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I A Nieduszynski

Publications and source records attributed to I A Nieduszynski.

At least 37 records · Page 2Linked to original sources
PubMed1996

Characterization of a non-reducing terminal fragment from bovine articular cartilage keratan sulphates containing alpha(2-3)-linked sialic acid and alpha(1-3)-linked fucose. A sulphated variant of the VIM-2 epitope.

Alkaline-borohydride-reduced keratan sulphate chains were isolated from bovine articular cartilage (6-8-year-old animals) and digested with keratanase II, an endo- beta-N-acetylglucosaminidase. The resulting oligosaccharides were borohydride-reduced and fractionated on a strong anion-exchange column. 1H-NMR spectroscopic analysis of the products revealed one containing both alpha(2-3)-linked sialic acid and alpha(1-3)-linked fucose which was shown to have the structure (I) shown. This structure is a sulphated variant of the VIM-2 epitope (CD65), a putative ligand of E-selectin. No oligosaccharide containing the sialyl-Le(+) structure [NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-] was identified in this study. [equation: see text]

Acetylglucosaminidase↗
PubMed1996

Multiple non-reducing chain termini isolated from bovine corneal keratan sulfates.

Keratan sulfate-containing proteoglycans were isolated from bovine cornea (15-month-old to 3-year-old animals) and digested with the enzyme, keratanase II. The released oligosaccharides, which included non-reducing termini and repeat region oligosaccharides but not linkage regions, were reduced with alkaline borohydride and fractionated on a Spherisorb column. These oligosaccharides were examined by 600-MHz 1H NMR spectroscopy using one- and two-dimensional methods and, in addition to some oligosaccharide alditols previously recovered from skeletal keratan sulfate, the following new capping structures were identified: NeuAcalpha2-6Galbeta1-4GlcNAc(S)-ol, NeuAcalpha2-3Gal(S)beta1-4GlcNAc(S)beta1-3Galbeta1-4GlcNAc(S )-ol, NeuGcalpha2-6Galbeta1-4GlcNAc(S)beta1-3Galbeta1-4Gl cNA c(S)-ol, NeuGcalpha2-3Galbeta1-4GlcNAc(S)beta1-3Galbeta1-4Gl cNA c(S)-ol, NeuGcalpha2-3Gal(S)beta1-4GlcNAc(S)beta1-3Galbeta1-4GlcNAc(S )-ol, NeuGcalpha2-3Gal(S)beta1-4GlcNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)-o l, Galalpha1-3Galbeta1-4GlcNAc(S)beta1-3Galbeta1-4GlcNAc( S)-ol, Galalpha1-3Galbeta1-4GlcNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)- ol, GlcNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)-ol, and GalNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)-ol. These structures represent seven families of capping residues, whose relative molar proportions are given in parentheses: NeuAcalpha(2-3)- (12%), NeuAcalpha(2-6)- (41%), NeuGcalpha(2-3)- and NeuGcalpha(2-6)- families (12%), Galalpha(1-3)- (26%), GalNAc(S)beta(1-3)- (5%), and GlcNAc(S)beta(1-3)- (4%). It is not clear, at present, where each of these structures occurs on the bi-antennary N-linked corneal keratan sulfate chains, which themselves occur within three keratan sulfate proteoglycan species. However, examination of the relative proportions of the capping to the repeat structures and knowledge of the average molecular size suggests that the sum of these non-reducing termini represents the caps of two antennae.

Acetylglucosaminidase↗
PubMed1996

Cell surface-associated keratan sulfate on normal and migrating corneal endothelium.

PURPOSE: To investigate cell surface-associated keratan sulfate on the corneal endothelium. METHODS: Immunolabeling techniques were used at the light, scanning, and transmission electron microscopic level to localize keratan sulfate on the corneal endothelium. The investigation included human, bovine, and rabbit corneal endothelia. A quantitative study of the relationship between cell size and keratan sulfate levels was conducted on normal bovine corneal endothelium. Changes in the distribution of keratan sulfate and chondroitin sulfate on endothelial cell surfaces were investigated on organ cultured bovine corneas during endothelial wound healing. Changes in the levels of keratan sulfate during endothelial wound healing were investigated in organ cultured human corneas and in vivo in rabbit corneas. Inhibition-enzyme-linked immunosorbent assay also was used to detect keratan sulfate in the aqueous humor. RESULTS: A variegated distribution of keratan sulfate was revealed on normal human, bovine, and rabbit corneal endothelia. Some cells had high levels of keratan sulfate on their surfaces whereas others, sometimes immediately adjacent, had little or none. Wound healing experiments resulted in changes of keratan sulfate levels on the migrating endothelial cells in bovine, human, and rabbit. In wounded organ cultured bovine corneas, there was a decrease in keratan sulfate levels and an increase in chondroitin sulfate levels on migrating endothelial cells. Keratan sulfate was detected in bovine aqueous humor. CONCLUSIONS: The pattern of occurrence of keratan sulfate and chondroitin sulfate on the corneal endothelial cells in normal and wounded cornea suggests that these glycosaminoglycans have differing roles in endothelial adhesion and migration.

