Structural characterization of fucose-containing oligosaccharides by high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Eight pyridylamino (PA) derivatives of fucose-containing oligosaccharides, which occur as free oligosaccharides in human milk and also are derived from glycosphingolipids, have been analyzed by high-performance liquid chromatography (HPLC) on normal-phase and reversed-phase columns, and by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Six out of eight PA-oligosaccharides were clearly separated by both normal- and reversed-phase HPLC at a column temperature of 40 degrees C, but two PA-oligosaccharides, lacto-N-fucopentaose II [Gal beta1-3(Fuc alpha1-4)GlcNAc beta1-3Gal beta1-4GIcPA] and lacto-N-fucopentaose III [Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-3Gal beta1-4GIcPA], were not separated. The two unresolved PA-oligosaccharides were finally separated by reversed-phase HPLC at a column temperature of 11 degrees C. MALDI-TOF mass spectra of PA-oligosaccharides demonstrated pseudo-molecular ions as the predominant signals, therefore information about the molecular mass of each PA-oligosaccharide was easily obtained. Post-source decay (PSD) MALDI-TOF mass spectra of PA-oligosaccharides gave information about the carbohydrate sequences and carbohydrate species of each PA-oligosaccharide by detecting the ions responsible for the cleavage of the glycosidic bonds. The detection limits of the PA-oligosaccharides by HPLC, MALDI-TOF mass spectrometry, and PSD MALDI-TOF mass spectrometry were 20 fmol, 20 fmol, and 2 pmol, respectively. These results suggest that a system including HPLC and MALDI-TOF mass spectrometry or HPLC and PSD MALDI-TOF mass spectrometry is quite useful for the structural characterization of sub-pmol or pmol levels of fucose-containing oligosaccharides, and that these methods could be used for the analysis of various types of oligosaccharides derived from glycoproteins and glycosphingolipids.     

Cartilage degradation by hyaluronate lyase and chondroitin ABC lyase: a MALDI-TOF mass spectrometric study

Matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI-TOF MS) has been used to investigate degradation products of two selected polysaccharides of cartilage (chondroitin sulfate and hyaluronic acid). Testicular hyaluronate lyase and chondroitin ABC lyase were used for enzymic digestion of both polysaccharides as well as of cartilage specimens. Polysaccharide solutions and cartilage supernatants were assayed by positive and negative MALDI-TOF MS. Especially chondroitin ABC lyase produced high amounts of digestion products (unsaturated di- and tetrasaccharides) from polysaccharides as well as from cartilage, clearly monitored by MALDI-TOF MS. It is concluded that MALDI-TOF MS provides a precise and fast tool for the determination of oligosaccharides since no previous derivatization is required.     

Structure elucidation of arabinoxylan isomers by normal phase HPLC-MALDI-TOF/TOF-MS/MS

Normal phase-high performance liquid chromatography (NP-HPLC) coupled to matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry is evaluated for the detailed structural characterization of various isomers of arabinoxylan (AX) oligosaccharides produced from endo-beta-(1-->4)-xylanase (endoxylanase) digestion of wheat AX. The fragmentation characteristics of these oligosaccharides upon MALDI-TOF/TOF high-energy collision induced dissociation (CID) were investigated using purified AX oligosaccharide standards labeled at the reducing end with 2-aminobenzoic acid (2-AA). A variety of cross-ring cleavages and 'elimination' ions in the fragment ion spectra provided extensive structural information, including Araf substitution patterns along the xylan backbone and comprehensive linkage assignment. The off-line coupling of this MALDI-CID technique to capillary normal phase HPLC enabled the separation and identification of isomeric oligosaccharides (DP 4-8) produced by endoxylanase digestion of AX. Furthermore, this technique was used to characterize structurally different isomeric AX oligosaccharides produced by endoxylanase enzymes with different substrate specificities.     

Structural determination of five novel tetrasaccharides containing 3-O-sulfated D-glucuronic acid and two rare oligosaccharides containing a beta-D-glucose branch isolated from squid cartilage chondroitin sulfate E

Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAbeta1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing D-Glc. Their structures were determined by fast atom bombardment mass spectrometry and (1)H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but rather interspersed in the CS-E polysaccahride chains, being preferentially located in the highly sulfated sequences. The predominant structure on the nearest nonreducing side of a GlcA(3-O-sulfate) residue was GalNAc(4-O-sulfate) (80%), whereas that on the reducing side was GalNAc(4,6-O-disulfate) (59%). The structural variety in the vicinity of the GlcA(3-O-sulfate) residue might represent the substrate specificity of the unidentified chondroitin GlcA 3-O-sulfotransferase. The results also revealed a trisaccharide and a pentasaccahride sequence, both of which contained a beta-d-Glc branch at the C6 position of the constituent GalNAc residue. Approximately 5 mol % of all disaccharide units were substituted by Glc in the CS-E preparation used.     

