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PubMed1985

Both acidic-type and neutral-type asparaginyl-oligosaccharides of host-cell glycoproteins are altered in Rous-sarcoma-virus-transformed chick-embryo fibroblasts.

In comparisons of [3H]mannose-labelled glycopeptides from chick-embryo fibroblasts infected and transformed with non-defective Prague C Rous-sarcoma virus and from untransformed fibroblasts infected with a transformation-defective derivative of Prague C Rous-sarcoma virus, we have detected transformation-dependent alterations in both the acidic-type and the neutral-type asparagine-linked oligosaccharides of cellular glycoproteins. Pronase-digested glycopeptides were analysed by the combined techniques of gel filtration, exo- and endo-glycosidase digestion and concanavalin A-agarose affinity chromatography. The transformed cell glycoproteins contained more sialic acid and were enriched for more highly branched (versus biantennary) acidic-type structures compared with the untransformed cell glycoproteins, similarly to previously reported transformation-dependent alterations. In addition, the glycopeptides from the virus-transformed cells contained several neutral-type structures that were apparently absent from the untransformed cells: small neutral-type oligosaccharides (Man3GlcNAc2) that were sensitive to endo-beta-N-acetylglucosaminidase D but resistant to endo-beta-N-acetylglucosaminidase H, and oligosaccharides with the property of 'truncated' precursor oligosaccharides (endoglycosidase-resistant, alpha-mannosidase-sensitive). Endoglycosidase-released oligosaccharides with the properties of hybrid-type structures were derived from the glycoproteins of both transformed and untransformed cells.

Acetylglucosaminidase↗
PubMed1990

Molecular-mass heterogeneity of Griffonia simplicifolia lectin IV subunits. Differences in the oligosaccharide moieties in the N-terminal region.

Lectin IV of Griffonia simplicifolia (Mr approximately 56,000), which has a strong affinity for both the Lewis b and Y blood-group determinants, is a dimeric protein of two subunits, alpha (29 kDa) and beta (27 kDa), separable by SDS/PAGE and containing covalently linked oligosaccharide. After digestion with N-glycanase, the protein migrates as a single band with a mobility identical with that of the beta-subunit. After cleavage with hydroxylamine of 3H-labelled, but otherwise intact, lectin, the radioactively labelled oligosaccharide was found to be associated with two blocked N-terminal peptides separable by h.p.l.c. and having identical amino acid compositions. One of these had three or four glucosamine residues per molecule, whereas the other had only one or two. Sequence analyses of these, as well as of a 21 kDa hydroxylamine-cleaved fragment and of the intact lectin pretreated with pyroglutamate aminopeptidase, have provided a unique sequence for residues 1-62 of the two subunits. Evidence is presented for two sites of N-linked oligosaccharide attachment at Asn-5 and Asn-18. Whereas the alpha-subunit has oligosaccharide linked to both sites, the beta-subunit has carbohydrate associated with only one (Asn-18). Sugar analyses of the whole lectin reveal a monosaccharide composition of (Xyl)3(Fuc)2(Man)10(GlcNAc)6, representing 6.4% of the mass of the molecule. Taken together with the susceptibility of the Asn-5 linkage (but not of Asn-18) to N-glycanase digestion, the observations indicate that the structures of the oligosaccharides at residues 5 and 18 are different.

Amino Acid Sequence↗
PubMed1992

Characterization and cellular distribution of acidic peptide and oligosaccharide metal-binding compounds from kidneys.

