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

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.

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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.

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The co-polymeric structure of pig skin dermatan sulphate. Distribution of L-iduronic acid sulphate residues in co-polymeric chains

Biochemical Journal ◽

10.1042/bj1450379 ◽

1975 ◽

Vol 145 (2) ◽

pp. 379-389 ◽

Cited By ~ 24

Author(s):

L Cöster ◽

A Malmström ◽

I Sjöberg ◽

L Fransson

Keyword(s):

Periodate Oxidation ◽

Low Molecular Weight ◽

Dermatan Sulphate ◽

Radioactive Product ◽

Chondroitinase Abc ◽

Pig Skin ◽

Iduronic Acid ◽

Polymeric Chains ◽

Complete Digestion ◽

Testicular Hyaluronidase

1. Pig skin dermatan sulphate was degraded by periodate oxidation followed by alkaline elimination or by chondroitinase-ABC to quantify irregular repeating units, i.e. those containing D-GlcUA (D-glucuronic acid) and L-IdUA-SO4 (sulphated iduronic acid). 2. Previous results of periodate oxidation (Fransson, 1974) indicated repeating sequences in pig skin dermatan sulphate containing, on average, 3D-GlcUA, 9 L-IdUA-SO4 or 28 L-IdUA units in addition to N-acetylgalactosamine sulphate. However, complete digestion with chondroitinase-ABC yielded, at the most, 3-4 disulphated disaccharides/chain. Consequently, more than one-half of the L-IdUA-SO4 residues were present in monosulphated periods, i.e. IdUA-(SO4)-GalNAc. 3. To determine the location of L-IdUA-SO4 residues along the copolymeric chain dermatan sulphate was digested with testicular hyaluronidase. (This enzyme cleaves GalNAc-GlcUA bonds within block regions containing D-GlcUA.) By NaB3H4 reduction GalNAc residues located in the reducing end of the fragments were converted into [3H]GalNAcOH (N-acetylgalactosaminitol). Finally, the radioactive product was fragmented by periodate oxidation followed by alkaline elimination. The bulk of the radioactivity was associated with periodate-resistant oligosaccharides indicating that clusters of GlcUA-GalNAc-SO4 periods are often adjacent to a varying number of (n = 1-4) of L-IdUA-SO4-containing periods. 4. To study the distribution of L-IdUA-SO4-containing periods in relation to blocks of IdUA-GalNAc-SO4 periods different fractions of hyaluronidase-degraded dermatan sulphate were degraded separately. In all types of fragments (mol. wts. 1,500-10,000) L-IdUA-SO4-containing periods were demonstrated. In short fragments reducing terminal GalNAc-6-SO4 (6-sulphated N-acetylgalactosamine) was found confirming that these sequences were joined to relatively long D-GlcUA-containing block sequences via GalNAc-6-SO4. Moreover, low-molecular-weight oligosaccharides composed of alternating sequences were encountered. An octasaccharide derived from the carbohydrate sequence -GalNAc-GlcUA-GalNAc-IdUA-GalNAc-GlcUA-GalNAc-IdUA-GalNAc-GlcUA-GalNAc (- indicates the position of cleavage by hyaluronidase) was identified.

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Synthesis of glycosaminoglycans by human embryonic lung fibroblasts. Different distribution of heparan sulphate, chondroitin sulphate and dermatan sulphate in various fractions of the cell culture

Biochemical Journal ◽

10.1042/bj1670383 ◽

1977 ◽

Vol 167 (2) ◽

pp. 383-392 ◽

Cited By ~ 28

Author(s):

Ingrid Sjöberg ◽

Lars-Åke Fransson

Keyword(s):

