Glycobiology Advance Access originally published online on April 19, 2006
Glycobiology 2006 16(8):693-701; doi:10.1093/glycob/cwj117
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N-Acetylgalactosamine 4,6-O-sulfate residues mediate binding and activation of heparin cofactor II by porcine mucosal dermatan sulfate
2 Division of Hematology, Department of Medicine, Campus Box 8125, and 3 Division of Laboratory Medicine, Department of Pathology and Immunology, Campus Box 8118, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110
1 To whom correspondence should be addressed; e-mail: tollefsen{at}im.wustl.edu
Received on November 10, 2005; revised on March 31, 2005; accepted on April 18, 2006
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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Dermatan sulfate (DS) accelerates the inhibition of thrombin by heparin cofactor II (HCII). A hexasaccharide consisting of three L-iduronic acid 2-O-sulfate (IdoA2SO3)
N-acetyl-D-galactosamine 4-O-sulfate (GalNAc4SO3) subunits was previously isolated from porcine skin DS and shown to bind HCII with high affinity. DS from porcine intestinal mucosa has a much lower content of this disaccharide but activates HCII with potency similar to that of porcine skin DS. Therefore, we sought to characterize oligosaccharides from porcine mucosal DS that interact with HCII. DS was partially depolymerized with chondroitinase ABC, and oligosaccharides containing 2–12 monosaccharide units were isolated. The oligosaccharides were then fractionated by anion-exchange and affinity chromatography on HCII-Sepharose, and the disaccharide compositions of selected fractions were determined. We found that the smallest oligosaccharides able to bind HCII were hexasaccharides. Oligosaccharides 6–12 units long that lacked uronic acid (UA)2SO3 but contained one or two GalNAc4,6SO3 residues bound, and binding was proportional to both oligosaccharide size and number of GalNAc4,6SO3 residues. Intact DS and bound dodecasaccharides contained predominantly IdoA but little D-glucuronic acid. Decasaccharides and dodecasaccharides containing one or two GalNAc4,6SO3 residues stimulated thrombin inhibition by HCII and prolonged the clotting time of normal but not HCII-depleted human plasma. These data support the hypothesis that modification of IdoAGalNAc4SO3 subunits in the DS polymer by either 2-O-sulfation of IdoA or 6-O-sulfation of GalNAc can generate molecules with HCII-binding sites and anticoagulant activity. Key words: blood coagulation / dermatan sulfate / heparin cofactor II / O-sulfation / thrombin
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin and has been implicated in regulation of blood coagulation, atherogenesis, and neointima formation (Tollefsen, 2004
). Inhibition occurs by the typical serpin mechanism, in which proteolytic cleavage of the reactive site peptide bond of HCII causes a conformational change that traps thrombin in a stable acyl ester complex (Huntington, 2003). Dermatan sulfate (DS), heparin, and certain other polyanions accelerate the thrombin-HCII reaction more than 1000-fold (Colwell et al., 1999). Binding of the polyanion to HCII appears to induce allosteric effects that make both the reactive site and an N-terminal acidic region of the inhibitor more accessible to thrombin (Baglin et al., 2002). In addition, the polyanion may bind to both HCII and thrombin simultaneously, forming a bridge between the two proteins that facilitates inhibition (Verhamme et al., 2004).
HCII-deficient mice develop normally and do not show evidence of spontaneous thrombosis, but they form occlusive thrombi in their carotid arteries faster than wild-type mice after photochemically induced endothelial cell injury (He et al., 2002). Intravenous administration of DS results in dose-dependent prolongation of the carotid artery occlusion time in wild-type mice, an effect that is not observed in HCII-deficient animals (Vicente et al., 2004). It seems likely that endogenous DS regulates HCII activity in vivo and that the distribution of DS molecules capable of binding to HCII determines the sites at which HCII is active.
