acetylated GM3s containing 9-O-acetyl sialic acid and 6-O-acetyl galactose in equine erythrocytes

Novel mono-O-acetylated GM3s, one containing 9-O-acetylN-glycolyl neuraminic acid and another containing 6-O-acetyl galactose, were isolated as a mixture from equine erythrocytes, and the structures were characterized by one- and two-dimensional proton nuclear magnetic resonance (NMR) and fast atom bombardment-mass spectrometry (FAB-MS). The position of theO-acetyl residue was identified by the downfield shift of the methylene protons at C-9 ofN-glycolyl neuraminic acid (9-O-Ac GM3) and C-6 of galactose (6-O-Ac GM3) in the NMR spectrum, in comparison to the respective non-acetylated counterparts. To confirm the presence of 6-O-Ac GM3, theO-acetylated GM3 mixture was desialylated withArthrobacter neuraminidase, giving 6-O-acetyl galactosyl glucosylceramide, the structure of which was estimated by NMR and FAB-MS, together with non-acetylated lactosylceramide with a ratio of 1:1. Abbreviations: Ac, acetyl; Gc, glycolyl; NeuGc,N-Gc neuraminic acid; GM3 (Gc), GM3 containing NeuGc (II³NeuGc-LacCer); 4-O-Ac GM3 (Gc), GM3 containing 4-O-Ac NeuGc; 9-O-Ac GM3 (Gc), GM3 containing 9-O-Ac NeuGc; 6-O-Ac GM3 (Gc), GM3 containing 6-O-Ac Gal; 1D-NMR, one-dimensional nuclear magnetic resonance spectrometry; 2D-COSY, two-dimensional chemical shift-correlated spectrometry; FAB-MS, fast atom bombardment-mass spectrometry; GLC, gas-layer chromatography; GC-MS, gas chromatography-mass spectrometry; TLC, thin-layer chromatography; Ggl, ganglioside; Cer, ceramide; CMH, monohexosylceramide; LacCer, lactosylceramide; 6-O-Ac LacCer, LacCer containing 6-O-Ac Gal; Me₂SO-d₆,²H₆-dimethylsufloxide; CMW, chloroform-methanol-water; Nomenclature and abbreviations of glycosphingolipids follow the system of Svennerholm (J Neurochem [1963]10: 613–23) and those recommended by the IUPAC-IUB Nomenclature Commission (Lipids [1977]12: 455–68).     

Characterization of sheep hemopexin glycovariants

The hemopexin phenotype HpxB1 isolated from sheep serum, yields three major bands when subjected to starch gel and/or polyacrylamide gel electrophoresis which are here designated as subcomponents HpxB1-I, HpxB1-II and HpxB1-III. Electrospray mass spectrometric analysis of samples of the isolated subcomponents prepared by ion exchange chromatography showed that each was composed of three glycoproteins and that the major difference between the subcomponents was due to their constituent glycoproteins possessing different numbers of sialic acid residues. Combined analysis of the ESI-MS data and of the overall carbohydrate compositional data obtained by colorimetric procedures, leads to the composition of the glycan of each glycoprotein, and a combined methylation and 400 MHz H-NMR analysis of the alkaline cleaved glycans identified them as being of only the biantennaryN-acetyllactosamine type. Taking into account the molecular mass, the carbohydrate content and structure it may be concluded that each of the constituent glycoproteins contain fiveN-glycosidically linked glycans.Abbreviations HpxB1 hemopexin phenotype B1 - Man mannose - Gal galactose - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - GlcNAc-ol N-acetylglucosaminitol     

SYNTHESIS OF GLYCOPROTEINS IN BRAIN: IDENTIFICATION, PURIFICATION AND PROPERTIES OF GLYCOSYL TRANSFERASES FROM PURIFIED SYNAPTOSOMES OF GUINEA PIG CEREBRAL CORTEX

