TY - JOUR

T1 - Oligosaccharides Derived by Keratanase II Digestion of Bovine Articular Cartilage Keratan Sulphates.

AU - Brown, Gavin

AU - Huckerby, T. N.

AU - Nieduszynski, I. A.

PY - 1994

Y1 - 1994

N2 - 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 II. Using 1H-NMR spectroscopy, 25 reduced oligosaccharides deriving from keratan sulphate were shown to have the following structures [GlcNAc(6S)-ol represents N -acetylglucosaminitol 6-O -sulphate]: Galβ1–4-GlcNAc(6S)-ol, Galβ1-4GlcNAc(6S) β1–3Galβ1–4GlcNAc(6S)-ol, Gal(6S) β1–4GlcNAc(6S)-ol, Gal-(6S) β1–4GlcNAc(6S) β-1–3 Galβ-4GlcNAc(6S)-ol, Galβ1–4GlcNAc(6S) β1–3Gal(6S) β1–4GlcNAc(6S)-ol, Gal(6S) β1–4GlcNAc(6S) β1–3Gal(6S)1–4GlcNAc(6S)-ol, Galβ1–4(Fuca1–3)GlcNAc(6S)-ol, Galβ–4-(Fucα1–3)GlcNAc(6S) β1–3Galβ1–4(Fcα1–3)G1cNAc(6S)-ol, Galβ1–4GlcNAc(6S) β1–3Galβ1–4(Fucα1–3)-GlcNAc(6S)-ol, Galβ1–3(Fucα1–3)GlcNAc(6S) β1–3Galβ1–4GlcNAc(6S)-ol, Gal(6S) β1–4GlcNAc-(6S) β1–3Galβ1–4(Fucα1–3)GlcNAc(6S)-ol, Galβ1–4(Fucα1–3)GlcNAc(6S) β1–3Gal(6S) β1–4GlcNAc(6S)-ol, Galβ1–4 GlcNAc(6S) β1–6(Galβ1–3)GalNAc-ol, Galβ1–4 GlcNAc(6S) β1–6(NeuAc2–3Galβ1–3)Gal-Nac-ol, Galβ1–4GclNAc(6S) β1–3Galβ1–4GlcNAc(6S) β1–4GlcNAc(6S) β1–6(Galβ1–3)GalNAc-ol, Gal(6S) β1–4GlcNAc-(6S) β1–6(Galβ1–3)GalNAc-ol. Galβ1–4GlcNAc(6S) β1–3Galβ1–4GlcNAc (6S) β1–6(NeuAc2–3Galβ1–3)-GalNAc-ol. Gal(6S) β1–4GlcNAc-(6S) β1–6(NeuAcα2–3Galβ1–3)GalNAc-ol. Gal(6S) β1–4GlcNAc-(6S) β1–3Galβ1–4GlcNAc(6S) β1–6(Galβ1–3)GalNAc-ol, Gal(6S) β1–4GlcNAc(6S)β1–3Galβ1–4GlcNAc- (6S) β1–6(NeuAcα2–3Galβ1–3)GalNAc-ol,NeuAcα2–6Galβ1–4 GalNAc(6S) β1–3Galβ1–4 GlcNAc(6s)-ol, NecAcα2–3Galβ1–4GlcNAC(6s)β1–3Galβ1–4GlcNAc(6S)-ol, NeuAcα2–6Galβ1–4GlcNAc(6S)β1–3Gal-(6S)β1–4GlcNAc(6S)-ol, NeuAcα2–3Galβ1–4GlcNAc(6S) β1–3Gal(6S)β1–4GlcNAc(6S)-ol and Neu-Acα2–3Gal(6S) β1–4GlcNAc(6S)β1–3Gal(6Sβ)1–4GlcNAc(6S)-ol. Proton chemical shifts for these oligosaccharides were assigned using one- and two-dimensional NMR spectroscopic methods. These results confirm the findings of Nakazawa et al. [Nakazawa, K., Ito, M., Yamagata, T. and Suzuki, S. (1989) in Keratan sulphate: chemistry, biology and chemical pathology (Greiling, H. and Scott, J. E., eds) pp. 99–110, The Biochemical Society, London], namely that keratanase II cleaves the O -glycosidic bond of a β (1–3)-linked 6-O -sulphated N -acetylglucosamine. 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 N -acetylglucosamine residue as fucose residues confer resistance to both keratanase and hydrazino-lysisnitrous acid fragmentation.

