OLIGOSACCHARIDES DERIVED FROM BOVINE ARTICULAR-CARTILAGE KERATAN SULFATES AFTER KERATANASE-II DIGESTION - IMPLICATIONS FOR KERATAN SULFATE STRUCTURAL FINGERPRINTING

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., and Suzuki , S. (1989) in Keratan Sulphate: Chemistry, Biology and Chemical Patho logy (Greiling, H., and Scott, J.E., Eds.) pp 99-110, The Biochemical Society, London. Twenty-five borohydride-reduced oligosaccharides wer e purified chromatographically and characterized by one- and two-dimen sional NMR spectroscopy. From the structures of these oligosaccharides the following conclusions can be drawn about the mode of action of ke ratanase II: (1) The enzyme cleaves the beta(1-->3)-glycosidic bond be tween 6-O-sulfated N-acetyl-glucosamine and galactose, the major produ cts being mono- and disulfated disaccharides. (2) Larger oligosacchari des containing keratanase II susceptible bonds are produced which are resistant to further degradation, e.g., tetrasaccharides from the sulf ated poly(N-acetyllactosamine) repeat sequence, fucose-containing pent a- and hexasaccharides, and hexa- and heptasaccharides from the linkag e region. (3) The enzyme cleaves the beta(1--> 3)-glycosidic bond of a fucosylated 6-O-sulfated N-acetylglucosamine. (4) Sialic acid-contain ing capping fragments are always recovered as pentasaccharides, despit e the presence of an apparently susceptible bond. Two new elements of skeletal keratan sulfate structure, namely, the highly sulfated cap Ne uAc 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 identif ied. A structural model for articular cartilage keratan sulfate is pro posed. The potential of the enzyme keratanase II for the structural fi ngerprinting of subnanogram quantities both of keratan sulfates and of sulfated oligosaccharide selectin ligands is discussed.