Characterisation by mass spectrometry and 500-MHz proton nuclear magnetic resonance spectroscopy of penta- and hexasaccharide chains of human foetal gastrointestinal mucins (meconium glycoproteins)

Structural studies using liquid secondary ion mass spectrometry, gas liquid chromatography/mass spectrometry and 500-MHz 1H NMR are described of the major penta- and hexasaccharides of a fraction of human foetal gastrointestinal mucins. Glycoproteins from a blood group H active meconium pool were studied after depletion of Ii antigenic activities by immunoaffinity chromatography and treatment with mild acid hydrolysis to reduce the chain heterogeneity. Oligosaccharides were released by mild alkali/borohydride degradation and purified by Bio-Gel P4 chromatography and HPLC. Eleven penta- and hexasaccharides have been fully characterised as a result of this study and one previous report [Hounsell et al. (1988) Biochem. J. 256, 397-401] and information obtained on additional oligosaccharides present in small amounts. These oligosaccharides show the following features: (table; see text) Sequences in these oligosaccharides not commonly found in mucins so far studied are chain-terminating GlcNAc alpha 1-4Gal, repeating-type-I (Gal beta 1-3GlcNAc) backbones, the backbone branch GlcNAc beta 1-6(GlcNAc beta 1-3)Gal and the backbone sequence GlcNAc beta 1-6Gal beta 1- in the absence of a substituent at C3 of galactose.     

Development of an apparatus for rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in chondroitin sulfate-type proteoglycans

An apparatus, AutoGlycoCutter (AGC), was developed as a tool for rapid release of O-linked-type glycans under alkaline conditions. This system allowed rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in proteoglycans (PGs). After digestion of PGs with chondroitinase ABC, the oligosaccharides at the linkage region were successfully released from the protein core by AGC within 3 min. The reducing ends of the released oligosaccharides were labeled with 2-aminobenzoic acid and analyzed by a combination of capillary electrophoresis (CE) and matrix-assisted laser desorption time-of-flight mass spectrometry. In addition, the unsaturated disaccharides produced by chondroitinase ABC derived from the outer parts of the glycans were labeled with 2-aminoacridone and analyzed by CE to determine the disaccharide compositions. We evaluated AGC as a method for structural analysis of glycosaminoglycans in some chondroitin-sulfate-type PGs (urinary trypsin inhibitor, bovine nasal cartilage PG, bovine aggrecan, bovine decorin, and bovine biglycan). Recoveries of the released oligosaccharides were 57-73% for all PGs tested in the present study. In particular, we emphasize that the use of AGC achieved ca. 1000-fold rapid release of O-glycans compared with the conventional method.     

Structural determination of novel tetra- and hexasaccharide sequences isolated from chondroitin sulfate H (oversulfated dermatan sulfate) of hagfish notochord

Oversulfated chondroitin sulfate H (CS-H) isolated from hagfish notochord is a unique dermatan sulfate consisting mainly of IdoAalpha1-3GalNAc(4S,6S), where IdoA, GalNAc, 4S and 6S represent L-iduronic acid, Nacetyl-D-galactosamine, 4-O-sulfate and 6-O-sulfate, respectively. Several tetra- and hexasccharide fractions were isolated from CS-H after partial digestion with bacterial chondroitinase B to investigate the sequential arrangement of the IdoAalpha1-3GalNAc(4S,6S) unit in the CS-H polysaccharide. A structural analysis of the isolated oligosaccharides by enzymatic digestions, mass spectrometry and 1H NMR spectroscopy demonstrated that the major tetrasaccharides shared the common disulfated core structure delta4,5HexAalpha1-3GalNAc(4S)beta1-4IdoAalpha1-3 GalNAc (4S) with 0 approximately 3 additional O-sulfate groups, where delta4,5HexA represents 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid. The major hexasaccharides shared the common trisulfated core structure delta4,5HexAalpha1-3 GalNAc(4S)beta1-4 IdoAalpha1-3 GalNAc(4S)beta1-4IdoAalpha1-3 GalNAc(4S) with 1 approximately 4 additional O-sulfate groups. Some extra sulfate groups in both tetra- and hexasaccharides were located at the C-2 position of a delta4,5HexA or an internal IdoA residue, or C-6 position of 4-O-sulfated GalNAc residues, forming the unique disulfated or trisulfated disaccharide units, IdoA (2S)-GalNAc(4S), IdoA-GalNAc(4S,6S) and IdoA (2S)-GalNAc(4S,6S), where 2S represents 2-O-sulfate. Of the demonstrated sequences, five tetra- and four hexasaccharide sequences containing these units were novel.     

