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Green seaweeds of the genus Ulva are rich in the bioactive sulfated polysaccharide ulvan. Herein we characterise ulvan from Ulva species collected from the Bay of Plenty, Aotearoa New Zealand. Using standardised procedures, we quantified, characterised, and compared ulvans from blade (U. australis, U. rigida, U. sp. B, and Ulva sp.) and filamentous (U. flexuosa, U. compressa, U. prolifera, and U. ralfsii) Ulva species. There were distinct differences in composition and structure of ulvans between morphologies. Ulvan isolated from blade species had higher yields (14.0-19.3 %) and iduronic acid content (IdoA = 7-18 mol%), and lower molecular weight (Mw = 190-254 kDa) and storage moduli (G' = 0.1-6.6 Pa) than filamentous species (yield = 7.2-14.6 %; IdoA = 4-7 mol%; Mw = 260-406 kDa; G' = 22.7-74.2 Pa). These results highlight the variability of the physicochemical properties of ulvan from different Ulva sources, and identifies a morphology-based division within the genus Ulva., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Extracellular matrix, either produced by cancer cells or by cancer-associated fibroblasts, influences angiogenesis, invasion, and metastasis. Chondroitin/dermatan sulfate (CS/DS) proteoglycans, which occur both in the matrix and at the cell surface, play important roles in these processes. The unique feature that distinguishes DS from CS is the presence of iduronic acid (IdoA) in DS. Here, we report that CS/DS is increased five-fold in human biopsies of esophagus squamous cell carcinoma (ESCC), an aggressive tumor with poor prognosis, as compared with normal tissue. The main IdoA-producing enzyme, DS epimerase 1 (DS-epi1), together with the 6-O- and 4-O-sulfotransferases, were highly upregulated in ESCC biopsies. Importantly, CS/DS structure in patient tumors was significantly altered compared with normal tissue, as determined by sensitive mass spectrometry. To further understand the roles of IdoA in tumor development, DS-epi1 expression, and consequently IdoA content, was downregulated in ESCC cells. IdoA-deficient cells exhibited decreased migration and invasion capabilities in vitro, which was associated with reduced cellular binding of hepatocyte growth factor, inhibition of pERK-1/2 signaling, and deregulated actin cytoskeleton dynamics and focal adhesion formation. Our findings show that IdoA in DS influences tumorigenesis by affecting cancer cell behavior. Therefore, downregulation of IdoA by DS-epi1 inhibitors may represent a new anticancer therapy.

Glycosaminoglycans were extracted from both young rabbit growth plate (GRP) and articular (ART) cartilage tissues and enzymatically treated to selectively eliminate chondroitin sulfates and hyaluronic acid. The procedure avoided any fractionation step that could enrich the extract with over- or under-sulfated species. Isolated heparan sulfate (HS) was characterized by mono- and bidimensional nuclear magnetic resonance (NMR) spectroscopy to quantify their specific structural features and/or by mass spectrometry to establish the disaccharide composition. Both GRP and ART HSs, despite differing in their yield (GRP at least 100 times greater than ART), exhibited a surprisingly high degree of sulfation. Quantitative two-dimensional heteronuclear single-quantum coherence-NMR analysis of GRP HS revealed unusually high N-sulfated glucosamine and 2-O-sulfated iduronic acid contents, similar to heparin. The unique pentasaccharide sequence of the binding site for antithrombin was also detected in a significant amount. High-performance liquid chromatography mass spectrometry analysis of the enzymatic digests with a cocktail of heparin lyases of both cartilaginous HSs confirmed the NMR results. As well as the discovery of an unusual HS structure in the two different types of rabbit cartilage, the feasibility of the analytical method adopted here has been demonstrated within this study. Such a method can be used to isolate and analyze HS from both normal and pathologic tissues. Characterization of healthy and pathological HS structures will contribute to improve the understanding of diseases related to malfunctions of HS biosynthesis and/or metabolism.

The amniotic membrane (AM) is the innermost layer of fetal membranes and possesses various biological activities. Although the mechanism underlying these biological activities remains unclear, unique components seem to be involved. AM contains various extracellular matrix components such as type I collagen, laminin, fibronectin, hyaluronan, and proteoglycans bearing chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycan side chains. Since CS/DS have been implicated in various biological processes, we hypothesized that CS/DS in AM may play a major role in the biological activities of AM. Therefore, the structure and bioactivity of the CS/DS chains from porcine fetal membranes (FM-CS/DS) were investigated. A compositional analysis using various chondroitinases revealed that the characteristic DS domain comprised of iduronic acid-containing disaccharide units is embedded in FM-CS/DS, along with predominant disaccharide units, GlcA-GalNAc, GlcA-GalNAc(4-O-sulfate), and GlcA-GalNAc(6-O-sulfate), where GlcA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. The average molecular mass of FM-CS/DS chains was unusually large and estimated to be 250 - 300 kDa. The FM-CS/DS chains showed neurite outgrowth-promoting activity, which was eliminated by digestion with chondroitinase ABC of the CS/DS chains. This activity was suppressed by antibodies against growth factors including pleiotrophin, midkine, and fibroblast growth factor-2, suggesting the involvement of these growth factors in the neurite outgrowth-promoting activity. The binding of these growth factors to FM-CS/DS was also demonstrated by surface plasmon resonance spectroscopy.

Determination of the pK(a) values of heparin disaccharide functional groups can provide insights into the nature of glycosaminoglycan (GAG)-protein interactions and prove useful for optimization of the charged-based separations typically used in GAG analysis. In order to gain a better understanding into the capillary electrophoresis (CE) separation process, the pK(a) values of the carboxylate and primary amine moieties of 11 heparin disaccharide standards were determined through (1)H NMR detected pH titrations. These pK(a) values were used to calculate the effective net charge of each disaccharide and compared to the electrophoretic mobilities measured by CE. Although a different migration order had been reported by other researchers, our results indicate a strong positive correlation between the two measurements, consistent with the migration order observed in our CE separations. The effect of mutarotation was also examined by (1)H NMR. Mutarotation equilibrium constants favored the alpha anomer over the beta conformation. pK(a) values determined for both anomers of the four disaccharide standards containing a GlcN primary amine indicated that the beta anomer of the GlcN residue was more acidic. Partial separation of these anomers was achieved in CE separations using either formic acid or phosphate buffer.