Aged↗
PubMed1995

13C-NMR spectroscopy of keratan sulphates. Assignments for four poly(N-acetyllactosamine)-repeat-sequence tetrasaccharides derived from bovine articular cartilage keratan sulphate by keratanase II digestion.

Skeletal keratan sulphate has been fragmented using the enzyme keratanase II, and the complete 13C chemical shift data are reported for the four tetrasaccharides which derived from the repeat region. They have the structures: Gal beta(1-4)GlcNAc(6S)beta(1-3)Gal beta(1-4)GlcNAc(6S)-ol, Gal(6S)beta(1-4)GlcNAc(6S)beta(1-3)Gal(1-4)GlcNAc(6S)-ol, Gal beta(1-4)GlcNAc(6S)beta(1-3)Gal(6S)beta(1-4)GlcNAc(6S)-ol, and Gal(6S)beta(1-4)GlcNAc(6S)(1-3)Gal(6S)beta(1-4)GlcNAc(6S)-ol, where GlcNAc(6S)-ol represents N-acetyl-glucosaminitol-6-O-sulphate. The value of these data for microstructural analysis of undegraded keratan sulphate samples using 13C-NMR spectroscopy is discussed with respect to galactose sulphation levels and the block sulphation structure.

Animals↗
PubMed1995

Keratanase digestion of keratan sulphates: characterization of large oligosaccharides from the N-acetyllactosamine repeat sequence and from the non-reducing terminal chain caps.

Keratan sulfate (KS) chains prepared from both bovine tracheal rings and bovine femoral head cartilage were digested with the enzyme keratanase from Pseudomonas species; large repeat-sequence and non-reducing terminal oligosaccharides were fractionated and purified using high-performance ion-exchange chromatography. The main beta-linked pentasulfated hexasaccharide repeat segment, [R6], GlcNAc(6S)1-1-3Gal(6S)1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S)1-3Gal-ol and the asialo beta-linked capping pentasulfated heptasaccharide, [C7], Gal1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S)1-3Gal(6S)1-4GlcNAc(6S) 1-3Gal-ol have been completely characterized by high-field NMR spectroscopy using one- and two-dimensional methods. Partial 1H assignments are summarized for three homologous series of higher oligosaccharides: GlcNAc(6S)[1-3Gal(6S)1-4GlcNAc(6S)]2-5(1-3)Gal-0l [R8,R10,R12] Gal1-4GlcNAc(6S)[1-3Gal(6S)1-4GlcNAc(6S)]3-5(1-3)Gal-ol [C9,C11,C13] NeuAc alpha 2-3Gal1-4GlcNAc(6S)[1-3Gal(6S)1-4GlcNAc(6S)]2-4(1-3)Gal-ol [C8,C10,C12] obtained from keratan sulfate by keratanase cleavage. The first shows that the unsulfated galactose residues within the repeat sequence region of KS may be separated by fully sulfated segments which have a wide distribution of lengths. The others, viz. those with sialylated caps, and the related galactose capped asialo-segments (derived from a KS digestion in which the keratanase also exhibited sialidase activity) represent an homologous series of epitopes in which the first internal unsulfated galactose is located at a position which may be up to five or more fully sulfated N-acetyllactosamine disaccharide repeat units along from the non-reducing terminus of the KS polymer.

Animals↗
PubMed1995

Spectroscopic characterisation of disaccharides derived from keratan sulfates.

