Domain structure of chondroitin sulfate E octasaccharides binding to type V collagen.

We demonstrated previously that chondroitin sulfate E (ChS-E) binds to type V collagen (Munakata, H., Takagaki, K., Majima, M., and Endo, M. (1999) Glycobiology 9, 1023--1027). In this study, we investigated the structure and binding of ChS-E oligosaccharides. Eleven oligosaccharides were isolated from ChS-E by gel filtration chromatography and anion-exchange high performance liquid chromatography after hydrolysis with testicular hyaluronidase. Separately, seven oligosaccharides were custom synthesized using the transglycosylation reaction of testicular hyaluronidase. Structural analysis was performed by enzymatic digestions in conjunction with high performance liquid chromatography and mass spectrometry. This library of 18 oligosaccharides was used as a source of model molecules to clarify the structural requirements for binding to type V collagen. Binding was analyzed by a biosensor based on surface plasmon resonance. The results indicated that to bind to type V collagen the oligosaccharides must have the following carbohydrate structures: 1) octasaccharide or larger in size; 2) a continuous sequence of three GlcAbeta1--3GalNAc(4S,6S) units; 3) a GlcAbeta1--3GalNAc(4S,6S) unit, GlcAbeta1--3GalNAc(4S) unit or GlcAbeta1--3GalNAc(6S) unit at the reducing terminal; 4) a GlcAbeta1--3GalNAc(4S,6S) unit at the nonreducing terminal. It is likely that these characteristic oligosaccharide sequences play key roles in cell adhesion and extracellular matrix assembly.     

Application of liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin

High-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS) were applied to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin (EPO) used as a model of the sialylated glycoprotein. N-linked oligosaccharides were released from recombinant human EPO expressed in Chinese hamster ovary cells enzymatically and reduced with NaBH(4). Many different sialylated oligosaccharides of EPO were separated and characterized by LC/MS equipped with a graphitized carbon column (GCC). Glycosylation sites and the preliminary glycosylation pattern at each glycosylation site were determined by LC/MS of endoproteinase Glu-C-digested EPO. The detailed site-specific carbohydrate heterogeneity caused by the differences in the molecular weight, branch, linkage, and sequence was elucidated by GCC-LC/MS of the N-linked oligosaccharides released from the isolated glycopeptides. Structural details of the isomers were analyzed by LC/MS/MS, and it was indicated that di- and trisialylated tetraantennary oligosaccharides are attached to Asn24, 38, and 83, whereas their isomers, di- and trisialylated triantennary oligosaccharides containing N-acetyllactosamines, are combined with Asn24. Our method is useful for the determination of glycosylation sites, the site-specific carbohydrate heterogeneity of glycoproteins, and the carbohydrate structure.     

Separation of oligosaccharides by capillary supercritical fluid chromatography and analysis by direct coupling to high-resolution mass spectrometer: application to analysis of oligomannosidic N-glycans

Supercritical fluid chromatography separations and supercritical fluid chromatography chemical ionization mass spectrometry analysis of permethylated and pertrimethylsilylated oligosaccharides are reported. Supercritical fluid chromatography was carried out using a DB-5 coated capillary column with carbon dioxide as a mobile phase. Peralkylated oligosaccharides were detected by flame ionization and by chemical ionization mass spectrometry using the GC interface. Analysis of permethylated malto-oligosaccharides, as well as oligomannosides from mannosidosis, was achieved by chemical ionization mass spectrometry with ammonia and provided the pseudo-molecular ions (M+H)+ and (M+NH4)+, in addition to some other fragments which allow interpretations of the structure of different oligosaccharides. The good resolution and sensitivity obtained emphasize the potential of supercritical fluid chromatography mass spectrometry for rapid separations and analysis of complex glycan mixtures.     

