Structural requirements for heparan sulphate self-association

To investigate heparan sulphate self-association, various sub-fractions of beef-lung heparan sulphate have been subjected to affinity chromatography on heparan sulphate-agarose. A particular variant of heparan sulphate was chiefly bound to matrices substituted with the same or cognate heparan sulphates. N-desulphation and N-acetylation abolished the chain-chain interaction. Also, dermatan sulphates and chondroitin sulphates showed affinity for heparan sulphate-agarose. [3H]Heparan sulphates that were bound to a heparan sulphate-agarose were desorbed by elution with the corresponding heparan sulphate chains and also with unrelated heparan sulphates, heparin, and the galactosaminoglycans to various degrees. However, the corresponding heparan sulphate species was the most efficient at low concentrations. Dextran sulphate was unable to desorb bound heparan sulphate. When the corresponding heparan sulphate was N-desulphated/N-acetylated, carboxyl-reduced, or periodate-oxidised (D-glucuronate), the modified polymer was unable to displace [3H]heparan sulphate from heparan sulphate-agarose. The displacing ability of heparin was also destroyed by periodate oxidation. It is concluded that self-interaction between heparan sulphate chains is strongly dependent on the overall molecular conformation. The N-sulphate and carboxylate groups as well as the integrity of the D-glucuronate residue are all essential for maintaining the proper secondary structure.     

Isolation and characterization of basement membrane and cell proteoheparan sulphates from HR9 cells

The mouse teratocarcinoma cell line HR9 was investigated for proteoheparan sulphate production. Four species of proteoheparan sulphate molecules were isolated and purified to homogeneity. The proteoheparan sulphate isolated from the tissue-culture medium contains four heparan sulphate side-chains of 25 kDa each, and its core protein has an approximate molecular mass of 50 kDa. The proteoheparan sulphates associated with the cells were separated into three individual species: cell proteoheparan sulphate I exhibits structural characteristics which are very similar to the proteoheparan sulphate isolated from the tissue culture medium; cell proteoheparan sulphates II and III contain one heparan sulphate chain of 25 kDa and 20 kDa, and core proteins of approximately 30 kDa and 25 kDa respectively. Antisera, raised against the medium form, react specifically with basement membranes in various tissues by immunofluorescence. This staining pattern was compared to the pattern observed with an antiserum which we have obtained to a proteoheparan sulphate species isolated from the plasma membrane of bovine aortic endothelial cells. The structural and immunological data suggest that basement membrane and plasma membrane proteoheparan sulphates are different biosynthetic products and are not directly related to each other.     

Constant and variable domains of different disaccharide structure in corneal keratan sulphate chains.

Four peptidokeratan sulphate fractions of different Mr and degree of sulphation were cut from the pig corneal keratan sulphate distribution spectrum. After exhaustive digestion with keratanase, the fragments were separated on DEAE-Sephacel and Bio-Gel P-10 and analysed for their Mr, degree of sulphation and amino sugar and neutral sugar content. It was found that every glycosaminoglycan chain is constructed of a constant domain of non-sulphated and monosulphated disaccharide units and a variable domain of disulphated disaccharide units. Total neuraminic acid of the four peptidokeratan sulphates was recovered from their isolated linkage-region oligosaccharides. In kinetic studies, the four peptidokeratan sulphates were investigated for Mr distribution after various incubation times with keratanase. There was a continuous shift towards lower Mr and no appearance of a distinct intermediate-sized product at any degradation time. The linkage-region oligosaccharide was already being liberated after a very short incubation period. From the results of these kinetic investigations in connection with the results of neuraminic acid analyses it is suggested that there exists only one disaccharide chain per peptidokeratan sulphate molecule. A model of corneal keratan sulphate is postulated. One of the alpha-mannose residues in the linkage region is bound to an oligosaccharide consisting of a lactosamine and a terminal sialic acid. The other alpha-mannose residue is attached to the disaccharide chain. This chain contains one or two non-sulphated disaccharide units at the reducing end, followed by 10-12 monosulphated disaccharide units. The disulphated disaccharide moiety of variable length is positioned at the non-reducing end of the chain.     

A general method for the detection and mapping of submicrogram quantities of glycosaminoglycan oligosaccharides on polyacrylamide gels by sequential staining with azure A and ammoniacal silver.

