High-field n.m.r. studies of keratan sulphates. 1H and 13C assignments of keratan sulphate from shark cartilage.
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ABSTRACT:
SUBMITTER: Cockin GH
PROVIDER: S-EPMC1146928 | biostudies-other | 1986 Jun
REPOSITORIES: biostudies-other
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Project description:Keratan sulphate chains from bovine articular cartilage were fully digested with keratanase from Pseudomonas sp. and the products were reduced with alkaline borohydride. The resultant fragments were fractionated on a Nucleosil 5SB column and the earliest eluting fucose-containing oligosaccharides were isolated. Structural analysis using 1H n.m.r. spectroscopy (600 MHz) showed the two least-charged species to have the following structure: GlcNAc(6S) beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S) beta 1- 3Gal beta 1-4GlcNAc(6S) beta 1-3Gal-ol and GlcNAc(6S) beta 1-3Gal beta 1- 4(Fuc alpha 1-3)GlcNAc(6S) beta 1-3Gal beta 1-4GlcNAc(6S) beta 1-6(Gal beta 1- 3)GalNAc-ol. Both galactoses adjacent to the fucosylated N-acetylglucosamine residue are unsulphated. Therefore, it can be deduced from these structures that the presence of fucose on N-acetylglucosamine residues in keratan sulphates protects both of the adjacent unsulphated galactose residues from keratanase cleavage. This result has implications for the interpretation of keratanase fingerprints, because in articular cartilage keratan sulphates the keratanase-resistant blocks are not solely those with fully sulphated galactose residues, but also include the fucosylated sequences, which have unsulphated galactoses. It is, therefore, not possible to estimate their galactose sulphation or the size of the fully sulphated disaccharide-repeat sequences from keratan sulphates that contain fucose.
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Project description:The depsipeptide DNA-intercalating antibiotic luzopeptin was studied in solution by n.m.r. methods. Two-dimensional 1H double-quantum-filtered correlation spectroscopy (DQF-COSY) and nuclear-Overhauser-effect spectroscopy (NOESY) confirm the primary structure and twofold symmetry of luzopeptin and provide details of its three-dimensional conformation in solution. Trans-annular hydrogen bonds between the glycine NH groups and carbonyl oxygen atoms have been identified in the crystalline state [Arnold & Clardy (1981) J. Am. Chem. Soc. 103, 1243-1244], and are important in maintaining an antiparallel beta-sheet conformation. The n.m.r. data indicate that the glycine NH protons are appreciably shielded from the solvent molecules, which suggests that these hydrogen bonds are maintained in solution. The orientation of the quinoline chromophores is defined by two-dimensional NOE cross-peaks that position the N-methyl group of the L-beta-hydroxyvaline residue close in space to both the quinoline H-8 and serine NH proton. This pattern of NOEs is in accord both with the chromophore configuration found in the crystal and one where the quinoline rings are aligned in a parallel manner at right-angles to the depsipeptide ring. The n.m.r. data are consistent with a hydrogen bond between the quinoline hydroxy groups and the quinoline carbonyl oxygen atoms. The pyridazine acetylmethyl groups give NOEs to the C(alpha)H groups of the beta-hydroxy-N-methylvaline residues, showing that the acetyl groups, for at least some of the time, stretch over the depsipeptide ring, occluding one face of the molecule. Both of the latter features are also found in the crystal structure. Resonances in the 13C-n.m.r. spectrum of luzopeptin have been assigned by transferring 1H assignments to their covalently bonded carbon atoms via a heteronuclear shift-correlation experiment (HETCOR). The measurement of spin-lattice relaxation times and 1H-13C NOEs at specific sites in the molecule has led us to conclude that segmental motions within the depsipeptide ring are restricted and that the 13C relaxation data for luzopeptin's protonated carbon atoms are adequately described by isotropic tumbling in solution. Furthermore, relaxation data for the carbon atoms of the quinoline chromophores show that these rings exhibit similar motion to the depsipeptide ring and are not rotating rapidly with respect to it. Taken together all the data imply that luzopeptin is fairly rigid in solution, on the time scale of molecular tumbling, and has, or can readily attain, a staple-like structure suitable for bisintercalation.(ABSTRACT TRUNCATED AT 400 WORDS)
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Project description:Alkaline-borohydride-reduced keratan sulphate chains were isolated from bovine articular cartilage (6-8-year-old animals). Nine keratan sulphate fractions of increasing molecular weight were prepared by gel-permeation chromatography on a calibrated column of TSK 30 XL. The samples were analysed for fucose and galactose contents (% by wt. of keratan sulphate) and fucose/galactose ratio. The fucose content increased with molecular size, but the galactose content remained constant. It was concluded that the alpha(1----3)-linked fucose [Thornton, Morris, Cockin, Huckerby, Nieduszynski, Carlstedt, Hardingham & Ratcliffe (1989) Biochem. J. 260, 277-282] was located within the poly-N-acetyl-lactosamine repeat sequence of articular-cartilage keratan sulphate.
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