Very-high-field n.m.r. studies of bovine lung heparan sulphate oligosaccharides produced by nitrous acid deaminative cleavage. 13C-n.m.r. study of methylene resonances: degree and positions of C-6 sulphation.

Oligosaccharides with the general structure UA-(GlcNAc-GlcUA-)m-aManOH (m = 1-5) (where UA represents uronic acid, GlcNAc N-acetylglucosamine, GlcUA glucuronic acid and aManOH anhydromannitol) were prepared from low-sulphated heparan sulphates of bovine lung origin by nitrous acid deaminative cleavage followed by reduction. Analysis of the methylene signals in the 100 MHz 13C-n.m.r. spectrum of the tetrasaccharide (m = 1) shows that, whereas the extent of C-6 O-sulphation in the GlcNAc is approx. 65%, in the aManOH [formerly a GlcNSO3 (N-sulphoglucosamine) residue in the parent heparan sulphate] it is only approx. 10%. In the higher oligosaccharides (m = 2-5) the gross extent of C-6 O-sulphation of GlcNAc residues falls systematically with increasing oligosaccharide size, whereas that in the aManOH residues remains below 10%. There is also evidence that the C-6 O-sulphation of the GlcNAc residues is confined to the GlcNAc residue adjacent to the non-reducing terminal uronic acid residue. It is therefore tentatively proposed that the GlcNAc in the sequence -GlcNSO3-UA-GlcNAc- might be a favoured substrate for the 6-O-sulphotransferase. It is concluded that in the low-sulphated heparan sulphates GlcNSO3 residues that do not occur in (GlcNSO3-UA-)n blocks tend to have a significantly smaller extent of C-6 O-sulphation than do GlcNAc residues that occur in -GlcNSO3-UA-GlcNAc-GlcUA-GlcNSO3-sequences.     

Interaction between heparan sulphate chains II. Structural characterization of iduronate- and glucuronate-containing sequences in aggregating chains

A heparan sulphate fraction (uronic acid composition: 20% sulphated iduronate, 15% iduronate and 65% glucuronate of total uronate) was separated into aggregating and non-aggregating chains by gel chromatography. ¹³C-NMR analyses revealed that non-aggregating chains had a higher degree of sulphation than did aggregating chains. In aggregating chains, there was more N-acetyl-glucosamine than N-sulphamidoglucosamine; the extent of C-6 sulphation of the latter moiety was low and most of the iduronate residues were non-sulphated. In non-aggregating chains, the N-acetyl-to-N-sulphate ratio was approx. 2 : 1, the N-sulphated glucosamines were also largely C-6 sulphated and the sulphated iduronates were concentrated to these species.Both preparations were subjected to deaminative cleavage which produces fragments like uronate-(N-acetylglucosamine-uronate)_n-anhydromannose. Tetrasaccharides (n = 1) were further fractionated into non-, mono-, di- and trisulphated species by ion-exchange chromatography. The tetrasaccharides have the general carbohydrate structure uronate-N-acetylglucosamine-glucuronate-anhydromannose. Non-reducing terminal glucuronate was removed by β-glucuronidase. The results showed that saccharides containing glucuronate in both positions were more prevalent in the products of aggregating chains. The β-glucuronidase-resistant saccharides (carrying either sulphated or non-sulphated iduronate in non-reducing terminal position) were oxidised with periodate under conditions where non-sulphated residues are degraded, whereas sulphated residues are resistant. Mono-sulphated and di-sulphated tetrasaccharides from aggregating chains were extensively degraded indicating that iduronate-N-acetylglucosamine-glucuronate-anhydromannose was the major sequence.In saccharides from non-aggregating chains iduronate was frequently sulphated. The results of this and previous investigations (Fransson, L.-Å., Nieduszynski, I.A. and Sheehan, J.K. (1980) Biochim. Biophys. Acta 630, 287–300) indicate that an alternating arrangement of iduronate and glucuronate in aggregating chains is present both in N-sulphated block regions and in regionsthat carry alternating N-acetyl- and N-sulphated glucosamine.     

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

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.     

