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

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.

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).