Department of Medical Biochemistry and Microbiology, Box582, The Biomedical Center, Uppsala University, S-751 23 Uppsala,Sweden and 2Department of VeterinaryMedical Chemistry, Box 575, The Biomedical Center, The Swedish Universityof Agricultural Science, S-751 23 Uppsala, Sweden
Received on March 1, 1999. revisedon May 17, 1999; accepted on May 17, 1999.
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Abstract |
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Introduction |
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A model system was developed based on the previously establishedmechanism of thrombin inhibition by AT, in the presence of heparin.Heparin (and HS) bind to AT via a unique pentasaccharide sequence,-GlcNSO3(6-OSO3)-GlcA-GlcNSO3(3,6-OSO3)-IdoA(2-OSO3)-GlcNSO3(6-OSO3)-,and this interaction is sufficient to greatly increase the rateof inhibition of Factor Xa (3Bourin and Lindahl,1993). Similar potentiation of the inhibition of thrombinrequires binding of this proteinase as well as of AT, adjacent toeach other, to a saccharide sequence that must consist of at least18 monosaccharide units. Information relating to purely syntheticoligosaccharides predicted that the reducing-terminal portion ofsuch a sequence should contain the AT-binding pentasaccharide sequence, whereasthe nonreducing-terminal, thrombin-binding, portion could lack thisregion (28
van Boeckel et al.,1994; 8
Grootenhuis etal., 1995). This information was used to validateour novel strategy to generate regio-specific neo-GAG conjugates,starting from natural heparin oligosaccharides.
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Results |
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The formation of Fragment A (Figures 1, 2) was initiated by partial deaminative cleavageof beef lung heparin with nitrous acid essentially as describedpreviously (19Pejler et al.,1988). This reaction resulted in the generation of variouslysized oligosaccharides with a reducing-terminal 2,5-anhydromannose unit(23
Shively and Conrad, 1976). Fractionationof these oligosaccharides by gel chromatography on Biogel P-10 yielded a10-mer fraction and a 12-mer fraction (data not shown) that wererecovered and desalted. The aldehyde group of the reducing-terminal2,5-anhydromannose residue was substituted by reductive amination(see 9
Hoffman et al., 1983)with cystamine·2HCl in the presence of NaB3H3CN,thus introducing a 3H label at the reducing end of theoligomers. The newly introduced amino group was acetylated by reactionwith acetic anhydride (Figure 2), yieldinga product that failed to react with ninhydrin (5
Doi et al., 1981) (data not shown). The substituted12-mers were separated by affinity chromatography on AT-Sepharose(10
Höök et al., 1976)into a major nonbound fraction, that was unretained by the immobilizedAT in 50 mM NaCl, a low-affinity (LA) fraction (~16.2 % ofthe initial oligosaccharide mass) and a high-affinity(HA) fraction (~5.4 % of the initial oligosaccharide mass)by elution with a linear gradient from 0.05 M to 3.0 M NaCl (datanot shown).
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Analysis of anticoagulant activity
A dissociation constant of 13 ± 0.1(range; n = 2) nM was measured for the complex of the LA-HA(nonreducing-reducing domain) neo-heparin conjugate with AT at ionicstrength 0.05 by titrations, monitored by tryptophan fluorescence,of AT with the saccharide. This ionic strength was chosen to increasethe AT affinity of the conjugate, compared with the affinity atI 0.15, because of the small amounts of conjugate available. Theinteraction thus appears somewhat weaker than that between nativeHA-heparin or the HA decasaccharide and AT, for which dissociationconstants of 4.8 ± 0.4 (SE; n = 3) and5.3 ± 1.2 (SE; n = 5) nM, respectively,were measured in parallel assays. Nevertheless, the LA-HA conjugatepromoted the inhibition of thrombin by AT about twice as efficientlyas authentic HA-heparin (Figure 5). As expected,neither the LA-LA neoconjugate nor the unconjugated HA-10-mer showed anyapparent ability to accelerate the AT-thrombin reaction. These resultsconform to the notion that the specific AT-binding pentasaccharideis essential but not sufficient for acceleration of thrombin inactivationand that an additional thrombin-binding sequence outside the pentasaccharideregion is required for such an effect.
