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

1989 ◽  
Vol 260 (1) ◽  
pp. 277-282 ◽  
Author(s):  
D J Thornton ◽  
H G Morris ◽  
G H Cockin ◽  
T N Huckerby ◽  
I A Nieduszynski ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Articular Cartilage ◽  
High Performance ◽  
Gel Permeation Chromatography ◽  
Spectroscopic Studies ◽  
Progressive Increase ◽  
Exchange Chromatography ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
Femoral Head 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.

scholarly journals Fucose content of keratan sulphates from bovine articular cartilage

1991 ◽  
Vol 273 (2) ◽  
pp. 307-310 ◽  
Author(s):  
G H Tai ◽  
G M Brown ◽  
H G Morris ◽  
T N Huckerby ◽  
I A Nieduszynski
Keyword(s):  
Molecular Weight ◽  
Articular Cartilage ◽  
Molecular Size ◽  
Gel Permeation Chromatography ◽  
Repeat Sequence ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
Gel Permeation ◽  
Fucose Content ◽  
Galactose Content

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.

scholarly journals 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

1993 ◽  
Vol 291 (3) ◽  
pp. 889-894 ◽  
Author(s):  
G H Tai ◽  
T N Huckerby ◽  
I A Nieduszynski
Keyword(s):  
Articular Cartilage ◽  
Structural Analysis ◽  
Spectroscopic Studies ◽  
Pseudomonas Sp ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
Charged Species ◽  
Repeat Sequences

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.

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

1992 ◽  
Vol 286 (1) ◽  
pp. 231-234 ◽  
Author(s):  
G H Tai ◽  
H G Morris ◽  
G M Brown ◽  
T N Huckerby ◽  
I A Nieduszynski
Keyword(s):  
Affinity Chromatography ◽  
Gel Permeation Chromatography ◽  
Similar Proportion ◽  
Lectin Affinity Chromatography ◽  
Sambucus Nigra ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
Acetylneuraminic Acid ◽  
Sambucus Nigra Agglutinin ◽  
Femoral Head Cartilage

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.

scholarly journals Age-related changes in the structure of the keratan sulphate chains attached to fibromodulin isolated from articular cartilage

1998 ◽  
Vol 330 (2) ◽  
pp. 753-757 ◽  
Author(s):  
M. Robert LAUDER ◽  
N. Thomas HUCKERBY ◽  
A. Ian NIEDUSZYNSKI ◽  
H. K. Anna PLAAS
Keyword(s):  
Articular Cartilage ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
Acetylneuraminic Acid ◽  
Age Related ◽  
Keratanase Ii ◽  
Age Related Changes ◽  
Related Increase

Bovine articular cartilage fibromodulin has been isolated from animals aged 3 months to 8 years, and the attached keratan sulphate (KS) chains digested with keratanase II. The oligosaccharides generated have been reduced, examined by high-pH anion-exchange chromatography and their structures identified by comparison with standards. It has been shown that in fibromodulin from young articular cartilage, the KS chains do not possess either non-reducing terminal (α2-6)-linked N-acetylneuraminic acid or fucose (α1-3)-linked to sulphated N-acetylglucosamine residues. However, an age-related increase has been observed in the abundance of both (α2-6)-linked N-acetylneuraminic acid and (α1-3)-linked fucose, neither of which is found in KS isolated from non-articular cartilage, irrespective of the age of the source. Interestingly, the KS chain length remains constant as a function of age, which possibly relates to a role in collagen fibril assembly. In addition, no significant age-related changes were identified in levels of galactose sulphation.