Two low-molecular-mass Ni-binding fractions first isolated from human kidneys [Templeton & Sarkar (1985) Biochem. J. 230, 35-42] are further characterized. Both components are acidic and are readily separated from each other by gel chromatography on Bio-Gel P-2. After equilibration with 63Ni the largest complex constitutes about 30% of the radioactive 63Ni and is an approx. 3.5 kDa peptide and the smallest species comprise short oligosaccharides containing 70% of the radioactivity. Both of these components are found in human, bovine and porcine kidneys as well as in a porcine proximal tubule-like cell line LLC-PK1. There is a small variation in amino acid composition between species. The oligosaccharides are reducing sugars and contain sulphate, glucosamine, glucuronic acid and iduronic acid with two to four overall negative charges. The monosaccharide composition was determined by h.p.l.c. with pulsed amperometric detection of the acid hydrolysates and by gas chromatography. In the LLC-PK1 cell line the acidic peptide is both intracellular and extracellular, whereas the oligosaccharides are only intracellular. The concentration of extracellular peptide, as measured by 63Ni binding, is found to increase after exposure of the cells to low micromolar concentrations of Ni, whereas the oligosaccharide concentrations, also measured by 63Ni binding, remain constant. The oligosaccharide component is decreased by 40% in the presence of NH4Cl, suggesting that is derived from degradation of internalized heparan sulphate.

Aged↗
PubMed1992

Hyaluronan-binding region of aggrecan from pig laryngeal cartilage. Amino acid sequence, analysis of N-linked oligosaccharides and location of the keratan sulphate.

The hyaluronan-binding region (HABR) was prepared from pig laryngeal cartilage aggrecan and the amino acid sequence was determined. The HABR had two N-termini: one N-terminal sequence was Val-Glu-Val-Ser-Glu-Pro (367 amino acids in total), and a second N-terminal sequence (Ala-Ile-Ser-Val-Glu-Val; 370 amino acids in total) was found to arise due to alternate cleavage by the signal peptidase. The N-linked oligosaccharides were analysed by examining their reactivity with a series of lectins. It was found that the N-linked oligosaccharide on loop A was of the mannose type, while that on loop B was of the complex type. No reactivity was detected between the N-linked oligosaccharide on loop B' and any of the lectins. The location of keratan sulphate (KS) in the HABR was determined by Edman degradation of the immobilized KS-containing peptide. The released amino acid derivatives were collected and tested for the presence of epitope to antibody 5-D-4. On the basis of 5-D-4 reactivity and sequencing yields, the KS chains are attached to threonine residues 352 and 357. There is no KS at threonine-355. This site is not in fact in G1, but about 16 amino acid residues into the interglobular domain. Comparison of the structure of the KS chain from the HABR and from the KS domain of pig laryngeal cartilage aggrecan was made by separation on polyacrylamide gels of the oligosaccharides arising from digestion with keratanase. Comparison of the oligosaccharide maps suggests that the KS chains from both parts of the aggrecan molecule have the same structure.

Aggrecans↗
PubMed1994

A novel oligosaccharide, GlcA beta 1-4Xyl beta 1-(4-methylumbelliferone), synthesized by human cultured skin fibroblasts.

Human skin fibroblasts were cultured in the presence of 4-methylumbelliferyl-beta-D-xyloside (Xyl-MU) using a mass-culture system with a microcarrier. The structures of Xyl-MU-induced sugars purified from the dialysable fraction of the incubation medium were investigated. In addition to glycosaminoglycans the elongation of which initiated by Xyl-MU has already been reported, and oligosaccharides similarly initiated by Xyl-MU, such as Gal-Gal-Xyl-MU, Gal-Xyl-MU and SA-Gal-Xyl-MU, a novel Xyl-MU-induced oligosaccharide was detected. This oligosaccharide was identified as GlcA beta 1-4Xyl beta 1-(4-methylumbelliferone) using sugar composition analysis, enzyme digestion, mass spectrometry and Smith degradation. Using this culture system, the amount of the new oligosaccharide produced increased with the incubation time, even after the production of glycosaminoglycan initiated by Xyl-MU and Gal-Xyl-MU had reached a plateau. These results suggest that this oligosaccharide may be involved in terminating the elongation of glycosaminoglycan chains that is initiated by Xyl-MU.

Benzoates↗
PubMed1997

Glycosylation differences between pig gastric mucin populations: a comparative study of the neutral oligosaccharides using mass spectrometry.