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Discussion |
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The group protection strategy applied involved blocking of thefree hydroxyl groups of Fragment B through peracetylation (Figure 2). The O-acetyl groups could not be readilyremoved following formation of the conjugate, since deesterification wouldbe expected to break up the newly formed linkage between FragmentsA and B. However, previous studies indicated that the weakeningof the interaction between heparin and AT caused by the presenceof O-acetyl groups should be relatively modest (1Barzu et al., 1993). This expectation is verifiedby the only moderately lower observed affinity of the conjugateas compared to that of HA-heparin or the HA-decasaccharide for AT.Still, we were surprised to find that the ability of the LA-HA conjugateto accelerate AT inhibition of thrombin was about twice as high,on a molar basis, as that of authentic HA-heparin (Figure 5). We ascribe this finding to the optimal positioningof the AT- and thrombin-binding domains of the LA-HA conjugate,as opposed to the random location of the AT-binding region in theHA-heparin chain (18
Oscarsson et al., 1989) which thus could accommodate thrombinon the "wrong" side of the AT molecule. Nevertheless,deacetylation may be prerequisite to studies of interactions involvingother proteins. To ensure general applicability the method shouldbe modified to allow removal of the protecting groups without the riskof cleaving the conjugate linkage.
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Materials and methods |
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Functionalization at the reducing end of heparin-derived oligosaccharides(type A fragment)
Bovine lung heparin (100 mg) was subjected to partial deaminativecleavage, essentially as described previously (19Pejler et al., 1988). The sample was dissolvedin 10 ml of deionized water at +4°C,and the solution was acidified to pH 1.5 by dropwise addition of1.0 M H2SO4. Following the addition of 2 mgsodium nitrite in 50 µl deionized water,under vigorous stirring, the reaction mixture was maintained atpH 1.5, in an ice bath, for 3 h. The sample was then adjusted topH 7.0 with 1 M sodium bicarbonate, concentrated to 1 ml, and thenfractionated by gel chromatography on a column (1 x 150cm) of Biogel P-10 in 0.5 M NaCl. Effluent fractions of 1.0 ml were collectedat a rate of 1.4 ml/h and analyzed for hexuronic acid bythe carbazole reaction (2
Bitter and Muir,1962). Both 10-mer and 12-mer fractions, correspondingto 8.4% and 5.2% of the starting material, respectively,were recovered, dialyzed in dialysis bags with molecular weightcut-off 1000 (Spectra) at +4°Cagainst deionized water, and then lyophilized to dryness.
A sample (2.5 mg) of 12-mer heparin deamination product in 1ml of 0.5 M phosphate buffer (pH 7.0) was mixed with 83 mg of cystamine2HClin 300 µl of the same buffer, and themixture was vortexed and was then incubated at room temperature. After30 min, 0.5 mCi of NaB3H3CN (10 mCi/mmol)was added in the fume hood, and incubation was continued for another2 h. To ensure complete reductive amination, excess unlabeled NaBH3CN(67 mg) in 100 µl of 0.5 M phosphate buffer(pH 7.0) was added, and incubation was continued at room temperatureovernight. The pH was then adjusted to 4.0 by addition of 4 M aceticacid to eliminate any excess NaBH3CN, after which thesolution was concentrated to ~1 ml and passed through a 1 x 180cm column of Sephadex G-15 in 0.2 M ammonium bicarbonate. A partof the resultant oligosaccharide fraction (840 µg;11,100 d.p.m. 3H/µg),dissolved in 100 µl 0.1 M sodium bicarbonate,was cooled in an ice-bath and N-acetylated by treatment with aceticanhydride (100 µl). After incubationat +4°C for 30 min (no freeprimary amine detectable by the ninhydrin reaction (5
Doi et al., 1981)) the sample was again desaltedby gel chromatography on Sephadex G-15, and was then lyophilized.
