Ian A. Nieduszynski

Human aggrecan keratan sulfate undergoes structural changes during adolescent development

Alkaline borohydride-reduced keratan sulfate chains were isolated from human articular cartilage aggrecan from individuals of various ages (0–85 years old). The chains were structurally characterized using 1H NMR spectroscopy, gel permeation chromatography, and oligosaccharide profiling (after digestion with the enzymes keratanase and keratanase II). The results show that from birth to early adolescence (0–9 years) the levels of α(1–3)-fucosylation, α(2–3)-sialylation, and galactose sulfation increase. Also, the weight-average molecular weight of the chains increases. During maturation (9–18 years) the levels of fucosylation and galactose sulfation continue to increase and α(2–6)-sialylation of the chains occurs. In adult life (18–85 years) there is little change in the weight-average molecular weight of the chains, and the levels of fucosylation, sialylation, and sulfation remain fairly constant.

Fast-atom-bombardment mass-spectrometric strategies for sequencing sulphated oligosaccharides

Abstract A strategy is presented for the structural analysis of sulphated oligosaccharides. The oligosaccharides are permethylated to leave sulphate groups intact, and the products examined by negative f.a.b.-m.s. The fragmentation observed from such compounds in the negative mode is described for the first time. The sulphates are then chemically replaced by acetyl groups, so producing a derivative that is examined in the positive mode. This procedure yields sequence data and defines the residues on which the sulphates were originally located. The strategy is illustrated using glycosaminoglycan fragments.

Human Corneal Keratan Sulfates

The keratan sulfate-containing proteoglycans were isolated from fourteen pooled human corneas (thirteen from 61- to 86-year-olds, plus one from a 12-year-old). These proteoglycans were subjected to digestion with the enzyme keratanase II, and the released oligosaccharides, which included nonreducing termini and repeat region oligosaccharides but not linkage regions, were reduced with alkaline borohydride and identified on two separate ion-exchange columns. Both of the latter had been calibrated with samples, most of which had been derived from bovine corneal keratan sulfate (Tai, G.-H., Huckerby, T. N., and Nieduszynski, I. A. (1996) J. Biol. Chem. 271, 23535–23546) and all of which had been fully characterized by NMR spectroscopic analysis. The capping structures identified in human corneal keratan sulfates occurred in the relative proportions: NeuAcα(2-6)- >NeuAcα(2-3)- >GalNAc(S)β(1-3)-. The other groups of capping structures which had been identified in bovine corneal keratan sulfate, i.e. NeuGcα(2-3)-, NeuGcα(2-6)-, GlcNAc(S)β(1-3)- were absent, although the possibility of the presence of some Galα(1-3)- structures could not be excluded. In addition, the human sample showed significantly higher levels of α(1-3)-fucosylated repeat region structures than did the bovine sample, and it is not clear whether this reflects a species or age dependence as the bovine corneas were from young animals, whereas the human corneas were predominantly from an older group. The charge densities and keratan sulfate chain sizes of the human and bovine keratan sulfate-containing proteoglycans were seen to be similar.

Interactions between dermatan sulpahte chains. III Light-scattering and viscometry studies of self-association

Abstract 1. 1.Two dermatan sulphate preparations from pig skin, DS-18 and DS-36 with L-iduronic acid and D-glucuronic acid ratios of 90 : 10 and 75 : 25, respectively, were studied by light scattering and viscometry. 2. 2.In 0.15 M NaCl, both preparations yielded high particle weights (224 00 and 322 000, respectively) and viscometry at very low shear also yielded abnormally high intrinsic viscocities (108 and 128 ml/g, respectively). However, the two dermatan sulphate preparations affored ‘monomeric’ weight-average molecular weights (30 000 and 25 000) and normal intrinsic viscosities (49 and 35 ml/g) in 0.15 M KCl. Gel chromatography experiments further confirmed that dermatan sulphate chains have a tendency to self-associate in the presence of Na + . 3. 3.Dissociation of these ‘superaggregates’ in NaCl solutions could be accomplished by: (i) increasing the ionic strength to 0.5 M NaCl; (ii) increasing the shear rate to 10–20 s −1 . 4. 4.When these ‘superaggregates’ were dissociated in 0.15 M NaCl, weight-average particle weights of 32 000 and 41 000 were found for DS-18 and DS-36 respectively. It is suggested that a further level of self-association, probably dimerisation, occurs in ‘copolymeric’ dermatan sulphates that have a high glucuronic acid content, as previously proposed (Fransson, L-A. (1976) Biochim. Biophys. Acta 437, 106–115).

Increased incidence of unsulphated and 4-sulphated residues in the chondroitin sulphate linkage region observed by high-pH anion-exchange chromatography.