DS is synthesized by addition of alternating D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) residues to a core protein structure (Trowbridge and Gallo, 2002). Many of the GlcA residues become epimerized at C-5 to yield L-iduronic acid (IdoA). Subsequently, O-sulfation occurs at the C-4 position of many GalNAc residues to produce the predominant IdoAGalNAc4SO3 repeating disaccharide unit characteristic of DS. Less frequently, O-sulfation occurs at the C-6 position of GalNAc or at the C-2 position of IdoA. Because the epimerization and sulfation reactions are variable and incomplete, structural heterogeneity occurs within the mature DS polymer. Moreover, the extent of these biosynthetic modifications, which determine the disaccharide composition of DS, depends on both the species and the tissue of origin (Mascellani et al., 1994).
Previous studies examined the structural basis for interaction of DS with HCII. Maimone and Tollefsen (1990) found that HCII preferentially binds to a minor hexasaccharide sequence in porcine skin DS composed of three IdoA2SO3GalNAc4SO3 disaccharide subunits. Subsequently, Pavão and others (1995, 1998) showed that invertebrate DS polymers composed mainly of IdoA2SO3GalNAc4SO3 potently activate HCII, whereas polymers composed mainly of IdoA2SO3GalNAc6SO3 are 1000 times less active. These results suggest that 4-O-sulfation of GalNAc is important for HCII activation. DS from porcine intestinal mucosa has less IdoA2SO3GalNAc4SO3 and has a much higher content of IdoAGalNAc4,6SO3 in comparison with porcine skin DS, yet the two preparations activate HCII with similar potency (Mascellani et al., 1994). Therefore, it is unclear what role the 2-O-sulfate group in IdoA and the 6-O-sulfate group in GalNAc play in HCII binding and activation.
In this study, we isolated oligosaccharides from partial enzymatic digests of porcine mucosal DS, determined their disaccharide compositions, and studied their ability to bind and activate HCII. We found that hexasaccharides that lack uronic acid (UA)2SO3 but contain one or two UAGalNAc4,6SO3 subunits interact with HCII. Together with previous results, these data support the hypothesis that modification of UAGalNAc4SO3 subunits in the DS polymer by either 2-O-sulfation of IdoA or 6-O-sulfation of GalNAc can generate HCII-binding sites.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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DS from porcine intestinal mucosa was partially degraded with chondroitinase ABC to produce oligosaccharides of varying chain length, each having a
4,5-unsaturated uronic acid (4,5UA) derived from IdoA or GlcA at the nonreducing end. Under the conditions of this experiment, about 25% of the glycosidic linkages were cleaved as determined by measurement of absorbance at 232 nm. The resulting oligosaccharide mixture was fractionated according to size using a P-10 gel-filtration column. This method gave good separation of oligosaccharides 2–12 monosaccharide units in length (Figure 1). Mass spectra obtained for each peak confirmed that the assignment of oligosaccharide chain lengths was correct. Each P-10 peak appeared homogeneous with regard to oligosaccharide length but contained a mixture of species that differed in the number of sulfate groups present. For each size fraction, the strongest signals by far in the mass spectrum were those of molecules containing one sulfate per disaccharide (data not shown).
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Hexasaccharides and larger fragments pooled from the P-10 column were fractionated by strong anion-exchange chromatography using a CarboPac PA200 high-performance liquid chromatography (HPLC) column. In each case, at least three broad peaks were eluted with the NaCl gradient. These peaks were named peaks I, II, III, and so on, as indicated in Figure 2. The compositions of individual peaks from the CarboPac PA200 column were determined by complete digestion with chondroitinase ABC and identification of the resulting disaccharides by anion-exchange HPLC (Table I). The compositions of intact DS and the tetrasaccharides pooled from the P-10 column were also determined in this manner. Hexasaccharide peak I contained one major disaccharide, 4,5UAGalNAc4SO3, indicating that most of these molecules were uniformly sulfated polymers of three IdoA (or GlcA)GalNAc4SO3 subunits (Figure 3). Hexasaccharide peak II contained two major disaccharides 4,5UAGalNAc4SO3 and 4,5UA GalNAc4,6SO3 in the ratio 2:1, whereas hexasaccharide peak III contained predominantly 4,5UAGalNAc4SO3 and 4,5UAGalNAc4,6SO3 in a ratio close to 1:2. Therefore, peaks I, II, and III contained three, four, and five sulfate groups per hexasaccharide, respectively.