HSEPI (glucuronyl C5-epimerase) catalyzes the conversion of d-glucuronic acid to l-iduronic acid in heparan sulfate (HS) biosynthesis. Disruption of the Hsepi gene in mice yielded a lethal phenotype with selective organ defects but had remarkably little effect on other organ systems. We have approached the underlying mechanisms by examining the course and effects of FGF2 signaling in a mouse embryonic fibroblast (MEF) cell line derived from the Hsepi(-)(/)(-) mouse. The HS produced by these cells is devoid of l-iduronic acid residues but shows up-regulated N- and 6-O-sulfation compared with wild type (WT) MEF HS. In medium fortified with 10% fetal calf serum, the Hsepi(-)(/)(-) MEFs proliferated and migrated similarly to WT cells. Under starvation conditions, both cell types showed attenuated proliferation and migration that could be restored by the addition of FGF2 to WT cells, whereas Hsepi(-)(/)(-) cells were resistant. Moreover, ERK phosphorylation following FGF2 stimulation was delayed in Hsepi(-)(/)(-) compared with WT cells. Assessment of HS-growth factor interaction by nitrocellulose filter trapping revealed a strikingly aberrant binding property of FGF2 and glia-derived neurotropic factor to Hsepi(-)(/)(-) but not to WT HS. glia-derived neurotropic factor has a key role in kidney development, defective in Hsepi(-)(/)(-) mice. By contrast, Hsepi(-)(/)(-) and WT HS interacted similarly and in conventional mode with FGF10. These findings correlate defective function of growth factors with their mode of HS interaction and may help explain the partly modest organ phenotypes observed after genetic ablation of selected enzymes in HS biosynthesis.

Electron detachment dissociation (EDD) Fourier transform mass spectrometry has recently been shown to be a powerful tool for examining the structural features of sulfated glycosaminoglycans (GAGs). The characteristics of GAG fragmentation by EDD include abundant cross-ring fragmentation primarily on hexuronic acid residues, cleavage of all glycosidic bonds, and the formation of even- and odd-electron product ions. GAG dissociation by EDD has been proposed to occur through the formation of an excited species that can undergo direct decomposition or ejects an electron and then undergoes dissociation. In this work, we perform electron-induced dissociation (EID) on singly charged GAGs to identify products that form via direct decomposition by eliminating the pathway of electron detachment. EID of GAG tetrasaccharides produces cleavage of all glycosidic bonds and abundant cross-ring fragmentation primarily on hexuronic acid residues, producing fragmentation similar to EDD of the same molecules, but distinctly different from the products of infrared multiphoton dissociation or collisionally activated decomposition. These results suggest that observed abundant fragmentation of hexuronic acid residues occurs as a result of their increased lability when they undergo electronic excitation. EID fragmentation of GAG tetrasaccharides results in both even- and odd-electron products. EID of heparan sulfate tetrasaccharide epimers produces identical fragmentation, in contrast to EDD, in which the epimers can be distinguished by their fragment ions. These data suggest that for EDD, electron detachment plays a significant role in distinguishing glucuronic acid from iduronic acid.

The relative proportion of L: -iduronic acid (IdoA) and D: -glucuronic acid (GlcA) is of great importance for the structure-function relationship of chondroitin sulfate (CS)/dermatan sulfate (DS). However, determination of the isotypes of uronic acid residues in CS/DS is still a challenge, due to the instability of free uronic acid released by chemical degradation and its conversion to unsaturated uronic acid by digestion with bacterial eliminase. (1)H-Nuclear magnetic resonance (NMR) spectroscopy is a promising tool with which to address this issue, but the traditional method based on the assignment of the ring proton signals of IdoA and GlcA residues still has drawbacks such as the serious overlap of signals in the (1)H-NMR spectrum of CS/DS polysaccharides. We found that the proton signals of the N-acetyl group of N-acetyl-D: -galactosamines in CS and DS could be clearly distinguished and accurately integrated in the one-dimensional (1D) (1)H-NMR spectrum. Based on this finding, here we report a novel, sensitive, and nondestructive 1D (1)H-NMR-based method to determine the proportion of IdoA and GlcA residues in CS/DS hybrid chains.

Distinguishing the epimers iduronic acid (IdoA) and glucuronic acid (GlcA) has been a long-standing challenge for the mass spectrometry analysis of glycosaminoglycan (GAG) oligosaccharides. In this work, electron detachment dissociation (EDD) and Fourier transform ion cyclotron resonance mass spectrometry is shown to provide mass spectral features that can distinguish GlcA from IdoA in heparan sulfate (HS) tetrasaccharides. EDD of HS tetrasaccharide dianions produces a radical species that fragments to produce information-rich glycosidic and cross-ring product ions which can be used to determine the sites of acetylation/sulfation. More significantly, EDD of HS tetrasaccharide epimers produces diagnostic product ions that can be used to distinguish IdoA from GlcA. These diagnostic product ions are not observed in the tandem mass spectra obtained by collisionally activated dissociation or infrared multiphoton dissociation of the tetrasaccharides, suggesting a radical-initiated mechanism for their formation. Differences in the observed product ions obtained by EDD of the tetrasaccharide epimers can be rationalized by simple alpha-cleavage of an oxy radical located at C2 or C3 or a radical at C3 or C4. These radicals are proposed to arise from a hydrogen rearrangement in which a hydrogen atom is transferred from the C2 or C3 hydroxyl group or C3 or C4 to a carboxy radical at C5. This hydrogen transfer depends on the proximity of the carboxy radical to the hydroxyl group on C2 or C3 or the hydrogen on C3 or C4 and is thus influenced by C5 stereochemistry. These epimer-sensitive fragmentations should allow this approach to be applied to the structural analysis of a wide variety of GAG oligosaccharides.

This study was designed to identify the specific proteoglycans and glycosaminoglycans (GAGs) in the leaflets and chordae of the mitral valve and to interpret their presence in relation to the tensile and compressive loads borne by these tissues. Leaflets and chordae from normal human mitral valves (n = 31, obtained at autopsy) were weighed and selected portions digested using proteinase K, hyaluronidase, and chondroitinases. After fluorescent derivatization, fluorophore-assisted carbohydrate electrophoresis was used to separate and quantify the derivatized saccharides specific for each GAG type. In addition, the lengths of the chondroitin/dermatan sulfate chains were determined. Proteoglycans were identified by western blotting. The regions of the valve that experience tension, such as the chordae and the central portion of the anterior leaflet, contained less water, less hyaluronan, and mainly iduronate and 4-sulfated N-acetylgalactosamine with chain lengths of 50-70 disaccharides. These GAGs are likely associated with the small proteoglycans decorin and biglycan, which were found in abundance in the tensile regions. The valve regions that experience compression, such as the posterior leaflet and the free edge of the anterior leaflet, contained significantly more water, hyaluronan, and glucuronate and 6-sulfated N-acetylgalactosamine with chain lengths of 80-90 disaccharides. These GAGs are likely components of water-binding versican aggregates, which were abundant in the compressive loading regions. The relative amounts and distributions of these GAGs are therefore consistent with the tensile and compressive loads that these tissues bear. Finally, the concentrations of total GAGs and many different chondroitin/dermatan sulfate subclasses were significantly decreased with advancing age.