Glucuronic Acid ◽

Chondroitin Sulphate ◽

Heparan Sulphate ◽

Periodate Oxidation ◽

Exchange Chromatography ◽

Lung Fibroblasts ◽

Relative Distribution ◽

Dermatan Sulphate ◽

Human Embryonic Lung ◽

Iduronic Acid

Foetal human lung fibroblasts, grown in monolayer, were allowed to incorporate 35SO42− for various periods of time. 35S-labelled macromolecular anionic products were isolated from the medium, a trypsin digest of the cells in monolayer and the cell residue. The various radioactive polysaccharides were identified as heparan sulphate and a galactosaminoglycan population (chondroitin sulphate and dermatan sulphate) by ion-exchange chromatography and by differential degradations with HNO2 and chondroitinase ABC. Most of the heparan sulphate was found in the trypsin digest, whereas the galactosaminoglycan components were largely confined to the medium. Electrophoretic studies on the various 35S-labelled galactosaminoglycans suggested the presence of a separate chondroitin sulphate component (i.e. a glucuronic acid-rich galactosaminoglycan). The 35S-labelled galactosaminoglycans were subjected to periodate oxidation of l-iduronic acid residues followed by scission in alkali. A periodate-resistant polymer fraction was obtained, which could be degraded to disaccharides by chondroitinase AC. However, most of the 35S-labelled galactosaminoglycans were extensively degraded by periodate oxidation–alkaline elimination. The oligosaccharides obtained were essentially resistant to chondroitinase AC, indicating that the iduronic acid-rich galactosaminoglycans (i.e. dermatan sulphate) were composed largely of repeating units containing sulphated or non-sulphated l-iduronic acid residues. The l-iduronic acid residues present in dermatan sulphate derived from the medium and the trypsin digest contained twice as much ester sulphate as did material associated with the cells. The content of d-glucuronic acid was low and similar in all three fractions. The relative distribution of glycosaminoglycans among the various fractions obtained from cultured lung fibroblasts was distinctly different from that of skin fibroblasts [Malmström, Carlstedt, Åberg & Fransson (1975) Biochem. J.151, 477–489]. Moreover, subtle differences in co-polymeric structure of dermatan sulphate isolated from the two cell types could be detected.

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A method for the sequence analysis of dermatan sulphate

Biochemical Journal ◽

10.1042/bj2690381 ◽

1990 ◽

Vol 269 (2) ◽

pp. 381-388 ◽

Cited By ~ 42

Author(s):

L Å Fransson ◽

B Havsmark ◽

I Silverberg

Keyword(s):

Periodate Oxidation ◽

Amino Group ◽

Serine Residue ◽

Dermatan Sulphate ◽

Pig Skin ◽

Complete Digestion ◽

Chondroitin Ac Lyase ◽

Beta Galactosidase ◽

Selective Periodate Oxidation ◽

Testicular Hyaluronidase

We are attempting to develop methods for the sequencing of glycosaminoglycans from their reducing end. Here we describe a procedure for the analysis of dermatan sulphate from pig skin. The glycosaminoglycan is released from its parent proteoglycan by exhaustive proteolysis by using both endo- and exo-peptidases. The amino group of the residual serine residue is conjugated with a p-hydroxyphenyl group, which in turn is iodinated with 125I (the Bolton-Hunter reagent, BHR). The ion-exchange-purified end-labelled dermatan sulphate is then degraded partially or completely by various enzymic or chemical means to yield fragments extending from the labelled serine residue to the point of cleavage. The various products are separated by gradient PAGE, detected by autoradiography and quantified by videodensitometry. Complete digestion with chondroitin ABC lyase affords the labelled fragment delta HexA-GalNAc(-SO4)-GlcA-Gal-Gal-Xyl-Ser(-BHR). The structure was confirmed by sequential degradation from the non-reducing end by chondroitin AC lyase, HgCl2, and beta-galactosidase. Periodate oxidation cleaves most of the Xyl even without treatment with alkaline phosphatase, showing that Xyl is not substituted with phosphate. Results from partial and selective periodate oxidation indicate that most of the non-sulphated IdoA residues are located towards the non-reducing end. Partial or complete digestions with testicular hyaluronidase (in the presence of an excess of beta-glucuronidase) or chondroitin AC lyase identify the positions of GlcA residues. The results confirm that HexA next to Gal is always GlcA. Moreover, GlcA is common in the first three disaccharide repeats. Results with testicular hyaluronidase indicate that the distribution of clustered GlcA-GalNAc repeats is periodic and peaks at positions 1-3, 8-9 and around 25. Although there must be chains that contain IdoA in nearly all of the available positions, regions that have not been fully processed during biosynthesis are markedly non-random.