The cystamine-substituted, N-acetylated oligosaccharides werefractionated with regard to affinity for AT by chromatography onAT-Sepharose, essentially as described previously (10Höök et al., 1976). The sample (~2 mg) wasapplied to a 5 ml column of the affinity matrix, equilibrated with50 mM NaCl. The NA (unretained) and LA (weakly bound) fractions(subsequently combined into one "LA" pool) andthe HA component were recovered following elution with a lineargradient from 0.05 M to 3.0 M NaCl and were then desalted. The LA12-mers (300 µg) were reduced by treatmentwith 200 µl of 0.1 M dithiothreitolcontaining 1 mM EDTA at room temperature overnight. 3H-Labeledoligosaccharides positive to Ellmans reagent were recoveredafter passage through a column (1 x 100cm) of Sephadex G-15 in 40 mM NaCl containing 1 mM EDTA.
Functionalization at the nonreducing end of heparin-derived oligosaccharides(type B fragment)
For partial lyase cleavage, bovine lung heparin (100 mg) was dissolvedin 100 ml of 100 mM sodium acetate buffer (pH 6.5), 10 mM CaCl2,0.1 mg/ml bovine serum albumin at 30°C,and heparinase I (12 units) was added. Following incubation at 30°C for 24 h the products were fractionatedby gel chromatography on a column (1 x 180cm) of Biogel P-10 in 0.5 M sodium chloride. Effluent fractionscorresponding to 10-mers and 12-mers were combined separately anddesalted by extensive dialysis in dialysis bags with molecular weightcut-off 1000 (Spectra) against deionized water. A sample (5 mg)of the product was reduced with 0.25 mCi NaB3H4 (29Ci/mmol) in 200 µl of water,adjusted to pH 8.0 with Na2CO3. After 30 min atroom temperature 5 mg of unlabeled NaBH4 was added, and incubationwas continued for another 5 h. The mixture was acidified to pH 4by adding 4 M acetic acid, and was then adjusted to pH 8 with Na2CO3.Labeled oligosaccharides were recovered following desalting by passagethrough Sephadex G-15, and were then lyophilized.
Before O-peracetylation (21Petitou et al., 1992), the reduced oligosaccharides(either 10-mers or 12-mers) were converted to the free acid formby passage through a column (1 x 10cm) of AG 50W-X4 (H+ form) at +4°C, and the effluent was immediatelyneutralized with n-tributylamine. The product was lyophilized, dissolvedin 3 ml dry N,N'-dimethylformamide, evaporatedto dryness and redissolved in 1.4 ml dry N,N'-dimethylformamide.N,N'-Dimethylpyridine (25 mg), acetic anhydride(100 µl), and n-tributylamine (200 µl) were added, and the mixture wasincubated at 37°C overnight. The reaction wasinterrupted by the addition of 100 µlH2O, and the peracetylated oligosaccharides were desaltedby passage through a PD-10 column.
Removal of the 4,5-unsaturated, nonreducing-terminal hexuronicacid unit was effected by treating the peracetylated oligosaccharide(1 mg) with 75 mM mercuric acetate in 400 µl acetatebuffer (pH 5.0) (14Ludwigs etal., 1987). After incubation at room temperaturefor 2 h, the reaction mixture was passed through a column (1 x 10 cm) of AG 50W-X4 (H+ form),and effluent fractions were neutralized with Na2CO3. Theoligosaccharides were extensively dialyzed against deionized water.
The modified heparin 11-mers were affinity fractionated on AT-Sepharose,as described for the Fragment A preparation, and the resultant LAand HA fractions (4.4% and 3.3% of the initial oligosaccharidemass, respectively) were recovered separately and desalted. Finally,the modified heparin 11-mers were acylated by reaction with iodoaceticanhydride. Samples (200 µg)of each species were converted to the free acid form, convertedto the n-tributylamine salts and evaporated to dryness in the presenceof dry N,N'-dimethylformamide. The residues wereredissolved in 1.2 ml of dry N,N'-dimethylformamide/n-tributylamine(v/v, 5/1). p-N,N'-Dimethylaminopyridine (100 µg) and iodoacetic anhydride (10 µg) were added, and the reaction mixtureswere left at room temperature with shaking for 36 h. Following theaddition of 100 µl deionized water thederivatized oligosaccharides were recovered by passage through PD-10 columns.
