Characterization of the Heparin Binding Properties of Annexin II Tetramer*

(Received for publication, November 26, 1996, and in revised form, January 27, 1997)

Geetha Kassam , Akhil Manro , Carol E. Braat , Peter Louie , Sandra L. Fitzpatrick and David M. Waisman Dagger

From the Cell Regulation Research Group, Department of Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 4N1, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

In this report, we have characterized the interaction of heparin with the Ca2+- and phospholipid-binding protein annexin II tetramer (AIIt). Analysis of the circular dichroism spectra demonstrated that the Ca2+-dependent binding of AIIt to heparin caused a large decrease in the alpha -helical content of AIIt from ~44 to 31%, a small decrease in the beta -sheet content from ~27 to 24%, and an increase in the unordered structure from 20 to 29%. The binding of heparin also decreased the Ca2+ concentration required for a half-maximal conformational change in AIIt from 360 to 84 µM. AIIt bound to heparin with an apparent Kd of 32 ± 6 nM (mean ± S.D., n = 3) and a stoichiometry of 11 ± 0.9 mol of AIIt/mol of heparin (mean ± S.D., n = 3). The binding of heparin to AIIt was specific as other sulfated polysaccharides did not elicit a conformational change in AIIt. A region of the p36 subunit of AIIt (Phe306-Ser313) was found to contain a Cardin-Weintraub consensus sequence for glycosaminoglycan recognition. A peptide to this region underwent a conformational change upon heparin binding. Other annexins contained the Cardin-Weintraub consensus sequence, but did not undergo a substantial conformational change upon heparin binding.


INTRODUCTION

The annexins are a family of ~13 proteins that bind to acidic phospholipids and biological membranes in a Ca2+-dependent manner (see Refs. 1-3 for reviews). These proteins are expressed in a wide range of organisms such as slime molds, higher plants, invertebrates, and vertebrates. Studies of the amino acid sequence of the annexins have established the homology of these proteins. All annexins contain four repeats (eight repeats in the case of annexin VI) of ~70 amino acids that are highly homologous. In contrast, the N terminus of each of the annexins is unique and displays the greatest variation in sequence and length. The crystal structure of several of the annexins has been reported (4-6) and has established that the annexins are composed of two distinct sides. The convex side faces the biological membrane and contains the Ca2+- and phospholipid-binding sites The concave side faces the cytosol and contains the N and C termini.

Annexin II (p36) contains three distinct functional regions, the N-terminal region, the C-terminal region, and the core region. The core region of p36 contains the Ca2+- and phospholipid-binding sites, whereas the C-terminal region contains the 14-3-3 homology domain (7) and the plasminogen-binding domain (8). The N terminus of annexin II (p36) contains two important regulatory domains, the L and P domains. The L domain consists of the first 14 residues of the N terminus and contains a high affinity binding site for the p11 protein (reviewed in Ref. 9). The P domain of p36 contains the phosphorylation sites for protein kinase C (Ser25) and pp60src (Tyr23). The N-terminal L and P domains play regulatory roles; activation of the phosphorylation sites of annexin II tetramer results in an increase in the A0.5(Ca2+) for chromaffin granule aggregation and F-actin binding, whereas binding of the p11 subunit decreases the A0.5(Ca2+) for these activities. The heterotetrameric complex (p362·p112) formed by the binding of p11 to p36, referred to as annexin II tetramer (AIIt),1 is the predominant form in most cells (reviewed in Ref. 9).

AIIt has been shown to be present at both the cytosolic and extracellular surfaces of the plasma membrane of many cells (9). Extracellular AIIt has been proposed to function as a cell adhesion factor (10, 11), a receptor for plasminogen and tissue plasminogen activator (8, 12), and a receptor for tenascin-C (13, 14).

In a previous study (15), we reported that AIIt bound to a heparin affinity column and that the phosphorylation of AIIt on tyrosine residues blocked the heparin-binding activity of the protein. In this report, we have characterized the interaction of AIIt with heparin. Our results identify AIIt as a specific, high affinity heparin-binding protein. Furthermore, we show that the Ca2+-dependent binding of heparin to AIIt causes a dramatic conformational change in the protein. Last, we show that the p36 subunit of AIIt contains a Cardin-Weintraub glycosaminoglycan recognition site (16) and that a peptide to this region of AIIt binds heparin.


