The structure of the keratan sulphate chains attached to fibromodulin from human articular cartilage

The small keratan sulphate proteoglycan, fibromodulin, has been isolated from pooled human articular cartilage. The main chain repeat region and the chain caps from the attached N-linked keratan sulphate chains have been fragmented by keratanase II digestion, and the oligosaccharides generated have been reduced and isolated. Their structures and abundance have been determined by high pH anion-exchange chromatography. These regions of the keratan sulphate from human articular cartilage fibromodulin have been found to have the following general structure: Significantly, both α(2-6)- and α(2-3)-linked N-acetyl-neuraminic acid have been found in the capping oligosaccharides. Fucose, which is α(1-3)-linked as a branch to N-acetylglucosamine, has also been found along the length of the repeat region and in the capping region. The chains, which have been found to be very highly sulphated, are short; the length of the repeat region and chain caps is ca. nine disaccharides. These data demonstrate that the structure of the N-linked keratan sulphate chains of human articular cartilage fibromodulin is similar, in general, to articular cartilage derived O-linked keratan sulphate chains. Further, the general structure of the keratan sulphate chains attached to human articular cartilage fibromodulin has been found to be generally similar to that of both bovine and equine articular cartilage fibromodulin. Abbreviations: KS, keratan sulphate; IEC, ion-exchange chromatography; ELISA, enzyme linked immunosorbent assay; Gal, β-D-galactose; Fuc, α-L-Fucose; GlcNAc, N-acetylglucosamine (2-acetamido-β-D-glucose); GlcNAc-ol, N-acetylglucosaminitol (2-acetamido-D-glucitol); NeuAc, N-acetyl-neuraminic acid; 6S/(6S), O-ester sulphate group on C6 present/sometimes present; NMR -nuclear magnetic resonance; HPAE, high pH anion-exchange; PED, pulsed electrochemical detection; HPLC, high performance liquid chromatography This revised version was published online in November 2006 with corrections to the Cover Date.     

Keratan sulfates from bovine tracheal cartilage structural studies of intact polymer chains using H and 13C NMR spectroscopy.

Intact keratan sulfate chains derived from bovine tracheal cartilage have been examined using both one-dimensional methods and the two-dimensional experiments COSY-45 and TOCSY for homonuclear shift correlations and a modified COLOC (correlated spectroscopy for long-range couplings) approach for 13C-1H shift correlations. Partial 1H and 13C NMR signal assignments for residues within the intact polymer chain are reported; data derived from the repeat region signals and from chain cap residues are assigned by comparison with published data derived from oligosaccharides obtained through cleavage of keratan sulfate polymer chains using keratanase and keratanase II and are discussed in detail. The one-dimensional spectra for both 1H and 13C nuclei contain highly crowded signal clusters for which data analysis is not directly possible. COSY-45 analysis allow the correlation and assignment of many proton resonances located within the 3.4-4.8 p.p.m. chemical shift region while from the C/H correlation spectrum data are assignable for some signals within the complex set of carbon resonances which fall in the region between 68 and 86 p.p.m., This work using material from tracheal cartilage has permitted the first detailed combined 1H and 13C NMR examination of the primary keratan sulfate polymer structure; this sequence forms the basis for the more complex members of the keratan sulfate family present in other tissues such as articular cartilage and cornea where further residues such as (alpha1-3)-linked fucose and (alpha2-6)-linked N-acetylneuraminic acid are also present. This nondestructive method of analysis complements the currently available degradative methods for structure determination which may then subsequently be utilized.     

Structural and immunological studies of keratan sulphates from mature bovine articular cartilage.

Two populations of alkaline-borohydride-reduced keratan sulphate (KS) chains were prepared from the two peptido-keratan sulphate trypsin fragments of proteoglycan aggregates isolated from bovine femoral head cartilage (6-year-old animals). Each population was separated by high-performance ion-exchange chromatography on a Pharmacia Mono-Q column into eight pools, Q1-Q8. These were analysed by gel permeation chromatography, radioimmunoassay with the monoclonal antibody MZ15, and 500 MHz 1H n.m.r. spectroscopy. Upon chromatography on Sephadex G-75 the Mono-Q fractions were shown to increase in hydrodynamic size progressively from Q1 to Q8 for both KS populations. For each population the strongest antigenic response with the anti-KS monoclonal antibody MZ15 was expressed by the two fractions of greatest size and charge density, Q7 and Q8. Proton n.m.r. spectroscopic studies on the two series of fractions demonstrated: (i) a progressive increase in the level of galactose sulphation from Q1 to Q8, (ii) the presence of approximately one alpha(1-3)-linked fucose residue per chain in every sample, and (iii) the presence of N-acetylneuraminic acids in three discrete environments, two alpha(2-3)- and one alpha(2-6)-linked in every sample. The results are discussed in terms of a possible heterogeneity in the carbohydrate-protein linkage region of keratan sulphates from bovine articular cartilage.     