Five mucin populations were isolated from the cardiac region,corpus and antrum of pig gastric mucosa. The released neutral oligosaccharides were permethylated and analysed using high-temperature gas chromatography-mass spectrometry (GC-MS) as well as matrix-assisted laser-desorption mass spectrometry (MALDI-MS). Thirty different oligosaccharides with up to six monosaccharide residues were characterized using both techniques, but the presence of an additional 49 structures was suggested on the basis of their molecular mass by MALDI-MS. Oligosaccharides based on core-1 (Galbeta1-3GalNAcalpha1-) and core-2 [Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-] structures were widely distributed, whereas core-3 structures (GlcNAcbeta1-3GalNAcalpha1-) were present only in mucins from the cardiac region and corpus, and core-4 structures [GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-] were present exclusively in mucins from the cardiac region. Furthermore the oligosaccharides from one of the mucins from the corpus were significantly longer than those from the other populations. The results illustrate vast structural diversity, but the relative abundances show only a few dominating structures, suggesting that many oligosaccharides may be quite rare in pig gastric mucins. Well-defined mucin populations with distinctly different glycosylation can thus be identified in pig stomach, suggesting that glycosylation of the large secreted mucins from this tissue is not a random event.

Animals↗
PubMed2002

Comparison of the in vitro bifidogenic properties of pectins and pectic-oligosaccharides.

AIMS: To compare the in vitro fermentation properties of pectins and oligosaccharides derived from them in pure and mixed faecal cultures. METHODS AND RESULTS: Specific growth rates of selected bacterial genera were calculated in pure culture. Bifidobacterium angulatum, B. infantis and B. adolescentis had higher growth rates on pectic oligosaccharides (POS I) derived from high methylated pectin (HMP) than on HMP and B. pseudolongum and B. adolescentis on pectic oligosaccharides (POS II) derived from low methylated pectin than on HMP. Controlled pH batch mixed faecal cultures were then carried out and a prebiotic index was calculated as a mean to compare the fermentation properties of the different substrates. In general, greater fermentation selectivity was obtained with lower degrees of methylation (PI24(-HMP) = -0.11, PI24(-LMP) = 0.033; PI24(-POS I) = 0.071 and PI24(-POS II) = 0.092). An effect of size on prebiotic potential was observed, with the oligosaccharides having more selective fermentation properties than the pectins they derived from. CONCLUSIONS: The degree of methylation plays an important role in the fermentation properties of pectins. Pectic-oligosaccharides are a better prebiotic candidate than the pectins, although their bifidogenic effect is low compared to oligofructose. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of size on prebiotic potential was demonstrated. Non-selectively fermented polysaccharides like pectin can have their bifidogenic properties improved by partial hydrolysis.

Bacteria↗
PubMed1998

The high degree of internal flexibility observed for an oligomannose oligosaccharide does not alter the overall topology of the molecule.

The conformational properties of oligosaccharides are important in determining their biological properties, such as recognition by proteins. The structural and dynamic properties of many oligosaccharides are poorly understood both because of a lack of experimental data (usually obtained from solution NMR parameters) and because of gross approximations frequently invoked in theoretical models. To characterise the oligomannose oligosaccharide Man,GlcNAc2 we have acquired a more extensive NMR data set and performed the first unrestrained molecular dynamics (MD) simulation in water of this large oligosaccharide (employing the GLYCAM_93 parameter set with the AMBER force field). Good agreement is seen between the computed dynamics data and the results of both an isolated spin pair (ISPA) analysis of short mixing time NOE data and NOE build-up curves for mixing times from 100 to 2000 ms. The number of experimental conformational constraints obtained in this study are in principle sufficient to fully define a rigid structure. The fact that this could not be done indicates a high degree of internal flexibility and/or the presence of multiple conformations about the glycosidic linkages. Independently, the same conclusions are reached from an analysis of the MD results. In addition, the theoretical results allow the overall topology of the molecule and its intra-molecular and solvent-mediated hydrogen bonding pattern to be defined. Extensive re-organisation of solvent and inter-residue hydrogen bonds is shown to be required for significant conformational changes to occur, resulting in relatively long life-times for distinct glycosidic linkage conformations, despite the high local flexibility of the glycosidic linkages. This factor is also seen in the overall topology of the molecule, where the considerable internal flexibility is not translated into gross changes in structure. The control exerted by the solvent over both the flexibility and overall topology of an oligosaccharide has important implications for recognition processes and for the conformational properties of glycans attached to glycoproteins.