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Disaccharide analyses of octa-, deca-, and dodecasaccharide peaks I, II, and III gave comparable results. In each case, peak I contained only monosulfated disaccharides, peak II one disulfated disaccharide, and peak III two disulfated disaccharides. The most abundant monosulfated disaccharide was 4,5UAGalNAc4SO3, whereas 4,5UA GalNAc4,6SO3 was the predominant disulfated disaccharide (Table I). Minor amounts of the monosulfated disaccharide 4,5UAGalNAc6SO3 and the disulfated disaccharide 4,5UA2SO3GalNAc4SO3 were also present.
Oligosaccharides pooled from the P-10 column were reduced with sodium [3H]borohydride and applied to an HCII-Sepharose affinity column equilibrated with 50 mM NaCl, 50 mM Tris–HCl (pH 7.4). The smallest oligosaccharides that bound to the HCII-Sepharose column were hexasaccharides, of which 4% bound (Figure 4, panel A). For molecules 6–12 monosaccharide units in length, a greater proportion of binding was observed with larger size. None of the flow-through material bound when reapplied to the column, indicating that the column had not been overloaded. To examine the relationship between binding and charge, we labeled hexa-, octa-, deca-, and dodecasaccharides with [3H]borohydride and fractionated them on the CarboPac PA200 column. The resulting peaks I, II, and III were then applied to the HCII-Sepharose column. For oligosaccharides containing 6–12 monosaccharide units, a greater percentage of binding of peaks II and III in comparison with peak I was observed (Figure 4, panel B). The ionic strength at which the bound fragments eluted did not increase with increasing size or charge (data not shown). Essentially none of the hexasaccharides from peak I bound to HCII-Sepharose, but 21% of peak II and 55% of peak III hexasaccharides bound and were subsequently eluted with 0.4–0.6 M NaCl (Figure 5). When bound material from hexasaccharide peak II was reapplied to the HCII column, essentially all bound the second time, but none of the reapplied flow-through material bound.
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The data presented above indicate that oligosaccharides containing GalNAc4,6SO3 bind to HCII. To characterize the UA present in the DS starting material, we performed complete digestions with chondroitinases ABC, AC, and B. Chondroitinase ABC cleaves the ß1,4 linkages of both GlcA and IdoA in DS. By contrast, chondroitinases B and AC are specific for IdoA and GlcA, respectively. Exhaustive digestion of DS with chondroitinase ABC or B produced similar amounts of 4,5UA as determined by absorbance at 232 nm, whereas digestion with chondroitinase AC produced only about 5% as much 4,5UA. These results indicate that not more than 5% of the UA in porcine intestinal mucosa DS is GlcA. Mass spectrometry of both the chondroitinase AC and B digests showed that most of the disaccharide products were monosulfated, the rest being mainly disulfated (data not shown).
We considered the possibility that GlcA residues are clustered in the HCII-binding oligosaccharides. To address this issue, we digested aliquots of HCII-bound dodecasaccharides with chondroitinases ABC, AC, or B and then analyzed the digests by capillary HPLC-coupled mass spectrometry. Examination of the mass spectrum of each HPLC-absorbance peak allowed us to distinguish oligosaccharides from contaminants. Peaks containing monosulfated disaccharides (m/z = 458), disulfated disaccharides (m/z = 538), and dodecasaccharides with seven (m/z = 471) or eight (m/z = 485) sulfates are indicated in Figure 6. Intact dodecasaccharides were virtually absent in the chondroitinase ABC and B digests, the major products being mono- and disulfated disaccharides. Trace amounts of di- and trisulfated tetrasaccharides and trisulfated hexasaccharides were also detectable in the chondroitinase B digest as minor peaks between 23 and 25 min. The bound dodecasaccharides remained largely intact after exhaustive chondroitinase AC digestion, although minor amounts of tetrasaccharides were observed. The relative ion strengths of the dodecasaccharide and tetrasaccharide peaks suggested that roughly 15% of HCII-bound dodecasaccharides were cleaved by chondroitinase AC. These findings indicate that HCII-bound dodecasaccharides, like the DS starting material, contain predominantly IdoA.