Skeletal keratan sulfates have been degraded by three independent techniques and the resultant, borohydride-reduced, disaccharides have been characterised by NMR spectroscopy. The 1H and 13C (where available) chemical shifts are reported for the following substances, where GalNAc-ol represents N-acetyl-galactosaminitol, GlcNAc-ol represents N-acetyl-glucosaminitol, GlcNAc(6S)-ol represents N-acetyl-glucosaminitol 6-O-sulfate and 2,5AnMan(6S)-ol represents 2,5-anhydro-D-mannitol 6-O-sulfate. (a) GlcNAc(6S)beta(1-3)Gal-ol, isolated after keratanase (from Pseudomonas sp.) digestion. (b) Gal beta(1-4)GlcNAc(6S)-ol and Gal(6S)beta(1-4) GlcNAc(6S)-ol, the 1H chemical shifts have been reported previously [Brown, G. M., Huckerby, T. N., Morris, H. G., Abram, B. L. & Nieduszynski, I. A. (1994) Biochemistry 33, 4836-4846; Brown, G. M., Huckerby, T. N. & Nieduszynski, I. A. (1994) Eur. J. Biochem. 224, 281-308], GlcNAc(6S)beta(1-6)GalNAc-ol, [formula: see text], [formula: see text], all isolated after keratanase II digestion. (c) Gal beta(1-4)2,5AnMan(6S)-ol and Gal(6S)beta(1-4)2,5AnMan(6S)-ol, isolated after hydrazinolysis and nitrous acid digestion. In addition, the model compounds Gal beta(1-4)GlcNAc-ol and Gal beta(1-6)GlcNAc-ol have also been examined by 1H and 13C NMR spectroscopy. The value of these data for microstructural analysis of keratan sulfate samples is discussed.

Animals↗
PubMed1995

The structure of the keratan sulphate chains attached to fibromodulin isolated from bovine tracheal cartilage: oligosaccharides generated by keratanase II digestion.

The repeat region and chain caps of the N-linked keratan sulphates attached to bovine tracheal cartilage fibromodulin were fragmented by digestion with keratanase II, and the oligosaccharides generated were isolated by strong anion-exchange chromatography. Each of these oligosaccharides has been examined by both HPAE chromatography and high field 1H-NMR spectroscopy. All of the capping oligosaccharides isolated terminated with alpha(2-6)-linked N-acetyl-neuraminic acid chain terminators, nor fucose alpha(1-3)-linked to N-acetylglucosamine were found. The keratan sulphate chains were short, with average lengths of five to seven disaccharides, and the level of galactose sulphation varied along the length of the chain. The repeat region and chain cap were confirmed as having the following general structure: [formula: see text]. This study has identified a novel structure in fibromodulin, namely a cap containing a sulphated galactose adjacent to a non-reducing terminal N-acetyl-neuraminic acid. We have also confirmed that the general structure of the repeat units and chain caps of N-linked keratan sulphate attached to fibromodulin isolated from bovine tracheal cartilage, is similar to that of O-linked keratan sulphate chains attached to aggrecan from non-articular cartilage. However, there are important differences in chain lengths and sulphation patterns.

Acetylglucosaminidase↗
PubMed1995

Skeletal keratan sulphate structural analysis using keratanase II digestion followed by high-performance anion-exchange chromatography.

A semi-quantitative fingerprinting method has been developed for the structural analysis of skeletal keratan sulphates. This involves the digestion of the parent keratan sulphate chains with the enzyme keratanase II (Bacillus sp.), followed by reduction of the resulting oligosaccharides with sodium borohydride and chromatography on a Dionex AS4A-SC column. This column has been calibrated for the elution positions of 26 previously characterized oligosaccharides (Brown et al., Biochemistry, 33, 4836-4846, 1994; Brown et al., Eur. J. Biochem., 224, 281-308, 1994). The technique permits sample analysis with pulsed electrochemical detection (sensitive to approximately 5 ng of oligosaccharide) or by monitoring [3H] or [35S] radiolabel (potentially sensitive to approximately 1 pg or less of an oligosaccharide); thus permitting the study of sub-microgram amounts of keratan sulphates. Skeletal keratan sulphates from a number of sources have been examined in this chromatographic system and their structural features are discussed.

Acetylglucosaminidase↗
PubMed1994

Structure of the keratan sulphate chains attached to fibromodulin isolated from bovine tracheal cartilage. Oligosaccharides generated by keratanase digestion.

The structure of the repeat region and chain caps of the N-linked keratan sulphate chains attached to bovine tracheal cartilage fibromodulin has been examined. The chains were fragmented by keratanase digestion, the resultant oligosaccharides isolated by strong anion-exchange chromatography, and their structures determined using high-field 1H-n.m.r. spectroscopy. The chains were found to possess the following general structure: [formula: see text] All of the capping oligosaccharides isolated terminate with alpha(2-3)-linked N-acetylneuraminic acid. No alpha(2-6)-linked N-acetylneuraminic acid chain terminators, nor any fucose, alpha (1-3)-linked to N-acetylglucosamine along the repeat region, were detected. This work demonstrates that the structure of the repeat region and chain caps of N-linked keratan sulphate attached to fibromodulin isolated from bovine tracheal cartilage is identical with that of O-linked keratan sulphate chains attached to aggrecan derived from non-articular cartilage.