Acetylglucosamine↗
PubMed2000

Fluorescent neoglycolipids. Improved probes for oligosaccharide ligand discovery.

A second generation of lipid-linked oligosaccharide probes, fluorescent neoglycolipids, has been designed and synthesized for ligand discovery within highly complex mixtures of oligosaccharides. The aminolipid 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE), which has been used extensively to generate neoglycolipids for biological and structural studies, has been modified to incorporate a fluorescent label, anthracene. This new lipid reagent, N-aminoacetyl-N-(9-anthracenylmethyl)-1, 2-dihexadecyl-sn-glycero-3-phosphoethanolamine (ADHP), synthesized from anthracenaldehyde and DHPE gives an intense fluorescence under UV light. Fluorescent neoglycolipids derived from a variety of neutral and acidic oligosaccharides by conjugation to ADHP, by reductive amination, can be detected and quantified by spectrophotometry and scanning densitometry, and resolved by TLC and HPLC with subpicomole detection. Antigenicities of the ADHP-neoglycolipids are well retained, and picomole levels can be detected using monoclonal carbohydrate sequence-specific antibodies. Among O-glycans from an ovarian cystadenoma mucin, isomeric oligosaccharide sequences, sialyl-Lea- and sialyl-Lex-active, could be resolved by HPLC as fluorescent neoglycolipids, and sequenced by liquid secondary-ion mass spectrometry. Thus the neoglycolipid technology now uniquely combines high sensitivity of immuno-detection with a comparable sensitivity of chemical detection. Principles are thus established for a streamlined technology whereby an oligosaccharide population is carried through ligand detection and ligand isolation steps, and sequence determination by mass spectrometry, enzymatic sequencing and other state-of-the-art technologies for carbohydrate analysis.

Amination↗
PubMed1977

Comparative rates of transfer of lipid-linked oligosaccharides to endogenous glycoprotein acceptors in vitro.

We have shown previously that particulate preparations of Nil 8 fibroblasts catalyze the synthesis of two oligosaccharide-lipids: one composed of N-acetylglucosamine and mannose residues and the other containing, in addition to mannose and N-acetylglucosamine, one or two glucose residues. These two oligosaccharide-lipids were purified and added to fresh microsomal preparations. In comparative studies, we find that the glucose-containing lipid-linked oligosaccharide is transferred much more rapidly to endogenous protein acceptors than the glucose-free compound. With materials of comparable specific activities, as much as 41% of the glucose-containing oligosaccharide was transferred to protein as compared to 5% for the glucose-free compound. These results suggest that the attachment of glucose to mannosyl lipid-linked oligosaccharide serves an important role in the transfer of these compounds from lipid carrier to protein acceptor.

Cells, Cultured↗
PubMed1982

Inhibition of formation of complex oligosaccharides by the glucosidase inhibitor bromoconduritol.

The alpha-glucosidase inhibitor bromoconduritol (6-bromo-3,4,5-trihydroxycyclohex-1-ene) inhibits trimming of the innermost glucose residue from the Glc3Man9GlcNAc2 precursor of high-mannose and complex oligosaccharides. This inhibition occurs both in intact cells and with a microsomal enzyme preparation. The formation of lipid-linked oligosaccharides was increased in glucosidase-inhibited cells. Inhibition of transfer of high-mannose oligosaccharides to protein was not observed. In bromoconduritol-treated virus-infected cells, trimming of mannose can occur despite incomplete removal of glucose. The glucosylated high-mannose oligosaccharides GlcMan9GlcNAc, GlcMan8GlcNAc, and GlcMan7GlcNAc were released from viral glycoproteins after digestion with Pronase and endo-beta-N-acetylglucosaminidase H. The formation of complex oligosaccharides was concomitantly inhibited. The release of infectious fowl plague virus particles (an influenza virus) was inhibited from bromoconduritol-treated infected chicken-embryo cells.

Animals↗
PubMed1984

Sulfation of lutropin oligosaccharides with a cell-free system.

