Heparinase I Research Articles

Structural Analysis of Glycosaminoglycans inDrosophila and Caenorhabditis elegans and Demonstration That tout-velu, a Drosophila Gene Related to EXT Tumor Suppressors, Affects Heparan Sulfate in Vivo

We have devised a sensitive method for the isolation and structural analysis of glycosaminoglycans from two genetically tractable model organisms, the fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. We detected chondroitin/chondroitin sulfate- and heparan sulfate-derived disaccharides in both organisms. Chondroitinase digestion of glycosaminoglycans from adult Drosophila produced both nonsulfated and 4-O-sulfated unsaturated disaccharides, whereas only unsulfated forms were detected in C. elegans. Heparin lyases released disaccharides bearing N-, 2-O-, and 6-O-sulfated species, including mono-, di-, and trisulfated forms. We observed tissue- and stage-specific differences in both chondroitin sulfate and heparan sulfate composition in Drosophila. We have also applied these methods toward the analysis of tout-velu, an EXT-related gene in Drosophila that controls the tissue distribution of the growth factor Hedgehog. The proteins encoded by the vertebrate tumor suppressor genes EXT1 and 2, show heparan sulfate co-polymerase activity, and it has been proposed that tout-velu affects Hedgehog activity via its role in heparan sulfate biosynthesis. Analysis of total glycosaminoglycans from tout-velu mutant larvae show marked reductions in heparan sulfate but not chondroitin sulfate, consistent with its proposed function as a heparan sulfate co-polymerase.

Purification and characterization of heparan sulphate proteoglycan from bovine brain

A heparan sulphate proteoglycan was purified from adult bovine brain tissues and its structure was characterized. The major heparan sulphate proteoglycan from whole bovine brain had a molecular mass of >200 kDa on denaturing SDS/PAGE and a core protein size of 66 kDa following the removal of glycosaminoglycan chains. Fractionation on DEAE-Sephacel showed that this proteoglycan consisted of three major forms having high, intermediate and low overall charge. All core proteins were identical in size and reacted with heparan sulphate proteoglycan-stub antibody and an antibody made to a synthetic peptide based on rat glypican. The three forms of proteoglycans had identical peptide maps and their amino acid compositional analysis did not match any of the known glypicans. The internal sequence of a major peptide showed only 37.5% sequence similarity with human glypican 5. The glycosaminoglycan chain sizes of the three forms of this proteoglycan, determined after beta-elimination by PAGE, were identical. The disaccharide compositional analysis on the heparan sulphate chains from the three forms of the proteoglycan, determined by treatment with a mixture of heparin lyases followed by high-resolution capillary electrophoresis, showed that they differed primarily by degree of sulphation. The most highly sulphated proteoglycan isolated had a disaccharide composition similar to heparan sulphate glycosaminoglycans found in brain tissue. Based on their sensitivity to low pH nitrous acid treatment, the N-sulphate groups in these proteoglycans were found to be primarily in the smaller glycosaminoglycan chains. The heparan sulphate proteoglycans were also heavily glycosylated with O-linked glycans and no glycosylphosphatidylinositol anchor could be detected.

Heparan Sulfate d-Glucosaminyl 3-O-Sulfotransferase-3A SulfatesN-Unsubstituted Glucosamine Residues

3-O-Sulfation of glucosamine by heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST-1) is the key modification in anticoagulant heparan sulfate synthesis. However, the heparan sulfates modified by 3-OST-2 and 3-OST-3A, isoforms of 3-OST-1, do not have anticoagulant activity, although these isoforms transfer sulfate to the 3-OH position of glucosamine residues. In this study, we characterize the substrate specificity of purified 3-OST-3A at the tetrasaccharide level. The 3-OST-3A enzyme was purified from Sf9 cells infected with recombinant baculovirus containing 3-OST-3A cDNA. Two 3-OST-3A-modified tetrasaccharides were purified from the 3-O-(35)S-sulfated heparan sulfate that was digested by heparin lyases. These tetrasaccharides were analyzed using nitrous acid and enzymatic degradation combined with matrix-assisted laser desorption/ionization-mass spectrometry. Two novel tetrasaccharides were discovered with proposed structures of DeltaUA2S-GlcNS-IdoUA2S-[(35)S]GlcNH(2)3S and DeltaUA2S-GlcNS-IdoUA2S-[3-(35)S]GlcNH(2)3S6S . The results demonstrate that 3-OST-3A sulfates N-unsubstituted glucosamine residues, and the 3-OST-3A modification sites are probably located in defined oligosaccharide sequences. Our study suggests that oligosaccharides with N-unsubstituted glucosamine are precursors for sulfation by 3-OST-3A. The intriguing linkage between N-unsubstituted glucosamine and the 3-O-sulfation by 3-OST-3A may provide a clue to the potential biological functions of 3-OST-3A-modified heparan sulfate.

