scholarly journals The acid lability of the glycosidic bonds of l-iduronic acid residues in glycosaminoglycans

1980 ◽  
Vol 191 (2) ◽  
pp. 355-363 ◽  
Author(s):  
H E Conrad
Keyword(s):  
Acid Hydrolysis ◽  
Glucuronic Acid ◽  
Heparan Sulphate ◽  
Neutral Sugar ◽  
Uronic Acids ◽  
Dermatan Sulphate ◽  
Glycosidic Bonds ◽  
Iduronic Acid ◽  
Quantitative Analyses ◽  
Kinetics Of

Heparan sulphate, heparin and dermatan sulphate were hydrolysed in 0.5M-H2SO4 at 100 degrees C. At intervals portions of the hydrolysate were removed and treated with HNO2 at pH 4.0 to cleave the glycosidic bonds of the N-unsubstituted hexosamine residues and to convert both free and combined hexosamines into anhydrohexoses. These hydrolysis/deamination mixtures were reduced with NaB3H4 and analysed by radiochromatography for alpha-L-iduronosylanhydrohexose, beta-D-glucuronosylanhydrohexose, and the free uronic acids and anhydrohexose. These data gave a kinetic profile of the cleavage of the alpha-L-iduronosyl and the beta-D-glucuronosyl bonds in these glycosaminoglycans. The beta-D-glucuronosyl bonds showed the expected resistance to acid hydrolysis, but the alpha-L-iduronosyl bonds were found to be as labile to acid as some neutral sugar glycosides. This unusual lability of alpha-D-iduronosyl-anhydromannitol and beta-D-glucuronosylanhydromannitol. The procedures used to follow the kinetics of glycosaminoglycan hydrolysis can also be sued to obtain quantitative analyses of L-iduronic acid, D-glucuronic acid and hexosamine in these polymers.

scholarly journals 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

1977 ◽  
Vol 167 (2) ◽  
pp. 383-392 ◽  
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.

scholarly journals 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

1980 ◽  
Vol 191 (1) ◽  
pp. 103-110 ◽  
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.

scholarly journals Specific association of iduronic acid-rich dermatan sulphate with the extracellular matrix of human skin fibroblasts cultured on collagen gels

1983 ◽  
Vol 215 (1) ◽  
pp. 107-116 ◽  
Author(s):  
J T Gallagher ◽  
N Gasiunas ◽  
S L Schor
Keyword(s):  
Human Skin ◽  
Heparan Sulphate ◽  
Surface Membrane ◽  
Skin Fibroblasts ◽  
Collagen Gels ◽  
Human Skin Fibroblasts ◽  
Dermatan Sulphate ◽  
Cellular Functions ◽  
Iduronic Acid ◽  
Sulphated Glycosaminoglycans

Human skin fibroblasts cultured on collagen gels produced two dermatan sulphate species, one, enriched in iduronic acid residues, that bound specifically to the collagenous fibres of the gel, the other, enriched in glucuronic acid, that accumulated in the culture medium. Collagen-binding and collagen-non-binding dermatan sulphates were also produced by cells grown on plastic surfaces, but in these cultures each constituent was released into the growth medium. Net synthesis of dermatan sulphate was 3-fold higher in cells maintained on collagen gels. In contrast, heparan sulphate synthesis was not influenced by the nature of the culture surface. The concentration of heparan sulphate in surface-membrane extracts was similar for cells grown on plastic and on collagen gels, but cells cultured on collagen showed a notable increase in the content of surface-membrane dermatan sulphate. The patterns of synthesis and distribution of sulphated glycosaminoglycans observed in skin fibroblasts maintained on collagen gels may reflect differentiated cellular functions.

SECMA 1®, a mitogenic hexapeptide from Ulva algeae modulates the production of proteoglycans and glycosaminoglycans in human foreskin fibroblast

1998 ◽  
Vol 17 (1) ◽  
pp. 18-22 ◽  
Author(s):  
R Ennamany ◽  
D Saboureau ◽  
N Mekideche ◽  
E E Creppy
Keyword(s):  
Glucuronic Acid ◽  
De Novo ◽  
Heparan Sulphate ◽  
Guanidine Hydrochloride ◽  
Sodium Deoxycholate ◽  
Effective Concentration ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblasts ◽  
Dermatan Sulphate ◽  
Salt Extract