Sulfate is covalently linked to the oligosaccharides on the alpha and beta subunits of bovine lutropin (luteinizing hormone; LH) but not to those on human chorionic gonadotropin (hCG). Since the amino acid sequences of the pituitary and placental alpha subunits are homologous, comparison of their asparagine-linked sugars can provide information regarding tissue specificity of oligosaccharide maturation. To characterize this post-translational modification, we have developed a reconstituted cell-free sulfation system. Sulfate is incorporated into exogenously added glycoproteins by sulfotransferases from Triton X-100-lysed Golgi membranes in the presence of 3'-phosphoadenosine 5'-phospho[35S]sulfate, which is generated from [35S]sulfate by a ribosome-free supernate from Krebs ascites tumor cells. LH is sulfated by pituitary and liver membranes but not by those from placenta. Desialylated hCG (AshCG) is sulfated by membranes from placenta and pituitary, but not liver, while hCG is not sulfated by any of these membranes. Endoglycosidase F releases all the incorporated sulfate from LH in the form of a heterogeneous mixture of mono- and disulfated oligosaccharides. In contrast, the sulfate added to AshCG is apparently attached to peptide rather than oligosaccharide. As found with the cell-free system, sulfate metabolically incorporated into LH by pituitary cells is present on a heterogeneous population of mono- and disulfated oligosaccharides. Thus the cell-free sulfation system accurately duplicates the in vivo process.

Animals↗
PubMed1993

Biotinylated diaminopyridine: an approach to tagging oligosaccharides and exploring their biology.

Fluorescent tagging of free oligosaccharides by reductive amination permits sensitive detection and fractionation of these molecules. To expand the scope of this approach, we have synthesized a fluorescent reagent, 2-amino-(6-amidobiotinyl)pyridine. This reagent can tag oligosaccharides under nondegradative conditions with high efficiency. The resulting adducts show excellent fractionation by reverse-phase HPLC with sensitive detection in the low picomole range. When combined with sequential exoglycosidase digestion, stepwise sequencing of the sugar chains is possible. The biotinyl group can also be used to recover the sugar chain from reaction mixtures. The high-affinity interaction of the biotinyl group with multivalent avidin or streptavidin can be used to create the functional equivalent of neoglycoproteins carrying multiple copies of oligosaccharides of defined structure. These complexes allow the production of IgG antibodies directed against the oligosaccharide chain. They can also harness the power of (strept)avidin-biotin technology for the detection and isolation of oligosaccharide-specific receptors from native sources of recombinant libraries.

Alkaline Phosphatase↗
PubMed1995

HNK-1-reactive novel oligosaccharide, sulfate-O-3GlcA beta 1-4Xyl beta 1-(4-methylumbelliferone), synthesized by cultured human skin fibroblasts.

4-Methylumbelliferyl-beta-D-xyloside (Xyl-MU) was added to the medium of cultured human skin fibroblasts. After incubation, the culture medium was pooled, and the Xyl-MU-induced oligosaccharides in the medium were purified by gel filtration chromatography. A novel Xyl-MU derivative was obtained, in addition to the previously reported Xyl-MU derivatives such as Gal-Gal-Xyl-MU, Gal-Xyl-MU, Sia-Gal-Xyl-MU, GlcA-Xyl-MU, and Xyl-Xyl-MU. The novel Xyl-MU derivative was purified using gel-filtration chromatography and high performance liquid chromatography and then subjected to carbohydrate composition analysis, enzymic digestion, Smith degradation, and ion spray mass spectrometric analysis. The results indicated that it was sulfate-O-3GlcA beta 1-4Xyl beta 1-MU. The structure of the nonreducing terminal of this Xyl-MU-induced oligosaccharide was the same as that of the oligosaccharide chain of a human peripheral nerve-derived glycolipid, reactive with the mouse monoclonal antibody HNK-1, and this Xyl-MU-induced oligosaccharide also reacted with HNK-1. These results suggest that the oligosaccharide, which is structurally identical to that of human peripheral nerve-derived glycolipid synthesized by nervous tissue and related to cell adhesion, is synthesized also by mesenchymal cells.