A novel heparan sulphate with high degree of N-sulphation and high heparin cofactor-II activity from the brine shrimp Artemia franciscana

With the aid of heparinase and heparitinases from Flavobacterium heparinum and 13C and IH NMR spectroscopy it was shown that the heparan sulphate isolated from the brine shrimp Artemia franciscana exhibits structural features intermediate between those of mammalian heparins and heparan sulphates. These include an unusually high degree of N-sulphation (with corresponding very low degree of N-acetylation), a relatively high content of iduronic acid residues (both unsulphated and 2-O-sulphated) and a relatively low degree of 6-O-sulphation of the glucosamine residues. The major sequences (glucuronic acid-->N-sulphated glucosamine and glucuronic acid-->N, 6-disulphated glucosamine) are most probably arranged in blocks. Although exhibiting negligible anticlotting activity in the APTT and anti-factor Xa assays the A. franciscana heparan sulphate has a high heparin cofactor-II activity (about 1/3 that of heparin).     

Molecular organization of heparan sulphate from human skin fibroblasts.

The molecular structure of human skin fibroblast heparan sulphate was examined by specific chemical or enzymic depolymerization and high-resolution separation of the resulting oligosaccharides and disaccharides. Important features of the molecular organization, disaccharide composition and O-sulphate disposition of this heparan sulphate were identified. Analysis of the products of HNO2 hydrolysis revealed a polymer in which 53% of disaccharide units were N-acetylated and 47% N-sulphated, with an N-/O-sulphate ratio of 1.8:1. These two types of disaccharide unit were mainly located in separate domains. Heparitinase and heparinase scission indicated that the iduronate residues (37% of total hexuronate) were largely present in contiguous disaccharide sequences of variable size that also contained the majority of the N-sulphate groups. Most of the iduronate residues (approx. 70%) were non-sulphated. About 8-10% of disaccharide units were cleaved by heparinase, but only a minority of these originated from contiguous sequences in the intact polymer. Trisulphated disaccharide units [alpha-N-sulpho-6-sulphoglucosaminyl-(1----4)-iduronate 2-sulphate], which are the major structural units in heparin, made up only 3% of the disaccharide units in heparan sulphate. O-Sulphate groups (approx. 26 per 100 disaccharide units) were distributed almost evenly among C-6 of N-acetylglucosamine, C-2 of iduronate and C-6 of N-sulphated glucosamine residues. The results indicate that the sulphated regions of heparan sulphate have distinctive and potentially variable structural characteristics. The high content of non-sulphated iduronate in this heparan sulphate species suggests a conformational versatility that could have important implications for the biological properties of the polymer.     

(1)H nuclear magnetic resonance spectroscopic analysis for determination of glucuronic and iduronic acids in dermatan sulfate, heparin, and heparan sulfate

(1)H NMR spectroscopy has been established for the determination of uronate residues in glycosaminoglycans (GAGs) such as dermatan sulfate (DS), heparin (HP), and heparan sulfate (HS). Because of variation in the sulfonation positions in DS, HP, or HS, interpretation of spectra is difficult. Solvolysis was applied to remove O-sulfo groups from these GAG chains in dimethyl sulfoxide containing 10% methanol at 80 degrees C for 5 h. In the cases of HP and HS, N-sulfo groups on glucosamine residues were also removed under the same conditions. The resulting unsubstituted amino groups in HP and HS chains were re-N-acetylated using acetic anhydride to obtain homogeneous core structure with the exception of the variation of uronate residues. The contents of glucuronate and iduronate residues in the chemically modified DS, HP, and HS samples were analyzed by 600-MHz (1)H NMR spectroscopy. These methods were applied to compositional analysis of uronate residues in GAGs isolated from various sources.     

Interactions between glycosaminoglycans and blood coagulation factors: 1. Affinity chromatography of glycosaminoglycans on thrombin-substituted agarose

Various glycosaminoglycans have been subjected to affinity chromatography on immobilized bovine thrombin. Chondroitin sulphate, dermatan sulphate and heparan sulphate variants with a sulphate-to-hexosamine molar ratio of ～ 1 exhibited weak affinities. Heparan sulphate/heparin fractions of higher sulphate content could be separated into material with high and low affinity for thrombin. Removal of N-sulphate followed by N-acetylation did not affect binding, whereas oxidation and cleavage of non-sulphated hexuronate abolished the interaction. Heparan-related molecules of high thrombin-affinity comprised sequences where large blocks of sulphated iduronate-containing repeats were joined via a few repeats carrying non-sulphated iduronate or glucuronate to form continuous segments that were larger than decasaccharide.     