Glycosaminoglycan composition of the large freshwater mollusc bivalve Anodonta anodonta

In this paper, glycosaminoglycans from the body of the large freshwater mollusc bivalve Anodonta anodonta were recovered at about 0.6 mg/g of dry tissue, composed of chondroitin sulfate (approximately 38%), nonsulfated chondroitin (about 21%), and heparin (41%). This last polysaccharide was found to consist of a large percentage (approximately 88%) of a fast-moving species possessing a lower molecular mass and sulfate group amount and about 12% of a more sulfated, slow-moving component having a greater molecular mass. The chondroitin sulfate was composed of approximately 28% of the 6-sulfated disaccharide, 46% of the 4-sulfated disaccharide, and about 26% of the nonsulfated disaccharide, with a charge density value of 0.74. Heparin was subjected to the oligosaccharide mapping after treatment with heparinase and then separation of the resulting unsaturated oligosaccharides by SAX-HPLC. A heparin sample from Anodonta anodonta showed a degree of sulfation similar to that of bovine mucosal heparin because of the presence of approximately the same mol % of the trisulfated disaccharide (DeltaUA2S(1-->4)-alpha-D-GlcN2S6S), a slight modification of the other oligosaccharides, and a significant increase of the disaccharide bearing the sulfate group in position 3 of the N-sulfoglucosamine 6-sulfate (-->4)-beta-D-GlcA(1-->4)-alpha-D-GlcN2S3S6S(1-->) part of the ATIII-binding region. However, the anticoagulant activity of mollusc heparin was quite similar to that of pharmaceutical grade heparin. The data obtained again emphasize the heterogeneity of GAGs from molluscs.     

Structure of sialylated fucosyl lactosaminoglycan isolated from human granulocytes

Sialylated fucosyl lactosaminoglycan was isolated from human neutrophilic granulocytes and its structure was elucidated. The lactosaminoglycan glycopeptides were digested by endo-beta-galactosidase and "the core portion" and released oligosaccharides were analyzed by permethylation, fast atom bombardment mass spectrometry, and exoglycosidases. In addition, lactosaminoglycan saccharides were obtained by hydrazinolysis and the structures of fractionated sialyl oligosaccharides were analyzed by fast atom bombardment mass spectrometry and permethylation coupled with exoglycosidase treatment. The structure of one of the major components was found to be: (Formula: see text). This structure is unique in that 1) four linear polylactosaminyl side chains are attached to the core portion, 2) the side chain arising from position 4 of 2,4-linked mannose contains one or more alpha 1----3 fucosyl residues, 3) the side chain arising from position 6 of 2,6-linked mannose is terminated with NeuNAc alpha 2----3Gal(Fuc alpha 1----3)GlcNAc, sialyl Lex, and 4) the side chain arising from position 2 of 2,4-linked mannose is terminated with sialic acid through alpha 2----6 linkage.     

Depolymerization of the capsular polysaccharide from Vibrio cholerae O139 by a lyase associated with the bacteriophage JA1.

We have studied the interaction between the Vibrio cholerae O139 specific phage JA1, belonging to the Podoviridae family, and the capsular polysaccharide (CPS) of the parent strain from which the phage was isolated. Upon incubation of the JA1 phage with the CPS, oligosaccharides were isolated and purified. The oligosaccharides derived from one (shown below) and two repeating units of the CPS were characterized using NMR spectroscopy, mass spectrometry and sugar analysis (structure: see text). The cleavage was found to occur by beta-elimination at the 4-substituted alpha-linked galacturonic acid, which results in a 4-deoxy-beta-L-threo-hex-4-enopyranosyl uronic acid group (Sug). The enzyme associated with the JA1 phage responsible for the depolymerization of the V. cholerae O139 CPS is thus a lyase.     

Structural analysis of oligosaccharides from lipopolysaccharide (LPS) of Escherichia coli K12 strain W3100 reveals a link between inner and outer core LPS biosynthesis