Heparin, a natural glycosaminoglycan (GAG), is widely used for the treatment of thrombotic diseases. Most of its side effects are related to its ability to bind to different proteins, thus interfering with its target biological activity. To gain insight into structure-activity relationships, we investigated the interaction of a homogeneous series of sulfated polysaccharides, derived from controlled desulfation of a supersulfated low-molecular-weight heparin (LMWH) with the target enzymes human antithrombin (AT) and thrombin (T). In addition, we analyzed the activation process of the serpin AT against T and factor Xa (FXa). A nonlinear correlation between the strength of the AT-heparin complex and the polysaccharide sulfation degree was observed, whereas only a modest modulation of T binding to heparin occurred. The efficiency of the LMWH derivatives in activating AT toward the proteases was generally high for derivatives exhibiting a low dissociation constant. Only the supersulfated heparin showed a serpin activation ability higher than expected from the affinity studies. Examination of the sulfation pattern in the light of the above results suggests a key role of the substitution of the iduronic acid residue in the heparin-mediated serpin binding and activation processes. Indeed, sulfation at position 2 of the uronic acid is beneficial, whereas 2,3-disubstitution generates unfavorable contacts between the GAG and AT. Glucosamine sulfation at position 6 appears to grant increased catalytic efficiency. These results indicate that chemical modification of the heparin sulfation pattern can be used to modulate binding specificity and activity toward its biological targets.

The glycosaminoglycan, heparan sulfate (HS), binds proteins to modulate signaling events in embryogenesis. All identified protein-binding HS epitopes contain l-iduronic acid (IdoA). We report that targeted disruption of the murine d-glucuronyl C5-epimerase gene results in a structurally altered HS lacking IdoA. The corresponding phenotype is lethal, with renal agenesis, lung defects, and skeletal malformations. Unexpectedly, major organ systems, including the brain, liver, gastrointestinal tract, skin, and heart, appeared normal. We find that IdoA units are essential for normal kidney, lung, and skeletal development, albeit with different requirement for 2-O-sulfation. By contrast, major early developmental events known to critically depend on heparan sulfate apparently proceed normally even in the absence of IdoA.

Biglycan is widely distributed in the extracellular matrix and is a member of the small proteoglycan family characterized by a core protein with leucine-rich repeat motifs. We show in this paper for the first time that biglycan from human lung fibroblasts can be expressed as different isoforms. These isoforms can be separated from the predominant form of biglycan by hydrophobic interaction chromatography, where the more hydrophobic isoforms are retarded. The newly found isoforms of biglycan have a smaller core protein substituted with smaller glycosaminoglycan chains, migrating on SDS/PAGE at between 110 and 200 kDa. These molecules were identified as biglycan using MALDI-TOF MS. Identification of C-terminal peptides together with glycosylation of the N-terminal glycosaminoglycan sites excludes the possibility of terminal proteolytic cleavage. The biglycan isoforms are N-glycosylated, which demonstrates that a lack in N-glycosylation is not the reason for a smaller core. Two components revealed by RT-PCR indicate alternative splicing, which could be located in regions of the protein that have not been identified, with the exclusion of sites of glycosylations. Analyses of glycosaminoglycan chain length of the isoforms show that besides the normally occurring glycosaminoglycan chains, there is a mixture of shorter glycosaminoglycan chains. Structural analysis shows that these glycosaminoglycan chains contain a lower proportion of iduronic acid (61%) relative to glucuronic acid when compared to the glycosaminoglycan chain of the predominant form of biglycan (71%). We can anticipate that variation in structure of biglycan can cause changes in the connective tissue formation depending on its ability to bind matrix molecules, as well as cytokines.

Galactosaminoglycans, isolated from decalcified chicken eggshell by papain digestion and ion-exchange chromatography, were fractionated by selective precipitation at varying concentrations of ethanol and characterized by chemical and enzymatic methods. The eggshell contained 0.15 microg galactosaminoglycan uronic acid/mg dry weight. Most (to approximately 87% of total) galactosaminoglycans were found to be chondroitin sulfate-dermatan sulfate copolymers with iduronic acid contents being approximately 20 to 30% of uronic acid. The remaining (to approximately 12% of total) galactosaminoglycans were chondroitin sulfate-dermatan sulfate copolymers with higher iduronic acid contents averaging 59% of uronic acid. Results of chondroitinase-ABC digestion demonstrated 4-sulfated disaccharides to be the major repeating units in the chicken eggshell galactosaminoglycans.

(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., (Copyright 2001 Academic Press.)

Galactosaminoglycans, i.e. dermatan sulfate (DS) and chondroitin sulfate, are linear heteropolysaccharides consisting of repeating disaccharide units of L-iduronic acid (L-IdoA) or D-glucuronic acid (D-GlcA) residues linked to N-acetyl-galactosamine. High-performance capillary electrophoresis (HPCE or CE) has been successfully used for determining the disaccharide composition of glycosaminoglycans. However, only limited information is available on how to identify oligomeric domains rich in D-GlcA or L-IdoA. The aim of this study was therefore to develop a rapid and accurate CE procedure by which such oligosaccharides can be determined together with the variously sulfated disaccharides. Isolated dermatan sulfates of human origin were separately digested with chondroitinases ABC, AC and B and the enzymic products were derivatized with 2-aminoacridone. CE analysis of these products was performed using a phosphate buffer, pH 3.0, and reversed polarity at 30 kV. The derivatization enabled their detection with laser-induced fluorescence (LIF) and UV at 260 nm at much higher sensitivity than the detection of nonderivatized delta-saccharides at 232 nm and therefore components undetectable at 232 nm were nicely detected after derivatization. Except for delta-disaccharides, altogether five distinct oligosaccharides with differences in charge density were identified. Depending on the lyase that produced these oligomers, information on the presence of L-IdoA- or D-GlcA-containing domains within the DS chain and the sulfation pattern of these oligomeric domains was obtained. This CE method could also be useful in studying the functional oligomeric domains in galactosaminoglycan chains.