EXPERIMENTAL PROCEDURES

Materials

Annexin II tetramer and annexin II monomer were prepared from bovine lung as described (17) and stored at -70 °C in 40 mM Tris-HCl, pH 7.5, 1.0 mM DTT, 0.1 mM EGTA, and 150 mM NaCl. Carbohydrates were obtained from Sigma and were the purest grade available. Heparin (bovine lung) was obtained from Calbiochem and had an average molecular mass of 17 kDa and an activity of 149 USP units/mg. Porcine intestinal mucosa heparin (6 kDa, 70 USP units/mg), bovine intestinal mucosa heparin (3 kDa, 50 USP units/mg), and bovine kidney heparan sulfate (7.5 kDa) were obtained from Sigma.

Measurement of Protein Secondary Structure by Circular Dichroism

CD measurements were performed with a Jasco J-715 spectropolarimeter. The spectropolarimeter was calibrated with an aqueous solution of recrystallized ammonium camphor sulfonate-d10. At a concentration of 1 mg/ml and in a 1-mm cell, this compound had a Delta epsilon of 2.36 at its CD maximum of 290.5 nm. AIIt (2.2 µM) was incubated in either buffer A (10 mM Tris-HCl, pH 7.5, 0.1 mM DTT, 0.15 M NaCl, and 1.0 mM CaCl2) or buffer B (10 mM Tris-HCl, pH 7.5, 0.1 mM DTT, 150 mM NaCl, and 0.5 mM EGTA) in the presence or absence of ligand (0.5-50 µM) for 20 min at room temperature. Samples (0.1 ml) were scanned in a quartz cuvette (0.5-mm path length) from 178 to 260 nm at a rate of 10 nm/s, using a bandwidth of 1 nm and a response time of 4 s. CD spectra of proteins were obtained by averaging four wavelength scans and were corrected by subtracting buffer scans or, where appropriate, scans of ligand in buffer. The measurements at 222 nm were obtained using the time scan mode of the software, and each point was the average of a minimum of 20 × 1-s measurements (at a bandwidth of 1 nm). Results are expressed as Delta epsilon (M-1 cm-1).

Analysis of CD Spectra

CD spectra of AIIt were analyzed by the self-consistent method of Sreerama and Woody (18). The analysis was performed with the computer program SELCON, which was a generous gift from Dr. Robert W. Woody (Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO). Typically, scans from six individual experiments were analyzed, and these values were averaged. The root mean square difference between the predicted CD data and the actual CD data was always <0.15.

AIIt Affinity Chromatography

Approximately 1.4 g of CNBr-activated Sepharose 4B was washed with 400 ml of 1 mM HCl, followed by extensive washing in coupling buffer (0.1 M NaHCO3, pH 8.3, and 0.5 M NaCl). AIIt (10 mg) was extensively dialyzed against coupling buffer, mixed with 5.0 ml of washed CNBr-activated Sepharose 4B resin in a final volume of 20 ml, and incubated overnight at room temperature. The resin was then washed with coupling buffer; incubated with 1 M ethanolamine, pH 8.5, in a final volume of 50 ml for 2 h; and equilibrated with buffer C (20 mM Tris-HCl, pH 7.5, 1 mM DTT, and 1 mM CaCl2) containing 25 mM NaCl.

Heparin was dissolved in buffer C containing 25 mM NaCl and applied to a 5-ml AIIt affinity column. The column was washed with buffer C containing 25 mM NaCl, followed by step elution with buffer C containing 150 and 500 mM NaCl. The heparin concentration of these fractions was determined by the uronic acid/carbazole reaction (19).

Heparin Binding Assays

Heparin binding experiments were performed in a total volume of 200 µl in a reaction mixture containing 25 mM Hepes, pH 7.5, 1 mM DTT, 150 mM NaCl, 2.0 µM AIIt, and variable concentrations of heparin. The reaction mixture was incubated at room temperature for 20 min and then centrifuged at 400,000 × g for 30 min. The pellet was extracted with Laemmli SDS disruption buffer (20) and resolved by SDS-polyacrylamide gel electrophoresis. Protein bands were visualized with Coomassie Brilliant Blue R-250, and the p36 subunit protein band (36 kDa) was cut from each lane of the polyacrylamide gel, extracted overnight with pyridine (25%), and then quantified spectrophotometrically by measuring the absorbance at 605 nm as described (21).