Animals↗
PubMed1994

Oligosaccharides derived by keratanase II digestion of bovine articular cartilage keratan sulphates.

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]: Gal beta 1-4-GlcNAc(6S)-ol, Gal beta 1-4GlcNAc(6S)beta 1-3Gal beta 1-4GlcNAc(6S)-ol, Gal(6S)beta 1-4GlcNAc(6S)-ol, Gal-(6S)beta 1-4GlcNAc(6S) beta 1-3Gal beta 1-4GlcNAc(6S)-ol, Gal beta 1-4GlcNAc(6S)beta 1-3Gal(6S)beta 1-4GlcNAc(6S)-ol, Gal(6S)beta 1-4GlcNAc(6S)beta 1-3Gal(6S)1-4GlcNAc(6S)-ol, Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S)-ol, Gal beta 1-4-(Fuc alpha 1-3)GlcNAc(6S)beta1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S)-ol, Gal beta 1-4GlcNAc(6S)beta 1-3Gal beta 1-4(Fuc alpha 1-3)-GlcNAc(6S)-ol, Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S)beta 1-3Gal beta 1-4GlcNAc(6S)-ol, Gal(6S) beta 1-4GlcNAc-(6S)beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S)-ol, Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S)beta 1-3Gal(6S)1-4GlcNAc(6S)-ol, Gal beta 1-4GlcNAc(6S)beta 1-6(Gal beta 1-3)GalNAc-ol, Gal beta 1-4GlcNAc(6S) beta1-6(NeuAc2-3Gal beta 1-3)Gal-NAc-ol, Gal beta 1-4GlcNAc(6S)beta 1-3Gal beta 1-4GlcNAc(6S)beta 1-6(Gal beta 1-3) GalNAc-ol, Gal(6S)beta 1-4GlcNAc-(6S)beta 1-6(Gal beta 1-3)GalNAc-ol, Gal beta 1-4GlcNAc(6S)beta 1-3Gal beta 1-4GlcNAc(6S)beta 1-6(NeuAc2-3Gal beta 1-3)-GalNAc-ol, Gal(6S)beta 1-4GlcNAc(6S)beta 1-6(NeuAc alpha 2-3Gal beta 1-3)GalNAc-ol, Gal(6S) beta 1-4GlcNAc-(6S)beta 1-3Gal beta 1-4GlcNAc(6S)beta 1-6(Gal beta 1-3)GalNAc- ol, Gal(6S)beta 1-4GlcNAc(6S)beta 1-3Gal beta 1-4GlcNAc-(6S)beta 1-6(NeuAc alpha 2-3Gal beta 1-3)GalNAc-ol, NeuAc alpha 2-6Gal beta 1-4GlcNAc(6S)beta 1-3Gal beta 1-4GlcNAc(6S)-ol, NeuAc alpha 2-3Gal beta 1-4GlcNAc(6S)beta 1-3Gal beta 1-4GlcNAc(6S)-ol, NeuAc alpha 2-6Gal beta 1-4GlcNAc(6S)beta 1-3Gal-(6S)beta 1-4GlcNAc(6S)-ol, NeuAc alpha 2-3Gal beta 1-4GlcNAc(6S)beta 1-3Gal(6S)beta 1-4GlcNAc(6S)-ol and Neu-Ac alpha 2-3Gal(6S)beta 1-4GlcNAc(6S)beta 1-3Gal(6S beta)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 beta(1-3)-linked 6-O-sulphated N-acetylglucosamine.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylglucosaminidase↗
PubMed1994

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., & 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.

Acetylglucosaminidase↗
PubMed1993

N.m.r. spectroscopic studies of fucose-containing oligosaccharides derived from keratanase digestion of articular cartilage keratan sulphates. Influence of fucose residues on keratanase cleavage.

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.

Acetylglucosamine↗
PubMed1992

A sub-population of keratan sulphates derived from bovine articular cartilage is capped with alpha(2-6)-linked N-acetylneuraminic acid residues. Affinity chromatography using immobilized Sambucus nigra lectin and characterization using 1H n.m.r. spectroscopy.