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DS oligosaccharides from the P-10 column were tested for the ability to stimulate inhibition of thrombin by purified HCII. Although the hexasaccharide pool had no activity under the conditions of our assay, the octasaccharides had some activity at concentrations above 0.05 mg/mL, and the deca- and dodecasaccharides had much greater activity (data not shown). To investigate the relationship between sulfate content and activation of HCII, we tested deca- and dodecasaccharide peaks I, II, and III for their ability to activate HCII. For both oligosaccharides, peak I had limited activity at concentrations greater than 0.1 mg/mL, peak II was active at concentrations between 0.01 and 0.1 mg/mL, and peak III was active at concentrations between 0.001 and 0.01 mg/mL (Figure 7). The deca- and dodecasaccharides from the P-10 column also prolonged the activated partial thromboplastin time (aPTT) of normal pooled plasma, although they were about one-tenth as active as intact DS (Figure 8). Minimal prolongation of the aPTT occurred when these oligosaccharides were added to HCII-depleted plasma, confirming that their anticoagulant activity depends on the presence of HCII. Consistent with their ability to activate purified HCII, peak III deca- and dodecasaccharides had the greatest activity in the aPTT assay, whereas peak I had the least (Figure 9).
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), II (
), and III (
). Dodecasaccharide peaks I (
), II (
), and III (
).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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Maimone and Tollefsen (1990)
prepared oligosaccharides by deaminative cleavage and subsequent borohydride reduction of partially N-deacetylated porcine skin DS. They found that the smallest oligosaccharides that bound to HCII-Sepharose in the presence of 0.05 M NaCl were hexasaccharides. Hexasaccharides that contained three sulfate groups per molecule (
94% of the total hexasaccharides) did not bind to HCII, but less abundant hexasaccharides that contained four, five, or six sulfate groups bound with increasing affinity. The hexasaccharide having the highest affinity contained six sulfate groups, being composed of three IdoA2SO3GalNAc4SO3 disaccharide units with the reducing terminal GalNAc4SO3 converted to anhydrotalitol 4-sulfate by the cleavage/reduction procedure.
Scully and others (1986, 1988) showed that HCII could be activated by several glycosaminoglycans composed of disaccharide subunits that contain GalNAc4,6SO3, including chondroitin sulfate E (GlcAGalNAc4,6SO3), chondroitin sulfate H (IdoAGalNAc4,6SO3), and polysulfated DS (IdoA2SO3 or 3SO3GalNAc4,6SO3). Mascellani and others (1993) suggested that GalNAc4,6SO3 contributes to HCII activation by porcine mucosal DS. DS from this source contained a higher amount of IdoAGalNAc4,6SO3 and a lower amount of IdoA2SO3GalNAc4SO3 compared with DS from either porcine skin or bovine mucosa. Despite these differences in composition, DS preparations from all three sources activated HCII with similar potency (Mascellani et al., 1993, 1994). In other experiments, chemical oversulfation of porcine skin DS, which raised the content of IdoAGalNAc4,6SO3 and IdoA2SO3GalNAc4,6SO3 subunits, substantially increased its activity with HCII (Mascellani et al., 1996). Finally, Linhardt and others (1994) reported that a low-molecular-weight bovine intestinal mucosal DS fraction with a high content of 4,6- and 2,4-disulfated disaccharides had enhanced in vivo antithrombotic activity in comparison with fractions containing lower amounts of these disulfated disaccharides.
We took advantage of the unusual composition of porcine mucosal DS to investigate the roles of IdoA2SO3 and GalNAc4,6SO3 in HCII binding and activation. We observed that the smallest enzymatically derived oligosaccharides from porcine mucosal DS that bind to HCII were hexasaccharides. This finding is consistent with the work of Maimone and Tollefsen (1990), in which deaminative cleavage was used to generate oligosaccharides of porcine skin DS. Also in agreement with the previous study, we found that hexasaccharides composed exclusively of UAGalNAc4SO3 disaccharide subunits (hexasaccharide peak I) did not bind HCII and that incorporation of one or more additional O-sulfate groups was required for binding. It is important to note that none of the tetrasaccharides bound to HCII, even though some of them contained a disulfated disaccharide (Figure 4A and Table I). Tetrasaccharides containing four sulfate groups were not detected by mass spectrometry (data not shown). Thus, the minimum structure required for binding is a hexasaccharide with at least one disulfated disaccharide subunit.