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1H-n.m.r. investigation of naturally occurring and chemically oversulphated dermatan sulphates. Identification of minor monosaccharide residues

Biochemical Journal ◽

10.1042/bj2670625 ◽

1990 ◽

Vol 267 (3) ◽

pp. 625-630 ◽

Cited By ~ 30

Author(s):

V Bossennec ◽

M Petitou ◽

B Perly

Keyword(s):

Glucuronic Acid ◽

Nuclear Overhauser Effect ◽

Double Quantum ◽

Dermatan Sulphate ◽

Proton Proton ◽

Pig Skin ◽

Overhauser Effect ◽

Naturally Occurring ◽

Iduronic Acid ◽

Nuclear Overhauser

The 1H-n.m.r. spectra of various dermatan sulphate preparations present, besides the major signals of the basic disaccharide unit, several other minor signals. We have assigned most of them by n.m.r., using two-dimensional proton-proton double-quantum-correlation and nuclear-Overhauser-effect spectroscopy experiments. This allowed us to identify 2-O-sulphated L-iduronic acid and D-glucuronic acid residues as well as 6-sulphated N-acetylgalactosamine (presumably 4-O-sulphated as well). 2-O-Sulphated iduronic acid was present to similar extents (6-10% of total uronic acids) in pig skin dermatan sulphate and pig intestine dermatan sulphate, whereas glucuronic acid represented 17% of the uronic acid of pig skin dermatan sulphate and was virtually absent (1%) from the other preparation. 6-O-Sulphated N-acetylgalactosamine was present in minor amounts in pig intestine dermatan sulphate only. The influence of sulphation of iduronic acid units on their conformation was assessed by using chemically oversulphated pig intestine dermatan sulphate. Introduction of sulphate groups in this unit in dermatan sulphate tends to shift the conformational equilibrium towards the 1C4 conformer.

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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.

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Structural studies on heparan sulphate from human lung fibroblasts. Characterization of oligosaccharides obtained by selective periodate oxidation of d-glucuronic acid residues followed by scission in alkali

Biochemical Journal ◽

10.1042/bj1910103 ◽

1980 ◽

Vol 191 (1) ◽

pp. 103-110 ◽

Cited By ~ 15

Author(s):

Ingrid Sjöberg ◽

Lars-Ȧke Fransson

Keyword(s):