Data Analysis

Nonlinear least-squares fitting was performed with the computer program SigmaPlot (Jandel Scientific). Titration data were analyzed as detailed by the SigmaPlot reference manual with the four-parameter logistic equation f = (a - d)/(1 + (x/c)b) + d, where a = asymptotic maximum, b = slope parameter, c = value at inflection point (A0.5), and d = asymptotic minimum. The nonlinear least-squares curve fitting was then iterated by allowing the four fitting parameters to float while utilizing the Marquardt method for the minimization of the sum of the squared residuals. The Kd and n values for the binding of heparin to AIIt were derived from plots of the fraction of AIIt bound versus the total heparin concentration ([HEP]T) using the quadratic equilibrium binding equation (Equation 1),
&agr; = <FR><NU><AR><R><C>(n[<UP>AIIt</UP>]<SUB>T</SUB>+[<UP>HEP</UP>]<SUB>T</SUB>+K<SUB>d</SUB>−</C></R><R><C><RAD><RCD>(n[<UP>AIIt</UP>]<SUB>T</SUB>+[<UP>HEP</UP>]<SUB>T</SUB>+K<SUB>d</SUB>)<SUP>2</SUP>−4n[<UP>AIIt</UP>]<SUB>T</SUB>[<UP>HEP</UP>]<SUB>T</SUB></RCD></RAD></C></R></AR></NU><DE>2n[<UP>AIIt</UP>]<SUB>T</SUB></DE></FR> (Eq. 1)
where alpha  = fractional binding (AIIt pelleted/total AIIt), n = mol of heparin/mol of AIIt, and Kd = dissociation constant. The nonlinear least-squares curve fitting was then iterated by allowing the Kd and n parameters to float while utilizing the Marquardt method for the minimization of the sum of the squared residuals. For this analysis, it was assumed that there was only a single class of heparin-binding sites on AIIt.

Miscellaneous Techniques

AIIt concentration was determined spectrophotometrically using an extinction coefficient of A280 nm = 0.68 for 1 mg/ml AIIt. Ca2+/nitrilotriacetic acid buffers were prepared according to Ref. 22. The Ca2+ concentration of CaCl2 stock solutions was determined by atomic absorption. The final Ca2+ concentrations of Ca2+/nitrilotriacetic acid buffers were verified by Ca2+ electrode and FURA-2 measurement. Peptides were synthesized by the University of Calgary Peptide Facility and were purified by high pressure liquid chromatography. Phospholipid liposomes were prepared from mixtures of 50 µl of 20 mg/ml phosphatidylserine, phosphatidylethanolamine, and cholesterol in chloroform, which were dried in an N2 stream and hydrated with 1 ml of buffer containing 30 mM Hepes, pH 7.5, and 2 mM MgCl2. The mixture was sonicated (4 × 15-s bursts at 75 watts with a Braun probe sonicator), and the phospholipid liposomes were stored at room temperature prior to their use.


RESULTS

CD Spectra of the AIIt-Heparin Complex

Consistent with a previous report (23), we also found that the binding of Ca2+ to AIIt caused a 10% reduction in intensity of the CD spectrum of AIIt (Fig. 1A). In contrast, the binding of heparin to AIIt in the presence of Ca2+ resulted in a decrease in the intensity of the CD spectrum at 222 nm of ~40%. Furthermore, the CD spectrum of AIIt lost intensity and changed shape as the protein bound heparin in the presence of Ca2+ (Fig. 1B). In the absence of Ca2+, heparin did not produce a dramatic change in the intensity and shape of the CD spectrum of AIIt. We also observed that in the absence of Ca2+, the CD spectrum of AIIt measured in the presence of heparin was slightly less intense (a decrease of ~4% at 222 nm) than the CD spectrum of AIIt measured in the absence of both heparin and Ca2+.


Fig. 1. CD spectra of the AIIt·Ca2+ and AIIt-heparin complexes. A, wavelength scans were conducted at 20 °C in buffer A (dotted line) or buffer B (solid line). The protein concentration of AIIt was 2.2 µM. B, wavelength scans were conducted in the presence of 0.56 µM heparin and 2.2 µM AIIt in the presence of buffer A (solid line) or buffer B (dotted line).
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Quantitative assessment of the secondary structure components of the CD spectra of AIIt was made as described (18). The results are summarized in Table I. In the presence of Ca2+, the binding of heparin to AIIt resulted in a decrease in the alpha -helical content of AIIt from ~44 to 31%, a decrease in the beta -sheet content from ~27 to 24%, an increase in the content of beta -turns from ~12 to 14%, and an increase in the unordered structure from ~20 to 29%.