Conjugation
A solution of Fragment A (12-mers, ~200 µg,550 x 103 d.p.m. 3H)mixed with Fragment B (11-mers, ~100 µg,160x103 d.p.m.) in a 1.5ml microfuge tube was evaporated to dryness. The residue was dissolvedin 100 µl 10 mM EDTA, pH 7.8, and thesolution was kept at room temperature overnight. The reaction productswere separated by gel chromatography on a column (1 x 150cm) of Biogel P-10 in 0.5 M ammonium bicarbonate. Effluent fractionswere analyzed for 3H radioactivity. Fractions correspondingto the putative neo-GAG conjugate were combined and lyophilized.
Interaction of AT with neo-GAG conjugate
Stoichiometries and affinities of heparin, HA-decasaccharide orneo-GAG conjugate binding to AT at 25°Cwere measured by titrations, monitored by the increase of AT tryptophanfluorescence induced by the interaction, as in previous work (17Olson et al., 1993; 27
Turk et al., 1997). Thebuffer was 0.02 M sodium phosphate, 0.1 mM EDTA, 0.1 % (w/v)poly(ethyleneglycol), pH 7.4.
The ability of heparin, HA-decasaccharide or neo-GAG conjugateto potentiate the AT-dependent inhibition of thrombin was assessedat 25°C in 0.02 M sodium phosphate,0.1 M NaCl, 0.1 mM EDTA, 0.1% (w/v) poly(ethyleneglycol),pH 7.4, essentially as described previously (27Turk et al., 1997). AT and saccharide, at finalconcentrations of 100 nM and 00.5 nM, respectively, weremixed with thrombin at a final concentration of 10 nM. The concentrationsof HA-heparin, HA-decasaccharide, and LA-HA neo-GAG conjugate werethose calculated from the measured stoichiometries of binding to AT,whereas concentrations measured by carbazole analyses were usedfor the LA-LA neo-GAG conjugate. After varying times, portions ofthe reaction mixtures were diluted tenfold into a cuvette, containingthe chromogenic thrombin substrate, D-Phe-Pip-Arg-p-nitroanilinide (Chromogenix, Mölndal,Sweden) at a final concentration of 100 mM, and the residual enzymeactivity was obtained from the linear rate of the absorbance increaseat 405 nm. The time-dependent loss of enzyme activity was fittedby nonlinear regression to a single exponential function with anendpoint of complete inactivation to give the observed pseudo-first-orderrate constant, kobs (17
Olson et al., 1993). The accelerating effect ofthe saccharides on the AT-thrombin reaction was evaluated by plottingkobs vs. saccharide concentration.
Additional analytical procedures
Uronic acid was determined by the carbazole method (2Bitter and Muir, 1962). A standard curveof absorbance at 530 nm vs. µg of heparinwas made using a standard heparin (from bovine lung) solution. Radioactivitywas measured by liquid scintillation counting using a Beckman modelLS 3800 liquid scintillation spectrometer. Free sulfrylhydryl groupswere determined by a microscale assay as described previously (6
Ellman, 1959).
Analysis of heparin oligosaccharides by polyacrylamidegel electrophoresis
Polyacrylamide gel electrophoresis was done in a solution of 0.89M Tris base, 0.89 M boric acid, and 20 mM EDTA, pH 8.3, on 20% acrylamidecontaining 0.53% bisacrylamide. A 10 ml volume of monomersolution was poured onto glass plates and allowed to polymerizefor 1 h. The minigel was pre-run at 120 V for 1 h, to achieve aconstant current. Oligosaccharide samples (1 to 30 µgof oligosaccharides in 5 µl deionized water)were mixed with 0.5 µl 2.0 M sucrosein electrophoresis buffer. After electrophoresis was performed at100 V for 23 h, the gel was stained with 5% Alcianblue, or further by silver staining essentially as described (16Möller et al., 1993).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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