A full assignment of proton resonances for an alpha(1-3)-linked fucose residue in keratan sulphate from bovine articular cartilage.

Full proton NMR assignments have been achieved for the alpha(1-3)-linked fucose residues contained in alkaline borohydride reduced keratan sulphate chains derived from bovine articular cartilage. This involved 500 MHz spectroscopy at 60 degrees C and included COSY and RELAYED-COSY determinations.

Structural and immunological studies of keratan sulphates from mature bovine articular cartilage.

Two populations of alkaline-borohydride-reduced keratan sulphate (KS) chains were prepared from the two peptido-keratan sulphate trypsin fragments of proteoglycan aggregates isolated from bovine femoral head cartilage (6-year-old animals). Each population was separated by high-performance ion-exchange chromatography on a Pharmacia Mono-Q column into eight pools, Q1-Q8. These were analysed by gel permeation chromatography, radioimmunoassay with the monoclonal antibody MZ15, and 500 MHz 1H n.m.r. spectroscopy. Upon chromatography on Sephadex G-75 the Mono-Q fractions were shown to increase in hydrodynamic size progressively from Q1 to Q8 for both KS populations. For each population the strongest antigenic response with the anti-KS monoclonal antibody MZ15 was expressed by the two fractions of greatest size and charge density, Q7 and Q8. Proton n.m.r. spectroscopic studies on the two series of fractions demonstrated: (i) a progressive increase in the level of galactose sulphation from Q1 to Q8, (ii) the presence of approximately one alpha(1-3)-linked fucose residue per chain in every sample, and (iii) the presence of N-acetylneuraminic acids in three discrete environments, two alpha(2-3)- and one alpha(2-6)-linked in every sample. The results are discussed in terms of a possible heterogeneity in the carbohydrate-protein linkage region of keratan sulphates from bovine articular cartilage.

Structural studies of two populations of keratan sulphate chains from mature bovine articular cartilage.

Two discrete peptido-keratan sulphate fragments were isolated via chondroitinase ABC and trypsin digestion of a proteoglycan aggregate fraction prepared from bovine femoral head cartilage (six year old animals). The larger fragments (K(av) = 0.07, CL-6B) contained peptides substituted with several keratan sulphate (KS) chains from the KS-rich region of the proteoglycan and the smaller fragments (K(av) = 0.5, CL-6B) contained peptides with, perhaps, only one KS chain and the stubs of post-chondroitinase-treated chondroitin sulphate chains. The two peptido-KS samples and the KS chains derived from these by alkaline borohydride reduction were characterised by 13C-NMR spectroscopy. The two populations of KS chains were also examined by chromatography (Sephadex G-75), and keratanase digestion followed by chromatography on Bio-Gel P-10. From the results it was concluded that the KS chains from the two major trypsin-derived peptido-KS fragments had similar sulphation levels, distributions of hydrodynamic sizes and susceptibilities to keratanase.

High-field n.m.r. studies of keratan sulphates. 1H and 13C assignments of keratan sulphate from shark cartilage.

Keratan sulphate was extracted from a shark/whale cartilage preparation and examined by 400 MHz 1H- and 100 MHz 13C-n.m.r. spectroscopy. Assignment of the majority of the resonances was facilitated by two-dimensional 13C-1H correlation by using a modified COLOC procedure and a COSY-45 experiment. The spectra are consistent with an N-acetyl-lactosamine repeating unit that is predominantly sulphated at C-6 of both galactose and N-acetylglucosamine. Gel chromatography of a keratanase digest of the shark keratan sulphate confirmed the high degree of galactose sulphation.