Antigens, CD↗
PubMed1995

Carbohydrate-binding property of peptide: N-glycanase from mouse fibroblast L-929 cells as evaluated by inhibition and binding experiments using various oligosaccharides.

Carbohydrate binding to peptide: N-glycanase from mouse fibroblast L-929 cells (L-929 PNGase) and inhibition by oligosaccharides of its catalytic activity were studied. L-929 PNGase was found to bind strongly with oligosaccharides having triomannosido-N,N'-diacetyl-chitobiosyl (Man3GlcNAc2) structure (Kd = approximately 10 microM). This binding was inhibited by mannotriose (Man3; Man alpha 1-->3[Man alpha 1-->6]Man) but not by N,N'-diacetylchitobiose (GlcNAc2; GlcNAc beta 1-->4GlcNAc). Scatchard analysis indicated that there exist two binding sites for Man3 on a homodimeric form of a 105-kDa subunit. Oligosaccharides having Man3GlcNAc2 structure were also shown to be strong inhibitors for the PNGase-catalyzed reaction (Ki = approximately 10 microM). The minimum structural requirements for inhibition of the PNGase activity were Man3 and GlcNAc2. Enzyme kinetic studies showed that the mechanism of inhibition by the oligosaccharides and Man3 fits well with a model wherein two inhibitor binding sites reside on L-929 PNGase. The conformity of Kd with IC50 values may be taken as an evidence for inhibition of the catalytic activity by the oligosaccharides and Man3 through the occupation of the binding sites with these molecules. On the other hand, inhibition by GlcNAc2 followed the simple competitive mode. Since the minimum substrate for the L-929 PNGase was shown to be Man beta 1-->4GlcNAc beta 1-->4GlcNAc beta 1-->peptide, GlcNAc2 may be directly accessible to the catalytic site in competition with substrate. Interestingly, alkylation of -SH group in L-929 PNGase caused complete loss of the catalytic activity, but the carbohydrate binding activity was completely retained, indicating that the catalytic site(s) is discriminated from the carbohydrate-binding sites in the active site of this enzyme. The carbohydrate-binding property seems to be unique to soluble PNGases from mammals and may be associated not only with regulation of the enzyme activity, but also with receptor and carrier functions for glycoconjugates in certain intracellular processes.

Amidohydrolases↗
PubMed1995

Unusual anionic N-linked oligosaccharides from bovine lung.

We previously described a diverse family of sulfated anionic N-linked oligosaccharides released by peptide: N-glycosidase F (PNGaseF) from calf pulmonary artery endothelial (CPAE) cells (Roux, L., Holoyda, S., Sundblad, G., Freeze, H.H., and Varki, A. (1988) J. Biol. Chem. 263, 8879-8889). Since a major fraction of the intact lung consists of endothelial cells, we reasoned that bovine lung might be a rich source of similar molecules. Total N-linked oligosaccharides from bovine lung acetone powder were released by PNGaseF, labeled by [3H]NaBH4 reduction, and the anionic fractions were studied with a variety of techniques. The sugar chains with lesser negative charge (designated Class I) share several properties of conventional multiantennary complex-type chains. However, unlike the case with CPAE cells, sialic acids account only for a minority of the anionic properties and only a small proportion carry sulfate esters. A variety of different treatments indicate that most of the unexplained negative charge is due to multiple carboxylic acid groups. Resistance to beta-glucuronidase and alpha-iduronidase suggests that these may be previously undescribed modifications of mammalian oligosaccharides. The most highly charged N-linked chains (designated Class II) are more similar in general structure to the corresponding ones from CPAE cells, although relatively more abundant. Their high charge is primarily due to chondroitin sulfate, heparin/heparan sulfate, or keratan sulfate glycosaminoglycan chains. Sequential digestion studies suggest that a significant proportion of these molecules have more than one type of glycosaminoglycan chain associated with them. Compositional analysis indicates the presence of xylose residues in Class II, but not Class I molecules. However, unlike the case with conventional glycosaminoglycans, these residues are not at the reducing terminus. Most previously reported structures of complex-type N-linked oligosaccharides are derived from the glycoproteins of blood cells, plasma, or the secretions of cultured mammalian cells. This library of N-linked oligosaccharides from an intact mammalian organ (lung) contains a high proportion of novel anionic sugar chains whose structures are different from conventional complex-type sialylated chains and only partially related to those from CPAE cells. Further exploration of the N-linked chains of intact mammalian tissues seems warranted.