IdoA2SO3GalNAc4SO3 was the only disulfated disaccharide identified in porcine skin DS in the previous study. By contrast, UAGalNAc4,6SO3 was the predominant disulfated disaccharide in our porcine mucosal DS preparation, comprising 9% of the total disaccharides. We found that hexasaccharides lacking UA2SO3 but containing one or two UAGalNAc4,6SO3 subunits (hexasaccharide peaks II and III, respectively) interacted with HCII. A greater percentage of hexasaccharide peak III (55%) bound to HCII in comparison with peak II (21%). The ability of some molecules in each peak, but not others, to bind HCII suggests that the arrangement of the UAGalNAc4,6SO3 subunits within the hexasaccharide is important. We observed that longer oligosaccharides composed only of UAGalNAc4SO3 subunits (i.e. peak I octa-, deca-, and dodecasaccharides) did not bind to HCII, even though they contained as many or more sulfates per molecule as did the HCII-binding hexasaccharides. HCII binding was proportional to both the oligosaccharide length and the number of 4,6-disulfated residues. The longest oligosaccharide tested, the dodecasaccharide, needed just one extra sulfate group to result in binding of >90% of molecules to HCII (cf. dodecasaccharide peaks I and II in Figure 4). The disaccharide analysis of peak II dodecasaccharides (Table I) suggests that each of these molecules contained four UAGalNAc4SO3 and one UAGalNAc4,6SO3 subunits. Selective depolymerization of both the DS starting material and the HCII-bound dodecasaccharides with chondroitinase ABC, AC, and B indicated that most of the UA in these preparations is IdoA.
Earlier work from our laboratory and others showed that DS oligosaccharides at least 14–16 residues in length are required for maximum stimulation of the thrombin-HCII reaction (Tollefsen et al., 1986; Mascellani et al., 1993; Sié et al., 1993). The smallest oligosaccharide reported to stimulate this reaction was the high-affinity hexasaccharide isolated from porcine skin DS (Maimone and Tollefsen, 1990). However, this hexasaccharide was at least 20 times less potent than unfractionated DS on the basis of weight, implying that longer chains are required for efficient stimulation of the thrombin-HCII reaction. Although we did not detect any stimulatory activity with porcine mucosal DS hexasaccharides, we found significant activity in the deca- and dodecasaccharide fractions, which increased with the number of GalNAc4,6SO3 residues (Figure 6). The relatively weak but detectable activity of the deca- and dodecasaccharides containing only UAGalNAc4SO3 subunits (peak I) may indicate binding to HCII that was not detectable by HCII-Sepharose chromatography. Porcine mucosal DS deca- and dodecasaccharides also prolonged the clotting time (aPTT) of human plasma in an HCII-dependent manner (Figure 7). The anticoagulant activity of these oligosaccharides also increased with their GalNAc4,6SO3 content.
In conclusion, our study indicates that DS molecules present in porcine skin and intestinal mucosa contain different structures capable of binding and activating HCII. Together with previous results, our data support the hypothesis that modification of IdoAGalNAc4SO3 subunits in the DS polymer by either 2-O-sulfation of IdoA or 6-O-sulfation of GalNAc can generate HCII-binding sites. The presence of these modifications in specific tissues may serve to localize the thrombin inhibitory activity of HCII.