Generation of anti-factor Xa active, 3-O-sulfated glucosamine-rich sequences by controlled desulfation of oversulfated heparins.

In the framework of a project aimed at generating heparin-like sulfation patterns and biological activities in biotechnological glycosaminoglycans, different approaches have been considered for simulating the alpha(1-->4)-linked 2-O-sulfated L-iduronic acid (IdoA2SO(3))-->N,6-O-sulfated D-glucosamine (GlcNSO(3)6SO(3)) disaccharide sequences prevalent in mammalian heparins. Since the direct approach of sulfating totally O-desulfated heparins, taken as model compounds for C-5-epimerized sulfaminoheparosan (N-deacetylated, N-sulfated Escherichia coli K5 polysaccharide), preferentially afforded heparins O-sulfated at C-3 instead than at C-2 of the iduronate residues, leading to products with low anticoagulant activities, the problem of re-generating a substantial proportion of the original IdoA2SO(3) residues was circumvented by performing controlled solvolytic desulfation (with 9:1 v/v DMSO-MeOH) of extensively sulfated heparins. The order of desulfation of major residues of heparin GlcN and IdoA and of the minor one D-glucuronic acid was: GlcNSO(3)>GlcN6SO(3)>IdoA3SO(3) congruent with GlcA2SO(3) congruent with GlcN3SO(3)>IdoA2SO(3) congruent with GlcA3SO(3). Starting from a 'supersulfated' low-molecular weight heparin, we obtained products with up to 40% of iduronate residues O-sulfated exclusively at C-2 and up to 40% of their glucosamine residues O-sulfated at both C-6 and C-3. Upon re-N-sulfation, these products displayed an in vitro antithrombotic activity (expressed as anti-factor Xa units) comparable with those of current low-molecular weight heparins.     

Conformational equilibria of alpha-L-iduronate residues in disaccharides derived from heparin.

The disaccharides IdoA(2SO3)-anManOH(6SO3) and IdoA-anManOH (where IdoA represents alpha-L-iduronate, anManOH represents 2,5-anhydro-D-mannitol and SO3 represents sulphate ester) were prepared from bovine lung heparin using HNO2 depolymerization, borohydride reduction and desulphation, and were examined by 400 MHz 1H-n.m.r. spectroscopy. Three-bond proton-proton coupling constants around the IdoA ring were determined under a range of experimental conditions. For unsulphated IdoA all four proton-proton coupling constants varied markedly as a function of temperature, pH and solvent, providing clear evidence for a rapid conformational equilibrium. These data were analysed in terms of the three most energetically stable IdoA conformers: 1C4, 4C1, and 2S0. Predicted coupling constants for these conformers were determined using a modified Karplus-type relationship. For unsulphated IdoA in dimethyl sulphoxide the equilibrium was provoked strongly in favour of a slightly distorted 4C1 'chair' IdoA conformer for which coupling constants have not previously been reported. For sulphated IdoA in aqueous conditions and at low pH the equilibrium is strongly in favour of the alternative 1C4 chair conformer. Under many conditions, however, significant contributions from all three conformers occur for the non-reducing terminal IdoA in these disaccharides.     

Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E(1), and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of (35)SO(4) (2-) into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of (35)SO(4) (2-) or [(14)C]glucosamine into heparan sulphate was decreased to 40-50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse-chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by beta-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, (35)SO(4) (2-) incorporation was decreased to 5%. Addition of p-nitrophenyl beta-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl beta-d-xylosides. Incorporation of [(3)H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.     

Synthesis and immunoadjuvant activity of 2-acetamido-1,5-anhydro-2-deoxy-3-O-[(R)-2-propanoyl-l-alanyl-d-isoglutamine]-d-glucitol (“1-deoxymuramoyl dipeptide”) and its 6-(2-behenoyloxyisobutyrate)

Two heparin-related preparations from beef lung and pig mucosa are able to inhibit the enzymic activity of the clotting factor X_a. These preparations were subjected to deaminative cleavage and periodate oxidation-alkaline elimination. The following structural features were observed: (a) N-acetylated and glucuronate-rich regions are short and frequently intercalated between N-sulphated and iduronate-rich segments of deca- to hexadeca-saccharide size; (b) in the latter segments, sulphated iduronate occurs together with non-sulphated iduronate and glucuronate in a random fashion. These characteristics are distinctly different from those of regular heparan sulphate and of archetypal heparin.     