Sulfation of the tumor cell surface sialomucin of the 13762 rat mammary adenocarcinoma

ASGP-1, the major cell surface sialomucin of the 13762 ascites rat mammary adenocarcinoma, is at least 0.5% of the total ascites cell protein and has sulfate on 20% of its O-linked oligosaccharide chains. We have used this system to investigate the O-glycosylation pathway in these cells and to determine the temporal relationship between sulfation and sialylation. The two major sulfated oligosaccharides (S-1 and S-2) were isolated as their oligosaccharitols by alkaline borohydride elimination, anion exchange HPLC, and ion-suppression HPLC. From structural analyses S-1 is proposed to be a branched, sulfated trisaccharide -O4S-GlcNAc beta 1,6-(Gal beta 1,3)-GalNAc and S-2 its sialylated derivative -O4S-GlcNAc beta 1,6-(NeuAc alpha 2,3-Gal beta 1,3)-GalNac. Pulse labeling with sulfate indicated that sulfation occurred primarily on a form of ASGP-1 intermediate in size between immature and mature sialomucin. Pulse-chase analyses showed that the intermediate could be chased into mature ASGP-1. The concomitant conversion of S-1 into S-2 had a half-time of less than 5 min. Monensin treatment of the tumor cells led to a 95% inhibition of sulfation with the accumulation of unsulfated trisaccharide GlcNAc beta 1,6-(Gal beta 1,3)-GalNAc and sialylated derivative GlcNAc beta 1,6-(NeuAc alpha 2,3-Gal beta 1,3)-GalNAc. These data suggest that sulfation of ASGP-1 is an intermediate synthetic step, which competes with beta-1,4-galactosylation for the trisaccharide intermediate and thus occurs in the same compartment as beta-1,4-galactosylation. Moreover, sulfation precedes sialylation, but the two are rapidly successive kinetic events in the oligosaccharide assembly of ASGP-1.     

Lectin affinity chromatography of articular cartilage fibromodulin: Some molecules have keratan sulphate chains exclusively capped by α(2-3)-linked sialic acid

Fibromodulin from bovine articular cartilage has been subjected to lectin affinity chromatography by Sambucus nigra lectin which binds α(2-6)- linked N-acetylneuraminic acid, and the structure of the keratan sulphate in the binding and non-binding fractions examined by keratanase II digestion and subsequent high pH anion exchange chromatography. It has been confirmed that the keratan sulphate chains attached to fibromodulin isolated from bovine articular cartilage may have the chain terminating N-acetylneuraminic acid residue α(2-3)- or α(2-6)-linked to the adjacent galactose residue. Although the abundance of α(2-6)-linked N-acetylneuraminic acid (ca. 22%) is such that this could cap one of the four chains in almost all fibromodulin molecules, it was found that ca. 34% of the fibromodulin proteoglycan molecules from bovine articular cartilage were capped exclusively with α(2-3)-linked N-acetylneuraminic acid. The remainder of the fibromodulin proteoglycans, which bound to the lectin had a mixture of α(2-3)- and α(2-6)-linked N-acetylneuraminic acid capping structures. The keratan sulphates attached to fibromodulin molecules capped exclusively with α(2-3)- linked N-acetylneuraminic acid were found to have a higher level of galactose sulphation than those from fibromodulin with both α(2-3)- and α(2-6)-linked N-acetylneuraminic acid caps, which bound to the Sambucus nigra lectin. In addition, both pools contained chains of similar length (ca. 8–9 disaccharides). Both also contained α(1-3)-linked fucose, showing that this feature does not co-distribute with α(2-6)-linked N-acetylneuraminic acid, although these two features are present only in mature articular cartilage. These data show that there are discrete populations of fibromodulin within articular cartilage, which may have differing impacts upon tissue processes.     