Functional interactions of heparan sulfate (HS) with selected proteins depend on distinct saccharide sequences which are generated during biosynthesis of the polysaccharide. Glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs) catalyze both the N-deacetylation and N-sulfation reactions that initiate the modification of the (GlcNAc-GlcA)(n) polysaccharide backbone. The N-acetyl/N-sulfate exchange is restricted to certain regions of the polysaccharide chains, and only these can be further modified by glucuronyl C5-epimerization and O-sulfation at various positions. To investigate whether NDST isoforms influenced differently the structure of HS, murine NDST-1 was overexpressed in human kidney 293 cells, and the structure of the HS produced was compared to HS from NDST-2 overexpressing cells [Cheung, W. F., Eriksson, I., Kusche-Gullberg M., Lindahl, U., and Kjellén, L. (1996) Biochemistry 35, 5250-5256]. The level of N-sulfation increased from 40% in control cells to 60% and 80%, respectively, in NDST-1 and NDST-2 transfected cells. Interestingly, the increase in N-sulfation was accompanied by an increased chain length, while no effect on IdoA content or O-sulfation was seen. The most extended N-sulfated domains were found in HS synthesized by NDST-2 transfected cells. Since both the N-deacetylase and the N-sulfotransferase activities were lower in these cells than in the NDST-1 overexpressing cells, we conclude that, in addition to the level of enzyme expression, the NDST isoform also is important in determining the N-sulfation pattern in HS.

The structural characteristics of oversulfated chondroitin/dermatan sulfates (C/DSs) in the fibrous lesions of the rat liver with cirrhosis were examined. Long-Evans Cinnamon rats were subjected to the present study as the model animals with cirrhosis. The serial polyester wax sections of liver with cirrhosis were processed into the fibrous lesions and the nonfibrous lesions. The oversulfated C/DSs in the tissue sections on a glass slide were degraded to unsaturated disaccharides by chondroitinase ABC and ACII digestion in the presence of bacterial collagenase. Subsequently, the resulting unsaturated disaccharides were determined by the reversed-phase ion-pair high-performance liquid chromatography with fluorometric postcolumn derivatization using 2-cyanoacetamide as a reagent. Through these in situ investigations, we found some facts as follows: (i) in the fibrous lesion, the remarkable increase of the oversulfated C/DSs content and the decrease of the oversulfation degree of the C/DSs were observed compared with those in the nonfibrous lesion, (ii) the proportion of the iduronic acid content in the C/DSs in the fibrous lesion was significantly low compared with that in the nonfibrous lesion, and (iii) in the nonfibrous lesion close to the fibrous lesion, both quantitative and qualitative alterations of C/DSs were not observed at all. These findings indicate that the oversulfated C/DSs with low iduronic acid content are possible marker for the fibrogenesis of liver with cirrhosis., (Copyright 1999 Academic Press.)

The non-reducing ends of the preponderant dermatan sulfates of adult human skin (DS18 and DS28) can have D-GalNAc, D-GlcA and L-1doA. D-GlcA of DS18 and D-GalNAc of both DS18 and DS28 are sulfated.

A natural low molecular weight heparin (8.5 kDa), with an anticoagulant activity of 95 IU/mg by the USP assay, was isolated from the shrimp Penaeus brasiliensis. The crustacean heparin was susceptible to both heparinase and heparitinase II from Flavobacterium heparinum forming tri- and di-sulfated disaccharides as the mammalian heparins. (13)C and (1)H NMR spectroscopy revealed that the shrimp heparin was enriched in both glucuronic and non-sulfated iduronic acid residues. The in vitro anticlotting activities in different steps of the coagulation cascade have shown that its anticoagulant action is mainly exerted through the inhibition of factor Xa and heparin cofactor II-mediated inhibition of thrombin. The shrimp heparin has also a potent in vivo antithrombotic activity comparable to the mammalian low molecular weight heparins.

To study how the pattern of sulfation along a heparan sulfate chain affects its turnover, we examined heparan sulfate catabolism in wild-type Chinese hamster ovary cells and mutant pgsF-17, defective in 2-O-sulfation of uronic acid residues (Bai, X., and Esko, J. D. (1996) J. Biol. Chem. 271, 17711-17717). Heparan sulfate from the mutant contains normal amounts of 6-O-sulfated glucosamine residues and iduronic acid and somewhat higher levels of N-sulfated glucosamine residues but lacks any 2-O-sulfated iduronic or glucuronic acid residues. Pulse-chase experiments showed that both mutant and wild-type cells transport newly synthesized heparan sulfate proteoglycans to the plasma membrane, where they shed into the medium or move into the cell through endocytosis. Internalization of the cell-associated molecules leads to sequential endoglycosidase (heparanase) fragmentation of the chains and eventual lysosomal degradation. In wild-type cells, the chains begin to degrade within 1 h, leading to the accumulation of intermediate (10-20-kDa) and small (4-7-kDa) oligosaccharides. Mutant cells did not generate these intermediates, although internalization and intracellular trafficking of the heparan sulfate chains appeared normal, and the chains degraded with normal kinetics. This difference was not due to defective heparanase activities in the mutant, since cytoplasmic extracts from mutant cells cleaved wild-type heparan sulfate chains in vitro. Instead, the heparan sulfate chains from the mutant were relatively resistant to degradation by cellular heparanases. These findings suggest that 2-O-sulfated iduronic acid residues in heparan sulfate are important for cleavage by endogenous heparanases but not for the overall catabolism of the chains.

This study presents a comparison of heparan sulphate chains isolated from various porcine and bovine tissues. 1H-NMR spectroscopy (500 MHz) was applied for structural and compositional studies on intact heparan sulphate chains. After enzymic digestion of heparan sulphate using heparin lyase I (EC 4.2.2.7) II and III (EC 4.2.2.8), the compositions of unsaturated disaccharides obtained were determined by analytical capillary electrophoresis. Correlations between the N-sulphated glucosamine residues and O-sulphation and between iduronic acid content and total sulphation were discovered using the data obtained by NMR and disaccharide analysis. Heparan sulphate chains could be classified into two groups based on the sulphation degree and the iduronic acid content. Heparan sulphate chains with a high degree of sulphation possessed also a significant number of iduronic acid residues and were isolated exclusively from porcine brain, liver and kidney medulla. The presence and amount of N-unsubstituted glucosamine residues (GlcNp) was established in all of the heparan sulphates examined. The structural context in which this residue occurs was demonstrated to be: high sulphation domain --> 4)-beta-D-GlcAp-(1 --> 4)-alpha-D-GlcNp-(1 --> 4)-beta-D-GlcAp-(1 --> low sulphation domain (where GlcNp is 2-amino-2-deoxyglucopyranose, and GlcAp is glucopyranosyluronic acid), based on the isolation and characterization of a novel, heparin lyase III-derived, GlcNp containing tetrasaccharide and hexasaccharide. The results presented suggest that structural differences may play a role in important biological events controlled by heparan sulphate in different tissues.