Lipopolysaccharide (LPS) from Escherichia coli K12 W3100 is known to contain several glycoforms, and the basic structure has been investigated previously by methylation analyses (Holst, O. (1999) in Endotoxin in Health and Disease (Brade, H., Opal, S. M., Vogel, S. N., and Morrison, D., eds) pp. 115-154; Marcel Dekker, Inc., New York). In order to reveal dependences of gene activity and LPS structure, we have now determined the composition of de-O-acylated LPS by electrospray ionization-Fourier transform ion cyclotron-mass spectrometry (ESI-FT-MS) and identified 11 different LPS molecules. We have isolated the major glycoforms after de-O- and de-N-acylation and obtained four oligosaccharides that differed in their carbohydrate structure and phosphate substitution. The main oligosaccharide accounted for approximately 70% of the total and had a molecular mass of 2516 Da according to ESI-FT-MS. The dodecasaccharide structure (glycoform I) as determined by NMR was consistent with MS and compositional analysis. One minor oligosaccharide (5%) of the same carbohydrate structure did not contain the 4'-phosphate of the lipid A. Two oligosaccharides contained the same phosphate substitution but differed in their carbohydrate structure, one (5%) which contained an additional beta-D-GlcN in 1-->7 linkage on a terminal heptose residue (glycoform II) which was N-acetylated in LPS. A minor amount of a molecule lacking the terminal L-alpha-D-Hep in the outer core but otherwise identical to the major oligosaccharide (glycoform III) could only be identified by ESI-FT-MS of the de-O-acylated LPS. The other oligosaccharide (20%) contained an alpha-Kdo-(2-->4)-[alpha-l-Rha-(1-->5)]-alpha-Kdo-(2-->4)-alpha-Kdo branched tetrasaccharide connected to the lipid A (glycoform IV). This novel inner core structure was accompanied by a truncation of the outer core in which the terminal disaccharide L-alpha-D-Hep-(1-->6)-alpha-D-Glc was missing. The latter structure was identified for the first time in LPS and revealed that changes in the inner core structure may be accompanied by structural changes in the outer core.     

Structural analysis of N-linked oligosaccharides from glycoproteins secreted by Dictyostelium discoideum. Identification of mannose 6-sulfate

The N-linked oligosaccharides found on the lysosomal enzymes from Dictyostelium discoideum are highly sulfated and contain methylphosphomannosyl residues (Gabel, C. A., Costello, C. E., Reinhold, V. N., Kurtz, L., and Kornfeld, S. (1984) J. Biol. Chem. 259, 13762-13769). Here we report studies done on the structure of N-linked oligosaccharides found on proteins secreted during growth, a major portion of which are lysosomal enzymes. Cells were metabolically labeled with [2-3H]Man and 35SO4 and a portion of the oligosaccharides were released by a sequential digestion with endoglycosidase H followed by endoglycosidase/peptide N-glycosidase F preparations. The oligosaccharides were separated by anion exchange high performance liquid chromatography into fractions containing from one up to six negative charges. Some of the oligosaccharides contained only sulfate esters or phosphodiesters, but most contained both. Less than 2% of the oligosaccharides contained a phosphomonoester or an acid-sensitive phosphodiester typical of the mammalian lysosomal enzymes. A combination of acid and base hydrolysis suggested that most of the sulfate esters were linked to primary hydroxyl groups. The presence of Man-6-SO4 was demonstrated by the appearance of 3,6-anhydromannose in acid hydrolysates of base-treated, reduced oligosaccharides. These residues were not detected in acid hydrolysates without prior base treatment or in oligosaccharides first treated by solvolysis to remove sulfate esters. Based on high performance liquid chromatography quantitation of percentage of 3H label found in 3,6-anhydromannose, it is likely that Man-6-SO4 accounts for the majority of the sulfated sugars in the oligosaccharides released from the secreted glycoproteins.     

High-pH anion-exchange chromatography with pulsed amperometric detection and molar response factors of human milk oligosaccharides

A method is described to separate and characterize neutral and acidic lactose-derived oligosaccharides without prior derivatization or reduction by high-pH anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD). This method has been applied to human milk oligosaccharides from donors with different blood group specificity (A, Le^a and A, Le^b). Neutral and acidic components were separated from each other by anion-ecchange chromatography. A distinct separation of individual components was obtained by size-exclusion chromatography on Fractogel TSK HW 50S (acidic oligosaccharides) or Fractogel TSK HW 40S (neutral oligosaccharides containing up to 6 monomers) and Bio-Gel P-4 size exclusion (neutral oligosaccharides containing more than 6 monomers). Furthermore, the molar response factors after HPAEC-PAD have been determined for 8 components.     