Alkaline borohydride-reduced keratan sulphate (KS) chains derived from bovine femoral head cartilage were fractionated by lectin affinity chromatography with Sambucus nigra agglutinin (SNA) into binding and non-binding populations. Analysis of the SNA-binding and non-binding KS chains using 600 MHz 1H n.m.r. spectroscopy showed that the former population contained alpha(2-6)-N-acetylneuraminic acid residues and the latter contained primarily alpha(2-3)-N-acetylneuraminic acid residues as chain terminators. Both populations contained a similar proportion of alpha(2-3)-N-acetylneuraminic acid residues within their protein-linkage regions, and similar sulphation and fucosylation levels. Analysis of these two fractions by gel-permeation chromatography (g.p.c.) on a TSK-30 XL column showed them to have the same size distributions. It was concluded from the n.m.r. spectra and g.p.c. data that the populations differed primarily in the mode of linkage of the chain-terminating sialic acids.

Animals↗
PubMed1992

Degradation of articular cartilage keratan sulphates using hydrazinolysis and nitrous acid. Environment of fucose residues.

Alkaline borohydride-reduced keratan sulphate (KS) chains from bovine articular cartilage (6-8-year-old animals) were fragmented by an anhydrous hydrazine/nitrous acid procedure, previously used on KS by Hopwood & Elliott to isolate the major disaccharides from the poly-N-acetyl-lactosamine repeat sequence [Hopwood & Elliott (1983) Carbohydr. Res. 117, 263-274]. The resulting oligosaccharides were reduced with NaB3H4 or NaBH4 and subjected to ion-exchange chromatography on a Nucleosil 5SB column. In addition to the major disaccharides, two fucose-containing oligosaccharides were examined by high-field 1H n.m.r. spectroscopy, and shown to have the following structures (where AnManOH is 2,5-anhydro-D-mannitol): [formula: see text] It is evident that the presence of fucose protects the N-acetylglucosamine residue from de-N-acetylation, and therefore fragments are produced which preserve the immediate environment of the fucose residue. It may be of biosynthetic significance that these two oligosaccharides contain an unsulphated galactose on the non-reducing side of the fucose residue. The hydrazine/nitrous acid/NaB3H4 method followed by h.p.l.c. provides a sensitive fingerprinting technique for the assay of KS composition and sub-populations.

Animals↗
PubMed1992

Skeletal keratan sulphate chains isolated from bovine intervertebral disc may terminate in alpha(2----6)-linked N-acetylneuraminic acid.

Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (2-year-old animals) after digestion with chondroitin ABC lyase followed by digestion with diphenylcarbamoyl chloride-treated trypsin of A1D1 proteoglycans and gel-permeation chromatography on Sepharose CL-6B. The peptido-keratan sulphate fragments were subjected to alkaline borohydride reduction. The reduced chains were treated with keratanase in the presence of the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, and the digest was subjected to alkaline borohydride reduction. This produced oligosaccharides with galactitol at their reducing ends. This reduced digest was chromatographed on a Nucleosil 5 SB anion-exchange column and individual oligosaccharides were isolated. One of these was shown by 600 MHz 1H-n.m.r. spectroscopy to have the following structure: NeuAc alpha 2-6Gal beta 1-4GlcNAc(6-SO4)beta 1-3Gal-ol The structure of this oligosaccharide shows that keratan sulphate chains from bovine intervertebral disc have non-reducing termini with N-acetylneuraminic acid linked alpha(2----6) as well as alpha(2----3) to an unsulphated galactose.

Animals↗
PubMed1991

A non-reducing terminal fragment from tracheal cartilage keratan sulphate chains contains alpha (2-3)-linked N-acetylneuraminic acid.

Keratan sulphate chains were isolated from bovine tracheal ring cartilage (15-18-month-old animals) after papain digestion of the tissue followed by ethanol fractionation, chondroitinase ABC digestion and alkaline borohydride reduction. The keratan sulphate chains were further purified by anion-exchange chromatography on a Pharmacia Mono-Q column in order to remove any contaminating chondroitin sulphate and O-linked oligosaccharides. The chains were then treated with keratanase and the digest was subjected to alkaline borohydride reduction, producing oligosaccharides with galactitol at their reducing ends. The reduced digest was chromatographed on a Nucleosil 5 SB anion-exchange column and individual oligosaccharides were isolated. One of these, oligosaccharide (I), was shown by 500 MHz 1H-n.m.r. spectroscopy to have the following structure: NeuAc alpha 2-3Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (I) The structure of this oligosaccharide shows that keratan sulphate chains from bovine tracheal ring cartilage may be terminated with N-acetylneuraminic acid linked alpha (2-3) to an unsulphated galactose. Keratan sulphate chains were also isolated from bovine femoral head cartilage (15-18-month-old animals) using an identical protocol, but with keratanase which was subsequently shown to have sialidase activity. This yielded oligosaccharide (II), the unsialyated version of (I): Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (II).

Animals↗
PubMed1991

Fucose content of keratan sulphates from bovine articular cartilage.

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.

Animals↗