Table I. Effect of heparin on the secondary structure of annexin II tetramer

Data are presented as means ± S.D. (n = 6). CD scans were conduced at 20 °C in buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2.2 µM AIIt, 0.2 mM DTT, 0.56 µM heparin, and either 1 mM CaCl2 or 0.5 mM EGTA. Predicted secondary structure assignments were obtained from analysis of CD spectra using the computer program SELCON (19).

Structure Ca2+ Ca2+, heparin EGTA EGTA, heparin

 alpha -Helix 43.8  ± 1.63 31.4  ± 2.2 44.6  ± 1.0 40.1  ± 0.6
 beta -Sheet 26.6  ± 4.2 24.3  ± 3.9 20.9  ± 3.8 26.5  ± 6.2
 beta -Turns 11.5  ± 2.0 13.8  ± 0.6 11.9  ± 1.0 14.8  ± 1.5
Unordered 20.0  ± 3.6 29.4  ± 4.4 22.3  ± 7.3 18.4  ± 4.0

In the absence of Ca2+, heparin induced a more moderate change in the conformation of AIIt. Of interest was the heparin-induced increase in the beta -sheet from ~21 to 27% and decrease in unordered structure from 22 to 18%.

These results establish that in the presence of Ca2+, heparin induces a large conformational change in AIIt, resulting in a substantial change in the conformation of the protein. However, heparin can also interact with AIIt in the absence of Ca2+ and, to a much smaller degree, affect the conformation of the protein.

Dose Dependence of the Heparin-induced Conformational Change in AIIt

Fig. 2 presents the dose dependence of the heparin-induced conformational change in AIIt as determined by the changes in the CD spectrum at 222 nm. Increasing the heparin concentration decreased the intensity of the CD spectrum of AIIt until a maximum was reached at ~0.59 µM heparin. At the optimal concentration of heparin, the CD intensity decreased by ~36%. The heparin concentration required for a half-maximal decrease in the CD intensity of 2.2 µM AIIt was ~0.10 µM. Increasing the heparin concentration in excess of 0.59 µM resulted in an increase in the CD intensity.


Fig. 2. Concentration dependence of the heparin-induced conformational change in AIIt. AIIt (2.2 µM) was incubated for 20 min at 20 °C in buffer A with variable concentrations of heparin. The circular dichroism spectrum was then measured at 222 nm. Data shown are expressed as means ± S.D. (n = 3).
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Ca2+ Dependence of the Heparin-induced Conformational Change in AIIt

AIIt binds Ca2+ with a Kd(Ca2+) of ~0.5 mM. When AIIt is bound to chromaffin granules, phospholipid liposomes, or F-actin, the Kd(Ca2+) is reduced to µM values (9). The decrease in the Kd(Ca2+) upon phospholipid binding is due to a conformational change in the protein resulting from the binding of the phospholipid to the protein (6). We therefore used CD to examine the Ca2+ requirement for the changes in the conformation of AIIt observed upon heparin binding. As shown in Fig. 3, when assayed in the presence of saturating heparin, the half-maximal change in the CD spectrum of AIIt at 222 nm required 0.084 ± 0.008 µM Ca2+ (mean ± S.D., n = 3). In contrast, the half-maximal change in the CD spectrum of AIIt at 222 nm in the absence of heparin required 0.361 ± 0.149 µM Ca2+ (mean ± S.D., n = 3). This result therefore establishes that the binding of heparin to AIIt increases the affinity of the protein for Ca2+.


Fig. 3. Ca2+ dependence of the heparin-induced conformational change in AIIt. AIIt (2.2 µM) was incubated for 20 min at 20 °C in buffer (10 mM Tris-HCl, pH 7.5, 0.1 mM DTT, and 0.15 M NaCl) containing variable concentrations of Ca2+ in the absence (open circle ) or presence (bullet ) of 0.56 µM heparin. The circular dichroism spectra were measured at 222 nm. Data shown are expressed as means ± S.D. (n = 3). The conformational change at 222 nm is plotted as a function of the maximal conformational change in AIIt at 222 nm, where the maximal conformational change (100% of maximum) was determined by averaging the Delta epsilon 222 nm value for AIIt in the presence of 2 mM Ca2+ and saturating heparin. The line through the points is a nonlinear least-squares curve fit of the data points calculated from computer modeling of data to the four-parameter logistic equation (see "Experimental Procedures") and four fitting parameters, a (asymptotic maximum), b (slope parameter), c (value at inflection point, A0.5), and d (asymptotic minimum), which were allowed to float during the computer iterations.
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Specificity of the Heparin-induced Conformational Change in AIIt