Teneurin-1, a vertebrate homologue of the Drosophila pair-rule gene ten-m, is a neuronal protein with a novel type of heparin-binding domain.

The Drosophila gene ten-m is the first pair-rule gene not encoding a transcription factor, but an extracellular protein. We have characterized a highly conserved chicken homologue that we call teneurin-1. The C-terminal part harbors 26 repetitive sequence motifs termed YD-repeats. The YD-repeats are most similar to the core of the rhs elements of Escherichia coli. Related repeats in toxin A of Clostridium difficile are known to bind specific carbohydrates. We show that recombinantly expressed proteins containing the YD-repeats of teneurin-1 bind to heparin. Furthermore, heparin lyase treatment of extracts of cells expressing recombinant YD-repeat protein releases this protein from high molecular mass aggregates. In situ hybridization and immunostaining reveals teneurin-1 expression in neurons of the developing visual system of chicken and Drosophila. This phylogenetic conservation of neuronal expression from flies to birds implies fundamental roles for teneurin-1 in neurogenesis. This is supported by the neurite outgrowth occurring on substrates made of recombinant YD-repeat proteins, which can be inhibited by heparin. Database searches resulted in the identification of ESTs encoding at least three further members of the teneurin family of proteins. Furthermore, the human teneurin-1 gene could be identified on chromosome Xq24/25, a region implied in an X-linked mental retardation syndrome.

Rapid and sensitive analysis of disaccharide composition in heparin and heparan sulfate by reversed-phase ion-pair chromatography on a 2 μm porous silica gel column

A rapid and sensitive method was developed for the analysis of disaccharide composition in heparin (HP) and heparan sulfate (HS) by reversed-phase ion-pair chromatography on a 2 μm porous silica gel column. HP and HS were digested with heparin lyase I, II and III in combination, and the produced unsaturated disaccharides were separated within 15 min. Calibration graphs were linear in the range 1 ng–1 μg with the fluorometoric post-column detection using 2-cyanoacetamide.

New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli and 2-O-desulfated heparin.

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.

In Vivo Clearance of Ternary Complexes of Vitronectin-Thrombin-Antithrombin Is Mediated by Hepatic Heparan Sulfate Proteoglycans

Thrombin is inhibited by its cognate plasma inhibitor antithrombin, through the formation of covalent thrombin-antithrombin (TAT) complexes that are found as ternary complexes with vitronectin (VN-TAT). To determine whether the metabolism of VN-TAT ternary complexes is different from that previously reported for binary TAT complexes, plasma clearance studies were done in rabbits using human VN-TAT. 125I-VN-TAT was shown to be cleared rapidly from the circulation (t1/2alpha = 3.8 min) in a biphasic manner mainly by the liver. 125I-TAT had a similar initial clearance (t1/2alpha = 5.3 min) but had a significantly faster beta-phase clearance (t1/2beta = 42.8 min versus 85.4 min for VN-TAT; p = 0.005). Protamine sulfate and heparin abolished the rapid initial alpha-phase of 125I-VN-TAT clearance and reduced its liver-specific association and in vivo degradation. Heparin also reduced the alpha-phase clearance of 125I-TAT and was associated with the appearance of high molecular weight complexes, suggesting enhanced complex formation between VN and TAT. 125I-VN-TAT binding to HepG2 cells was reduced by competition with VN-TAT or heparin but to a much lesser extent in the presence of TAT. The binding of VN-TAT to HepG2 cells was not inhibited by competition with the low density lipoprotein receptor-related protein ligand, methylamine-alpha2-macroglobulin. 125I-VN-TAT binding was also inhibited by treating HepG2 cells with heparinase or by growing the cells in the presence of beta-D-xyloside. Finally, both heparin and chloroquine, but not methylamine-alpha2-macroglobulin, reduced the internalization and degradation of VN-TAT by HepG2 cells. Taken together, these data indicate the importance of VN in TAT metabolism and demonstrate that VN-TAT binds to liver-associated heparan sulfate proteoglycans, which mediate its internalization and subsequent intracellular degradation.