A sensitive method has been developed for the visualization of nonradiolabeled glycosaminoglycan oligosaccharides resolved by polyacrylamide gel electrophoresis using fixation with azure A followed by staining with ammoniacal silver. This method, which can detect as little as 1-2 ng of a single oligosaccharide species, can be used to stain a few micrograms of a complex oligosaccharide mixture. The combination of gradient polyacrylamide gel electrophoresis and sequential azure A/silver staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate) and hyaluronate, as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes. This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited and/or where radiolabeling is impractical or undesirable.     

Domain structure of proteoheparan sulphate from confluent cultures of human embryonic skin fibroblasts.

Radiolabelled proteoheparan sulphates were isolated from confluent monolayers of fibroblasts and from their spent media. The cell-surface-associated proteoglycan (Mr 350 000) has a core protein of Mr 180 000 that is cleaved by reduction of disulphide bonds into polypeptides of Mr 90 000, both of which can bind transferrin [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661]. Thrombin digestion of the proteoglycan yielded two major fragments. The larger one contained the heparan sulphate chains and glycoprotein-type oligosaccharides, whereas the smaller one contained interchain disulphide bond(s) and had affinity for transferrin as well as for octyl-Sepharose. The larger thrombic fragment was cleaved by trypsin into fragments containing the heparan sulphate chains and the oligosaccharides respectively. The smaller proteoheparan sulphate derived from the culture medium (Mr 150 000) had a core protein of Mr 30 000, which contained heparan sulphate-attachment and oligosaccharide-attachment regions, but no domains for binding of transferrin or for hydrophobic interactions.     

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.     

Heparan sulphate/heparin glycosaminoglycans with strong affinity for the growth-promoter spermine have high antiproliferative activity

Depletion of intracellular polyamine pools inhibits cell proliferation.Polyamine pools are maintained by intracellular synthesis andby uptake from the extracellular environment. It may be expectedthat cationic polyamines are sequestered by the polyanionicglycosaminoglycan substituents of extracellular proteoglycans.Moreover, highly sulphated heparin-related glycans inhibit growthof human embryonic lung fibroblasts. We have therefore investigatedinteractions between polyamines and heparin-related glycosaminoglycans.Affinity chromatography of various polyamines on heparin-agaroseindicated that spermine was the only polyamine that bound efficientlyto this type of glycan. By using equilibrium dialysis we foundthat spermine binds to a highly sulphated heparan sulphate/heparinpreparation with a dissociation constant of 3.7x10^–5M.Enzymatic degradation of heparan sulphate using three differentheparan sulphate/heparin lyases, separately or in combinationand in the absence or presence of spermine, was used to generatespermine-binding and degradation-protected oligosaccharides.As indicated by chromato graphic and electrophoretic analysisa size- and charge-heterogeneous collection was obtained. However,protected oligosaccharides derived from antiproliferative heparansulphates were inactive. Highly sulphated, antiproliferativeheparan sulphates were subfractionated on spermine-agarose yieldinghigh-affinity material with increased antiproliferative activity.A very potent material was obtained from pig skin. Althoughthere was generally a clear correlation between high spermine-affinityand strong growth-inhibition, no correlation with sulphate contentor oligosaccharide mapping patterns could be detected. Beeflung heparan sulphate comprised naturally occurring fragmentsof eicosasaccharide size with substantially increased specificactivity. As these fragments were longer than oligosaccharidesgenerated by enzymatic degradation in the presence of spermine(hexa- to tetradecasaccharide), multiple spermine-binding sitesin tandem may be necessary to induce antiproliferative activity. heparan sulphate spermine interaction antiproliferation     

Interaction between heparan sulphate chains. I. A gel chromatographic, light-scattering and structural study of aggregating and non-aggregating chains