Conformational equilibrium of unsulphated iduronate in heparan sulphate tetrasaccharides

Proton-proton coupling constants for terminal -l-iduronate residues in tetrasaccharides obtained from heparan sulphates by complete nitrous acid deaminative cleavage were shown to vary with experimental conditions. It is proposed that the iduronate residue is in a conformational equilibrium between the¹C₄ chair and either the²S_o skewboat or possibly the²H₃ half-chair conformers. It was not possible to discriminate between the two non-chair forms empirically. The position of the equilibrium is sensitive to temperature, pH and sulphation of neighbouring residues. The likelihood of iduronate residues within glycosaminoglycans existing in the⁴C₁ conformer in addition to the¹C₄ and²S_o forms is discussed.     

Minimal heparin/heparan sulfate sequences for binding to fibroblast growth factor-1

The glycosaminoglycans heparin and heparan sulfate (HS) bind to fibroblast growth factor FGF1 and promote its dimerization, a proposed prerequisite for binding to a cellular receptor and triggering mitogenic signals. The problem of minimal structural requirements for heparin/HS sequences to bind FGF1 was approached by surface plasmon resonance (SPR), NMR spectroscopy, and MALDI mass spectrometry studies using the three synthetic tetrasaccharides GlcNSO(3)6OR-IdoA2SO(3)-GlcNSO(3)6OR'-IdoA2SO(3)OPr (AA, R = R' = SO(3); BA, R = H, R' = SO(3); BB, R = R' = H; Pr, propyl). AA and BA significantly interact with the protein, whereas BB is practically inactive. The NMR spectra show that, whereas the interaction of AA primarily involves the GlcNSO(3)6SO(3)IdoA2SO(3) disaccharide moiety at its nonreducing end, residues at both the nonreducing (NR) and reducing side (R) appear to be involved in the weaker complex of BA. Furthermore, MALDI experiments show that, in addition to 1:1 protein:tetrasaccharide complexes, AA and BA are able to form 2:1 complexes, indicating that heparin/HS-induced dimerization of FGF1 requires only one 6-OSO(3) group per tetrasaccharide.     

Molecular distinctions between heparan sulphate and heparin. Analysis of sulphation patterns indicates that heparan sulphate and heparin are separate families of N-sulphated polysaccharides.

Heparan sulphate and heparin are chemically related alpha beta-linked glycosaminoglycans composed of alternating sequences of glucosamine and uronic acid. The amino sugars may be N-acetylated or N-sulphated, and the latter substituent is unique to these two polysaccharides. Although there is general agreement that heparan sulphate is usually less sulphated than heparin, reproducible differences in their molecular structure have been difficult to identify. We suggest that this is because most of the analytical data have been obtained with degraded materials that are not necessarily representative of complete polysaccharide chains. In the present study intact heparan sulphates, labelled biosynthetically with [3H]glucosamine and Na2(35)SO4, were isolated from the surface membranes of several types of cells in culture. The polysaccharide structure was analysed by complete HNO2 hydrolysis followed by fractionation of the products by gel filtration and high-voltage electrophoresis. Results showed that in all heparan sulphates there were approximately equal numbers of N-sulpho and N-acetyl substituents, arranged in a similar, predominantly segregated, manner along the polysaccharide chain. O-Sulphate groups were in close proximity to the N-sulphate groups but, unlike the latter, the number of O-sulphate groups could vary considerably in heparan sulphates of different cellular origins ranging from 20 to 75 O-sulphate groups per 100 disaccharide units. Inspection of the published data on heparin showed that the N-sulphate frequency was very high (greater than 80% of the glucosamine residues are N-sulphated) and the concentration of O-sulphate groups exceeded that of the N-sulphate groups. We conclude from these and other observations that heparan sulphate and heparin are separate families of N-sulphated glycosaminoglycans.     