Determination of the structure of oligosaccharides prepared from acharan sulfate.

The fine structure of acharan sulfate, a recently discovered glycosaminoglycan isolated from Achatina fulica , was examined. This glycosaminoglycan has a major disaccharide repeating unit of -->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S(1--> (where GlcNpAc is N -acetylglucosamine, IdoAp is iduronic acid, and S is sulfate) making it structurally related to both heparin and heparan sulfate. Using heparin lyases prepared from Flavobacterium heparinum and a newly isolated heparinase from Bacteroides stercoris , the controlled enzymatic depolymerization of acharan sulfate was undertaken to prepare a mixture of oligosaccharides. Fractionation of this mixture of oligosaccharides by strong-anion-exchange high performance liquid chromatography afforded oligosaccharides that capillary electrophoresis established were sufficiently pure for structural characterization. Electrospray ionization mass spectrometry identified two series of oligosaccharides, one derived from acharan sulfate's major repeating unit and a second minor group of undersulfated oligosaccharides. Proton nuclear magnetic resonance spectroscopy established the structure of these two classes of oligosaccharides to be DeltaUAp2S(1-->[4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S (1-->]n4)- D-GlcNpAcalpha,beta (where n = 0,1,2,3 and DeltaUAp is 4-deoxy-alpha-L- threo -hex-4-enopyranosyluronic acid) and DeltaUAp(1-->[4)- alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S(1-->]m-D-GlcNpAcal pha,beta (where m = 1,2,3). These results suggest the presence of minor sequence variants in acharan sulfate containing unsulfated iduronic acid having the structure -->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp(1-->.

Heparan sulfate proteoglycans mediate a potent inhibitory signal for migration of vascular smooth muscle cells.

Migration of vascular smooth muscle cells (SMCs) is a key step in vascular remodeling and formation of pathological lesions in diseased arteries and may be controlled by extracellular matrix (ECM) and by factors that regulate ECM composition, such as platelet-derived growth factor (PDGF). In culture, PDGF-AB and -BB enhance but PDGF-AA (although having no effect alone) suppresses SMC migration stimulated by other PDGF isoforms. To determine whether the migration-inhibitory mechanism of PDGF-AA was mediated by ECM composition, we examined baboon SMC migration in a Boyden chamber assay using filters coated with different ECM proteins. PDGF-AA suppressed the PDGF-BB-induced migration of baboon SMCs on a filter coated with basement membrane proteins (Matrigel) and fibronectin but failed to inhibit cell migration on a type I collagen (Vitrogen)-coated filter. Fibronectin and fibronectin fragments that contain heparin-binding domains permitted PDGF-AA inhibition of cell migration, but a fragment lacking heparin-binding domains did not. Treatment of SMCs with heparin lyases II and III, but not with chondroitin ABC lyase, diminished the PDGF-AA-mediated inhibition of migration. PDGF-AA stimulated accumulation of proteoglycan (PG) in the cell layer more potently than did PDGF-BB, whereas the turnover of cell layer PG was unaffected by either PDGF-AA or -BB. Northern blot analysis revealed that PDGF-AA increased syndecan-1 mRNA expression more than did PDGF-BB, whereas both PDGF isoforms decreased perlecan expression. The changes in cell migration and PG synthesis induced by PDGF-AA were accompanied by changes in the morphology of SMCs. PDGF-AA dramatically induced the spreading of SMCs, whereas the heparin lyase treatment of PDGF-AA-stimulated cultures diminished cell spreading. The data suggest that PDGF-AA selectively modifies heparan sulfate PG accumulation on SMCs and thereby influences the interactions of SMCs with heparin-binding ECM proteins. These interactions, in turn, generate signals that suppress SMC migration.