Acidic glycosphingolipids of human amnion

Six major acidic glycosphingolipids were isolated from human amnion using DEAE Sephadex A-25 and silica beads column chromatography. The structures of these glycosphingolipids were determined by methylation analysis, TLC immunostaining and/or negative ion FAB-MS, and were concluded to be II3 alpha NeuAcLacCer(GM3), IV3 alpha NeuAcnLc4-Cer (sialyl[alpha 2-3]paragloboside), IV6 alpha NeuAcnLc4Cer (sialyl[alpha 2-6]paragloboside), IV3 alpha NeuAcIII4 alpha FucLc4Cer (sialyl Lea), VI3 alpha NeuAcnLc6Cer (i-ganglioside) and II3 alpha (NeuAc alpha 2----8NeuAc)LacCer (GD3). In addition, several minor glycosphingolipids were detected with specific monoclonal antibodies, including glycolipids with NeuAc alpha 2-3Gal beta 1-4GlcNAc-beta 1- or NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1- determinant. Our results show that the glycosphingolipids of human amnion are characterized by having mainly type II chain analogues and onco-fetal antigens.     

Sulfation of sialyl lactosamine oligosaccharides by chondroitin 6-sulfotransferase

We have previously shown that chondroitin 6-sulfotransferase(C6ST) catalyzes transfer of sulfate not only to position 6of GalNAc residue of chondroitin but also to position 6 of Galresidue of keratan sulfate. In this study, we examined the sulfationof sialyl lactosamine oligosaccharides by C6ST. C6ST catalyzedtransfer of sulfate to NeuAc     

Structure determination by 1H NMR spectroscopy of (sulfated) sialylated N-linked carbohydrate chains released from porcine thyroglobulin by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F

The N-linked carbohydrate chains of porcine thyroglobulin were released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F (PNGase-F). The resulting oligosaccharides were fractionated by a combination of fast protein liquid chromatography and high performance liquid chromatography and analyzed by 500-MHz 1H NMR spectroscopy. The major acidic compounds are mono- and disialylated, fucosylated diantennary compounds terminated with alpha(2-6)-linked sialic acid on the Man alpha(1-3) branch. The Man alpha(1-6) branch shows a large heterogeneity. It can be terminated with Man-4', GlcNAc-5', or Gal-6', whereas the Gal-6' residue may be extended with Gal alpha(1-3), NeuAc alpha(2-3), or Sia alpha (2-6). In the major structures 8% of alpha(2-6)-linked sialic acid was found as NeuGc instead of NeuAc. The main compounds have sulfated homologues bearing a sulfate group (6-20%) at C-3 of Gal-6' or at C-6 of GlcNAc-5 as follows. [formula: see text]     

Carbohydrate structure of recombinant human uterine tissue plasminogen activator expressed in mouse epithelial cells

Recombinant human uterine tissue plasminogen activator (tPA), in part metabolically labeled with [6-3H]glucosamine or [35S]sulfate, was isolated from mouse epithelial cells (C127). Oligosaccharides present were liberated by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, methylation analysis and, in part, by acetolysis and 1H-NMR spectroscopy. Glycopeptides comprising individual glycosylation sites were identified by N-terminal amino acid sequencing. The results demonstrate that recombinant tPA from C127 cells carries at Asn117 oligomannosidic glycans with 5-8 mannose residues as well as small amounts of hybrid-type species. Asn184 is only partially glycosylated and substituted by fucosylated triantennary and small amounts of diantennary N-acetyllactosaminic glycans. Likewise, Asn448 carries predominantly fucosylated triantennary species, in addition to, small amounts of diantennary and tetraantennary oligosaccharides. As a characteristic feature, part of the triantennary glycans at Asn184 and Asn448 contain additional Gal(alpha 1-3) substituents and/or sulfate groups linked to position six of beta-galactosyl residues forming NeuAc(alpha 2-3)[HO3S-6]Gal(beta 1-4) units. Oligosaccharides attached to Asn448 are almost completely substituted by (alpha 2-3)- or (alpha 2-6)-linked sialic acid residues and carry the majority of sulfate groups present. Glycans at Asn184 were found to be less sialylated and sulfated.     