Hypertrophic scarring is characterized by disordered collagen fibrils. In order to determine whether this is, in part, a result of changes in the population of proteoglycans that are thought to be involved in regulation of collagen fibril formation, we have compared PGs from post-burn normal and hypertrophic scar tissue, as well as from human dermis and epidermis. Efforts to separate the two major iduronic acid-containing proteoglycans, decorin [PG(IdoA)-II] and biglycan [PG(IdoA)-I], for quantitation were not successful. The different N-terminal sequences of these two iduronic acid-containing proteoglycans PG(IdoA-I and -II were utilized to estimate the relative amounts in the above PG(IdoA) preparations. Normal scar, dermis and epidermis were all found to contain primarily decorin with low (< 10%) levels of biglycan relative to decorin. In contrast, iduronic acid-containing proteoglycans from hypertrophic scar were found to be approximately 30% biglycan [PG(IdoA)-I]. This may be a proximal cause of altered collagen fibrils, or may result in alterations in the sequestration of growth factors, which then results in changes in collagen that effect the appearance of the scar. 1966 Elsevier Science Ltd.

Heparan sulfate (HS) secreted into the medium of bovine aortic endothelial cell (BAEC) cultures was subjected to chemical and enzymatic degradation followed by analysis using gel-filtration and ion-exchange chromatography. Treatment with HNO2 showed that 41% of the disaccharides were N-sulfated. Degradation by Heparin lyases I (Hep I) showed that 8-9% of the disaccharides contained IdoA(2S) residues. Heparin lyase III (Hep III) degradation produced mainly disaccharides with 67% of the molecules glycosidic linkages susceptible to cleavage. Further degradation of Hep III-resistant fragments with Hep I showed that IdoA(2S) residues were predominantly positioned centrally within the repeating GlcNSO3(+/- 6S)alpha 1-4IdoA containing domains. Digestion with a mixture of Heparin lyases I, II and III degraded the molecule almost entirely to disaccharides, with small amounts of tetrasaccharides containing resistant linkages, suggesting the presence of 3-O sulfated GlcNSO3. Further analysis of the disaccharide products by ion-exchange chromatography and comparison with the data from single enzymatic digestion, allowed an estimate of the disaccharide composition to be made. The results suggest an ordered arrangement of structural domains; however, variations in the structure of these domains results in a heterogeneous population of HS chains. It is suggested that biosynthetic differences in HS structure may act as a regulator of bFGF induced cellular responses.

The glycosaminoglycan (GAG) and uronic acid (UA) composition of human hip articular cartilage from patients with femoral neck fractures [assumed osteoporosis (OP); n = 12], from patients with osteoarthritis (OA; n = 12) and from normal controls (n = 9) was determined. Full depth tissue samples from the control and OP groups were analysed from the superior, inferior, anterior and posterior regions, while the OA tissue was from cystic (tissue growing on top of cystic bone lesions) and osteophytic regions, from normal and fibrillated resident cartilage and from regions immediately adjacent to eburnated bone. The total sulphated GAG and UA content was reduced in the inferior region of control cartilage compared to the other regions and the values of all regions of the assumed OP group. Cystic regions and OA cartilage adjacent to the bone also showed lower GAG and UA levels than the other regions. The ratios of chondroitin 6-sulphate (C6S) to chondroitin 4-sulphate (C4S) indicated a similar pattern in the different regions of controls and the patient group with femoral neck fracture (OP group). The cystic and osteophytic cartilage of the OA group exhibited lower C6S/C4S ratios than any other region. The levels of dermatan sulphate (DS) in the cartilage of all regions of the OP and control groups were very similar and low, while the tissues of the OA group contained significantly higher amounts, particularly the cartilage from osteophytes. The previously presumed compositional similarity between normal aged and osteoporotic articular hip cartilage was essentially confirmed in a comparative analysis. Significant changes in GAG and UA composition of OA cartilage from distinct regions was also recorded.

Monosaccharides obtained by reduction and hydrolysis of galactosaminoglycan isomers, are entirely determined as their perbenzoyl derivatives by reversed phase HPLC, without removal of hexosamines prior to benzoylation. The method is suitable for the analysis of arterial proteoglycan constituent galactosaminoglycans, providing specific, precise and reproducible results. Moreover, synthesis and characterization of tri-O-benzoyl-1,6-L-anhydroidose and N-benzoyl-tetra-O-benzoyl-alpha- and -beta-D-galactosamine have been accomplished.

We have investigated interactions between spermine, a member of the growth promoting polyamine family, and various glycosaminoglycans. By using gel chromatography and equilibrium dialysis experiments we found that spermine binds to L-iduronic acid-rich dermatan sulfate (Kd, approximately 3.9 x 10(-4) M) with an affinity similar to that between spermine and DNA. By digesting spermine-dermatan sulfate complexes with chondroitin ABC lyase, the formation of oligosaccharide fragments (tetra-to-decasaccharides) was demonstrated by polyacrylamide gel electrophoresis. Chondroitin sulfate, which is deficient in L-iduronic acid, generates no spermine-protected fragments. Analysis of protected dermatan sulfate oligosaccharides indicates that the majority of the L-iduronic acid residue is non-sulfated and in a periodate-resistant conformation. The oligosaccharides also possess antiproliferative activity.

The effects of various glycosaminoglycans (GAGs) on the growth rate of normal fibroblasts and a fibrosarcoma cell line (HT 1080) were examined. Cells were grown in 96-well microplates in the absence or presence of serum mitogens, epidermal (EGF), platelet-derived (PDGF), acidic fibroblast (aFGF), or basic fibroblast growth factor (bFGF). Cell number was measured by using crystal violet to stain cell nuclei (Westergren-Thorsson, G., Onnervik, P.-O., Fransson, L.-A., and Malmström, A. J. Cell. Phys. 147, 523-530, 1991) and also by using a Coulter counter. In the presence of serum mitogens, L-iduronate (IdoA)-rich GAGs, such as dermatan sulfate, heparin, and highly sulfated heparan sulfate, inhibited proliferation of normal cells (25-35%), whereas HT 1080 cells were unaffected or slightly stimulated. Ham's F-12 supplemented with insulin and transferrin but without growth factors was able to support growth of both cell types. Under these conditions, the IdoA-rich GAGs still suppressed growth of normal cells (40-55%), whereas HT 1080 cells again responded poorly. When growth factors were added proliferation of normal fibroblasts was further stimulated, EGF being the most effective. In the presence of either EGF, PDGF, or bFGF, IdoA-rich GAGs had a sustained inhibitory effect on normal fibroblasts (30-50% at concentrations at or above 10 micrograms/ml). However, in the presence of aFGF, both IdoA-rich and IdoA-poor heparan sulfates enhanced growth (nearly twofold after prolonged exposure) suggesting a stabilization of this growth factor. In general, IdoA-rich GAGs appear to inhibit proliferation of normal cells irrespective of the type of growth factor used. Therefore, GAGs are likely to act directly on cell-derived regulatory components, either before or after internalization. As fibrosarcoma cells were much less sensitive to growth inhibition, they may contain altered receptors for GAGs.