The Short and Long Forms of Type XVIII Collagen Show Clear Tissue Specificities in Their Expression and Location in Basement Membrane Zones in Humans

Two N-terminal ends of human type XVIII collagen chains have recently been identified. The two chains have different signal peptides and variant N-terminal noncollagenous NC1 domains of 493 (NC1-493) and 303 (NC1-303) amino acid residues, respectively, but share 301 residues of their NC1 domains as well as the collagenous and C-terminal noncollagenous portions of the molecule. Antibodies were produced against the NC1 region common to both human alpha1(XVIII) chain variants and against NC1 sequences specific to the long variant and were used in combination with in situ hybridization to localize this collagen in a number of human tissues. They were also used for Western blotting, which resulted in detection of overlapping high-molecular weight bands above the 200-kd standard in a kidney extract. Heparin lyase II and heparin lyase III digestions of kidney and placenta extracts indicated that at least in these tissues, type XVIII collagen contains heparin sulfate glycosaminoglycan side chains. Type XVIII collagen was found to be a ubiquitous basement membrane component, occurring prominently at vascular and epithelial basement membranes throughout the body. Comparison of the expression of the NC1-493 and NC1-303 variants revealed marked differences. The short variant was found in most conventional basement membranes, including blood vessels and the various epithelial structures, and around muscular structures. The long variant was expressed very strongly in liver, where it was virtually the only variant in the liver sinusoids, and it occurred only in minor amounts elsewhere. Thus, the 192 N-terminal residues specific to the long variant apparently confer some functional property needed above all in the liver sinusoids, but also at certain other locations.

Conformational study of synthetic Δ 4-uronate monosaccharides and glycosaminoglycan-derived disaccharides

Sixteen Δ 4-uronate monosaccharides were chemically synthesized. Their carboxy group was protected as a methyl or benzyl ester, the anomeric hydroxyl group as a benzyl glycoside and the 2 and 3 hydroxyl groups were protected with different substitution patterns as both ester and ether derivatives. Disaccharides containing Δ 4-uronates were prepared from heparin using heparin lyases. Their carboxy group was unprotected or protected as a benzyl ester and the two hydroxyls in the uronate moiety were free, as O-sulfo derivatives or acylated. The conformation of these unsaturated uronate monosaccharide and disaccharide residues was studied using 1H NMR by examining interproton vicinal coupling constants. The Δ 4-uronate residue adopted either the 2H 1 or the 1H 2 conformations. The equilibrium between these two conformers was shown to be controlled by substitution pattern.

Characterization of aBacteroidesspecies from human intestine that degrades glycosaminoglycans

Polysaccharide lyases that can degrade glycosaminoglycans (GAGs) were identified in an anaerobic strain living in the human intestine. The strain was isolated from the stool of a healthy male and identified as Bacteroides sp. strain HJ-15. A detailed taxonomical study indicated the species is a strain of Bacteroides stercoris. The isolate was cultured and the polysaccharide lyase activity was partially purified. This enzyme preparation could act on GAGs containing either glucosamine or galactosamine suggesting the presence of both heparinases and chondroitinases. Various GAGs were incubated with the partially purified enzyme and the products formed were analyzed by strong anion-exchange high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. These studies demonstrated the presence of at least two types of polysaccharide lyases: heparin lyase and chondroitin sulfate lyase. The eliminative mechanism of these lyase enzymes was confirmed through the isolation of unsaturated disaccharide products. The heparin lyase acted on both heparin and acharan sulfate, a GAG recently isolated from Achatina fulica. The Bacteroides chondroitin lyase, acted on chondroitin sulfates A, B (dermatan sulfate), and C, resembling chondroitin lyase ABC. The presence of a GAG-degrading organism in human intestine may pose problems for the effective oral administration of GAG drugs.Key words: Bacteroides stercoris, glycosaminoglycan, nuclear magnetic resonance spectroscopy, polysaccharide lyase, heparinase, chondroitinase.