Studies on gangliosides with affinity for Helicobacter pylori: binding to natural and chemically modified structures

Helicobacter pylori, like many other microbes, has the ability to bind to carbohydrate epitopes. Several sugar sequences have been reported as active for the bacterium, including some neutral, sulfated, and sialylated structures. We investigated structural requirements for the sialic acid-dependent binding using a number of natural and chemically modified gangliosides. We have chosen for derivatization studies two kinds of binding-active glycolipids, the simple ganglioside S-3PG (Neu5Ac alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, sialylparagloboside) and branched polyglycosylceramides (PGCs) of human origin. The modifications included oxidation of the sialic acid glycerol chain, reduction of the carboxyl group, amidation of the carboxyl group, and lactonization. Binding experiments confirmed a preference of H. pylori for 3-linked sialic acid and penultimate 4-linked galactose. As expected, neolacto gangliosides (with Gal beta 4GlcNAc in the core structure) were active in our assays, whereas gangliosides with lacto (Gal beta 3GlcNAc) and ganglio (Gal beta 3GalNAc) carbohydrate chains were not. Negative binding results were also obtained for disialylparagloboside (with terminal NeuAc alpha 8NeuAc) and NeuAc alpha 6-containing glycolipids. Chemical studies revealed dependence of the binding on Neu5Ac and its glycerol and carboxyl side chains. Most of the derivatizations performed on these groups abolished the binding; however, some of the amide forms turned out to be active, and one of them (octadecylamide) was found to be an excellent binder. The combined data from molecular dynamics simulations indicate that the binding-active configuration of the terminal disaccharide of S-3PG is with the sialic acid in the anticlinal conformation, whereas in branched PGCs the same structural element most likely assumes the synclinal presentation.     

Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (alpha-series) gangliosides and novel sulfated Chol-1 analogs

Extended glycoconjugate binding specificities of three sialic acid-dependent immunoglobulin-like family member lectins (siglecs), myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), and sialoadhesin, were compared by measuring siglec-mediated cell adhesion to immobilized gangliosides. Synthetic gangliosides bearing the alpha-series determinant (NeuAc alpha2,6-linked to GalNAc on a gangliotetraose core) were tested, including GD1alpha (IV(3)NeuAc, III(6)NeuAc-Gg(4)OseCer), GD1alpha with modified sialic acid residues at the III(6)-position, and the "Chol-1" gangliosides GT1aalpha (IV(3)NeuAc, III(6)NeuAc, II(3)NeuAc-Gg(4)OseCer) and GQ1balpha (IV(3)NeuAc, III(6)NeuAc, II(3)(NeuAc)(2)-Gg(4)OseCer). The alpha-series gangliosides displayed enhanced potency for MAG- and SMP-mediated cell adhesion (GQ1balpha > GT1aalpha, GD1alpha > GT1b, GD1a > GM1 (nonbinding)), whereas sialoadhesin-mediated adhesion was comparable with alpha-series and non-alpha-series gangliosides. GD1alpha derivatives with modified sialic acids (7-, 8-, or 9-deoxy) or sulfate (instead of sialic acid) at the III(6)-position supported adhesion comparable with that of GD1alpha. Notably, a novel GT1aalpha analog with sulfates at two internal sites of sialylation (NeuAcalpha2,3Galbeta1,4GalNAc-6-sulfatebeta1, 4Gal3-sulfatebeta1,4Glcbeta1,1'ceramide) was the most potent siglec-binding structure tested to date (10-fold more potent than GT1aalpha in supporting MAG and SMP binding). Together with prior studies, these data indicate that MAG and SMP display an extended structural specificity with a requirement for a terminal alpha2, 3-linked NeuAc and great enhancement by nearby precisely spaced anionic charges.     

A new ganglioside in human meconium detected by antiserum against the human milk sialyloligosaccharide, LS-tetrasaccharide b

Antibodies directed against human milk sialyloligosaccharides [D. F. Smith and V. Ginsburg (1980) J. Biol. Chem. 255, 55-59] are used to identify human meconium gangliosides by radioimmuneoverlay-thin-layer chromatography or by direct binding on nitrocellulose filters of sialyl[3H]oligosaccharide alditols obtained from gangliosides after ozonolysis and alkali-fragmentation. Thin-layer chromatograms of meconium monosialylgangliosides immunostained with rabbit antisera specific for LS-tetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) or LS-tetrasaccharide b (Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) reveal their corresponding gangliosides, 6'-LM1 and a previously undescribed ceramide derivative of LS-tetrasaccharide b, respectively. The sialyl[3H]oligosaccharides derived from the monosialylganglioside fraction of meconium are separated by paper chromatography and assayed for binding to specific anti-sialyloligosaccharide sera. Antisera specific for LS-tetrasaccharide c and 3'-sialyllactose (NeuAc alpha 2-3Gal beta 1-4Glc) identify their corresponding 3H-labeled haptens released from the major meconium gangliosides 6'-LM1 and GM3, respectively. Binding of a ganglioside-derived sialyl[3H]oligosaccharide by anti-LS-tetrasaccharide b serum is consistent with the presence in meconium of a monosialylganglioside with the following proposed structure: (formula; see text)     