The unusual tetrasaccharide sequence GlcA{beta}-3GalNAc(4-sulfate)({beta}1-4GlcA(2-sulfate){beta}1-3GalNAc(6-sulfate) found in the hexasaccharides prepared by testicular hyaluronidase digestion of shark cartilage chondroitin sulfate D

Eight hexasaccharide fractions were isolated from commercialshark cartilage chondroitin sulfate D by means of gel nitrationchromatography and HPLC on an amine-bound silica column afterexhaustive digestion with sheep testicular hyaluronidase. Capillaryelectrophoresis of the enzymatic digests as well as one- andtwo-dimensional 500 MHz ¹H-NMR spectroscopy demonstrated thatthese hexasaccharides share the common core saccharide structureGlcAß1-3GalNAcß1-4GlcAß1-3GalNAcß1-4GlcAß1-3GalNAcwith three, four, or five sulfate groups in different combinations.Six structures had the same sulfation profiles as those of theunsaturated hexasaccharides isolated from the same source afterdigestion with chondroitinase ABC (Sugahara et al., Eur. J.Biochem., 293, 871–880, 1996) and the other two have notbeen reported so far. In the new components, a D disaccharideunit, GlcA(2-sulfate)ß1-3GalNAc(6-sulfate), characteristicof chondroitin sulfate D was arranged on the reducing side ofan A disaccharide unit, GlcAß1-3GalNAc(4-sulfate),forming an unusual A-D tetrasaccharide sequence, GlcAß1-3GalNAc(4-sulfate)-4GlcA(2-sulfate)ß1-3GaINAc(6-sulfate)which is known to be recognized by the monoclonal antibody MO225.These findings support the notion that the tetrasaccharide sequence,GlcAß1-3GalNAc(4-sulfate)ß1-4GlcAß1-3GalNAc(6-sulfate)is included in the acceptor site of a hitherto unreported 2-O-sulfotransferaseresponsible for its synthesis. The sulfated hexasaccharidesisolated in this study will be useful as authentic oligosaccharideprobes and enzyme substrates in studies of sulfated glycosaminogly-cans. sulfated hexasaccharides chondroitin sulfate D hyaluronidase ¹ H-NMR     

Structural characterization of multibranched oligosaccharides from seal milk by a combination of off-line high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and sequential exoglycosidase digestion

A complex mixture of diverse oligosaccharides related to the carbohydrates in glycoconjugates involved in various biological events is found in animal milk/colostrum and has been challenging targets for separation and structural studies. In the current study, we isolated oligosaccharides having high molecular masses (MW ∼ 3800) from the milk samples of bearded and hooded seals and analyzed their structures by off-line normal-phase-high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight (NP-HPLC-MALDI-TOF) mass spectrometry (MS) by combination with sequential exoglycosidase digestion. Initially, a mixture of oligosaccharides from the seal milk was reductively aminated with 2-aminobenzoic acid and analyzed by a combination of HPLC and MALDI-TOF MS. From MS data, these oligosaccharides contained different numbers of lactosamine units attached to the nonreducing lactose (Galβ1-4Glc) and fucose residue. The isolated oligosaccharides were sequentially digested with exoglycosidases and characterized by MALDI-TOF MS. The data revealed that oligosaccharides from both seal species were composed from lacto-N-neohexaose (LNnH, Galβ1-4GlcNAcβ1-6[Galβ1-4GlcNAcβ1-3]Galβ1-4Glc) as the common core structure, and most of them contained Fucα1-2 residues at the nonreducing ends. Furthermore, the oligosaccharides from both samples contained multibranched oligosaccharides having two Galβ1-4GlcNAc (N-acetyllactosamine, LacNAc) residues on the Galβ1-4GlcNAcβ1-3 branch or both branches of LNnH. Elongation of the chains was observed at 3-OH positions of Gal residues, but most of the internal Gal residues were also substituted with an N-acetyllactosamine at the 6-OH position.     

Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. II. Seven compounds containing 2 or 3 sulfate residues.

Shark cartilage proteoglycans bear predominantly chondroitin 6-sulfate. After exhaustive protease digestion, reductive beta-elimination and subsequent chondroitinase ABC digestion, 13 hexasaccharide alditols were obtained from the carbohydrate-protein linkage region and six of them contain 0 or 1 sulfate and/or 1 phosphate residue (Sugahara, K., Ohi, Y., Harada, T., de Waard, P., and Vliegenthart, J. F. G. (1992) J. Biol. Chem. 267, 6027-6035). The other seven compounds, which represent approximately 60% of the isolated linkage hexasaccharides, were analyzed by chondroitinase ACII digestion in conjunction with high performance liquid chromatography and by 500-MHz one- and two dimensional 1H NMR spectroscopy. All seven compounds have the following conventional structure in common. [formula: see text] Two disulfated compounds have an O-sulfate on C-6 of the Gal-2 residue attached to xylitol in combination with an O-sulfate on C-4 or on C-6 of the GalNAc residue. The third disulfated compound has O-sulfate on C-6 of Gal-2, and also on C-6 of Gal-3. Two of the trisulfated compounds also have O-sulfate on C-6 of both Gal-2 and Gal-3 with in addition sulfate on C-6 or C-4 of GalNAc. The other two trisulfated compounds have O-sulfate on C-6 of Gal-2 and on C-4 of Gal-3 in conjunction with sulfate on C-6 or C-4 of GalNAc.     

Novel sulfated octa- and decasaccharides from squid cartilage chondroitin sulfate E: sequencing and application for determination of the epitope structure of the monoclonal antibody MO-225

A mixture of octa- and decasaccharides obtained by the digestion with the hyaluronidase of chondroitin sulfate E derived from squid cartilage was subfractionated into 20 and 23 different components, respectively, by anion-exchange HPLC. MALDI-TOF/MS was used to assign the sugar and sulfate composition of the putative octa- and decasaccharides, and a disaccharide composition analysis revealed the building blocks to be A- [GlcUAbeta1-3GalNAc(4S)], C- [GlcUAbeta1-3GalNAc(6S)], and E- [GlcUAbeta1-3GalNAc(4S,6S)] units, where 4S and 6S represent 4-O- and 6-O-sulfate, respectively. The sequences of these octa- and decasaccharides were determined at low picomole amounts by a combination of enzymatic digestions with chondroitinases in conjunction with anion-exchange HPLC. Sequencing revealed that each fraction is a mixture of a major component together with one to three minor components, reflecting the heterogeneity of the parent polysaccharide. Among the 11 different octasaccharide sequences reported here, 8 are novel, while all of the 6 decasaccharide sequences are novel, and this is the first report of the sequencing of CS oligosaccharides longer than octasaccharides. The reactivity of the monoclonal antibody MO-225 with octa- and decasaccharides tested with an oligosaccharide microarray revealed that a CS-E decasaccharide is the minimal requirement for antibody recognition. Among the 6 decasaccharides, only E-E-E-E-C was recognized by MO-225, suggesting the requirement of a C-unit at the reducing end and also the importance of chain length, which in turn may indicate the importance of the conformation acquired by this specific sequence for antibody recognition.     

Structural analysis of sulfated N-linked oligosaccharides in erythropoietin.

We previously demonstrated that high-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column (GCC) is useful for the structural analysis of carbohydrates in glycoproteins. Using LC/MS with GCC, sulfated N-linked oligosaccharides were found in erythropoietin (EPO) expressed in baby hamster kidney cells. Sulfation occurs in a part of the N-linked oligosaccharides in the EPO. Sulfated monosaccharide residue in the sulfated N-linked oligosaccharide was determined by exoglycosidase digestion followed by sugar mapping by LC/MS. The linkage position and branch-location of the sulfate group in the tetraantennary oligosaccharide were analyzed by (1)H-nuclear magnetic resonance. It was suggested that sulfation occurs on the C-6 position of GlcNAc located in the GlcNAcbeta1-4Manalpha1-3 branch.     