Abstract— Four glycoprotein:glycosyl transferases (a fetuin:N-acetylglucosaminyl transferase; a bovine submaxillary mucin: N-acetylgalactosaminyl transferase; a collagen: glucosyl transferase and an orosomucoid: galactosyl transferase) were purified 34-, 45-, 37- and 47-fold, respectively, from synaptosomes prepared from guinea pig cerebral cortex. Purifications were achieved by centrifugation and by column chromatography on Sephadex G-100 and G-150 of 0 , 1% (w/v) Triton X-100 extractsof the purified cerebral cortical synaptosomes. The enzymes were separated from endogenous acceptors and were highly specific for specific macromolecular acceptors; small molecules were ineffective as acceptors. The fetuin: N-acetylglucosaminyl transferase functioned only with fetuin minus N-acetylneuraminic acid, galactose and N-acetylglucosamine; the bovine submaxillary mucin: N- acetylgalactosaminyl transferase with bovine submaxillary much minus N-acetylneuraminic acid and N-acetylgalactosamine; the collagen: glucosyl transferase with collagen minus glucose; and the orosomucoid: galactosyl transferase with either orosomucoid minus N-acetylneuraminic acid and galactose or fetuin minus N-acetylneuraminic acid and galactose. Each transferase required a specific (XDP)-monosaccharide for transfer. The transferases were entirely dependent on either Mn²⁺ or Mg²⁺ for activation and Fe²⁺ and Hg²⁺ inhibited each of the four enzymes. The optimum pH's for the enzymes were: for fetuin: N-acetylglucosaminyl transferase, 7 , 4–8.0; for bovine submaxillary mucin: N-acetylgalactosaminyl transferase, 7 , 7; for collagen: glucosyl transferase, 7 , 7 and for orosomucoid: galactosyl transferase, 6 , 6. The enzymes were distributed subsynaptosomally primarily in the synaptosomal plasma membrane and in the mitochondria of the synaptosome. The respective values for K_m (μM) and V_mex (pmoles/h/mg of protein) for the transferases were: fetuin: N-acetylglucosaminyl transferase, 12 and 143; for bovine submaxillary mucin: N-acetylgalactosaminyl transferase, 25 and 166; for collagen: glucosyl transferase, 4 and 10 and for orosomucoid:galactosyl transferase, 8 and 111.     

The structure of a trisialyl di-antennaryN-type glycopeptide obtained from rat plasma hemopexin

Rat hemopexin is a plasma glycoprotein that contains 18.3% carbohydrate consisting of onlyN-glycosidically-linked oligosaccharide chains. Glycopeptides obtained from hemopexin by Pronase^® digestion could be separated on Concanavalin A-Sepharose into three fractions. The lectin-binding fraction has been characterized as a mixture of monosialyl and disialyl di-antennary compounds ending inN-acetylneuraminic acid residues (2-6)-linked to galactose in the respective branches [Bernard N, Lombart C, Strecker G, Montreuil J, Van Halbeek H, Vliegenthart JFG (1983) Biochimie 65:185–92].The structures of the glycans in the Concanavalin A non-binding fractions were determined by a combination of methylation analysis and 500-MHz¹H-NMR spectroscopy. Some of them appeared to be tri-antennary glycans. However, the major component of these fractions possesses the following structure: This type of structure has been encountered before in some bovine blood coagulation factors as well as in rat -acid glycoprotein, but the¹H-NMR parameters for it are first reported here. Furthermore, by methylation analysis, the occurrence of the NeuAc2-8NeuAc disaccharide element was demonstrated in a minor part of the carbohydrate moiety of rat hemopexin. This element has also been reported previously for rat brain glycopeptides.     

GANGLIOSIDES OF PERIPHERAL NERVE MYELIN

—Gangliosides have been isolated from myelin obtained from three types of peripheral nerve: bovine spinal roots, bovine sciatic nerve and human sciatic nerve. Yields in most cases were 218–287 μg of lipid-bound sialic acid per g myelin, less than half that previously obtained from CNS myelin. Myelin accounted for approx 60% of total ganglioside present in whole spinal root. The human sample contained only N-acetylneuraminic acid but the two bovine preparations contained that as well as N-glycolylneuraminic acid; N-acetylglucosamine and N-acetylgalactosamine were both present in all three preparations. Sphingosine was the major long-chain base in each preparation while 4-eicosasphingenine (d20:1) comprised about 14% in the two bovine samples and 3% in the human sample. The major fatty acids in all preparations were 16:0, 18:0, 22:0, 24:0 and 24:1. Sialosylgalactosyl ceramide (G₇), a ganglioside characteristic of CNS myelin, was not detected in any of the PNS samples. The majority of gangliosides in bovine spinal root myelin were monosialo species, although the structures differed in some respects from those of CNS myelin. The molar concentration of lipid-bound sialic acid in PNS myelin is roughly equivalent to that of the P₁ basic protein.     

Purification and characterization of a sialidase fromClostridium chauvoei NC08596