A computational study of structure-reactivity relationships in Na-adduct oligosaccharides in collision-induced dissociation reactions

Elucidating the fragmentation mechanisms in oligosaccharides using theoretical calculations is useful in analyzing the experimentally obtained mass spectra. Semi-empirical and ab initio quantum mechanics calculations were used to study the relationship between the structure and reactivity and the chemical properties of oligosaccharides. In these calculations, sodium-cationized oligosaccharides were investigated to determine Na+ ion affinity at several binding positions; in addition, the dependence of the glycosidic bond cleavage on the Na+ position was examined. The calculated structures reported in this study are directed at interpreting experimentally observed fragment ions, resulting from the cleavage of the glycosidic bonds. The calculated results for oligosaccharides containing between three and five monosaccharide units (27 oligosaccharides) were compared with experimental data generated by matrix-assisted laser-desorption/ionization (MALDI) using a quadrupole ion trap (QIT) with a time-of-flight (TOF) mass spectrometer (MS).     

Molecular cloning and expression of endo-beta-N-acetylglucosaminidase D, which acts on the core structure of complex type asparagine-linked oligosaccharides

Endo-beta-N-acetylglucosaminidase D (Endo D) produced by Streptococcus pneumoniae cleaves the di-N-acetylchitobiose structure in asparagine-linked oligosaccharides. The enzyme generally acts on complex type oligosaccharides after removal of external sugars by neuraminidase, beta-galactosidase, and beta-N-acetylglucosaminidase. We cloned the gene encoding the enzyme and expressed it as a periplasm enzyme in Escherichia coli. The first 37 amino acids in the predicted sequence are removed in the mature enzyme, yielding a protein with a molecular mass of 178 kDa. The substrate specificity of the recombinant enzyme is indistinguishable from the enzyme produced by S. pneumoniae. Endo-beta-N-acetylglucosaminidase A (Endo A) from Arthrobacter protophormiae, the molecular mass of which is 72 kDa, had 32% sequence identity to Endo D, starting from the N-terminal sides of both enzymes, although Endo A hydrolyzes high-mannose-type oligosaccharides and does not hydrolyze complex type ones. Endo D is not related to endo-beta-N-acetylglucosaminidases H, F(1), F(2), or F(3), which share common structural motifs. Therefore, there are two distinct groups of endo-beta-N-acetylglucosaminidases acting on asparagine-linked oligosaccharides. The C-terminal region of Endo D shows homology to beta-galactosidase and beta-N-acetylglucosaminidase from S. pneumoniae and has an LPXTG motif typical of surface-associated proteins of Gram-positive bacteria. It is possible that Endo D is located on the surface of the bacterium and, together with other glycosidases, is involved in virulence.     

Stability of non-Watson-Crick G-A/A-G base pair in synthetic DNA and RNA oligonucleotides

A non-Watson-Crick G-A/A-G base pair is found in SECIS (selenocysteine-insertion sequence) element in the 3'-untranslated region of Se-protein mRNAs and in the functional site of the hammerhead ribozyme. We studied the stability of G-A/A-G base pair (bold) in 17mer GT(U)GACGGAAACCGGAAC synthetic DNA and RNA oligonucleotides by thermal melting experiments and gel electrophoresis. The measured Tm value of DNA oligonucleotide having G-A/A-G pair showed an intermediate value (58 degrees C) between that of Watson-Crick G-C/C-G base pair (75 degrees C) and that of G-G/A-A of non-base-pair (40 degrees C). Similar thermal melting patterns were obtained with RNA oligonucleotides. This result indicates that the secondary structure of oligonucleotide having G-A/A-G base pair is looser than that of the G-C type Watson-Crick base pair. In the comparison between RNA and DNA having G-A/A-G base pair, the Tm value of the RNA oligonucleotide was 11 degrees C lower than that of DNA, indicating that DNA has a more rigid structure than RNA. The stained pattern of oligonucleotide on polyacrylamide gel clarified that the mobility of the DNA oligonucleotide G-A/A-G base pair changed according to the urea concentration from the rigid state (near the mobility of G-C/C-G oligonucleotide) in the absence of urea to the random state (near the mobility of G-G/A-A oligonucleotide) in 7 M urea. However, the RNA oligonucleotide with G-A/A-G pair moved at an intermediate mobility between that of oligonucleotide with G-C/C-G and of the oligonucleotide with G-G/A-A, and the mobility pattern did not depend on urea concentration. Thus, DNA and RNA oligonucleotides with the G-A/A-G base pair showed a pattern indicating an intermediate structure between the rigid Watson-Crick base pair and the random structure of non-base pair. RNA with G-A/A-G base pair has the intermediate structure not influenced by urea concentration. Finally, this study indicated that the intermediate rigidity imparted by Non-Watson-Crick base pair in SECIS element plays an important role in the selenocysteine expression by UGA codon.     

Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast.

The N-glycans of purified recombinant middle surface protein (preS2+S) from hepatitis B virus, a candidate vaccine antigen expressed in a mnn9 mutant strain of Saccharomyces cerevisiae, have been characterized structurally. The glycans were released by N-glycanase treatment, isolated by size-exclusion chromatography on Sephadex G-50 and Bio-Gel P-4 columns, and analyzed by 500-MHz 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The mixture of oligosaccharides was fractionated by HPLC, the major subfractions were isolated, and their carbohydrate compositions were determined by high-pH anion-exchange chromatography with pulsed amperometric detection. The combined results suggest that high-mannose oligosaccharides account for all the N-glycans released from preS2+S: structures include Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 isomers in the ratios of 3:6:1. Approximately 80% of the oligosaccharides contain the C2,C6-branched trimannosyl structural element typical of yeast high-mannose oligosaccharides but not usually found in high-mannose oligosaccharides in animal glycoproteins.     

Structural studies on kappa-carrageenan derived oligosaccharides

Oligosaccharides were prepared through mild hydrochloric acid hydrolysis of kappa-carrageenan from Kappaphycus striatum carrageenan. Three oligosaccharides were purified by strong-anion exchange high-performance chromatography. Their structure was elucidated using mass spectral and NMR data. Negative-ion electrospray ionization (ESI) mass spectra at different fragmentor voltages provided the molecular weight of the compounds and unraveled the fragmentation pattern of the kappa-carrageenan oligosaccharides. 2D NMR techniques, including 1H-(1)H COSY, 1H-(1)H TOCSY and 13C-(1)H HMQC, were performed to determine the structure of a trisulfated pentasaccharide. 1D NMR and ESIMS were used to determine the structures of a kappa-carrageenan-derived pentasaccharide, heptasaccharide, and an undecasaccharide. All the oligosaccharides characterized have a 4-O-sulfo-D-galactopyranose residue at both the reducing and nonreducing ends.     

Oligosaccharide-Related Epitope Specific for a Brain-Specific Glycoprotein, 1D4 Antigen

The characteristics of glycosylation of a brain-specific glycoprotein, 1D4 antigen, and the epitope recognized by its monoclonal antibody were studied. Removal of high-mannose and hybrid types of N-linked oligosaccharides by treatment with endoglycosidase H converted the molecular mass of the 1D4 antigen from 89 kDa to 78 kDa, but did not affect its reactivity with the 1D4 monoclonal antibody. Removal of all types of N-linked oligosaccharides by treatment with glycopeptidase F or removal of both N- and O-linked oligosaccharides by chemical treatment caused both reduction of the molecular mass of the antigen to 63 kDa and loss of its reactivity with the monoclonal antibody. These results suggest that the 1D4 monoclonal antibody recognizes a complex-type oligosaccharide-related epitope specific for the 1D4 antigen. Results also showed that N-linked glycosylation was not responsible for the charge heterogeneity of the 1D4 antigen. The oligosaccharide chain-related epitope was detected in rat brain but not in mouse, rabbit, or bovine brain, but the 1D4 antigen was recognized in rat and mouse brains with antiserum (polyclonal antibodies). These findings indicate that the oligosaccharide-related epitope is species specific. Furthermore, results with neuraminidase-treated 1D4 antigen indicated that sialic acids were not involved in the oligosaccharide-related epitope. These findings suggest that the 1D4 antigen may have the oligosaccharide structure specific for rat brain and itself.     

The spectrum of N-linked oligosaccharide structures detected by enzymic microsequencing on a recombinant soluble CD4 glycoprotein from Chinese hamster ovary cells