Interaction of Secretory Leukocyte Protease Inhibitor with Heparin Inhibits Proteases Involved in Asthma

Protease inhibition by secretory leukocyte protease inhibitor (SLPI) is accelerated by the sulfated polysaccharides. The nature of the SLPI-polysaccharide interaction, explored with affinity chromatography, indicated that this interaction was sensitive to the charge and type of polysaccharide. Dextran and chondroitin had the lowest affinity for SLPI, followed by dermatan, heparan, and dextran sulfates. While heparin bound SLPI tightly, the highest affinity heparin chains unexpectedly contained a lower level of sulfation than more weakly interacting chains. Heparin oligosaccharides, prepared using heparin lyase I were SLPI-affinity fractionated. Surprisingly, undersulfated heparin oligosaccharides bound SLPI with the highest affinity, suggesting the importance of free hydroxyl groups for high affinity interaction. Isothermal titration calorimetry was used to determine the thermodynamics of SLPI interaction with a low molecular weight heparin, an undersulfated decasaccharide and a tetrasaccharide. The studies showed 12-14 saccharide units, corresponding to molecular weight of approximately 4,800, were required for a 1:1 (SLPI:heparin) binding stoichiometry. Furthermore, an undersulfated decasaccharide was able to bind SLPI tightly (Kd approximately 13 nM), resulting in its activation and the inhibition of neutrophil elastase and pancreatic chymotrypsin. The in vitro assessment of heparin and the decasaccharide and tetrasaccharide using stopped-flow kinetics suggested that heparin was the optimal choice to study SLPI-based in vivo protease inhibition. SLPI and heparin were co-administered by inhalation in therapy against antigen-induced airway hyperresponsiveness in a sheep bronchoprovocation model. Heparin, in combination with SLPI demonstrated in vivo efficacy reducing early and late phase bronchoconstriction. Heparin also increased the therapeutic activity of SLPI against antigen-induced airway hyperresponsiveness.

Characterization of a Bacteroides species from human intestine that degrades glycosaminoglycans.

Polysaccharide lyases that can degrade glycosaminoglycans (GAGs) were identified in an anaerobic strain living in the human intestine. The strain was isolated from the stool of a healthy male and identified as Bacteroides sp. strain HJ-15. A detailed taxonomical study indicated the species is a strain of Bacteroides stercoris. The isolate was cultured and the polysaccharide lyase activity was partially purified. This enzyme preparation could act on GAGs containing either glucosamine or galactosamine suggesting the presence of both heparinases and chondroitinases. Various GAGs were incubated with the partially purified enzyme and the products formed were analyzed by strong anion-exchange high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. These studies demonstrated the presence of at least two types of polysaccharide lyases: heparin lyase and chondroitin sulfate lyase. The eliminative mechanism of these lyase enzymes was confirmed through the isolation of unsaturated disaccharide products. The heparin lyase acted on both heparin and acharan sulfate, a GAG recently isolated from Achatina fulica. The Bacteroides chondroitin lyase, acted on chondroitin sulfates A, B (dermatan sulfate), and C, resembling chondroitin lyase ABC. The presence of a GAG-degrading organism in human intestine may pose problems for the effective oral administration of GAG drugs.

Sensitive high-performance liquid chromatographic method with fluorometric detection for the determination of heparin and heparan sulfate in biological samples: application to human urinary heparan sulfate

A sensitive high-performance liquid chromatographic method for the determination of unsaturated disaccharides produced from heparin and heparan sulfate is described. Heparan sulfate was depolymerized using a combination of heparin lyase I (EC 4.2.2.7), heparin lyase II and heparin lyase III (EC 4.2.2.8). Seven unsaturated disaccharides were separated under isocratic conditions within 25 min using acetonitrile–H 2O–0.2 M sodium phosphate buffer (pH 7.0)–3.0 M ammonium chloride (32:10:1:1) and were monitored by fluorescence detection using 2-cyanoacetamide as a post-column derivatizing reagent. As little as 2 pmol of a disaccharide could be detected with excitation at 346 nm and emission at 410 nm. This method was applied to the analysis of normal human urine. It was revealed that the concentration of normal human urinary heparan sulfate is 1.53±0.36 mg/mg creatinine ( n=4).

Detection of glycosaminoglycans as a copper(II) complex in high-performance liquid chromatography

Glycosaminoglycans including heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and hyaluronan were analyzed by high-performance gel filtration chromatography. Detection was achieved at 240 nm based on the formation of copper(II) complex in copper sulfate solution at low pH. Detection of the copper(II)-heparin complex is sensitive, permitting the analysis of as little as 10 −7 g. This method was also successfully applied to the analysis of the chemically derivatized glycosaminoglycans (de-N-acetylated and/or oversulfated chondroitin sulfate) that cannot be depolymerized by enzymes such as heparin and chondroitin lyases.