SECMA 1® is a polypeptide purified from a green algeae of the Ulva species by several gel chromatographies, showing the following sequence (Glu-Asp-Arg-Leu-Lys-Pro). In order to determine the effect of SECMA 1® on human skin fibroblasts extracellular matrix, proteoglycans (PGs) and glycosaminoglycans (GAGs) were assayed after 24 h incubation of 20 day-old foreskin fibroblasts at the 2nd passage. The results revealed that most of [35S]sulphate was associated with fibroblast membranes, which contained (67%) of the total de novo synthesized sulphated PGs, in two distinct forms: one hydrophilic (39%), and one hydrophobic (28%). The remaining `matrix' retained 5% of proteoglycans. The remaining 35S-label may represent the free label in the cytosol. After 24 h incubation of skin fibroblasts with different concentrations of SECMA 1® (2, 4 and 10 μg/ml), the [35S] sulphate incorporation into PGs of Salt-extract, sodium deoxycholate (DOC) extract and Guanidine hydrochloride (GuA-HCl)-extract was increased significantly ( P<0.005) with 4 μg/ml, as compared to untreated control. The most effective concentration (4 μg/ml) increased the different [35S]sulphate PGs extracts (NaCl, DOC and GuA-HCl) by respectively (66; 17 and 75%). The relative contents of iduronic and glucuronic acid in the GAG produced by skin fibroblasts were estimated. No effect of SECMA 1® on the incorporation of [35S]sulphate into Heparan sulphate was found. The incorporation of [35S]sulphate into (chondroïtine sulphate + heparan sulphate) and (chondroïtine sulphate + dermatan sulphate) was increased by respectively 37% and 11% by SECMA 1® (4 μg/ml).

URONIC ACIDS FROM WHITE SPRUCE (PICEA GLAUCA (MOENCH) VOSS)

1959 ◽  
Vol 37 (1) ◽  
pp. 29-34 ◽  
Author(s):  
G. A. Adams
Keyword(s):  
Acid Hydrolysis ◽  
Picea Glauca ◽  
Glucuronic Acid ◽  
White Spruce ◽  
Uronic Acids ◽  
Spruce Wood ◽  
Acidic Sugars ◽  
Hydrolysis Of ◽  
Acidic Components

Acid hydrolysis of extractive-free white spruce wood produced a number of neutral and acidic sugars and oligosaccharides. The acidic components were isolated and three of these were shown to be 4-O-methyl-D-glucuronic acid, 2-O-(4-O-methyl-α-D-glucopyranosyluronic acid)-D-xylose, and tentatively O-(4-O-methyl-α-D-glucopyranosyluronic acid)-(1→ 2)-O-β-D-xylopyranosyl-(1→ 4)-D-xylopyranose.

scholarly journals Substrate specificities of mouse heparan sulphate glucosaminyl 6-O-sulphotransferases

2003 ◽  
Vol 372 (2) ◽  
pp. 371-380 ◽  
Author(s):  
Emanuel SMEDS ◽  
Hiroko HABUCHI ◽  
Anh-Tri DO ◽  
Eva HJERTSON ◽  
Helena GRUNDBERG ◽  
...  
Keyword(s):  
Growth Factors ◽  
Glucuronic Acid ◽  
Heparan Sulphate ◽  
Sulphate Group ◽  
Functional Importance ◽  
Cellular Functions ◽  
Recombinant Enzymes ◽  
Substrate Specificities ◽  
Iduronic Acid ◽  
Target Sequences

Glycosaminoglycan heparan sulphate interacts with a variety of proteins, such as growth factors, cytokines, enzymes and inhibitors and, thus, influences cellular functions, including adhesion, motility, differentiation and morphogenesis. The interactions generally involve saccharide domains in heparan sulphate chains, with precisely located O-sulphate groups. The 6-O-sulphate groups on glucosamine units, supposed to be involved in various interactions of functional importance, occur in different structural contexts. Three isoforms of the glucosaminyl 6-O-sulphotransferase (6-OST) have been cloned and characterized [H. Habuchi, M. Tanaka, O. Habuchi, K. Yoshida, H. Suzuki, K. Ban and K. Kimata (2000) J. Biol. Chem. 275, 2859–2868]. We have studied the substrate specificities of the recombinant enzymes using various O-desulphated poly- and oligo-saccharides as substrates, and using adenosine 3′-phosphate 5′-phospho[35S]sulphate as sulphate donor. All three enzymes catalyse 6-O-sulphation of both -GlcA-GlcNS- and -IdoA-GlcNS- (where GlcA represents d-glucuronic acid, NS the N-sulphate group and IdoA the l-iduronic acid) sequences, with preference for IdoA-containing targets, with or without 2-O-sulphate substituents. 6-OST1 showed relatively higher activity towards target sequences lacking 2-O-sulphate, e.g. the -GlcA-GlcNS- disaccharide unit. Sulphation of such non-O-sulphated acceptor sequences was generally favoured at low acceptor polysaccharide concentrations. Experiments using partially O-desulphated antithrombin-binding oligosaccharide as the acceptor revealed 6-O-sulphation of N-acetylated as well as 3-O-sulphated glucosamine residues with each of the three 6-OSTs. We conclude that the three 6-OSTs have qualitatively similar substrate specificities, with minor differences in target preference.

scholarly journals Structure of heparan sulphate oligosaccharides and their degradation by exo-enzymes