Spectrum of sialylated and nonsialylated fuco-oligosaccharides bound by the endothelial-leukocyte adhesion molecule E-selectin. Dependence of the carbohydrate binding activity on E-selectin density.

Carbohydrate recognition by the human endothelial-leukocyte adhesion molecule, E-selectin, has been investigated by binding studies using 3H-labeled Chinese hamster ovary cells expressing different levels of the transfected full-length adhesion molecule and a series of structurally defined oligosaccharides linked to the lipid phosphatidylethanolamine dipalmitoate (neoglycolipids) and synthetic glycolipids chromatographed on silica gel plates or immobilized on plastic wells. Evidence is presented for density-dependent binding of the membrane-associated E-selectin not only to 3'-sialyl-lacto-N-fucopentaose II (3'-S-LNFP-II) and 3'-sialyl-lacto-N-fucopentaose III (3'-S-LNFP-III) which express the sialyl Le(a) and sialyl Le(x) antigens, respectively, but also to the nonsialylated analogue LNFP-II; there is a threshold density of E-selectin required for binding to these sialylated sequences, and binding to the nonsialylated analogue is a property only of cells with the highest density of E-selectin expression. The presence of fucose linked to subterminal rather than to an internal N-acetylglucosamine is shown to be a requirement for E-selectin binding, and although the presence of sialic acid 3-linked to the terminal galactose of the LNFP-II or LNFP-III sequences substantially enhances E-selectin binding, the presence of 6-linked sialic acid abolishes binding. E-selectin binding is unaffected in the presence of the blood group H fucose (alpha 1-2 linked to galactose to form the Le(b) antigen). However, the binding is abolished when in addition alpha 1-3-linked N-acetylgalactosamine to the galactose (blood group A antigen) is present. These results indicate that some E-selectin-mediated adhesive events may be influenced by blood group status.     

Identification of an O-glycosidic mannose-linked sialylated tetrasaccharide and keratan sulfate oligosaccharides in the chondroitin sulfate proteoglycan of brain

The chondroitin sulfate proteoglycan of rat brain was digested with Pronase, and after removal of glycosaminoglycans, the resulting glycopeptides were treated with alkaline borohydride to release O-glycosidically linked oligosaccharides. These were fractionated by ion exchange chromatography, gel filtration, and preparative thin layer chromatography, and their structural properties were studied by specific enzymatic degradations, methylation analysis, and gas-liquid chromatography-mass spectrometry of disaccharides as their trimethylsilylated and permethylated derivatives. In addition to the previously characterized N-acetyl-galactosamine-linked oligosaccharides and neutral mannitol-containing oligosaccharides [GlcNAc(beta 1-3) Manol and Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Manol] (where Fuc is fucose), we have now identified the sialylated tetrasaccharide NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc (beta 1-3)Manol, which accounts for approximately 20% of the mannitol-containing oligosaccharides. The proteoglycan also contains mannose-linked keratan sulfate chains (with a molecular size of 3,000 to 10,000 Da) composed of disaccharide repeating units consisting of Gal(beta 1-4)GlcNAc-6-O-SO4(beta 1-3), with a small proportion of branch points at C-6 of galactose residues. There is approximately one keratan sulfate chain per four chondroitin sulfate chains of 18,000-19,000 Da. After alkaline borohydride treatment of the neutral and monosialyl glycopeptide fractions, the combined decrease in mannose and N-acetylgalactosamine was very close to the observed destruction of serine + threonine and was accompanied by an equimolar increase in alanine and alpha-aminobutyric acid. One half of the mannose was destroyed by alkaline borohydride treatment of the glycopeptides and stoichiometrically converted to mannitol, while there were only small changes in the relative amounts of the other sugars and amino acids. The data demonstrate that over half of the carbohydrate-peptide linkages in the proteoglycan are of the mannosyl-O-serine/threonine type.     