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Materials and Methods |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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Materials
DS (chondroitin sulfate B from porcine intestinal mucosa) was purchased from Sigma (St. Louis, MO) and was treated with nitrous acid as described previously to degrade contaminating heparin (Teien et al., 1976
). Recombinant chondroitinase AC and chondroitinase B were purchased from IBEX Technologies, Inc. (Montreal, Québec). Chondroitinase ABC and unsaturated disaccharide standards were purchased from Seikagaku America, Inc. (East Falmouth, MA). 3H-labeled sodium borohydride was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Biogel P-10 was purchased from Bio-Rad Laboratories (Hercules, CA), Sephadex G 25-80 from Sigma, and PD-10 disposable columns from Amersham Biosciences (Piscataway, NJ). HCII-depleted plasma was purchased from Enzyme Research Laboratories (South Bend, IN).
Preparation of DS oligosaccharides
Twenty-five milligrams of DS in 2.5 mL lyase buffer (40 mM Tris–HCl [pH 8.0], 40 mM sodium acetate, 0.1 mg/mL bovine serum albumin) was digested with 100 mU chondroitinase ABC at 37°C. The reaction was monitored by measuring absorbance at 232 nm and was stopped by boiling at 100°C for 3 min when the absorbance reached about 25% of its maximum value. The digested material was fractionated by size using a 1.5 x 184 cm Bio-Gel P-10 column equilibrated in 10% ethanol containing 1 M NaCl. The column was eluted at 6 mL/h, and 2-mL fractions were collected. The amount of digested DS in each fraction was monitored by absorbance at 232 nm. Fractions corresponding to each peak were pooled and desalted using either a G 25–80 column or a PD-10 column. Desalted oligosaccharides were lyophilized and dissolved in H2O. The concentration was determined using the carbazole assay standardized with porcine mucosal DS (Bitter and Muir, 1962).
3H labeling of oligosaccharides
Oligosaccharides were radiolabeled by reduction with NaB3H4. The oligosaccharide (0.1 µmol) was dissolved in 90 µL of 50 mM sodium acetate buffer (pH 7.0). Ten microliters of a 10 mM NaB3H4 stock (10 Ci/mmol in 0.1 M NaOH) was added, and the reaction was incubated at 38°C for 15 min. Then, 2.5 µL of 1 M unlabeled NaBH4 was added, and the incubation continued for another 15 min at 38°C. Finally, 25 µL of acetic acid was added to stop the reaction. The mixture was desalted using a PD-10 column. One-microliter fractions were collected, and radioactivity was measured using liquid scintillation counting.
Fractionation of oligosaccharides by strong anion-exchange HPLC
Size-uniform oligosaccharides were separated by charge using a CarboPac PA200 analytical HPLC column (Dionex, Sunnyvale, CA) equilibrated with 70 mM NaH2PO4 (pH 3.0). The oligosaccharides were eluted with a linear gradient from 0.07 to 2.0 M NaH2PO4 over 2 h at a flow rate of 0.4 mL/min. Absorbance at 232 nm was measured, and 0.4-mL fractions were collected. Radiolabeled oligosaccharides were detected by liquid scintillation counting of a portion of each fraction.
Disaccharide analysis
Intact DS or oligosaccharides were digested to completion using chondroitinase ABC at room temperature. The reaction was monitored by absorbance at 232 nm. The enzyme was separated from the digested molecules using a Centricon YM-30 centrifugal filter unit (Millipore, Bedford, MA). The digest was then desalted using a PD-10 column. The disaccharide mixture was applied to a Supelco Spherisorb SAX 5-µm HPLC column (Waters, Milford, MA) equilibrated with H2O and adjusted to pH 3.5 with HCl. The column was eluted with a linear gradient from 0 to 1 M NaCl over 45 min at a flow rate of 0.5 mL/min. Peaks were detected by absorbance at 232 nm and quantified using EZ Start 7.2 software (Shimadzu, Torrance, CA).
HCII-affinity chromatography
HCII was purified from human plasma as previously described (Lian et al., 2001). Fifty-one milligrams of HCII was reacted with 2.2 mL of CNBr-activated Sepharose 4B (Amersham Biosciences) in 27 mL of 50 mM NaCl, 100 mM NaHCO3 (pH 8.2), according to the manufacturer’s instructions. N-Acetylated heparin (300 mg), prepared as described previously (Maimone and Tollefsen, 1990), was added to the coupling reaction to block free amino groups in the glycosaminoglycan-binding site of HCII. Approximately 41 mg of HCII was covalently bound to the resin. Oligosaccharides were loaded onto the HCII-Sepharose column (0.75 x 5 cm) equilibrated with 50 mM NaCl, 50 mM Tris–HCl (pH 7.4). The column was then washed with 50 mL of the equilibration buffer and eluted with a 30-mL linear gradient from 0.05 to 1 M NaCl at a flow rate of 1 mL/min. Two-milliliter fractions were collected. Oligosaccharides were detected by absorbance at 232 nm or liquid scintillation counting.