A solution of hog intestinal heparin (average M(r) 12,000, anti-clotting activity 168 USP units/mg) in 0.2 M phosphate buffer (pH 7.2), was incubated in the presence of Fe2+ for 20 h at 50 degrees under an O2 atmosphere to yield oxidative-reductively depolymerized heparin (ORD heparin, average M(r) 3,000, anti-clotting activity 34 USP units/mg). Chemical analysis of the ORD heparin showed a 22, 26, and 14% loss of hexosamine, uronic acid, and N-acetyl group, respectively, but no remarkable loss of both total and N-sulfate groups. 1H and 13C NMR spectroscopic analysis indicated no decrease in the amount of L-iduronic acid 2-sulfate, but a marked loss of nonsulfated uronic acid (73 and 39% loss of D-glucuronic acid and L-iduronic acids, respectively, the sum of which corresponds to the chemically determined loss of total uronic acid). The results indicated that the ORD reaction of heparin proceeds essentially by destruction of monosaccharide units, except L-iduronic acid 2-sulfate residues, due to oxygen-derived free radicals, followed by secondary hydrolytic cleavage of the resulting unstable residues.

Sulfuric acid hydrolysis according to the Saeman procedure, TFA hydrolysis, and methanolysis combined with TFA hydrolysis were compared for the hydrolysis of water-soluble uronic acid-containing polysaccharides originating from fungi, plants, and animals. The constituent sugar residues released were subsequently analyzed by either conventional GLC analysis of alditol acetates or high-performance anion-exchange chromatography with pulsed-amperometric detection. It was shown that TFA hydrolysis alone is not sufficient for complete hydrolysis. Sulfuric acid hydrolysis of these polysaccharides resulted in low recoveries of 6-deoxy-sugar residues. Best results were obtained by methanolysis combined with TFA hydrolysis. Methanolysis with 2 M HCl prior to TFA hydrolysis resulted in complete liberation of monosaccharides from pectic material and from most fungal and animal polysaccharides tested. Any incomplete hydrolysis could be assessed easily by HPAEC, by the detection of characteristic oligomeric products, which is difficult using alternative methods currently in use. Methanolysis followed by TFA hydrolysis of 20 micrograms water-soluble uronic acid containing polysaccharides and subsequent analysis of the liberated sugar residues by HPAEC allowed us to determine the carbohydrate composition of these polysaccharides rapidly and accurately in one assay without the need for derivatization.

The current study was undertaken to examine the reactivity of anti-chondroitin sulfate monoclonal antibody, CS-56, to the comb and wattle galactosaminoglycans containing varying proportions of L-iduronic acid. Galactosaminoglycans were isolated from mature rooster comb and wattle tissues and were separated into four fractions (I, II, III, and IV), which were precipitable with 18, 25, 40, and 50% ethanol, respectively. Each fraction was examined using an ELISA technique. The results demonstrated presence of CS-56 epitope in galactosaminoglycans from both tissues. Most (> 99%) of the epitope were found in Fractions III and IV, which were galactosaminoglycans rich in D-glucuronic acid. Fractions I and II, which were dermatan sulfates rich in L-iduronic acid, showed little or very weak antigenicities.

1. The structure of chondroitin/dermatan and heparan-sulphate chains from various proteoglycan populations derived from cultured human skin fibroblasts have been examined. Confluent cell cultures were biosynthetically labelled with [3H]-glucosamine and 35SO4(2-), and proteoglycans were purified according to buoyant density, size and charge density [Schmidtchen, A., Carlstedt, I., Malmström, A. & Fransson, L.-A. (1990) Biochem. J. 265, 289-300]. Some proteoglycan fractions were further fractionated according to hydrophobicity on octyl-Sepharose in Triton X-100 gradients. The glycosaminoglycan chains, intact or degraded by chemical or enzymic methods were then analysed by gel chromatography on Sepharose CL-6B, Bio-Gel P-6, ion exchange HPLC and gel electrophoresis. 2. Three types of dermatan-sulphate chains were identified on the basis of disaccharide composition and chain length. They were derived from the large proteoglycan, two small proteoglycans and a cell-associated proteoglycan with core proteins of 90 kDa and 45 kDa. Intracellular, free dermatan-sulphate chains were very similar to those of the small proteoglycans. 3. Heparan-sulphate chains from different proteoglycans had, in spite of small but distinct differences in size, strikingly similar compositional features. They contained similar amounts of D-glucuronate, L-iduronate (with or without sulphate) and N-sulphate groups. They all displayed heparin-lyase-resistant domains with average molecular mass of 10-15 kDa. The heparan-sulphate chains from proteoglycans with 250-kDa and 350-kDa cores were the largest greater than 50 kDa), containing an average of four or five domains, in contrast to heparan-sulphate chains from the small heparan-sulphate proteoglycans which had average molecular mass of 45 kDa and consisted of three or four such domains. Free, cell-associated heparan-sulphate chains were heterogeneous in size (5-45 kDa). 4. These results suggest that the core protein may have important regulatory functions with regard to dermatan-sulphate synthesis. On the other hand, synthesis of heparan sulphate may be largely controlled by the cell that expresses a particular proteoglycan core protein.