We report the isolation, characterization and quantification of five octasaccharides, four hexasaccharides and two tetrasaccharides, derived from the chondroitin sulphate (CS) linkage region of 6-8-year-old bovine articular cartilage aggrecan, following digestion with chondroitin ABC endolyase. Using a novel high-pH anion-exchange chromatography (HPAEC) method, in conjunction with one- and two-dimensional (1)H-NMR spectroscopy, we have identified the following basic structure for the CS linkage region of aggrecan: DeltaUA(beta1-3)GalNAc[0S/4S/6S](beta1-4)GlcA(beta1-3)GalNAc[0S/4S/6S](beta1-4)GlcA(beta1-3)Gal[0S/6S](beta1-3)Gal(beta1-4)Xyl, where DeltaUA represents 4,5-unsaturated hexuronic acid, and 4S and 6S represent an O-ester sulphate group on C-4 and C-6 respectively. The octa-, hexa- and tetra-saccharide linkage region fragments were used to develop a HPAEC fingerprinting method, with detection at A(232 nm), and a linear response to approx. 0.1 nmol of substance. The sulphation patterns of CS linkage regions, of up to octasaccharide in size, from articular and tracheal cartilage aggrecan were examined. The results show that in articular cartilage, for the majority (53%) of octasaccharides the 2-deoxy-2-N-acetyl amino-D-galactose (GalNAc) residues closest to the linkage region are both 6-sulphated; however, in a significant portion (34%), one or more of these GalNAc residues are unsulphated, and in 8% both are unsulphated. Approximately 10-18% of the chains have a 4-sulphated GalNAc in the first disaccharide, and 12% have a sulphated linkage region Gal residue. No evidence was found for uronic acid sulphation. These data show that there is a significant increase in the incidence of unsulphated and 4-sulphated GalNAc residues adjacent to the linkage region compared with the rest of the chain. Bovine tracheal cartilage linkage regions displayed very similar sulphation profiles to those from articular cartilage, despite the presence of a higher level of GalNAc 4-sulphation within the repeat region of the main CS chain.

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: ΔUA(β1–3)GalNAc4S(β1–4)l‐IdoA(α1–3)GalNAc4S(β1–4)l‐IdoA(α1–3)GalNAc4S‐ol, whereas one from bovine tracheal cartilage CS comprised only 6‐O‐sulfated residues and had the structure: ΔUA(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S‐ol. No oligosaccharide showed any uronic acid 2‐sulfation. One novel disaccharide was examined and found to have the structure: GalNAc6S(β1–4)GlcA‐ol. The trisaccharides isolated from the CS/DS chains were found to have the structures: ΔUA(β1–3)GalNAc4S(β1–4)GlcA‐ol and ΔUA(β1–3)GalNAc6S(β1–4)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(β1–4)GlcA(β1–3)GalNAc4S‐ol and GalNAc6S(β1–4)GlcA(β1–3)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.

Conformational equilibrium of unsulphated iduronate in heparan sulphate tetrasaccharides

Proton-proton coupling constants for terminal α-l-iduronate residues in tetrasaccharides obtained from heparan sulphates by complete nitrous acid deaminative cleavage were shown to vary with experimental conditions. It is proposed that the iduronate residue is in a conformational equilibrium between the1C4 chair and either the2So skewboat or possibly the2H3 half-chair conformers. It was not possible to discriminate between the two non-chair forms empirically. The position of the equilibrium is sensitive to temperature, pH and sulphation of neighbouring residues. The likelihood of iduronate residues within glycosaminoglycans existing in the4C1 conformer in addition to the1C4 and2So forms is discussed.

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

N.M.R. studies of the disulphated disaccharide obtained by degradation of bovine lung heparin with nitrous acid

The disulphated disaccharide IdoA(2SO3)-anManOH(6SO3) was prepared from bovine lung heparin by treatment with nitrous acid followed by borohydride reduction. The 1H- (400 MHz) and 13C-n.m.r. (100 MHz) spectra of this disaccharide derivative have been assigned completely using homonuclear spin-decoupling experiments, 13C-1H correlations, and a COSY-45 two-dimensional homonuclear correlation experiment. The 3JH,H values show that the IdoA(2SO3) residue exists in a single conformation throughout the temperature range 20-90 degrees.

On the mechanism of scission of alginate chains by periodate

Abstract The rapid, initial decrease in viscosity of alginate solutions containing periodate has been shown by light scattering and ultracentrifugation to be due to scission of the alginate molecule. This depolymerisation is not affected by ·OH scavengers such as 1-propanol or benzoate. 1-Propanol protects alginate against degradation by ·OH produced by pulse radiolysis. A slower process of degradation also goes on in periodate solutions, and this is suppressed by 1-propanol. The rapid-phase degradation is suggested to be due to scission of infrequent and specific components of the polymer chain.