Enzymatic degradation of HCII-bound dodecasaccharides
HCII-bound dodecasaccharides were dissolved in 200 µL of digestion solution containing 20 mM ammonium acetate (pH 7.0), 0.5 mM CaCl2, 0.1 mg/mL bovine serum albumin, and 1 mU chondroitinase ABC, AC, or B. Digestion was monitored by increased absorbance at 232 nm using a Spectra MAX M2 plate-reading spectrophotometer (Molecular Devices, Sunnyvale, CA), with the plate chamber set at 37°C. When the absorbance reached a plateau, the digests were incubated at 37°C overnight following addition of another 100 µL of digestion buffer and 1 mU enzyme. The digests were lyophilized and reconstituted in 10 µL water. Digested samples were analyzed by capillary HPLC-coupled mass spectrometry as described below.
Capillary HPLC-coupled mass spectrometry
An Agilent 1100 series capillary HPLC workstation (Agilent, Palo Alto, CA) with Chemstation software was used for data acquisition, analysis, and management. HPLC separations were performed on a 0.3 x 250 mm C18 column (Zorbax 300 SB, 5 µm, Agilent) using a binary solvent system composed of 5% methanol (eluent A) and 90% methanol in water (eluent B), both containing 3.5 mM dibutylamine, with pH adjusted to 5.5 with 2 M acetic acid. After injection of 0.2 µL of sample, the elution profile was 0% B for 7 min, 15% B for 9 min, 40% B for 11 min, and 100% B for 23 min. The flow rate was 5 µL per min, and absorbances at 232, 260, and 280 nm were monitored during each run. After each run, the column was washed with 90% B for 15 min and equilibrated with 100% A for 40 min. The capillary HPLC was directly coupled to the mass spectrometer. Mass spectra were acquired on a Mariner BioSpectrometry Workstation ESI time-of-flight mass spectrometer (PerSeptive Biosystems, Framingham, MA) in the negative-ion mode. Nitrogen was used as a desolvation gas as well as a nebulizer. Conditions were as follows: nebulizer flow, 1 L/min; nozzle temperature, 140°C; N2 flow, 0.1 L/min; spray tip potential, 2.8 kV; nozzle potential, 70 V; and skimmer potential, 9 V. Negative-ion spectra were generated by scanning the range of 150–1000 m/z. During analyses, the vacuum was 2.1 x 10–6 Torr. Total ion current chromatograms and mass spectra were processed with Data Explorer software version 3.0.
Inhibition of thrombin by HCII
Inhibition of thrombin by HCII in the presence of DS oligosaccharides was determined as previously described (Maimone and Tollefsen, 1990).
Activated partial thromboplastin time assay
Citrate-anticoagulated human plasma (90 µL) was mixed with 10 µL of a glycosaminoglycan solution and 100 µL of Alexin HS reagent (Sigma). After a 2-min pre-incubation at 37°C, 100 µL of 0.25 M CaCl2 was added, and the clotting time was determined with a fibrometer (Becton Dickinson, Sparks, MD).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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This work was supported by grants from the National Institutes of Health (HL55520 to D.M.T. and GM069968 to L.Z.). A.M.H. was a fellow of the American Heart Association.
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Abbreviations |
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aPTT, activated partial thromboplastin time; DS, dermatan sulfate; GalNAc, N-acetyl-D-galactosamine; GlcA, D-glucuronic acid; HCII, heparin cofactor II; HPLC, high-performance liquid chromatography; IdoA, L-iduronic acid; UA, uronic acid;
4,5UA, 4,5-unsaturated uronic acid |
References |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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