The sialidase secreted byClostridium chauvoei NC08596 was purified to apparent homogeneity by ion-exchange chromatography, gel filtration, hydrophobic interaction-chromatography, FPLC ion-exchange chromatography, and FPLC gel filtration. The enzyme was enriched about 10 200-fold, reaching a final specific activity of 24.4 U mg^–1. It has a relatively high molecular mass of 300 kDa and consists of two subunits each of 150 kDa. The cations Mn²⁺, Mg²⁺, and Ca²⁺ and bovine serum albumin have a positive effect on the sialidase activity, while Hg²⁺, Cu²⁺, and Zn²⁺, chelating agents and salt decrease enzyme activity. The substrate specificity, kinetic data, and pH optimum of the enzyme are similar to those of other bacterial sialidases.Abbreviations FPLC fast protein liquid chromatography - NCTC National Collection of Type Cultures - ATCC American Type Culture Collection - MU-Neu5Ac 4-methylumbelliferyl--d-N-acetylneuraminic acid - buffer A 0.02m piperazine, 0.01m CaCl₂, pH 5.5 - buffer B 0.02m piperazine, 0.01m CaCl₂, 1.0m NaCl, pH 5.5 - buffer C 0.1m sodium acetate, 0.01m CaCl₂, pH 5.5 - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - Neu5Ac N-acetylneuraminic acid - BSM bovine submandibular gland mucin - GD1a IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer - GM1 II³Neu5Ac-GgOse₄Cer - MU-Neu4,5Ac₂ 4-methylumbelliferyl--d-N-acetyl-4-O-acetylneuraminic acid - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - EDTA ethylenediamine tetraacetic acid - EGTA ethylene glycol bis(2-aminoethyl-ethen)-N,N,N,N-tetraacetic acid - BSA bovine serum albumin - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - IEF isoelectric focusing - IEP isoelectric point     

Detection of gangliosides by direct binding ofLimax flavus agglutinin to thin layer chromotograms

A simple and sensitive method for detecting gangliosides on TLC plates is described. Gangliosides are extracted by phase partition in chloroform/methanol, developed on TLC plates in chloroform/methanol/0.25% aqueous KCl (5/4/1 by vol) and identified by binding of¹²⁵I-labelled, sialic acid-specificLimax flavus agglutinin (LFA) autoradiography and scanning densitometry. The detection limit of the method is below 1 ng (0.5 pmol) for GM3, GM1 and GT1b, and below 0.3 ng (0.2 pmol) for GM2 and GD1a. Binding of¹²⁵I-LFA is not inhibited by 10⁶-fold molar excess concentrations ofN-acetylneuraminic acid or lactose but is decreased in a dose-dependent manner by eitherN-acetylneuraminyllactose or unlabelled lectin. Gangliosides were not detected after their treatment byClostridium perfringens sialidase in the presence of taurocholic acid. Ten gangliosides were detected in human brain and seven in normal human serum. Extracts from 0.2 mg of brain and 20 l of serum were sufficient for the detection of major gangliosides.Abbreviations LFA Limax flavus agglutinin - ELLA Enzyme Linked Lectin Assay - PIM Poly(isobutyl methacrylate) - PVP Polyvinylpyrrolidone mol.wt. 40,000 - PBS Phosphate buffered saline - BSA Bovine serum albumin     

Subcellular characterization of glycoproteins in the principal cells of human gallbladder

Gallbladder mucus is mainly composed of glycoproteins, which seem to play a critical role in cholesterol nucleation during gallstone formation. The biosynthetic pathway and sequential processing as well as the characterization of the oligosaccharide sidechains of human gallbladder secretory glycoproteins have not been completely defined. The aim of the present study is the subcellular characterization of the glycoproteins in the principal cells of human gallbladder. Principal cells of normal human gallbladder were studied by means of a variety of cytochemical techniques, including lectin histochemistry, enzyme and chemical treatments, immunocytochemistry and lectin-gold technology. Fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid residues were detected in mucous granules, Golgi apparatus and apical membrane of principal cells. Mannose residues were only observed in dense bodies. Oligosaccharide side-chains of the glycoproteins contained in the biliary mucus are synthesized in the Golgi apparatus of the principal cells of the gallbladder epithelium and are also contained in the mucous granules of these cells. Terminal N-acetylneuraminic acid(2-3)galactose(1-3)N-acetylgalactosamine, N-acetylneuraminic acid(2-3)galactose(1-4)N-acetylglucosamine and galactose(1-4)N-acetylglucosamine sequences are contained in the oligosaccharide chains of gallbladder mucus glycoproteins. The dense bodies detected in the cytoplasm of the principal cells contained N-linked glycoproteins. Mucin-type O-linked glycoproteins were the main components of the mucous granules although some N-linked chains were also detected.     

Deuterium isotope effects in carbohydrates revisited. Cryoprobe studies of the anomerization and NH to ND deuterium isotope induced 13C NMR chemical shifts of acetamidodeoxy and aminodeoxy sugars

Complete ¹H and ¹³C NMR chemical shift assignments have been generated from a series of acetamidodeoxy and aminodeoxy sugar derivatives. For free sugars, the enhanced sensitivity of an NMR cryoprobe allowed simple 1D and 2D NMR spectra to be obtained from essentially single anomers, before significant mutarotation had occurred. The NMR assignments have been used to characterize deuterium isotope effects on ¹³C chemical shifts measured under conditions of slow NH to ND exchange in single solutions. Within a range of 0 to −0.138 ppm, β, γ, δ, and ζ deuterium isotope effects have been observed, thus providing additional reference data for assignment of the ¹³C NMR spectra of nitrogenous saccharides.     