Structure of the carbohydrate moiety of human interferon-beta secreted by a recombinant Chinese hamster ovary cell line

The carbohydrate structure of the major oligosaccharide of human interferon-beta (IFN-beta) synthesized by a genetically engineered Chinese hamster ovary cell line has been determined. Analysis of the glycopeptidase F-released carbohydrates by sequential exoglycosidase treatment, methylation analysis, and fast atom bombardment-mass spectrometry revealed that 95% of the IFN-beta oligosaccharides had the following structure: (Formula: see text). The remaining 5% of the carbohydrates are probably tri- or higher antennary oligosaccharide chains. The major oligosaccharide of the recombinant IFN-beta is remarkably homogeneous with respect to terminal galactose sialylation. NeuAc, which is alpha 2-3-linked to galactose in the human IFN-beta secreted by Chinese hamster ovary cells, can be re-incorporated with an alpha 2-6 linkage in vitro, into enzymatically desialylated IFN-beta using rat liver Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase. The sugar chain is important for maintaining protein solubility as shown by the fact that IFN-beta protein precipitates after deglycosylation with glycopeptidase F.     

Sulfation of sialyl N-acetyllactosamine oligosaccharides and fetuin oligosaccharides by keratan sulfate Gal-6-sulfotransferase

We have previously cloned keratan sulfate Gal-6-sulfotransferase (KSGal6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of Gal residue of keratan sulfate. In this study, we examined whether KSGal6ST could transfer sulfate to sialyl N -acetyllactosamine oligosaccharides or fetuin oligo-saccharides. KSGal6ST expressed in COS-7 cells catalyzed transfer of sulfate to NeuAcalpha2-3Galbeta1-4GlcNAc (3'SLN), NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Gl cNAc (SL1L1), NeuAcalpha2-3Galbeta1-4(6-sulfo)GlcNAcbeta1-3(6-sulfo) Galbeta1-4(6-su lfo)GlcNAc (SL2L4), and their desialylated derivatives except for Galbeta1-4GlcNAc, but not to NeuAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc (SLex). When the sulfated product formed from 3'SLN was degraded with neuraminidase and reduced with NaBH(4), the resulting sulfated disaccharide alditol showed the same retention time in SAX-HPLC as that of [(3)H]Gal(6SO(4))beta1-4GlcNAc-ol. KSGal6ST also catalyzed sulfation of fetuin. When the sulfated oligosaccharides released from the sulfated fetuin after sequential digestion with proteinase and neuraminidase were subjected to a reaction sequence of hydrazin-olysis, deaminative cleavage and NaBH(4)reduction, the major product was co-eluted with [(3)H]Gal(6SO(4))beta1-4anhydromannitol in SAX-HPLC. These observations show that KSGal6ST is able to sulfate position 6 of Gal residue of 3'SLN and fetuin oligosaccharides. The relative rates of the sulfation of SL2L4 was much higher than the rate of the sulfation of keratan sulfate. These results suggest that KSGal6ST may function in the sulfation of sialyl N -acetyllactosamine oligosaccharide chains attached to glycoproteins.     

Isolation and NMR spectroscopic characterization of sialic acid compounds and hemofiltrate of chronic uremia patients

Eight sialyloligosaccharides have been isolated from the hemofiltrate of a patient with end stage renal disease using reverse osmosis, gel filtration, ion-exchange and high-performance liquid chromatography. The structures were predominantly elucidated by one- and two-dimensional 1H- and 13C-NMR spectroscopy: 1 NeuAc alpha 2-3Gal beta 1-4Glc; 2 NeuAc alpha 2-6Gal beta 1-4Glc; 3 NeuAc alpha 2-3Gal beta 1-4GlcNAc; 4 NeuAc-alpha 2-6Gal beta 1-4GlcNAc; 5 NeuAc alpha 2-3Gal beta 1-4-GlcNAc alpha 1-P; 6 NeuAc alpha 2-6Gal beta 1-4GlcNAc alpha 1-P; 7 NeuAc alpha 2-3Gal beta 1-3GalNAc alpha 1-P; 8 NeuAc alpha 2-8NeuAc. While compounds 1-7 are also components of normal human urine, di-N-acetyl-D-neuraminic acid (8) could be isolated for the first time from biological material. The origin and possible clinical relevance of these compounds have to be proved in further investigations.     