Uronic Acid ◽

Glucuronic Acid ◽

Human Lung ◽

General Structure ◽

Heparan Sulphate ◽

Periodate Oxidation ◽

Exchange Chromatography ◽

Lung Fibroblasts ◽

Human Lung Fibroblasts ◽

Iduronic Acid

1. 3H- and 35S-labelled heparan sulphate was isolated from monolayers of human lung fibroblasts and subjected to degradations by (a) deaminative cleavage and (b) periodate oxidation/alkaline elimination. Fragments were resolved by gel- and ion-exchange-chromatography. 2. Deaminative cleavage of the radioactive glycan afforded mainly disaccharides with a low content of ester-sulphate and free sulphate, indicating that a large part (approx. 80%) of the repeating units consisted of uronosyl-glucosamine-N-sulphate. Blocks of non-sulphated [glucuronosyl-N-acetyl glucosamine] repeats (3–4 consecutive units) accounted for the remainder of the chains. 3. By selective oxidation of glucuronic acid residues associated with N-acetylglucosamine, followed by scission in alkali, the radioactive glycan was degraded into a series of fragments. The glucuronosyl-N-acetylglucosamine-containing block regions yielded a compound N-acetylglucosamine–R, where R is the remnant of an oxidized and degraded glucuronic acid. Periodate-insensitive uronic acid residues were recovered in saccharides of the general structure glucosamine–(uronic acid–glucosamine)n–R. 4. Further degradations of these saccharides via deaminative cleavage and re-oxidations with periodate revealed that iduronic acid may be located in sequences such as glucosamine-N-sulphate→iduronic acid→N-acetylglucosamine. Occasionally the iduronic acid was sulphated. Blocks of iduronic acid-containing repeats may contain up to five consecutive units. Alternating arrangements of iduronic acid- and glucuronic acid-containing repeats were also observed. 5. 3H- and 35S-labelled heparan sulphates from sequential extracts of fibroblasts (medium, EDTA, trypsin digest, dithiothreitol extract, cell-soluble and cell-insoluble material) afforded similar profiles after both periodate oxidation/alkaline elimination and deaminative cleavage.

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The copolymeric structure of dermatan sulphate produced by cultured human fibroblasts. Different distribution of iduronic acid- and glucuronic acid-containing units in soluble and cell-associated glycans

Biochemical Journal ◽

10.1042/bj1510477 ◽

1975 ◽

Vol 151 (3) ◽

pp. 477-489 ◽

Cited By ~ 60

Author(s):

A Malström ◽

I Carlstedt ◽

L Åberg ◽

L Å Fransson

Keyword(s):

Glucuronic Acid ◽

Human Fibroblasts ◽

Periodate Oxidation ◽

Kinetic Studies ◽

Acid Sulphate ◽

Iduronic Acid ◽

Cultured Human Fibroblasts ◽

Structural Differences ◽

Extracellular Polymer ◽

Testicular Hyaluronidase

The structure of dermatan [35S]sulphate-chondroitin [35S]sulphate copolymers synthesized and secreted by fibroblasts in culture was studied. 35S-labelled glycosaminoglycans were isolated from the medium, a trypsin digest of the cells and the cell residue after 72h of 35SO42-incorporation. The galactosaminoglycan component (dermatan sulphatechondroitin sulphate copolymers) was isolated and subjected to various degradation procedures including digestion with testicular hyaluronidase, chondroitinase-AC and-ABC and periodate oxidation followed by alkaline elimination. The galactosaminoglycans from the various sources displayed significant structural differences with regard to the distribution of various repeating units, i.e. IdUA-GalNAc-SO4 (L-iduronic acid-N-acetyl-galactosamine sulphate), GlcUA-GalNAc-SO4 (D-glucuronic acid-N-acetylgalactosamine-sulphate) and IdUA(-SO4)-GalNAc (L-iduronosulphate-N-acetylgalactosamine). The galactosaminoglycans of the cell residue contained larger amounts of IdUA-GalNAc-SO4 than did those isolated from the medium or those released by trypsin. In contrast, the glycans from the latter 2 sources contained large proportions of periodate-resistant repeat periods [GlcUA-GalNAc-SO4 and IdUA(-SO4)-GalNAc]. Periods containing L-iduronic acid sulphate were particularly prominent in copolymers found in the medium. Kinetic studies indicated that the 35S-labelled glycosaminoglycan of the cell residue accumulated radioactivity more slowly than did the glycans of other fractions, indicating that the material remaining with the cells was not exclusively a precursor of the secreted polymers. The presence of copolymers rich in glucuronic acid or iduronic acid sulphate residues in the soluble fractions may be the result of selective secretion from the cells. Alternatively, extracellular, polymer-level modifications such as C-5 inversion of L-iduronic acid to D-glucuronic acid, or sulphate rearrangements, would yield similar results.

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