We have also examined the effect of a variety of charged polysaccharides on the CD spectra of AIIt at 222 nm. As shown in Table II, charged polysaccharides such as heparan sulfate, chondroitin sulfate, and dextran sulfate failed to induce a large conformational change in AIIt. Similarly, other amino monosaccharides such as N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and N-acetylneuraminic acid or other monosaccharides such as glucose, mannose, fucose, and galactose failed to induce a substantial change in the conformation of AIIt. We also examined the possibility that other carbohydrates could bind to the heparin-binding site of AIIt without producing a conformational change. This possibility was ruled out by our observation that 50 µM heparan sulfate or 50 µM N-acetylglucosamine failed to block the conformational change in AIIt induced by 0.5 µM heparin.

Table II. Specificity of the heparin-induced conformational change in annexin II tetramer

Results are expressed as means ± S.D. (n = 5). Percent change at Delta varepsilon 222 nm = Delta varepsilon 222 nm(AIIt) - Delta varepsilon 222 nm(AIIt + ligand)/Delta varepsilon 222 nm(AIIt) × 100. Annexin II tetramer (2.2 µM) was incubated in buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2 mM DTT, 1 mM CaCl2, and 50 µM carbohydrate, 5.0 µM low molecular mass heparin (3 and 6 kDa), or 0.56 µM heparin (17 kDa).

Polysaccharide Change at Delta varepsilon 222 nm

Heparin 41.0  ± 3.4
Heparan sulfate 3.3  ± 1.0
Chondroitin sulfate 2.3  ± 0.9
Dextran sulfate 6.9  ± 4.1
N-Acetylglucosamine 6.2  ± 1.7
N-Acetygalactosamine 5.1  ± 1.0
N-Acetylneuraminic acid 3.9  ± 1.0
Glucose 2.8  ± 0.1
Galactose 9.5  ± 0.1
Mannose 6.1  ± 0.2
Fucose 5.1  ± 1.0
Heparin (3 kDa) 40.9  ± 1.2
Heparin (6 kDa) 58.0  ± 2.3
Heparin + heparan sulfate 39.3  ± 2.4
Heparin + N-acetylglucosamine 35.3  ± 2.2

We have also observed that smaller molecular mass forms of heparin can induce a conformational change in AIIt. As shown in Table II, both 3- and 6-kDa forms of heparin could induce a conformational change in AIIt. However, we have been unable to detect a conformational change in AIIt in the presence of disaccharide heparin derivatives (data not shown). These results therefore suggest that the heparin-induced conformational change in AIIt requires the interaction of AIIt with a region of heparin larger than a disaccharide repeating unit.

Binding of Heparin to an AIIt Affinity Column

Heparin is an acidic polysaccharide characterized by a disaccharide repeating unit of hexosamine and uronic acid (L-iduronic or D-glucuronic acid) connected through 1-4-linkages. Commercially prepared heparin is heterogeneous due to the varying degree of modification of the functional groups in the disaccharide unit and also to variations in the size of the polysaccharide chains (24). This presented the possibility that the heterogeneous heparin preparation might contain subpopulations of heparin that might not bind to AIIt. We therefore examined the binding of the heparin preparation to an AIIt affinity column. As shown in Fig. 4, ~3% of the heparin applied to the AIIt affinity column flowed through the column, whereas ~85% was recovered when the AIIt column was washed with 150 mM NaCl. Washing the column with 1 M NaCl failed to further elute any heparin.