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

1992 ◽  
Vol 286 (1) ◽  
pp. 235-241 ◽  
Author(s):  
G M Brown ◽  
T N Huckerby ◽  
H G Morris ◽  
I A Nieduszynski
Keyword(s):  
Articular Cartilage ◽  
Nitrous Acid ◽  
High Field ◽  
Ion Exchange Chromatography ◽  
Repeat Sequence ◽  
Exchange Chromatography ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
Acid Environment ◽  
Fucose Residue

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.

scholarly journals Mature bovine articular cartilage contains abundant aggrecan that is C-terminally truncated at Ala719-Ala720, a site which is readily cleaved by m-calpain

2004 ◽  
Vol 382 (1) ◽  
pp. 253-259 ◽  
Author(s):  
Hidefumi OSHITA ◽  
John D. SANDY ◽  
Kiichi SUZUKI ◽  
Atsushi AKAIKE ◽  
Yun BAI ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Globular Domain ◽  
Cleavage Sites ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
The Matrix ◽  
A Site

Extracts of normal mature articular cartilage contain aggrecan molecules which bear the G1 domain (the N-terminal globular domain of aggrecan) and are C-terminally truncated by proteolysis at a number of sites. A proportion of these molecules are generated by an aggrecanase and/or matrix-metalloproteinase-mediated cleavage in the IGD (interglobular domain between the G1 and G2 domains of aggrecan). However, the proteinase(s) responsible for formation of the majority of the larger G1-G2 and glycosaminoglycan-bearing truncated species is (are) unknown. N-terminal sequencing of aggrecan core fragments generated by m-calpain digestion of bovine aggrecan has identified four novel cleavage sites: one within the CS (chondroitin sulphate)-1 domain (at one or more of the bonds Ser1229–Val1230, Ser1249–Val1250, Ser1287–Val1288, Gly1307–Val1308 and Ser1346–Val1347), two within the IGD (at bonds Ala474–Ala475 and Gly365–Gly366) and one within the KS (keratan sulphate) domain (at Ala719–Ala720). A new monoclonal antibody (SK-28) to the C-terminal neoepitope at M710VTQVGPGVA719 showed that aggrecan products generated by this cleavage are present in high abundance in mature bovine articular cartilage extracts. We conclude that m-calpain, or an unidentified proteinase with the capacity to cleave at the same site, is active during aggrecan biosynthesis/secretion by mature chondrocytes or in the matrix of mature bovine articular cartilage in vivo.

scholarly journals Two linkage-region fragments isolated from skeletal keratan sulphate contain a sulphated N-acetylglucosamine residue

1990 ◽  
Vol 269 (1) ◽  
pp. 55-59 ◽  
Author(s):  
J M Dickenson ◽  
T N Huckerby ◽  
I A Nieduszynski
Keyword(s):  
Gel Permeation Chromatography ◽  
Anion Exchange Chromatography ◽  
Ester Group ◽  
Intervertebral Discs ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Keratan Sulphate ◽  
Linkage Region ◽  
Gel Permeation ◽  
Electron Micro Probe Analysis

Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (6-year-old animals) after chondroitin ABC lyase digestion followed by digestion of A1D1 proteoglycans by diphenylcarbamoyl chloride-treated trypsin and gel-permeation chromatography on Sepharose CL-6B. Treatment of these peptido-keratan sulphate fragments with alkaline NaB3H4 yielded keratan sulphate chains with [3H]galactosaminitol end-labels, and these chains were further purified by gel-permeation chromatography on Sephadex G-50 and ion-exchange chromatography on a Pharmacia Mono-Q column in order to exclude any contamination with O-linked oligosaccharides. The chains were then treated with keratanase, and the digest was chromatographed on a Bio-Gel P-4 column followed by anion-exchange chromatography on a Nucleosil 5 SB column. Two oligosaccharides, each representing 18% of the recovered radiolabel, were examined by 500 MHz 1H-n.m.r. spectroscopy, and shown to have the following structures: [formula: see text] The structure of oligosaccharide (I) confirms the N-acetylneuraminylgalactose substitution at position 3 of N-acetylgalactosamine in the keratan sulphate-protein linkage region found by Hopwood & Robinson [(1974) Biochem. J. 141, 57-69] but additionally shows the presence of a 6-sulphated N-acetylglucosamine. Electron micro-probe analysis specifically confirmed the presence of sulphur in this sample. This sulphate ester group differentiates the keratan sulphate linkage region from similar structures derived from O-linked oligosaccharides [Lohmander, De Luca, Nilsson, Hascall, Caputo, Kimura & Heinegård (1980) J. Biol. Chem. 255, 6084-6091].

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