SynopsisA structure determination of the naturally occrirring marine algal polysaccharide polyp-D-mannuronic acid is described. The structure consists of l e -+ 4e linked o-mannuronic acid chains with the monosaccharide units in the C1 chair conformation. The X-ray fiber diffraction photograph obtained from bundles of fibers prepared forom Fucus vesiculosus !as been in$exed to an orthorhombic unit cell in which a = 7.6 A, b (fiber axis) = 10.4 A, c = 8.6 A, the unit cell containing two disaccharide chain segments with space group P212121.A sheet-like structure involving one intra-chain, one intra-sheet, and one inter-sheet hydrogen bond per monosaccharide is proposed. Features of the chain-packing arrangement are compared with mannan.

A previously published method for the analysis of glycosaminoglycan disaccharides by high pH anion exchange chromatography (Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994), Glycobiology, 4, 333-342) has been modified and calibrated for chondroitin and dermatan sulfate oligosaccharides up to hexasaccharide in size and hyaluronan oligosaccharides up to hexadecasaccharide. For hyaluronan oligosaccharides chain length controls elution position; however, for chondroitin and dermatan sulfate oligosaccharides elution times primarily depend upon the level of sulfation, although chain length and hence charge density plays a role. The sulfation position of GalNAc residues within an oligosaccharide is also important in determining its elution position. Compared to 4-sulfation a reducing terminal 6-sulfate retards elution; however, when present on an internal GalNAc residue it is the 4-sulfate containing oligosaccharide which elutes later. These effects allow discrimination between oligosaccharides differing only in the position of GalNAc sulfation. Using this simple methodology, a Dionex CarboPac PA-1 column with NaOH/NaCl eluents and detection by absorbance at 232 nm, a quantitative analytical fingerprint of a chondroitin/dermatan sulfate chain may be obtained, allowing a determination of the abundance of chondroitin sulfate, dermatan sulfate, and hyaluronan along with an analysis of structural features with a linear response to ∼0.1 nmol. The method may readily be calibrated using either commercial disaccharides or the di-and tetrasaccharide products of a limit digest of commercial chondroitin sulfate by chondroitin ABC endolyase. Commercially available and freshly prepared shark, whale, bovine, and human cartilage chondroitin sulfates have been examined by this methodology and we have confirmed that freshly isolated shark cartilage CS contains significant amounts of the biologically important GlcA2Sβ(1-3)GalNAc6S structure.

Keratan sulfate chains were isolated from bovine articular cartilage (6-8-year-old animals) and digested with keratanase II, an endo-beta-N-acetylglucosaminidase [Nakazawa, K., Ito, M., Yamagata, T., & Suzuki, S. (1989) in Keratan Sulphate: Chemistry, Biology and Chemical Pathology (Greiling, H., & Scott, J. E., Eds.) pp 99-110, The Biochemical Society, London]. Twenty-five borohydride-reduced oligosaccharides were purified chromatographically and characterized by one- and two-dimensional NMR spectroscopy. From the structures of these oligosaccharides the following conclusions can be drawn about the mode of action of keratanase II: (1) The enzyme cleaves the beta (1-->3)-glycosidic bond between 6-O-sulfated N-acetyl-glucosamine and galactose, the major products being mono- and disulfated disaccharides. (2) Larger oligosaccharides containing keratanase II susceptible bonds are produced which are resistant to further degradation, e.g., tetrasaccharides from the sulfated poly(N-acetyllactosamine) repeat sequence, fucose-containing penta- and hexasaccharides, and hexa- and heptasaccharides from the linkage region. (3) The enzyme cleaves the beta (1-->3)-glycosidic bond of a fucosylated 6-O-sulfated N-acetylglucosamine. (4) Sialic acid-containing capping fragments are always recovered as pentasaccharides, despite the presence of an apparently susceptible bond. Two new elements of skeletal keratan sulfate structure, namely, the highly sulfated cap NeuAc alpha 2-3Gal(6S) beta 1-4GlcNAc (6S) beta 1-3Gal(6S) beta 1-4GlcNAc (6S)-ol and the difucosylated sequence Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S)beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S)-ol, have been identified. A structural model for articular cartilage keratan sulfate is proposed. The potential of the enzyme keratanase II for the structural fingerprinting of subnanogram quantities both of keratan sulfates and of sulfated oligosaccharide selectin ligands is discussed.