Fig. 4. Binding of heparin to an AIIt affinity column. Heparin (0.5 mg) was dissolved in buffer C containing 25 mM NaCl and applied to an AIIt affinity column equilibrated against the same buffer. The column was washed with buffer C containing 50 mM NaCl and eluted with buffer C containing 150 mM NaCl. Fractions were analyzed for the presence of heparin with the uronic acid/carbazole reaction (see "Experimental Procedures").
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Characterization of the Binding of AIIt to Heparin

We observed that in the presence of Ca2+, the AIIt-heparin complex could be pelleted by centrifugation at 400,000 × g. This simple pelleting assay allowed an alternative estimation of the affinity and capacity of the binding of AIIt to heparin. As shown in Fig. 5, half-maximal binding of AIIt to heparin required 0.115 ± 0.021 µM heparin (mean ± S.D., n = 3). The asymptotic maximum of the plot was 1.904 µM AIIt, which suggested that ~97% of the total AIIt could be recovered in the pellet. Analysis of the binding data (Fig. 5, inset) estimated that AIIt bound to heparin with an apparent Kd of 32 ± 6 nM (mean ± S.D., n = 3) and a stoichiometry of 11 ± 0.9 mol of AIIt/mol of heparin (mean ± S.D., n = 3).


Fig. 5. Binding isotherm for the titration of AIIt with heparin. AIIt (1.971 µM) was incubated with 25 mM Hepes, pH 7.5, 1 mM DTT, 150 mM NaCl, 1 mM CaCl2, and variable concentrations of heparin at room temperature for 20 min. The reaction mixture was centrifuged at 400,000 × g for 30 min, and the amount of AIIt in the pellet was determined as described under "Experimental Procedures." The line through the points is a nonlinear least-squares curve fit of the data points calculated from computer modeling of data using the logistic equation and the following values: a = 1.904 µM (asymptotic maximum), b = 1.592 (slope parameter), c = 0.115 µM (value at inflection point, A0.5), and d = 0 (asymptotic minimum). Inset, the fractional saturation (mol of AIIt in pellet/total mol of AIIt) is plotted against the total heparin concentration. The line through the points is a nonlinear least-squares curve fit of the data points calculated from computer modeling of data to the quadratic binding equation (Equation 1 under "Experimental Procedures") with the assumption of a single class of heparin-binding sites on AIIt and two fitting parameters, Kd and n, which were allowed to float during the computer iterations. The convergent best fit for these experiments was determined for Kd = 32 nM and n = 0.091 mol of heparin/mol of AIIt.
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We also examined the Ca2+ dependence of AIIt-heparin complex formation. Fig. 6 presents a typical result. Half-maximal formation of the AIIt-heparin complex required pCa2+ = 4.259 ± 0.035. This value (55 µM Ca2+) was similar to the value of 84 µM Ca2+ determined for the half-maximal Ca2+ dependence of the heparin-induced conformational change in AIIt.


Fig. 6. Ca2+ dependence of AIIt-heparin binding. AIIt (1.9 µM) was incubated at room temperature for 20 min with 25 mM Hepes, pH 7.5, 1 mM DTT, 150 mM NaCl, 0.56 µM heparin, and 1 mM Ca2+/nitrilotriacetic acid buffers. The reaction mixture was centrifuged at 400,000 × g for 30 min, and the amount of AIIt in the pellet was determined as described under "Experimental Procedures." Data shown are means ± S.D. (n = 3). The line through the points is a nonlinear least-squares curve fit of the data points calculated from computer modeling of data to the four-parameter logistic equation (see "Experimental Procedures"), the parameters of which were allowed to float during the computer iterations.
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As discussed under "Experimental Procedures," AIIt can be purified by NaCl-dependent elution from a heparin affinity column. In the presence of Ca2+, a 500 mM NaCl wash was sufficient to elute the AIIt from the heparin affinity column. We therefore examined the dependence of the formation of the AIIt-heparin complex on ionic strength. As shown in Fig. 7, half-maximal dissociation of the AIIt-heparin complex required ~380 ± 99 mM NaCl. This result is consistent with the formation of AIIt-heparin complexes under physiological conditions.


Fig. 7. Salt dependence of AIIt-heparin binding. AIIt (1.2 µM) was incubated at room temperature for 20 min with 25 mM Hepes, pH 7.5, 1 mM DTT, 0.56 µM heparin, 1 mM Ca2+, and variable concentrations of NaCl. The reaction mixture was centrifuged at 400,000 × g for 30 min, and the amount of AIIt in the pellet was determined as described under "Experimental Procedures." Data shown are means ± S.D. (n = 3). The line through the points is a nonlinear least-squares curve fit of the data points calculated from computer modeling of data to the four-parameter logistic equation (see "Experimental Procedures") and four fitting parameters, a (asymptotic maximum), b (slope parameter), c (value at inflection point, A0.5), and d (asymptotic minimum), which were allowed to float during the computer iterations.
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Identification of a Heparin-binding Site in AIIt