A strategy that we originally used to identify an N-acetylated domain adjacent to the protein-linkage sequence of heparan sulphate proteoglycan (HSPG) [Lyon, Steward, Hampson & Gallagher (1987) Biochem. J. 242, 493-498] has been adapted for analysis of the location of GlcNSO3-HexA and GlcNSO3(+/- 6S)-IdoA(2S) units most proximal to the core protein. [3H]Glucosamine-labelled HSPG from human skin fibroblasts was depolymerized by using HNO2 or heparinase under conditions that allowed cleavage of all susceptible linkages. The degraded PG was coupled to Sepharose beads through the protein component, enabling specific recovery of protein-linked resistant oligosaccharides. These were released by treatment with alkaline borohydride and analysed by gel filtration and gradient PAGE. This strategy allowed investigation of the sequence of sugar residues along the chain relative to a common reference point (i.e. the reducing end of the chain). HNO2 scission confirmed the presence of a well-defined N-acetylated sequence predominantly 9-12 disaccharide units in length proximal to the core protein. Heparinase scission produced two classes of oligosaccharides (Mr approx. 7000 and 15,000) with the general formula: IdoA(2S)-GlcNSO3-[HexA-GlcNR]n-HexA-GlcNSO3-[Hex A-GlcNAc]9 12-GlcA-Gal-Gal-Xyl in which the average value for n is 1-2 for the 7000-Mr species and approx. 22 for the 15,000-Mr species. The latter oligosaccharides extend to about one-third of the total length of the HS chains (Mr approx. 45,000). HNO2 scission of these oligosaccharides enabled hypothetical models for their sequence to be proposed. The general arrangement of N-sulphated and N-acetylated disaccharides between the proximal GlcNSO3 and terminal IdoA(2S) residues of the 15,000-Mr fragment was similar to that in the original polysaccharide, suggesting the possibility of a tandemly repeating pattern in the sequence of HS.

We have modified a method (Gilles et al: Anal. Biochem., 159:109-113, 1986) for measuring cell number, that is based on the binding of crystal violet to cell nuclei and used it to assay effects of various glycosaminoglycans on growth of human lung fibroblasts. The procedure was modified by growing cells in microcultures (96 well microplates) and by measuring the amount of adsorbed dye using a microplate photometer after solubilisation of the cells with detergent. There was a linear relationship between absorbance and cell number measured by a Coulter Counter. The method is rapid and sensitive and can be used for screening many samples as well as measuring growth rates at low initial cell densities. Even the low growth rates obtained in the absence of serum can be detected. Human lung fibroblasts were growth arrested by serum deprivation and then grown for periods of up to 4 days in the presence of serum and exogenously added glycosaminoglycans (range, 0.1-100 micrograms/ml). Hyaluronan, chondroitin sulfate, and dextran sulfate were without effects, whereas dermatan sulfate, certain heparan sulfates, and heparin suppressed growth (20%-50% inhibition). The antiproliferative activity of dermatan sulfate increased with increasing iduronate content. Certain heparan sulfates, with moderately high sulfate and L-iduronate contents were better inhibitors than heparin despite the fact that the latter glycan has even higher sulfate and L-iduronate contents. The antiproliferative effect of exogenous glycans appeared after a lag period of 3-4 days, suggesting that they interfered with factors produced by the cells. Furthermore, the degree of inhibition was greater when the initial cell density was low. Above a certain level of seeded cells (approx. 10,000 cells/well), there was no inhibition by any of the glycosaminoglycans. It is possible that exogenous glycans cannot overcome endogenous growth-promoting effects in densely seeded cultures.

A high-performance liquid chromatography (HPLC) method with pulsed amperometric detection (PAD) was used for the determination of the acid hydrolysis products of L-iduronic acid containing oligosaccharides isolated from biological sources. This HPLC-PAD method was compared with gas chromatographic (GLC) methods. Since acid hydrolysis of oligosaccharides can produce a number of products, several uronic acid derivatives were prepared by chemical synthesis. These well characterized standards in conjunction with mass spectrometry allowed for the identification of most of the products of methanolysis or hydrolysis of glycosamino-glycans, which included chondroitin sulfates A and B (dermatan sulfate), heparin, and hyaluronic acid. (4 M) HCl in methanol 100 degrees C for 24 h was found to be optimum for GLC and 1 M aqueous HCl for 4 h at 100 degrees C for HPLC-PAD. All of the monosaccharides, hexosamines, and uronic acids could be separately identified in a single chromatographic step using either technique. Good resolution, high sensitivity (low microgram samples) and rapid analysis makes these methods particularly useful for the determination of small amounts of glycosaminoglycans and other glycoconjugates found in samples isolated from biological sources. These two techniques are specifically designed to allow the qualitative determination of the carbohydrate content and composition of samples whose carbohydrate composition and content is completely unknown.

The structure of human skin fibroblast heparan sulphate has been examined by depolymerization with heparinase, which specifically cleaves highly sulphated disaccharides of structure GlcNSO3 (+/-6S)-alpha 1,4IdoA(2S) [N-sulphated glucosamine (6-sulphate)-alpha 1,4-iduronic acid 2-sulphate]. Heparan sulphate contained only a small proportion (approximately 10%) of linkages susceptible to this enzyme. The major products of depolymerization with heparinase were large oligosaccharides with an average molecular mass of 10 kDa (dp approximately 40, where dp is degree of polymerization; for disaccharides, dp = 2 etc.) as assessed by gel filtration on Sepharose CL-6B, compared with a molecular mass of 45 kDa (dp approximately 200) for the intact chains. The large heparinase-resistant oligosaccharides were highly susceptible to depolymerization with the enzyme heparitinase, which cleaves heparan sulphate in areas of low sulphation, where N-acetylated disaccharides [GlcNAc-alpha 1,4GlcA (N-acetylglucosaminyl-alpha 1,4-glucuronic acid)] are the predominant structural unit. Further analysis of the location of the heparinase cleavage sites indicated that they were predominantly found in a central position in GlcNSO3-alpha 1,4IdoA repeat sequences of average length four to seven disaccharides (dp 8-14). These results indicate that heparinase cleaves heparan sulphate in approximately four or five N-sulphated domains, each domain containing a cluster of two or three susceptible disaccharides; the domains are separated by long N-acetyl-rich sequences that are markedly deficient in sulphate groups. On the basis of these findings a model is proposed which depicts heparan sulphate as an ordered polymeric structure composed of an alternate arrangement of sulphate-rich and sulphate-poor regions. The sulphate-rich regions are likely to be flexible areas of the chain because of their high content of the conformationally versatile IdoA and IdoA(2S) residues. The model has important implications for the biosynthesis and functions of heparan sulphate.