Structures of the N-linked oligosaccharides of a recombinant soluble form of human CD4 glycoprotein (sCD4) have been investigated by enzymic microsequencing. The glycoprotein has two N-glycosylation sites, Asn271 and Asn300, at both of which evidence for the presence of complex type biantennary sialo-oligosaccharides has been obtained previously by mass spectrometric analyses [Carr, S.A., Hemling, M.E., Folena-Wasserman, G., Sweet, R.W., Anumula, K., Barr, J.R., Huddleston, M.J. & Taylor, P. (1989) J. Biol. Chem. 264, 21,286-21,295]. Among oligosaccharides released from sCD4 by hydrazinolysis and labelled with NaB3H4, neutral (12.8%) and acidic (87.2%) oligosaccharides were detected by paper electrophoresis. The latter were rendered neutral following sialidase treatment indicating that acidity was due exclusively to the presence of sialic acid residues. By enzymic microsequencing of the sialidase-treated oligosaccharides (fractionated on affinity columns of Ricinis communis agglutinin 120 and concanavalin A) in conjunction with methylation data from the earlier study, 14 sequences were identified. These accounted for over 80% of the sialidase-treated oligosaccharides of sCD4 as follows: [formula: see text] where +/- indicates residues present on only a proportion of chains. The spectrum of oligosaccharide structures released from each glycosylation site was assessed as being similar to that of total oligosaccharides on the basis of their chromatographic profiles on the lectin columns and on Bio-Gel P-4.     

Glycoproteomics of milk: differences in sugar epitopes on human and bovine milk fat globule membranes

Oligosaccharides from human and bovine milk fat globule membranes were analyzed by LC-MS and LC-MS/MS. Global release of N-linked and O-linked oligosaccharides showed both to be highly sialylated, with bovine peak-lactating milk O-linked oligosaccharides presenting as mono- and disialylated core 1 oligosaccharides (Galbeta1-3GalNAcol), while human milk had core type 2 oligosaccharides (Galbeta1-3(GlcNAcbeta1-6)GalNAcol) with sialylation on the C-3 branch. The C-6 branch of these structures was extended with branched and unbranched N-acetyllactosamine units terminating in blood group H and Lewis type epitopes. These epitopes were also presented on the reducing terminus of the human, but not the bovine, N-linked oligosaccharides. The O-linked structures were found to be attached to the high molecular mass mucins isolated by agarose-polyacrylamide composite gel electrophoresis, where MUC1 and MUC4 were present. Analysis of bovine colostrum showed that O-linked core 2 oligosaccharides are present at the early stage (3 days after birth) but are down-regulated as lactation develops. This data indicates that human milk may provide different innate immune protection against pathogens compared to bovine milk, as evidenced by the presence of Lewis b epitope, a target for the Helicobacter pylori bacteria, on human, but not bovine, milk fat globule membrane mucins. In addition, non-mucin-type O-linked fucosylated oligosaccharides were found (NeuAc-Gal-GlcNAc1-3Fuc-ol in bovine milk and Gal-GlcNAc1-3Fuc-ol in human milk). The O-linked fucose structure in human milk is the first to our knowledge to be found on high molecular mass mucin-type molecules.     

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.     

A defect in exodegradative pathways provides insight into endodegradation of heparan and dermatan sulfates

Within cells, dermatan sulfate (DS) and heparan sulfate (HS) are degraded in two steps. The initial endohydrolysis of these polysaccharides is followed by the sequential action of lysosomal exoenzymes to reduce the resulting oligosaccharides to monosaccharides and inorganic sulfate. Mucopolysaccharidosis (MPS) type II is a lysosomal storage disorder caused by a deficiency of the exoenzyme iduronate-2-sulfatase (I2S). Consequently, partially degraded fragments of DS and HS have been shown to accumulate in the lysosomes of affected cells and are excreted in the urine. Di- to hexadecasaccharides, isolated from the urine of a MPS II patient using anion exchange and gel filtration chromatography, were identified using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). These oligosaccharides were shown to have non-reducing terminal iduronate-2-sulfate residues by digestion with recombinant I2S. A pattern of growing oligosaccharide chains composed of alternating uronic acid and N-acetylhexosamine residues was identified and suggested to originate from DS. A series of oligosaccharides consisting of hexosamine/N-acetylhexosamine alternating with uronic acid residues was also identified and on the basis of the presence of unacetylated hexosamine; these oligosaccharides are proposed to derive from HS. The presence of both odd and even-length oligosaccharides suggests both endo-beta-glucuronidase and endo-N-acetylhexosaminidase activities toward both glycosaminoglycans. Furthermore, the putative HS oligosaccharide structures identified indicate that heparanase activities are directed toward regions of both low and high sulfation, while the N-acetylhexosaminidase activity acted only in regions of low sulfation in this polysaccharide.