Alkaline borohydride-reduced keratan sulphate chains from bovine articular cartilage (6 -8-year-old animals) were subjected to a limit digest with the enzyme keratanase 11. Using 'H-NMR spectroscopy, 25 reduced oligosaccharides deriving from keratan sulphate were shown to have the following structures [GlcNAc(6S)-ol represents N-acetylglucosaminitol 6-0-sulphate] : Galp-4- However, the internal sulphated N-acetylglucosamine in the sialylated capping oligosaccharides is not cleaved because of the proximity of the sialic acid residue. In addition, keratanase II is the only degradative method examined so far which can cleave the glycosidic bond of a fucosylated Nacetylglucosamine residue as fucose residues confer resistance to both keratanase and hydrazinolysishitrous acid fragmentation.Keratan sulphate (KS), a glycosaminoglycan, was first isolated from bovine cornea by Meyer et al. (1953); it has since been discovered in many tissues including nucleus pulposus (Gardell and Rastageldi, 1954) and human cartilage (Meyer et al., 1958). Its structure is known to be based upon Abbreviations. KS, keratan sulphate ; GlcNAc-ol, N-acetylglucosaminitol (2-acetamido-~-~-glucitol) ; GalNAc-ol, N-acetylgalactosaminitol (2-acetamido-P-~-galactitol) ; Le", Galpl -4(F~~a1-3)-GlcNAc ; NeuAc, N-acetylneuraminic acid ; (6S), 0-ester sulphate group on C6.Enzymes. Trypsin from bovine pancreas (EC 3.4.21.4); chondroitin ABC lyase from Proteus vulgaris (EC 4.2.2.4); keratanase I1from Bacillus sp., endo-P-N-acetylglucosaminidase (EC 3.2

synopsisA structural investigation of the marine algal polysaccharide poly-a-L-guluronic acid is described. The molecular chains consist. of 1 + 4 diaxially linked Irguluronic acid residues in the 1C chair conformation and are stabilized in a twofold helii conformation by an intra-molecular O(2)H. . . O(6)D hydrogen-bond. The X-ray fiber diffraction photograph has been indexed to an orthorhombic unit cell in which a = 8.6 & b (fiber axis) = 8.7 A, c = 10.7A.A structure corresponding to the space group P212121 is proposed, in which all intermolecular hydrogen bonds interact with water molecules and in which all oxygen atoms except for the inaccessible bridge oxygens are involved. The relationship between the shape and structure of the polyguluronic acid molecule and its biological function is discussed.

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 1 H 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 ␣(1-3)-fucosylation, ␣(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 ␣(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.

The disaccharides IdoA(2SO3)-anManOH(6SO3) and IdoA-anManOH (where IdoA represents alpha-L-iduronate, anManOH represents 2,5-anhydro-D-mannitol and SO3 represents sulphate ester) were prepared from bovine lung heparin using HNO2 depolymerization, borohydride reduction and desulphation, and were examined by 400 MHz 1H-n.m.r. spectroscopy. Three-bond proton-proton coupling constants around the IdoA ring were determined under a range of experimental conditions. For unsulphated IdoA all four proton-proton coupling constants varied markedly as a function of temperature, pH and solvent, providing clear evidence for a rapid conformational equilibrium. These data were analysed in terms of the three most energetically stable IdoA conformers: 1C4, 4C1, and 2S0. Predicted coupling constants for these conformers were determined using a modified Karplus-type relationship. For unsulphated IdoA in dimethyl sulphoxide the equilibrium was provoked strongly in favour of a slightly distorted 4C1 'chair' IdoA conformer for which coupling constants have not previously been reported. For sulphated IdoA in aqueous conditions and at low pH the equilibrium is strongly in favour of the alternative 1C4 chair conformer. Under many conditions, however, significant contributions from all three conformers occur for the non-reducing terminal IdoA in these disaccharides.

High-field 1H-n.m.r.-spectroscopic studies supported by chemical carbohydrate analyses show that skeletal keratan sulphates (KS-II) of bovine origin may be sub-classified into two groups. Keratan sulphate chains from articular and intervertebral-disc cartilage (KS-II-A) contain two structural features, namely alpha(1----3)-fucose and alpha(2----6)-linked N-acetyl-neuraminic acid residues, that are absent from keratan sulphates from tracheal or nasal-septum cartilage (KS-II-B).