Acetone↗
PubMed2000

3'-Azidothymidine potently inhibits the biosynthesis of highly branched N-linked oligosaccharides and poly-N-acetyllactosamine chains in cells.

Previous studies in our laboratory have characterized 3'-azido-3'-deoxythymidine (AZT) as a potent inhibitor of glycosphingolipid biosynthesis in cultured cells (Steet, R., Alizadeh, M., Melançon, P., and Kuchta, R. D. (1999) Glycoconj. J. 16, 237-245; Yan, J.-P., Ilsley, D. D., Frohlick, C., Steet, R., Hall, E. T., Kuchta, R. D., and Melançon, P. (1995) J. Biol. Chem. 270, 22836-22841). Here, we report that AZT treatment of K562 cells results in significant alterations in the profile of N-linked oligosaccharides. Fractionation of [(3)H]mannose-labeled oligosaccharides from AZT-treated K562 cells using lectin affinity chromatography revealed striking changes in the branching and processing of N-linked glycoconjugates. AZT treatment resulted in the production of fewer highly branched complex glycans (60% of control at 20 micrometer AZT) and a significant accumulation of core-fucosylated biantennary oligosaccharides. In addition, extension of branched oligosaccharides with multiple poly-N-acetyllactosamine repeats is nearly abolished by AZT concentrations as low as 2 micrometer. A shift from multiantennary to moderately branched oligosaccharides was also apparent in the melanoma cell line SK-MEL-30 upon AZT treatment. N-Linked glycans from both cell lines exhibited increased affinity for the beta-galactoside-binding lectin RCA-I in the presence of AZT, suggesting that the addition of terminal sialic acid is sensitive to the drug. These results demonstrate the ability of AZT to modulate strongly the processing of asparagine-linked glycoconjugates in whole cells and reveal a novel mechanism by which AZT treatment may cause anemia.

Humans↗
PubMed2001

Investigations of the in vitro transport of human milk oligosaccharides by a Caco-2 monolayer using a novel high performance liquid chromatography-mass spectrometry technique.

Complex lactose-derived oligosaccharides belong to the main components of human milk and are believed to exert multiple functions in the breast-fed infant. Therefore, we investigated the transepithelial transport of human milk oligosaccharides over Caco-2 monolayers. Main human milk oligosaccharides (HMOs) in the apical, basolateral, or intracellular compartment were separated by high performance liquid chromatography using a Hypercarb(TM) column and analyzed on line by mass spectrometry. This method allowed the identification and quantification of these components in intra- and extracellular fractions without prior purification. Using this technique we were able to show that acidic and neutral HMOs cross the epithelial barrier. The transepithelial flux of neutral, but not acidic, oligosaccharides was temperature-sensitive and partly inhibited by brefeldin A and bafilomycin A. Furthermore, net flux from the apical to the basolateral compartment was only observed for the neutral components. Similarly, apical cellular uptake was only found for neutral components but not for acidic oligosaccharides. Intracellular concentrations of neutral HMOs were significantly increased by inhibitors of transcytosis such as brefeldin A, N-ethylmaleimide, or bafilomycin A. The cellular uptake was saturable, and an apparent K(m) for lacto-N-fucopentaose I of 1.7 +/- 0.1 mmol/liter and for lacto-N-tetraose of 1.8 +/- 0.4 mmol/liter was determined. Furthermore, the uptake of lacto-N-fucopentaose I could be inhibited by the addition of the stereoisomer lacto-N-fucopentaose II but not by lacto-N-tetraose. These findings suggest that neutral HMOs are transported across the intestinal epithelium by receptor-mediated transcytosis as well as via paracellular pathways, whereas translocation of acidic HMOs solely represents paracellular flux.

Biological Transport↗