N-acetylneuraminic acid and sialoglycoconjugate metabolism in fibroblasts from a patient with generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from a patient suffering from generalized N-acetylneuraminic acid storage disease were found to accumulate large amounts (approx. 4.0 μmol/g fresh weight) of free N-acetylneuraminic acid in a lysosome-enriched subcellular fraction. However, there were no detectable deficiencies in lysosomal hydrolase activities (including neuraminidase), and the activities of CMP-N-acetylneuraminic acid synthetase and N-acetylneuraminic acid aldolase were within normal limits. The cellular glycoconjugate composition was normal, and pathologic fibroblasts labeled with either [³H]glucosamine-HCl or $\text{N-[}{}^3\text{H]acetylmannosamine}$ showed a marked accumulation of labeled free N-acetylneuraminic acid, along with elevated incorporation into sialoglycoconjugates. Neither normal nor pathologic fibroblasts secreted labeled free N-acetylneuraminic acid into the culture medium. These results are consistent with an inherited defect in N-acetylneuraminic acid reutilization, resulting in the lysosomal accumulation of the free monosaccharide in generalized N-acetylneuraminic acid storage disease.     

Inhibition of the biosynthesis ofN-acetylneuraminic acid by metal ions and seleniumin vitro

In liver homogenate the biosynthesis ofN-acetylneuraminic acid usingN-acetylglucosamine as precursor can be followed stepwise by applying different chromatographic procedures. In this cell-free system 16 metal ions (Zn²⁺, Mn²⁺, La³⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Ce³⁺, Cd²⁺, Fe²⁺, Fe³⁺, Al³⁺, Sn²⁺, Cs⁺ and Li⁺) and the selenium compounds, selenium(IV) oxide and sodium selenite, have been checked with respect to their ability to influence a single or possible several steps of the biosynthesis ofN-acetylneuraminic acid. It could be shown that the following enzymes are sensitive to these metal ions (usually applied at a concentration of 1 mmoll^–1):N-acetylglucosamine kinase (inhibited by Zn²⁺ and vandate), UDP-N-acetylglucosamine-2-epimerase (inhibited by zn²⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Cd²⁺, Fe³⁺, Cs⁺, Li⁺, selenium(IV) oxide and selenite), andN-acetylmannosamine kinase (inhibited by Zn²⁺, Cu²⁺, Cd²⁺, and Co²⁺). Dose dependent measurements have shown that Zn²⁺, Cu²⁺ and selenite are more efficient inhibitors of UDP-N-acetylglucosamine-2-epimerase than vanadate. As for theN-acetylmannosamine kinase inhibition, a decreasing inhibitory effect exists in the following order Zn²⁺, Cd²⁺, Co²⁺ and Cu²⁺. In contrast, La³⁺, Al³⁺ and Mn²⁺ (1 mmoll^–1) did not interfere with the biosynthesis ofN-acetylneuraminic acid. Thus, the conclusion that the inhibitory effect of the metal ions investigated cannot be regarded as simply unspecific is justified.Dedicated to Professor Theodor Günther on the occasion of his 60th birthday     

Negative-ion fast atom bombardment and electrospray ionization tandem mass spectrometry for characterization of sulfated and sialyl Lewis-type glycosphingolipids

Structural characterization of sulfated and sialyl Lewis (Le)-type glycosphingolipids performed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of acidic glycosphingolipids allowed identification of the sulfated or sialyl sugar, and provided information on the saccharide chain sequence. The negative-ion tandem FABMS of sulfated Le-type glycosphingolipids having the non-reducing end trisaccharide ion as the precursor can be used to differentiate the Le^a- and Le^X-type oligosaccharides. The ESI CID-MS/MS of multiple-charged ions provided even more detailed structural information, and some of the useful daughter ions appeared with higherm/z values than the precusor because of a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated and sialyl analogues.Abbreviations CID collision-induced dissociation - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - Le Lewis - Le^a Lewis^a - Le^X Lewis^X - MS/MS mass spectrometry/mass spectrometry - NeuAc N-acetylneuraminic acid - 3-SO₄-Le^a 3-sulfated Le^a pentaosyl ceramide - 3-SO₄-Le^X 3-sulfated Le^X pentaosyl ceramide - 2,3-SO₄-Le^X 2,3-disulfated Le^X pentaosyl ceramide - 3-S-Le^a 3-sialyl Le^a pentaosyl ceramide - 3-S-Le^x 3-sialyl Le^X heptaosyl ceramide - 3-S-Le^X-Le^X 3-sialyl-Le^x-Le^x octaosyl ceramide.