Interaction of Fibroblast Growth Factor-1 and Related Peptides with Heparan Sulfate and Its Oligosaccharides

Fibroblast growth factors (FGFs) are a family of angiogenic and mitogenic proteins that promote cell division. The binding of FGFs to the heparan sulfate of cell-surface-bound proteoglycans appears to be critical for their activity. The interaction of fibroblast growth factor-1 (FGF-1 or aFGF) using heparin lyase-derived oligosaccharides from heparan sulfate was investigated. FGF-1 was also shown to protect sequences in heparan sulfate from heparin lyase digestion and protected oligosaccharide products of octasaccharide and decasaccharide size were recovered by FGF-1 affinity chromatography, suggesting that the high-affinity binding of heparan sulfate to FGF-1 resides within an octasaccharide sequence. The FGF-1 binding affinity of heparan sulfate is reduced compared to heparin presumably due to the absence of 6-sulfate groups in heparan sulfate. Inspection of the FGF-1 heparan sulfate binding domain shows that the majority of interacting amino acids are contained within a 20-amino-acid sequence that folds back upon itself (because of three turns) forming a triangular shaped cup of positive charge. The importance of FGF-1 binding site topology was investigated using three synthetic peptide mimics of the FGF-1 glycosaminoglycan (GAG) binding site. Heparan sulfate affinity chromatography and isothermal titration calorimetry, used to measure binding thermodynamics, demonstrated that a synthetic peptide analogous to the GAG binding site in FGF-1 bound tightly to heparan sulfate. A peptide containing ad-proline in place ofl-proline bound with considerably reduced affinity, presumably due to the altered structure of the second turn in the binding site. A cyclic peptide, expected to be topologically most similar to the triangular GAG binding site in FGF-1, bound with the highest affinity to heparan sulfate. These data suggest the triangular topology of the GAG binding site in FGF is critical for its interaction with heparan sulfate. Analysis of known GAG binding sites in 25 proteins using the Chou–Fasman algorithm show that these sites commonly contain turns.

Chemical Microdetermination of Heparin in Plasma

A new, simple, and highly sensitive method for the determination of heparin has been established. Heparin was first converted into unsaturated disaccharides through the action of heparin lyases I, II, and III. A major trisulfated unsaturated disaccharide product results, consistent with structural analysis of a number of pharmaceutical heparins using one- and two-dimensional1H NMR spectroscopy. This disaccharide was analyzed by HPLC using fluorometric postcolumn derivatization. The correlation between the amount of this trisulfated unsaturated disaccharide and anticoagulant activity of heparin as measured by anti-IIa was determined. The analysis of these pharmaceutical heparins showed a linear correlation between both HPLC and bioassay. This HPLC method was then applied to a pharmacokinetic study of heparin intravenously administered to rabbits.

Heparan sulfate isomers in cerebral arteries of Japanese women with aging and with atherosclerosis—heparitinase and high-performance liquid chromatography determinations

Composition of heparan sulfate (HS) isomers from unaffected and atherosclerotic cerebral arteries (isolated by autopsy) of Japanese women, of various ages, was studied. HS isomers were separated as disaccharide units by high-performance liquid chromatography after degeneration with HS and heparin lyases. Heparitinase facilitated differentiation of eight unsaturated disaccharides (ΔDi-SHS) of vascular HS isomers. The HS isomers in the cerebral arteries consisted of approximately half the total glycosaminoglycans (GAGs). Both HS (and GAGs) tended to increase with the processes of aging but decreased with the advancing development of atherosclerosis. The HS isomers consisted of a higher proportion of non-sulfated disaccharide, about 2/3 of the total HS, followed by mono-sulfated and bis-sulfated saccharides; in addition, heparin existed, albeit in minute amounts. The proportion of ΔDi-SHS rich in sulfate compared with HS isomers tended to increase with aging but most decreased during formation of the atheroma. Putative functions of HS isomers in cerebral arteries are discussed, based on the characteristic distribution of HS components.

Structural differences and the presence of unsubstituted amino groups in heparan sulphates from different tissues and species

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.