Subunit-specific sulphation of oligosaccharides relating to charge-heterogeneity in porcine lutrophin isoforms.

Lutrophin (LH) consists of an array of isoforms with different charges and bioactivities. This study was undertaken to clarify specifically how oligosaccharides of alpha and beta subunits contribute to LH isoform charges. Porcine LH (pLH) was separated into four isoforms by isoelectric focusing (IEF), followed by subunit isolation. Their oligosaccharides were released by hydrazinolysis, labelled by reduction with NaB3H4, and fractionated by HPLC with a Mono Q column into five populations differing in the number of sulphate (S) and sialic acid (N) residues, designated as Neutral, N-1, S-1, S-N and S-2. Oligosaccharides were predominantly sulphated (S-1 and S-2) and infrequently sialylated (N-1 and S-N). Further analysis, including concanavalin A (Con A) affinity chromatography, desialylation, desulphation, sequential exoglycosidase digestion and methylation, clarified the structures of the acidic oligosaccharides. All were of the biantennary complex type. Their two peripheral branches were SO4-4GalNAc beta 1-4Glc-NAc and GalNAc beta 1-4GlcNAc or GlcNAc in S-1, SO4-4GalNAc beta 1-4GlcNAc and Sia alpha 2-6Gal beta 1-4GlcNAc in S-N, and (SO4-4GalNAc beta 1-4GlcNAc)2 in S-2 (where GalNAc is N-acetylgalactosamine and GlcNAc is N-acetylglucosamine). Ten percent of S-1 and of S-N had a bisecting GlcNAc residue. Sulphate residues occurred in nearly the same amount for both subunits; however, the alpha and beta subunits were sulphated differently. S-1 predominated in the alpha subunit, while S-1 and S-2 were major components in the beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative structural studies of urinary glycosaminoglycans in the Hurler and Hunter syndromes.

Some hitherto undetected differences in chemical and macromolecular structure between both dermatan sulphates and heparan sulphates excreted in the Hurler and Hunter syndromes are demonstrated. 1. Of Hunter dermatan sulphate, 37-43% is resistant to periodate oxidation, as opposed to 25% of the corresponding Hurler material. It is likely that the resistance is conferred by the presence of sulphate groups on carbon atoms 2 or 3 of the iduronate residues, correlating with the recently established deficiency of a sulphoiduronate sulphatase in Hunter fibroblasts. 2. Two distinct electrophoretic species of dermatan sulphate are found in Hunter urine, but only one in Hurler preparations. 3. Ion-exchange chromatography and gel filtration reveal that Hurler dermatan sulphate is more heterogeneous with respect to molecular weight distribution than the other. The dermatan sulphates were degraded by hyaluronidase to a limited extent. 4. Hurler heparan sulphate contains a higher proportion of sulphoamino-glucose than material from Hunter urine. Similar high levels in Sanfilippo patients, representing 65-78% of the total glucosamine suggest a direct correlation with mental deficiency.     

Structural features of the contact zones for heparan sulphate self-association

The self-association between heparan sulphate chains has been investigated by using heparan sulphate oligosaccharides for the competitive elution of [3H]heparan sulphate from heparan sulphate-agarose. Partial or complete periodate-oxidation followed by alkali-catalysed scission afforded oligomers having the general structure GlcN-(HexA-GlcN)n-R. Oligosaccharides with n greater than 5 were able to desorb bound heparan sulphate, provided that mixed or alternating arrangements of iduronate and glucuronate were present in these fragments. Longer fragments were more effective than shorter ones. The present results corroborate previous proposals that the highly copolymeric regions of heparan sulphate serve as contact zones for the chain-chain association.     

The asparagine-linked oligosaccharides at individual glycosylation sites in human thyrotrophin.