AB - 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 II. Using 1H-NMR spectroscopy, 25 reduced oligosaccharides deriving from keratan sulphate were shown to have the following structures [GlcNAc(6S)-ol represents N -acetylglucosaminitol 6-O -sulphate]: Galβ1–4-GlcNAc(6S)-ol, Galβ1-4GlcNAc(6S) β1–3Galβ1–4GlcNAc(6S)-ol, Gal(6S) β1–4GlcNAc(6S)-ol, Gal-(6S) β1–4GlcNAc(6S) β-1–3 Galβ-4GlcNAc(6S)-ol, Galβ1–4GlcNAc(6S) β1–3Gal(6S) β1–4GlcNAc(6S)-ol, Gal(6S) β1–4GlcNAc(6S) β1–3Gal(6S)1–4GlcNAc(6S)-ol, Galβ1–4(Fuca1–3)GlcNAc(6S)-ol, Galβ–4-(Fucα1–3)GlcNAc(6S) β1–3Galβ1–4(Fcα1–3)G1cNAc(6S)-ol, Galβ1–4GlcNAc(6S) β1–3Galβ1–4(Fucα1–3)-GlcNAc(6S)-ol, Galβ1–3(Fucα1–3)GlcNAc(6S) β1–3Galβ1–4GlcNAc(6S)-ol, Gal(6S) β1–4GlcNAc-(6S) β1–3Galβ1–4(Fucα1–3)GlcNAc(6S)-ol, Galβ1–4(Fucα1–3)GlcNAc(6S) β1–3Gal(6S) β1–4GlcNAc(6S)-ol, Galβ1–4 GlcNAc(6S) β1–6(Galβ1–3)GalNAc-ol, Galβ1–4 GlcNAc(6S) β1–6(NeuAc2–3Galβ1–3)Gal-Nac-ol, Galβ1–4GclNAc(6S) β1–3Galβ1–4GlcNAc(6S) β1–4GlcNAc(6S) β1–6(Galβ1–3)GalNAc-ol, Gal(6S) β1–4GlcNAc-(6S) β1–6(Galβ1–3)GalNAc-ol. Galβ1–4GlcNAc(6S) β1–3Galβ1–4GlcNAc (6S) β1–6(NeuAc2–3Galβ1–3)-GalNAc-ol. Gal(6S) β1–4GlcNAc-(6S) β1–6(NeuAcα2–3Galβ1–3)GalNAc-ol. Gal(6S) β1–4GlcNAc-(6S) β1–3Galβ1–4GlcNAc(6S) β1–6(Galβ1–3)GalNAc-ol, Gal(6S) β1–4GlcNAc(6S)β1–3Galβ1–4GlcNAc- (6S) β1–6(NeuAcα2–3Galβ1–3)GalNAc-ol,NeuAcα2–6Galβ1–4 GalNAc(6S) β1–3Galβ1–4 GlcNAc(6s)-ol, NecAcα2–3Galβ1–4GlcNAC(6s)β1–3Galβ1–4GlcNAc(6S)-ol, NeuAcα2–6Galβ1–4GlcNAc(6S)β1–3Gal-(6S)β1–4GlcNAc(6S)-ol, NeuAcα2–3Galβ1–4GlcNAc(6S) β1–3Gal(6S)β1–4GlcNAc(6S)-ol and Neu-Acα2–3Gal(6S) β1–4GlcNAc(6S)β1–3Gal(6Sβ)1–4GlcNAc(6S)-ol. Proton chemical shifts for these oligosaccharides were assigned using one- and two-dimensional NMR spectroscopic methods. These results confirm the findings of Nakazawa et al. [Nakazawa, K., Ito, M., Yamagata, T. and Suzuki, S. (1989) in Keratan sulphate: chemistry, biology and chemical pathology (Greiling, H. and Scott, J. E., eds) pp. 99–110, The Biochemical Society, London], namely that keratanase II cleaves the O -glycosidic bond of a β (1–3)-linked 6-O -sulphated N -acetylglucosamine. 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 N -acetylglucosamine residue as fucose residues confer resistance to both keratanase and hydrazino-lysisnitrous acid fragmentation.

U2 - 10.1111/j.1432-1033.1994.00281.x

DO - 10.1111/j.1432-1033.1994.00281.x

M3 - Journal article

VL - 224

SP - 281

EP - 308

JO - European Journal of Biochemistry

JF - European Journal of Biochemistry

SN - 1432-1033

IS - 2

ER -