Small proteoglycans (PGs), extracted from human keloid scar tissue with 4M guanidinium chloride and fractionated by DEAE-cellulose chromatography, were separated by ethanol precipitation into one L-iduronic acid-rich and one D-glucuronic acid-rich fraction. The size of the L-iduronic acid-rich PG was 102 kDa with a 27 kDa glycosaminoglycan chain, that of the D-glucuronic acid-rich PG was 90 kDa with a 26 kDa glycosaminoglycan chain, and the protein core of both PGs was 14.5 kDa. The two PGs carried sulfate groups mostly attached at C-4 of the 2-amino-2-deoxy-D-galactose units. The N-terminal amino acid sequence of both was similar to human bone PGII (decorin), normal and hypertrophic scar, and human dermal tissue PG.

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.

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.

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.

Sulfated galactosaminoglycans of mature bovine periodontal ligament were separated into four fractions by ethanol precipitation. Fractions I and II were dermatan sulfates with high contents of L-iduronate, but only small amounts of this hexuronic acid were present in fractions III and IV. Effects of digestion with testicular hyaluronidase or a periodate-alkali treatment showed that most if not all of the glycans in fractions I, II and III were hybrid chains containing both L-iduronate and D-glucuronate. The composition of fraction IV was less certain, but the chains strongly resembled fraction III hybrids in electrophoretic characteristics, not chondroitin sulfate. The total amount of the D-glucuronate-rich fractions III and IV in the ligament was similar to that of I plus II. In contrast, almost all of the sulfated galactosaminoglycans of mature skin were rich in L-iduronate. The more varied composition of the ligament glycosaminoglycans may be related to the mixed population of cells in this tissue.

Alkaline extraction of whale intestine, followed by pronase digestion and precipitation of heparin (omega-heparin) with dodecyltrimethylammonium chloride gave a supernatant fraction containing dermatan sulfate. Ethanol at 20% concentration precipitated dermatan sulfate from the supernatant fraction. The crude dermatan sulfate was further fractionated by ion-exchange column chromatography on Dowex-1 (C1- form), eluting stepwise with aqueous sodium chloride. The fractions eluted with 1.5M and 1.75M sodium chloride contained a typical dermatan sulfate. Chemical and enzymic studies of these preparations revealed that the sulfate groups were located solely at O-4 of the 2-acetamido-2-deoxy-D-galactose resides. L-Iduronic acid was assumed to be distributed uniformly in the backbone of the polysaccharide chain, with D-glucuronic acid being located in the linkage region to the protein core. A new method for determining the ratio of D-glucuronic to L-iduronic acid is also described.

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.

Bovine heparin was fractionated with respect to chain length and anticoagulant activity. Analysis of each of these fractions for iduronic and glucuronic acids demonstrated that active heparin has a greater amount of glucuronic acid than inactive heparin. The ratio of the uronic acids in the respective fractions was the same for heparin with different molecular weights. Thus, active heparin with longer chain lengths have more additional glucuronate residues than are required for the antithrombin-binding domain. The results indicate that the active and inactive heparin species differ in more than one structural characteristic and suggest a considerable divergence in their respective biosynthesis.

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.

The distribution of glucuronic and iduronic acid within the chains of anticoagulantly active and inactive beef lung heparin was investigated. A fraction with an average molecular weight of 19,500 was isolated from the heterodisperse mixture and then separated into active and inactive components by affinity chromatography. Each sample was linked through its reducing terminus to tyramine, reduced with sodium borotritide, and bound covalently to Sepharose via an azo bridge. The bound reduced heparin was treated with a limited amount of HNO2 and the degraded fragments were removed. The sections of the chain contiguous with the original reducing terminus were then detached from the insoluble matrix by reaction with sodium dithionite. The recovered polysaccharide was fractionated according to size on Sephadex G-200 and the amount of each uronic acid in the individual fractions was determined. Inactive heparin showed a constant percentage of glucuronic acid in all fragments, i.e. about 8.9% of the total uronic acid. With active heparin the percentage of glucuronic acid increased with the distance from the reducing terminus of the polysaccharide chain, ranging from 9.5 to 20% of the uronic acids. These results suggest that the biosynthesis of active heparin involves unique reactions or specific processing of the macromolecule.

1. Two proteodermatan sulphate species from bovine sclera (fractions PG-I and PG-II) separable by gel chromatography were studied by isopycnic centrifugations in CsCl and Cs(2)SO(4) both in an analytical and a preparative mode. 2. In CsCl, fraction PG-I formed a broad band at a density rho=1.75g/ml whereas fraction PG-II banded sharply at rho=1.64g/ml. However, in Cs(2)SO(4), fraction PG-II banded at rho=1.51g/ml and fraction PG-I at rho=1.40g/ml, a reversal of the relative banding positions of the two species in CsCl. 3. Preparative isopycnic centrifugations in the two caesium salts permitted further subfractionation of fractions PG-I and PG-II. In both CsCl and Cs(2)SO(4) fraction PG-I was split into subfractions that varied greatly in uronate/protein ratios but had very similar uronate composition. In contrast, isopycnic centrifugation of fraction PG-II in Cs(2)SO(4) gave rise to subfractions with similar uronate/protein ratios but markedly different uronate composition (iduronate content, 88-42%). 4. Subfractions of fractions PG-I and PG-II obtained in preparative centrifugations in CsCl or Cs(2)SO(4) were examined in the analytical ultracentrifuge. These subfractions banded at discrete positions in the gradient. Estimations of apparent molecular weight for these subfractions from data from the analytical isopycnic centrifugations gave values that were much higher (around 1x10(6)) than were those obtained previously for their ;monomeric' states (fraction PG-I 160000-410000, and fraction PG-II 70000-130000). 5. In CsCl, fraction PG-I may be subfractionated according to the number of side chains in the molecule. Fraction PG-I increased its net solvation (i.e. lowered its buoyant density) to a larger extent than did fraction PG-II in going from CsCl to Cs(2)SO(4) (i.e. from lower to higher water activity). It is proposed that the presence of large amounts of iduronate in fraction PG-II makes these molecules relatively less solvated in Cs(2)SO(4). Thus the uronate composition may be an important factor in determining the banding position of proteodermatan sulphates in density-gradient centrifugations.

Heparins from various sources and heparan sulfate from umbilical cords have been subjected to Smith-degradation and reaction with nitrites. These procedures were effective for providing data relating to the distribution of sulfated iduronic acid residues in the molecule. Results indicated that heparins may have, as a prominent structural feature of the molecule, non-sulfated uronic acid distributed in single sequences, much as had been shown previously for N-acetylglucosamine residues. Sulfated uronic acid, however, may occur in multiple sequences of up to 5 or 6 residues. Heparan sulfate was found to have a major proportion of its ester sulfate on iduronic acid rather than hexosamine units, thereby having sections similar to those in heparins, though in considerably lower proportion.