The asparagine-linked carbohydrate structures at each of the three glycosylation sites of human thyrotrophin were investigated by 400 MHz 1H-NMR spectroscopy. Highly purified, biologically active human thyrotrophin (hTSH) was dissociated into its subunits hTSH alpha (glycosylated at Asn 52 and Asn 78) and hTSH beta (glycosylated at Asn 23). The alpha-subunit was further treated with trypsin which gave two glycopeptides that were subsequently separated by reverse-phase HPLC and identified by amino acid sequence analysis. The oligosaccharides were liberated from hTSH alpha glycopeptides and from intact hTSH beta by hydrazinolysis, and were fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC preparatory to structural analysis. The N-glycans present on hTSH were mainly diantennary complex-type structures with a common Man alpha 1-3 branch that terminated with 4-O-sulphated GalNAc. The Man alpha 1-6 branch displayed structural heterogeneity in the terminal sequence, with chiefly alpha 2-3-sialylated Gal and/or 4-O-sulphated GalNAc. The relative amounts of the two major complete diantennary oligosaccharides and their core fucosylation differed according to glycosylation site; the sulphated/sialylated diantennary oligosaccharide was most abundant at the two sites on the alpha-subunit, whereas the disulphated, core-fucosylated oligosaccharide was more plentiful on the beta-subunit. Some interesting structural features, not previously reported for the N-glycans of hTSH, included 3-O-sulphated galactose (SO4-3Gal) and peripheral fucose (Fuc alpha 1-3GlcNAc) in the Man alpha 1-6 branch of some diantennary structures; the former suggests the presence of a hitherto uncharacterized galactose-3-O-sulphotransferase in thyrotroph cells of the human anterior pituitary gland.     

Initiation of altered heparan sulphate on beta-D-xyloside in rat hepatocytes

Inhibition of protein synthesis by cycloheximide 10(-3)M reduced the incorporation of [35S]sulphate into heparan sulphate to about 5% of untreated hepatocytes. Addition of rho-nitrophenyl beta-D-xyloside could partially revert this inhibitory effect. The sulphated material isolated from the cell layer or secretions of hepatocytes grown in presence of cycloheximide and rho-nitrophenyl beta-D-xyloside were shown to be mostly free heparan sulphate chains not bound to core protein. Covalent association of beta-xylosides to the heparan sulphates was demonstrated for heparan sulphate synthetized in the presence of [35S]sulphate, cycloheximide and the fluorogenic 4-methylumbelliferyl beta-D-xyloside. Beta-Xylosides served as an initiator of heparan sulphate chain synthesis in rat hepatocytes only in the absence of protein synthesis. Heparan sulphates primed on artificial beta-xylosides are slightly smaller in molecular size and are more sulphated than chains linked to core protein.     

Co-polymeric glycosaminoglycans in transformed cells. Transformation-dependent changes in the co-polymeric structure of heparan sulphate

1. Heparan sulphates from normal 3T3 fibroblasts are association-prone as indicated by their affinity for agarose gels substituted with cognate heparan sulphate species. Heparan sulphates from SV40-transformed or polyoma-virus-transformed cells have no affinity for the same gels. 2. Heparan sulphates from the medium, the pericellular and intracellular pools of normal, SV40-transformed and polyoma-transformed 3T3 cells were separated into four subfractions (HS1–HS4) by ion-exchange chromatography. In general, HS1–HS3 were found in cell-derived heparan sulphates, whereas HS3–HS4 were present in the medium. The heparan sulphates from transformed cells were more heterogeneous and of lower charge density than those from the normal counterpart. 3. Degradations via periodate oxidation/alkaline elimination yielded the oligomers glucosamine-(hexuronate–glucosamine)_n-R with n=1–5 and a large proportion of N-sulphate groups. There was a large contribution of fragments n=4–5 from heparan sulphates of normal cells. These fragments were less common in low-sulphated heparan sulphates of transformed cells. In the case of medium-drived heparan sulphates all species had a low content of fragments n=4–5. 4. The size distribution of (glucuronate–N-acetylglucosamine)_n regions was assessed after deaminative cleavage. It was broad and ranged from n=1–10 for all heparan sulphate species. In the case of medium-derived heparan sulphates there were distinct differences between normal and transformed cells. In the latter chains the N-acetyl-rich segments were both shorter and longer than in the normal case. The shape of the disaccharide peak was consistent with a lower content of O-sulphate in the heparan sulphates from transformed cells. 5. It was concluded that heparan sulphates from medium or transformed cells exhibit the greatest structural deviation from the normal case. The finding of lower proportions of extended, iduronate/glucuronate-bearing, N-sulphate-rich segments in heparan sulphates of transformed cells was particularly interesting in view of the fact that these elements have been associated with ability to self-interact.