1. Heparan sulphate from bovine lung was fractionated with cetylpyridinium chloride. Solubilisation of complexes was accomplished by increasing concentrations of NaCl in a step-wise manner. Fractions I-IV, which were low-sulphated, contained more D-glucuronic acid than L-iduronic acid, fraction V contained equal proportions while fraction VI was L-iduronic acid-rich. 2. Gel chromatography of heparan sulphates II-IV in 0.5 M sodium acetate yielded extremely asymmetric profiles, while fractions V, VI and heparin did not. 3. Heparan sulphate IV was separated into aggregatable and non-aggregatable species by gel chromatography in 0.5 M sodium acetate. The particle/molecular weights of the two species were determined by light scattering. In 0.15 M NaCl or KCl the aggregatable chains yielded particle weights of 60 000-100 000 while the molecular weight was 20 000 (in 4.0 M guanidine HCl). Non-aggregatable chains afforded 'monomeric' values in 0.15 M NaCl or KCl. 4. Periodate oxidation of D-glucuronic acid residues in N-acetylated block regions followed by scission in alkali was used to fragment aggregating and non-aggregating heparan sulphate IV. The former chains yielded, on average, shorter oligosaccharides than did the latter. Reoxidation of the remaining D-glucuronic acid residues (adjacent to N-sulphated amino sugars) in the oligosaccharides followed by alkaline cleavage resulted in distinctly different fragmentation patterns in the two cases. The iduronate-containing oligosaccharides derived from aggregatable chains were markedly degraded into fragments ranging from glucosamine-L-iduronic acid-glucosamine-(C-3 fragment) to higher saccharides. Only higher saccharides were obtained from fragments of non-aggregatable chains. 5. It is concluded that self-associating heparan sulphates comprise both D-glucuronic acid- and L-iduronic acid-containing repeating units and that these units are arranged in an alternating or mixed fashion. These characteristics are analogous to those observed with self-associating dermatan sulphate species (Fransson, L.-A. and C?ster, L. (1979) Biochim. Biophys. Acta 582, 132-144).     

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.     

Polyamine requirement for streptomycin action on protein synthesis in bacteria

Self-association between various heparan sulphate species and oligosaccharide fragments thereof have been studied by affinity chromatography. Polysaccharides or oligosaccharides were coupled to agarose and free chains were applied at low concentrations (less than or equal to 2 mg/ml) in 0.15 M NaCl to minimize self-association between free chains. The results show that the interaction may be specific. Heparan sulphate chains chiefly bind to gels substituted with cognate chains, i.e. the same kind or closely similar ones. Oligosaccharides of the general structure glucosamine-(iduronate/glucuronate-glucosamine)n--O--C(=CH2)--CHO were prepared by periodate oxidation/alkaline elimination and also coupled to agarose via the --CHO group. Cognate heparan sulphate chains were bound to this affinity matrix with the same affinity as in the case of heparan-sulphate--agarose. Free oligosaccharides were not bound to oligosaccharide-agarose, nor to the corresponding heparan-sulphate--agarose. Oligosaccharides of the same size and containing only iduronate were ineffective as affinity ligands. It is concluded that the segments comprising both iduronate and glucuronate may serve as contact zones in the heparan sulphate/heparan sulphate self-association and that the strength of binding is dependent on cooperative interactions between a number of such zones. The putative contact zones, as ligands on the matrix, showed an emerging lack of specificity as non-associating or unrelated and associating chains were bound to this gel. This is ascribed to a randomization of the contact zones which, in the polymeric chains, are placed in their proper register by the intervening (glucuronate-N-acetylglucosamine)n segments.     

Enzymatic placement of 6-O-sulfo groups in heparan sulfate

Heparan sulfate is a highly sulfated polysaccharide that exhibits important physiological and pathological functions. The glucosamine residue of heparan sulfate can carry sulfo groups at the 2-N, 3-O, and 6-O positions, leading to diverse polysaccharide structures. 6-O-Sulfation at the glucosamine residue contributes to a wide range of biological functions. Here, we report a method for controlling the positioning of 6-O-sulfo groups in oligosaccharides. This was achieved by synthesizing oligosaccharide backbones from a disaccharide building block utilizing glycosyltransferases followed by modifications using heparan sulfate N-sulfotransferase and 6-O-sulfotransferases. This method offers a viable approach for preparing heparan sulfate oligosaccharides with precisely located 6-O-sulfo groups.     

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.     

Biological implications of the structural, antithrombin affinity and anticoagulant activity relationships among vertebrate heparins and heparan sulphates.

