Chondroitinase ABC digestion of dermatan sulphate. N.m.r. spectroscopic characterization of the oligo- and poly-saccharides

P N Sanderson; T N Huckerby; I A Nieduszynski

doi:10.1042/bj2570347

Chondroitinase ABC digestion of dermatan sulphate. N.m.r. spectroscopic characterization of the oligo- and poly-saccharides

Biochemical Journal ◽

10.1042/bj2570347 ◽

1989 ◽

Vol 257 (2) ◽

pp. 347-354 ◽

Cited By ~ 26

Author(s):

P N Sanderson ◽

T N Huckerby ◽

I A Nieduszynski

Keyword(s):

Gel Permeation Chromatography ◽

Spectroscopic Characterization ◽

Repeat Sequence ◽

Chemical Shift Assignments ◽

Dermatan Sulphate ◽

Chondroitinase Abc ◽

Experimental Conditions ◽

Full Analysis ◽

Unsaturated Uronate

Dermatan sulphates, in which iduronate was the predominant uronate constituent, were partially digested by chondroitinase ABC to produce oligosaccharides of the following structure: delta UA-[GalNAc(4SO3)-IdoA]mGalNAc(4SO3) [where m = 0-5, delta UA represents beta-D-gluco-4-enepyranosyluronate, IdoA represents alpha-L-iduronate and GalNAc(4SO3) represents 2-acetamido-2-deoxy-beta-D-galactose 4-O-sulphate], which were fractionated by gel-permeation chromatography and examined by 100 MHz 13C-n.m.r. and 400/500 MHz 1H-n.m.r. spectroscopy. Experimental conditions were established for the removal of non-reducing terminal unsaturated uronate residues by treatment with HgCL2, and reducing terminal N-acetylgalactosamine residues of the oligosaccharides were reduced with alkaline borohydride. These modifications were shown by 13C-n.m.r. spectroscopy to have proceeded to completion. Assignments of both 13C-n.m.r. and 1H-n.m.r. resonances are reported for the GalNAc(4SO3)-IdoA repeat sequence in the oligosaccharides as well as for the terminal residues resulting from enzyme digestion and subsequent modifications. A full analysis of a trisaccharide derived from dermatan sulphate led to the amendment of published 13C-n.m.r. chemical-shift assignments for the polymer.

Download Full-text

The copolymeric structure of pig skin dermatan sulphate. Isolation and characterization of l-idurono-sulphate-containing oligosaccharides from copolymeric chains

Biochemical Journal ◽

10.1042/bj1430379 ◽

1974 ◽

Vol 143 (2) ◽

pp. 379-389 ◽

Cited By ~ 25

Author(s):

Lars-Åke Fransson ◽

Lars Cöster ◽

Birgitta Havsmark ◽

Anders Malmström ◽

Ingrid Sjöberg

Keyword(s):

Periodate Oxidation ◽

Exchange Chromatography ◽

Dermatan Sulphate ◽

Chondroitinase Abc ◽

Pig Skin ◽

Isolation And Characterization ◽

Smith Degradation ◽

Iduronic Acid ◽

Acidic Conditions

Dermatan sulphate was degraded by testicular hyaluronidase and an oversulphated fraction was isolated by ion-exchange chromatography. This preparation, which contained fairly long segments derived from the non-reducing terminal portion of the molecule, was subjected to periodate oxidation under acidic conditions. The oxidized iduronic acid residues were cleaved by reduction-hydrolysis (Smith-degradation) (Fransson & Carlstedt, 1974) or by alkaline elimination. The oligosaccharides so obtained contained both GlcUA (glucuronic acid) and IdUA-SO4 (sulphated iduronic acid) residues. Copolymeric oligosaccharides obtained after alkaline elimination were cleaved by chondroitinase-AC into disaccharide and higher oligosaccharides. Since the corresponding oligosaccharides obtained by Smith-degradation were unaffected by this enzyme, it was concluded that the carbohydrate sequences were GalNAc-(IdUA-GalNAc)n-GlcUA-GalNAc. The iduronic acid-containing sequences were resistant to digestion with chondroitinase-ABC. It was demonstrated that the presence of unsulphated N-acetylgalactosamine residues in these sequences could be responsible for the observed effect. This information was obtained in an indirect way. Chemically desulphated dermatan sulphate was found to be a poor substrate for the chondroitinase-ABC enzyme. Moreover, digestion with chondroitinase-ABC of chondroitinase-AC-degraded dermatan sulphate released periodate-resistant iduronic acid-containing oligosaccharides. It is concluded that copolymeric sequences of the following structure are present in pig skin dermatan sulphate: [Formula: see text] N-acetylgalactosamine moieties surrounding IdUA-SO4 residues are unsulphated to a large extent.