The phosphorylated and/or sulfated structure of the carbohydrate-protein-linkage region isolated from chondroitin sulfate in the hybrid proteoglycans of Engelbreth-Holm-Swarm mouse tumor.

The structure of the linkage region of chondroitin sulfate chains attached to the hybrid proteoglycans of the Engelbreth-Holm-Swarm mouse tumor was investigated. The peptidoglycan fraction which contains oversulfated chondroitin sulfate rich in the GlcA beta 1-3GalNAc-4,6-diO-sulfate unit and undersulfated heparan sulfate rich in GlcA beta 1-4GlcNAc and GlcA beta 1-4GlcN-2N-sulfate units was isolated after exhaustive protease digestion of the acetone powder of the tumor tissue, (GlcA, glucuronic acid; GalNAc, 2-deoxy-2-N-acetylamino-D-galactose). Glycosaminoglycans were released by beta-elimination using NaB3H4 and digested with chondroitinase ABC. The linkage region fraction was separated from heparan sulfate by gel filtration and fractionated by HPLC on an amine-bound silica column. Six radiolabeled compounds (L1-L6) were obtained and structurally analyzed by cochromatography with authentic hexasaccharide alditols recently isolated by us from the linkage region, and by digestion using chondroitinase ACII, alkaline phosphatase and beta-galactosidase in conjugation with HPLC. These compounds shared the conventional hexasaccharide backbone structure: delta GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl-ol, (delta GlcA, delta 4.5-GlcA or D-gluco-4-enepyranosyluronic acid). L1 was not sulfated or phosphorylated. L2 and L4 were monosulfated at C-6 and C-4 of the GalNAc residue, respectively. Upon alkaline phosphatase digestion, L3, L5 and L6 were converted to L1, L2 and L4, respectively. Analysis of the periodate oxidation products indicated that the phosphate group in L3, L5 and L6 is located at C-2 of Xyl-ol. These results suggest that Xyl-2-O-phosphate is associated with both 4-O-sulfated and 6-O-sulfated GalNAc units and does not directly determine the sulfation pattern of chondroitin sulfate.     

Characterization of O-glycosidically linked oligosaccharides of rat erythrocyte membrane sialoglycoproteins

The carbohydrate units of the rat erythrocyte membrane sialoglycoprotein rSGP-4 [Edge, A. S. B., & Weber, P. (1981) Arch. Biochem. Biophys. 209, 697-705] have been characterized. All of the carbohydrate of this Mr 19,000 glycoprotein occurs in O-glycosidic linkage to the peptide; following alkaline borohydride treatment and chromatography on Bio-Gel P-2, sialic acid containing oligosaccharides terminating in N-acetylgalactosaminitol were obtained. Their structures were determined by compositional analysis, exoglycosidase digestions, alkaline sulfite degradation, and periodate oxidation. The oligosaccharides were characterized for molecular weight and linkage by direct chemical ionization and gas-liquid chromatography/mass spectrometry, respectively. The structures are proposed to be NeuAc alpha 2----3Gal beta 1----3GalNAc-ol, Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, and NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-ol. Two of the N-acetylglucosamine-containing hexasaccharides were present per molecule of rSGP-4 along with two trisaccharides and seven tetrasaccharides.     

Enzymatic synthesis of natural and 13C enriched linear poly-N-acetyllactosamines as ligands for galectin-1

As part of a study of protein-carbohydrate interactions, linear N-acetyl-polyllactosamines [Galbeta1,4GlcNAcbeta1,3]nwere synthesized at the 10-100 micromol scale using enzymatic methods. The methods described also provided specifically [1-13C]-galactose-labeled tetra- and hexasaccharides ([1-13C]-Galbeta1,4GlcNAcbeta1,3Galbeta1,4Glc and Galbeta1, 4GlcNAcbeta1,3[1-13C]Galbeta1,4GlcNAcbeta1,3Galbeta 1,4Glc) suitable for NMR studies. Two series of oligosaccharides were produced, with either glucose or N-acetlyglucosamine at the reducing end. In both cases, large amounts of starting primer were available from human milk oligosaccharides (trisaccharide primer GlcNAcbeta1,3Galbeta1, 4Glc) or via transglycosylation from N-acetyllactosamine. Partially purified and immobilized glycosyltransferases, such as bovine milk beta1,4 galactosyltransferase and human serum beta1,3 N- acetylglucosaminyltransferase, were used for the synthesis. All the oligo-saccharide products were characterized by1H and13C NMR spectroscopy and MALDI-TOF mass spectrometry. The target molecules were then used to study their interactions with recombinant galectin-1, and initial1H NMR spectroscopic results are presented to illustrate this approach. These results indicate that, for oligomers containing up to eight sugars, the principal interaction of the binding site of galectin-1 is with the terminal N-acetyllactosamine residues.     

Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin sulfate proteoglycans of whale cartilage.

From the carbohydrate-protein linkage region of whale cartilage proteoglycans, which bear predominantly chondroitin 4-sulfate, one nonsulfated, two monosulfated and one disulfated hexasaccharide alditols were isolated after exhaustive digestions with Actinase E and chondroitinase ABC, and subsequent beta-elimination. Their structures were analyzed by chondroitinase ACII digestion in conjunction with HPLC and by 500-MHz 1H-NMR spectroscopy. The nonsulfated compound (A) had the following conventional structure: delta GlcA(beta 1-3)-GalNAc(beta 1-4)GlcA(beta 1-3)Gal(beta 1-4)Xylol, where GlcA, delta GlcA and GalNAc are glucuronic acid; 4,5-unsaturated glucuronic acid and 2-deoxy-2-N-acetylamino-D-galactose, respectively. The other compounds were sulfated derivatives of compound A. Two monosulfated compounds (B and C) had an ester sulfate on C4 or C6 of the GalNAc residue, respectively and the disulfated compound (D) had two ester sulfate groups, namely, one on C4 of the GalNAc and the other on C4 of the Gal residue substituted by GlcA. The molar ratio of A/B/C/D was 0.21:0.16:0.36:0.27. The compound containing Gal-4-O-sulfate was previously isolated by us in the form of a sulfated glycoserine [delta GlcA(beta 1-3)GalNAc(4-O- sulfate)(beta 1-4)GlcA(beta 1-3)Gal(4-O-sulfate)(beta 1-3)-Gal(beta 1- 4)Xyl beta 1-O-Ser] from the carbohydrate-protein linkage region of rat chondrosarcoma chondroitin-4-sulfate proteoglycans [Sugahara K., Yamashina, I., DeWaard, P., Van Halbeek, H. & Vliegenthart, J.F.G. (1988) J. Biol. Chem. 263, 10,168-10,174]. The discovery of this structure in the carbohydrate-protein linkage region of chondroitin 4-sulfate proteoglycans from nontumorous cartilage indicates that it is not a tumor-associated product but rather a physiological biosynthetic product since it represents a significant proportion. The biological significance of this structure is discussed in relation to glycosaminoglycan biosynthesis.     

Characterizing the non-reducing end structure of heparan sulfate

The reducing end of heparan sulfate has been known for a long time, but information on the non-reducing end has been lacking. Recent studies indicate that the non-reducing end of heparan sulfate might be the place where fibroblast growth factor signaling complex forms. The non-reducing end also changes with heparanase digestion and, thus, might serve as a marker for tumor pathology. Using high performance liquid chromatography-coupled mass spectrometry, we have identified and characterized the non-reducing end of bovine kidney heparan sulfate. We find that the non-reducing end region is highly sulfated and starts with a glucuronic acid (GlcA) residue. The likely sequence of the non-reducing end hexasaccharides is GlcA-GlcNS6S-UA+/-2S-GlcNS+/-6S-Ido2S-GlcNS+/-6S (where GlcNS is N-sulfate-D-glucosamine, S is sulfate, UA is uronic acid, and Ido is iduronic acid). Our data suggests that the non-reducing end of bovine kidney heparan sulfate is not trimmed by heparanase and is capable of supporting fibroblast growth factor signaling complex formation.     