1979 ◽  
Vol 183 (3) ◽  
pp. 711-720 ◽  
Author(s):  
A Linker
Keyword(s):  
Structural Analysis ◽  
Nitrous Acid ◽  
Uronic Acid ◽  
Glucuronic Acid ◽  
Heparan Sulphate ◽  
Major Fraction ◽  
Sulphate Content ◽  
Acid Degradation ◽  
Iduronic Acid ◽  
Heparan Sulphate Chain

Oligosaccharides obtained from heparan sulphate by nitrous acid degradation were shown to be degraded sequentially by beta-D-glucuronidase or alpha-L-iduronidase followed by alpha D-N-acetylglucosaminidase. Structural analysis of the tetrasaccharide fraction showed the following. (1) N-Acetylglucosamine is preceded by a non-sulphated uronic acid residue that can be either D-glucuronic of L-iduronic acid, but followed by a glucuronic acid residue. (2) The N-acetylglucosamine in the major fraction is sulphated. (3) Very few if any of the uronic acid residues are sulphated (4). The results indicate that the area of the heparan sulphate chain where disaccharides containing N-acetylglucosamine and N-sulphated glucosamine residues alternate is higher in sulphate content than expected and that the sulphate groups are mainly located on the hexosamine units.

scholarly journals Biosynthesis of dermatan sulphate. Defructosylated Escherichia coli K4 capsular polysaccharide as a substrate for the d-glucuronyl C-5 epimerase, and an indication of a two-base reaction mechanism

1996 ◽  
Vol 313 (2) ◽  
pp. 589-596 ◽  
Author(s):  
Helgi H. HANNESSON ◽  
Åsa HAGNER-McWHIRTER ◽  
Kerstin TIEDEMANN ◽  
Ulf LINDAHL ◽  
Anders MALMSTRÖM
Keyword(s):  
Escherichia Coli ◽  
Reaction Mechanism ◽  
Acid Hydrolysis ◽  
Capsular Polysaccharide ◽  
Glucuronic Acid ◽  
Microsomal Enzyme ◽  
Dermatan Sulphate ◽  
Mild Acid Hydrolysis ◽  
Mild Acid ◽  
Escherichia Coli K4

The capsular polysaccharide from Escherichia coli K4 consists of a chondroitin {[GlcA(β1→3)GalNAc(β1→4)]n} backbone, to which β-fructofuranose units are linked to C-3 of D-glucuronic acid (GlcA) residues. Removal of the fructose units by mild acid hydrolysis provided a substrate for the GlcA C-5 epimerase, which is involved in the generation of L-iduronic acid (IdoA) units during dermatan sulphate biosynthesis. Incubation of this substrate with solubilized fibroblast microsomal enzyme in the presence of 3H2O resulted in the incorporation of tritium at C-5 of hexuronyl units. A Km of 67×10-6 M hexuronic acid (equivalent to disaccharide units) was determined, which is similar to that (80×10-6 M) obtained for dermatan (desulphated dermatan sulphate). Vmax. was about 4 times higher with dermatan than with the K4 substrate. A defructosylated K4 polysaccharide isolated after incubation of bacteria with D-[5-3H]glucose released 3H2O on reaction with the epimerase, and thus could be used to assay the enzyme. Incubation of a K4 substrate with solubilized microsomal epimerase for 6 h in the presence of 3H2O resulted in the formation of about 5% IdoA and approximately equal amounts of 3H in GlcA and IdoA. A corresponding incubation of dermatan yielded approx. 22% GlcA, which contained virtually all the 3H label. These results are tentatively explained in terms of a two-base reaction mechanism, involving a monoprotic L-ido-specific base and a polyprotic D-gluco-specific base. Most of the IdoA residues generated by the enzyme occurred singly, although some formation of two or three consecutive IdoA-containing disaccharide units was observed.

Structure of Heparin and Related Glycosaminoglycans

1979 ◽  
Author(s):  
B. Casu
Keyword(s):  
Functional Groups ◽  
Plasma Proteins ◽  
Glucuronic Acid ◽  
The Other ◽  
Hybrid Structures ◽  
Dermatan Sulphate ◽  
Iduronic Acid

The structure of heparin is largely accounted for by disaccharide sequences of α1,4-linked 2-0-sulphated L-iduroic acid and N,6-0-disulphated D-glucosamine. However, the insertion of other residues (especially D-glucuronic acid, nor-sulphated L-iduronic acid and N-acetylated D-glucosamine) leads to hybrid structures. Also the other iduromc acid-containing glycosaminoglycans are structurally heterogeneous. Heparan sulphates contain variable amounts of heparin-like blocks, ana dermatan sulphate contains also chondroitin -like segments.Available evidence on the structure and conformation of the above glycosaminoglycans will be discussed in terms of the availability ot individual functional groups for interaction with plasma proteins.

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

1974 ◽  
Vol 143 (2) ◽  
pp. 369-378 ◽  
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.

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