Download Full-text

Elemental and spectroscopic characterization of humic acids fractionated by gel permeation chromatography

Agronomie ◽

10.1051/agro:2000144 ◽

2000 ◽

Vol 20 (5) ◽

pp. 491-504 ◽

Cited By ~ 13

Author(s):

Aaziz Ouatmane ◽

Valeria Dorazio ◽

Mohamed Hafidi ◽

Jean-Claude Revel ◽

Nicola Senesi

Keyword(s):

Humic Acids ◽

Gel Permeation Chromatography ◽

Spectroscopic Characterization ◽

Gel Permeation

Download Full-text

The copolymeric structure of pig skin dermatan sulphate. Characterization of d-glucuronic acid-containing oligosaccharides isolated after controlled degradation of oxydermatan sulphate

Biochemical Journal ◽

10.1042/bj1430369 ◽

1974 ◽

Vol 143 (2) ◽

pp. 369-378 ◽

Cited By ~ 15

Author(s):

Lars-Åke Fransson ◽

Lars Cöster ◽

Anders Malmström ◽

Ingrid Sjöberg

Keyword(s):

General Formula ◽

Glucuronic Acid ◽

Periodate Oxidation ◽

Dermatan Sulphate ◽

Chondroitinase Abc ◽

Pig Skin ◽

Iduronic Acid ◽

Unsaturated Disaccharide ◽

Selective Periodate Oxidation

Selective periodate oxidation of unsubstituted l-iduronic acid residues in copolymeric dermatan sulphate chains was followed by reduction-hydrolysis or alkaline elimination. By this procedure the glucuronic acid-containing periods were isolated in oligosaccharide form; general formula: [Formula: see text] Further degradation of these oligosaccharides with chondroitinase-AC yielded three types of products: (a) sulphated trisaccharide containing an unsaturated uronosyl moiety in the non-reducing terminal and a C4 fragment in the reducing terminal, ΔUA-GalNAc-(-SO4)-R; (b) monosulphated, unsaturated disaccharide, ΔUA-GalNAc-SO4 when n is greater than or equal to 2; and (c) N-acetylgalactosamine with or without sulphate. Oligosaccharides containing a single glucuronic acid residue (n=1) comprised more than half of the glucuronic acid-containing oligosaccharides. The terminal N-acetylgalactosamine moiety of the shortest oligosaccharide was largely 4-sulphated, whereas higher oligosaccharides primarily contained 6-sulphated or unsulphated hexosamine moieties in the same position. Moreover, IdUA-SO4-containing oligosaccharides were encountered. These oligosaccharides were resistant to the action of chondroitinase-ABC.

Download Full-text

Isolation and characterization of sulphated mucopolysaccharides from rat leukaemic (RBL-1) basophils

Biochemical Journal ◽

10.1042/bj1850367 ◽

1980 ◽

Vol 185 (2) ◽

pp. 367-372 ◽

Cited By ~ 18

Author(s):

D D Metcalfe ◽

S I Wasserman ◽

K F Austen

Keyword(s):

Mast Cell ◽

Salt Concentration ◽

Tumour Cells ◽

High Salt ◽

High Salt Concentration ◽

Dermatan Sulphate ◽

Chondroitinase Abc ◽

Isolation And Characterization

Proteoglycans of 300 000 mol.wt. were isolated from dispersed rat basophil tumour cells after labelling of the sulphated mucopolysaccharides with 35S in vitro:90% of the 35S-labelled mucopolysaccharides were extracted at high salt concentration. Alkali degradation of the 35S-labelled proteoglycans yielded glycosaminoglycan chains of 40 000 mol.wt. The composition of the salt-extracted 35S-labelled mucopolysaccharides, as defined by parallel or sequential degradation with chondroitinase AC, chondroitinase ABC and heparinase and resolution of the disaccharide-digestion products obtained with chondroitinase AC, was 48-61% chondroitin 4-sulphate, 20-30% dermatan sulphate, 10-15% heparin and 7-9% chondroitin 6-sulphate. Most of the salt-extracted 35S-labelled mucopolysaccharides were highly charged, with heparin and chondroitin 6-sulphate being relatively uniform in this regard, whereas chondroitin 4-sulphate and dematan sulphate exhibited a range of charge characteristics. The diversity of sulphated mucopolysaccharides present in the rat leukaemic basophil is in contrast with the predominance of heparin in the rat mast cell.

Download Full-text