We analysed the distribution, structural characteristics, antithrombin-III-binding properties and anticoagulant activities of heparins and heparan sulphates isolated from the tissues of a wide range of vertebrates. Heparin has a curiously limited distribution, since it was absent from lower aquatic vertebrate species, present in only certain organs such as intestine in many higher vertebrates, and completely absent from the rabbit among mammals examined. The heparins were structurally diverse, and they exhibited a broad range of anticoagulant activities, from approx. 50% to 150% of average commercial heparins. Although there was a rough correlation between the anticoagulant potency of the starting isolate and the proportional content of material exhibiting high-affinity binding to the proteinase inhibitor antithrombin III, activities of high-affinity fractions from heparins low in activity overlapped those of low-affinity fractions from highly active heparins. Heparan sulphates, which in contrast were isolated from nearly all vertebrate organs, contained high-affinity subfractions constituting up to 5% of the starting material and possessing anticoagulant potencies of 2-30 units/mg. In consideration of the heparin data, we infer that its biological function is either species-specific or may be served by other molecular elements, and that there exists considerable diversity in the antithrombin-III-binding sequence of heparin. The more-generally distributed glycosaminoglycan heparan sulphate possesses within its variable structure a small high-affinity subfraction with low anticoagulant potency, whether isolated from aorta or other tissues. Although heparan sulphate appears to have an essential function at the cellular level, we suggest that this is probably not that of providing heparin-like antithrombotic effects on vascular surfaces.     

ISOLATION AND CHARACTERIZATION OF GLYCOSAMINOGLYCANS FROM BRAIN OF CHILDREN WITH PROTEIN-CALORIE MALNUTRITION

Abstract— The uronic acid containing glycosaminoglycans (GAGs) were isolated from the brains of 1-year-old and 4-year-old kwashiorkor children and characterised by constituent analyses. A marked reduction is the total GAG concentration of brain was noticed in both cases of kwashiorkor. In the 1-year-old kwashiorkor brain, hyaluronic acid is the most predominant GAG (73.5 per cent) whereas heparan sulphate, chondroitin sulphates and low sulphated chondroitin sulphate constituted less than 10 per cent. In the 4-year-old kwashiorkor brain, the proportion of hyaluronic acid was 27.5 per cent, low sulphated chondroitin sulphate 31.2 per cent, chondroitin sulphates 28.3 per cent and heparan sulphate 10 per cent. This marked reduction in the concentration as well as qualitative changes in GAG in protein-calorie malnutrition as compared to the normal is discussed in relation to brain function.     

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.     

Examination of the substrate specificity of heparin and heparan sulfate lyases

We have examined the activities of different preparations of heparin and heparan sulfate lyases from Flavobacterium heparinum. The enzymes were incubated with oligosaccharides of known size and sequence and with complex polysaccharide substrates, and the resulting degradation products were analyzed by strong-anion-exchange high-performance liquid chromatography and by oligosaccharide mapping using gradient polyacrylamide gel electrophoresis. Heparinase (EC 4.2.2.7) purified in our laboratory and a so-called Heparinase I (Hep I) from a commercial source yielded similar oligosaccharide maps with heparin substrates and displayed specificity for di- or trisulfated disaccharides of the structure----4)-alpha-D-GlcNp2S(6R)(1----4)-alpha-L-IdoAp2S( 1----(where R = O-sulfo or OH). Oligosaccharide mapping with two different commercial preparations of heparan sulfate lyase [heparitinase (EC 4.2.2.8)] indicated close similarities in their depolymerization of heparan sulfate. Furthermore, these enzymes only degraded defined oligosaccharides at hexosaminidic linkages with glucuronic acid:----4)-alpha-D-GlcNpR(1----4)-beta-D-GlcAp(1----(where R = N-acetamido or N-sulfo). The enzymes showed activity against solitary glucuronate-containing disaccharides in otherwise highly sulfated domains including the saccharide sequence that contains the antithrombin binding region in heparin. A different commercial enzyme, Heparinase II (Hep II), displayed a broad spectrum of activity against polysaccharide and oligosaccharide substrates, but mapping data indicated that it was a separate enzyme rather than a mixture of heparinase and heparitinase/Hep III. When used in conjunction with the described separation procedures, these enzymes are powerful reagents for the structural/sequence analysis of heparin and heparan sulfate.     