Characterization of oligosaccharides from the chondroitin/dermatan sulfates. 1H-NMR and 13C-NMR studies of reduced trisaccharides and hexasaccharides

Chondroitin and dermatan sulfate (CS and DS) chains were isolated from bovine tracheal cartilage and pig intestinal mucosal preparations and fragmented by enzymatic methods. The oligosaccharides studied include a disaccharide and hexasaccharides from chondroitin ABC lyase digestion as well as trisaccharides already present in some commercial preparations. In addition, other trisaccharides were generated from tetrasaccharides by chemical removal of nonreducing terminal residues. Their structures were examined by high-field 1H and 13C NMR spectroscopy, after reduction using sodium borohydride. The main hexasaccharide isolated from pig intestinal mucosal DS was found to be fully 4-O-sulfated and have the structure: DeltaUA(beta1-3)GalNAc4S(beta1-4)L-IdoA(alpha1-3)GalNAc4S(beta1-4)L-IdoA(alpha1-3)GalNAc4S-ol, whereas one from bovine tracheal cartilage CS comprised only 6-O-sulfated residues and had the structure: DeltaUA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S-ol. No oligosaccharide showed any uronic acid 2-sulfation. One novel disaccharide was examined and found to have the structure: GalNAc6S(beta1-4)GlcA-ol. The trisaccharides isolated from the CS/DS chains were found to have the structures: DeltaUA(beta1-3)GalNAc4S(beta1-4)GlcA-ol and DeltaUA(beta1-3)GalNAc6S(beta1-4)GlcA-ol. Such oligosaccharides were found in commercial CS/DS preparations and may derive from endogenous glucuronidase and other enzymatic activity. Chemically generated trisaccharides were confirmed as models of the CS/DS chain caps and included: GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc4S-ol and GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S-ol. The full assignment of all signals in the NMR spectra are given, and these data permit the further characterization of CS/DS chains and their nonreducing capping structures.     

Preparation and structural determination of dermatan sulfate-derived oligosaccharides

Eight oligosaccharides were prepared from dermatan sulfate (DS) and their structures were elucidated. Porcine intestinal mucosal DS was subjected to controlled depolymerization using chondroitin ABC lyase (chondroitinase ABC). The oligosaccharide mixture formed was fractionated by low-pressure gel permeation chromatography (GPC). Size uniform mixtures of disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, and dodecasaccharides were obtained. Each size-fractionated mixture was then purified on the basis of charge by repetitive semi-preparative strong-anion-exchange (SAX) high-performance liquid chromatography (HPLC). This approach has led to the isolation of six homogeneous oligosaccharides. The size of the oligosaccharides were determined using GPC-HPLC. Treatment of tetrasaccharide and hexasaccharide fragments with Hg(OAc)2 afforded trisaccharide and pentasaccharide products, respectively. The purity of the oligosaccharides obtained was confirmed by analytical SAX-HPLC, and capillary electrophoresis (CE). The molecular mass and degree of sulfation of the eight purified oligosaccharides were elucidated using electrospray ionization (ESI) mass spectrometry and their structures were established with high field nuclear magnetic resonance (NMR) spectroscopy. These DS-oligosaccharides are currently being used to study for interaction of the DS with biologically important proteins.     

Determination of the distribution of constituent disaccharide units within the chain near the linkage region of shark-cartilage chondroitin sulfate C

A method for analyzing the distribution of constituent disaccharide units within the chain near the linkage region of chondroitin sulfate has been developed. The method consists of (a) chemical modification of the reducing terminal residue in the polysaccharide by a 2-(2,4-dinitrophenylamino)ethylamino (DNP-AEA) group, (b) controlled fragmentation of the DNP-AEA-labeled polysaccharide with chondroitinase AC-I, followed by separation of the digestion products into the DNP-AEA-labeled fragments and unlabeled fragments on octyl-Sepharose CL-4B gel, (c) fractionation of the DNP-AEA-labeled fragments into fractions having different chain-lengths on Sephadex G-100 (superfine), and (d) determination of the disaccharide unit composition of the de-dinitropheylated products (AEA-labeled fragments) by the method combining chondroitinase AC-II treatment with HPLC analysis. A preparation of shark cartilage chondroitin sulfate C, which had been characterized well with regard to molecular species (Mr 48,000; average number of repeating disaccharide units (dpav) 93-94; consisting of chondroitin 6-sulfated 66.8%, 4-sulfated 22.5%, disulfated (D type) 10.3%, and nonsulfated units 0.4%), was analyzed by the above method. On the basis of the data obtained, distribution features of the disaccharide units within the chain near the linkage region of the polysaccharide (dpav 27) were estimated. It was, however, difficult to propose a final primary sequence of the polysaccharide chain, although there was a definite trend towards an enrichment of 4-sulfated and nonsulfated disaccharide residues in the area close to the linkage region (dpav 3-9 or 11). This was apparent together with an enrichment of 6-sulfated and disulfated disaccharide residues in the area distant from the linkage region (dpav 11 or 13-27).