Directed evolution of lectins with sugar-binding specificity for 6-sulfo-galactose

6-sulfo-galactose (6S-Gal) is a prevalent motif observed in highly sulfated keratan sulfate, which is closely associated with the glioblastoma malignancy while acting as a critical determinant for endogenous lectins. However, facile detection of this unique glycoepitope is greatly hampered because of a lack of appropriate probes. We have previously reported tailoring an α2-6-linked sialic acid-binding lectin from a ricin-B chain-like galactose-binding protein, EW29Ch, by a reinforced ribosome display system following an error-prone PCR. In this study, we challenged the creation of novel lectins to recognize 6S-Gal-terminated glycans by incorporating a high-throughput screening system with a glycoconjugate microarray. After two rounds of selection procedures, 20 mutants were obtained and 12 were then successfully expressed in Escherichia coli, 8 of which showed a significant affinity for 6'-Sulfo-LN (6-O-sulfo-Galβ1-4GlcNAc), which the parental EW29Ch lacked. Analysis of two representative mutants by frontal affinity chromatography revealed a substantial affinity (K(d) ～3 μm) for a 6S-Gal-terminated glycan. On the basis of the observation that all eight mutants have a common mutation at Glu-20 to Lys, site-directed mutagenesis experiments were performed focusing on this aspect. The results clearly indicated that the E20K mutation is necessary and sufficient to acquire the specificity for 6S-Gal. We also confirmed a difference in binding between E20K and EW29Ch to CHO cells, in which enzymes to catalyze the synthesis of 6S-Gal were overexpressed. The results clearly demonstrate that these mutants have potential to distinguish between cells containing different amounts of 6S-Gal-terminated glycans. This new technology will be used to provide novel tools essential for sulfoglycomics.     

Structures of the major carbohydrates of natural human interleukin-2

Purified human interleukin-2 secreted by peripheral blood lymphocytes from healthy donors was found to exist in several forms. These forms were (partially) resolved by reversed-phase high-performance liquid chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Two major polypeptide species (interleukin-2 N1 and N2, 16.5 kDa) were shown to be glycosylated on the basis of [3H]galactose/[3H]glucosamine incorporation and determination of amino sugars after acid hydrolysis. A third component (interleukin-2 M, 14.5 kDa) represents a nonglycosylated form. The amino acid composition and the NH2-terminal sequence of both forms are consistent with the data deduced from the cDNA coding for interleukin-2 after removal of a leader peptide of 20 amino acids. Carbohydrates are O-linked to the IL-2 protein via threonine-3 of the polypeptide chain. The oligosaccharides were released by reductive beta-elimination and were purified by gel filtration and high-performance liquid chromatography. Applying methylation analysis, exoglycosidase digestion and fast atom bombardment mass spectrometry the following major carbohydrate structures were identified: N1, NeuAc(alpha 2-3)Gal(beta 1-3)GalNAc-ol; and N2, NeuAc(alpha 2-3)Gal(beta 1-3)[NeuAc(alpha 2-6)]GalNAc-ol.     

Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures.

The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans was investigated using bovine articular cartilage explant cultures maintained in medium containing 10(-6) M retinoic acid or 40 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha) and varying concentrations (1-1000 microg/ml) of sulfated glycosaminoglycans (heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate) and calcium pentosan polysulfate (10 microg/ml). In addition, the effect of the sulfated glycosaminoglycans and calcium pentosan polysulfate on the degradation of aggrecan by soluble aggrecanase activity present in conditioned medium was investigated. The degradation of 35S-labeled aggrecan and reduction in tissue levels of aggrecan by articular cartilage explant cultures stimulated with retinoic acid or rHuIL-1alpha was inhibited by heparin and heparan sulfate in a dose-dependent manner and by calcium pentosan polysulfate. In contrast, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate did not inhibit the degradation of 35S-labeled aggrecan nor suppress the reduction in tissue levels of aggrecan by explant cultures of articular cartilage. Heparin, heparan sulfate and calcium pentosan polysulfate did not adversely affect chondrocyte metabolism as measured by lactate production, incorporation of [35S]-sulfate or [3H]-serine into macromolecules by articular cartilage explant cultures. Furthermore, heparin, heparan sulfate and calcium pentosan polysulfate inhibited the proteolytic degradation of aggrecan by soluble aggrecanase activity. These results suggest that highly sulfated glycosaminoglycans have the potential to influence aggrecan catabolism in articular cartilage and this effect occurs in part through direct inhibition of aggrecanase activity.