Biosynthetic oligosaccharide libraries for identification of protein-binding heparan sulfate motifs. Exploring the structural diversity by screening for fibroblast growth factor (FGF)1 and FGF2 binding

Heparan sulfate is crucial for vital reactions in the body because of its ability to bind various proteins. The identification of protein-binding heparan sulfate sequences is essential to our understanding of heparan sulfate biology and raises the possibility to develop drugs against diseases such as cancer and inflammatory conditions. We present proof-of-principle that in vitro generated heparan sulfate oligosaccharide libraries can be used to explore interactions between heparan sulfate and proteins, and that the libraries expand the available heparan sulfate sequence space. Oligosaccharide libraries mimicking highly 6-O-sulfated domains of heparan sulfate were constructed by enzymatic O-sulfation of O-desulfated, end-group (3)H-labeled heparin octasaccharides. Acceptor oligosaccharides that were 6-O-desulfated but only partially 2-O-desulfated yielded oligosaccharide arrays with increased ratio of iduronyl 2-O-sulfate/glucosaminyl 6-O-sulfate. The products were probed by affinity chromatography on immobilized growth factors, fibroblast growth factor-1 (FGF1) and FGF2, followed by sequence analysis of trapped oligosaccharides. An N-sulfated octasaccharide, devoid of 2-O-sulfate but with three 6-O-sulfate groups, was unexpectedly found to bind FGF1 as well as FGF2 at physiological ionic strength. However, a single 2-O-sulfate group in the absence of 6-O-sulfation gave higher affinity for FGF2. FGF1 binding was also augmented by 2-O-sulfation, preferentially in combination with an adjacent upstream 6-O-sulfate group. These results demonstrate the potential of the enzymatically generated oligosaccharide libraries.     

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.     

Isolation and some structural analyses of a proteodermatan sulphate from calf skin.

A proteodermatan sulphate was isolated from 0.15 M-NaCl and 0.45 M-NaCl extracts of newborn-calf skin. The proteoglycan was separated from collagen and hyaluronic acid by precipitation with cetylpyridinium chloride and CsCl-density-gradient centrifugation. Further purification was performed by ion-exchange, affinity and molecular-sieve chromatography. The proteoglycan bound to concanavalin A-Sepharose in 1 M-NaCl. It gave a positive reaction with periodic acid/Schiff reagent and contained 8.3% of uronic acid. The dermatan sulphate, the only glycosaminoglycan component, was composed of 74% iduronosylhexosamine units and 26% glucuronosylhexosamine units. The Mr was assessed to be 15000-20000 by gel chromatography. The core protein was found to be a sialoglycoprotein that had O-glycosidic oligosaccharides with N-acetylgalactosamine at the reducing termini. The molar ratio of oligosaccharide chains to dermatan sulphate was approx. 3:1. From these results the proposed structure of proteodermatan sulphate is: one dermatan sulphate chain (average Mr 17500), three O-glycosidic oligosaccharide chains and probably N-glycosidic oligosaccharide chain(s) bound to one core-protein molecule (Mr 55000).     

An analytical system for the characterization of highly heterogeneous mixtures of N-linked oligosaccharides

A novel system for characterizing complex N-linked oligosaccharide mixtures that uses a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), capillary electrophoresis (CE), and high-performance liquid chromatography (HPLC) has been developed. In this study, oligosaccharides released from recombinant TNK-tPA (tissue plasminogen activator) were derivatized with 5-amino-2-naphthalenesulfonic acid (ANSA). The negative charge imparted by the ANSA label facilitated the analysis of the oligosaccharides by MALDI-TOF MS by allowing the observation of both neutral and sialylated oligosaccharides in a single negative ion mode spectrum. Labeling with ANSA was also determined to be advantageous in the characterization of oligosaccharides by both HPLC and CE. The ANSA label was demonstrated to provide superior resolution over the commonly used label 8-aminopyrene-1,3,6-trisulfonic acid (APTS) in both the CE and HPLC analysis of oligosaccharides. To date, no other labels that enable the analysis of complex oligosaccharide mixtures in a single mass spectral mode, while also enabling high-resolution chromatographic and electrophoretic separation of the oligosaccharides, have been reported. By integrating the structural information obtained by MALDI-TOF MS analysis with the ability of CE and HPLC to discriminate between structural isomers, the complete characterization of complex oligosaccharide mixtures is possible.