It has been suggested that many heparin-binding proteins contain consensus sequences for glycosaminoglycan recognition (16). Two consensus sequences, referred to as Cardin-Weintraub sequences, that have been identified are XBBBXXBX and XBBXBX, where B has the probability of a basic residue and X is a hydropathic residue. We have found that the p36 subunit of AIIt contains a Cardin-Weintraub sequence of the XBBBXXBX type. This region of the p36 subunit comprises 306FKKKYGKS314. As shown in Table III, in contrast to many heparin-binding proteins that contain the Cardin-Weintraub consensus sequence, this region of the p36 subunit conforms exactly to the Cardin-Weintraub consensus sequence.

Table III. Comparison of heparin-binding domains of heparin-binding proteins

Sequences for comparisons were obtained from Ref. 16. The Cardin-Weintraub consensus sequence (X-B-B-B-X-B-X) was derived from the usage frequency of basic (B) and hydropathic (X) residues in a data base of heparin-binding proteins (16).

     XBBBXXBX
Annexin II KIRSEFKKKYGKSLYY
Vnb QRFRHRNRKGYRSQRG
ApoB KFIIPSPKRPVKLLSG
bFGF GHFKDPKRLYCKNGGF
NCAM DGGSPIRHYLIKYKAK
PCI GLSEKTLRKWLKMFKK
AT-III KLNCRLYRKANKSSKL
ApoE SHLRKLRKRLLRDADD
 beta -TG PDAPRIKKIVQKKLAG
IGFBP-3 DKKGFYKKKQCRPSKG
Fibrin GHRPLDKKREEAPSLR
FGFR-1 AAPVAHLKKEMK

a Bold type indicates position in the consensus sequence of an invariant residue.
b Vn, vitronectin; bFGF, basic fibroblast growth factor; NCAM, neural cell adhesion molecule; PCI, protein C inhibitor; AT-III, antithrombin III; beta -TG, beta -thromboglobulin; IGFBP-3, insulin-like growth factor-binding protein-3; FGFR-1, fibroblast growth factor receptor-1.

To determine if the Cardin-Weintraub consensus sequence present in the p36 subunit of AIIt participates in heparin binding, we synthesized a peptide to this region of the p36 subunit and examined the potential interaction of the peptide with heparin. As shown in Fig. 8A, the CD spectrum of the peptide comprising 300LKIRSEFKKKYGKSLYY316 demonstrated a significant heparin-dependent conformational change. In contrast, only a slight change in conformation was observed when the consensus sequence peptide was incubated with N-acetylglucosamine. We also examined the interaction of two other peptides with heparin. Although heparin produced a conformational change in a peptide modeled to the actin-bundling region of the p36 subunit (286VLIRIMVSR294) (25), a similar conformational change was also induced by the interaction of this peptide with N-acetylglucosamine (Fig. 8B). Similarly, a peptide modeled to the phosphorylation sites of the p36 subunit (15), but containing an additional two lysines at the N terminus (KK9KLSLEGDHSTPPSAYGSVKAYT30), demonstrated conformational changes in the presence of both heparin and N-acetylglucosamine (Fig. 8C). Therefore, these results indicate that the region of the p36 subunit of AIIt that contains the Cardin-Weintraub consensus sequence probably participates in heparin binding.


Fig. 8. Heparin-induced conformational change in a peptide to the heparin-binding domain of AIIt. Wavelength scans were conducted at 20 °C in buffer A alone (solid line) or containing 0.56 µM heparin (dotted line) or 5 µM N-acetylglucosamine (dashed line). Peptides were added at a concentration of 100 µg/ml. A, heparin-binding site consensus peptide (300LKIRSEFKKKYGKSLYY316); B, actin-bundling site consensus peptide (286VLIRIMVSR294); C, phosphorylation site consensus peptide (KK9KLSLEGDHSTPPSAYGSVKAYT30).
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Heparin-dependent Conformational Changes in Other Annexins

Since the p36 subunit of AIIt contains the heparin-binding domain, it was reasonable to suspect that both AIIt and the isolated p36 subunit (annexin II) would undergo conformational changes upon heparin binding. This was not the case, however; and the p36 subunit did not undergo a significant conformational change in the presence of heparin (Table IV). Furthermore, of the six monomeric annexins examined, none demonstrated a specific conformational change in the presence of heparin. This was surprising because all these annexins were purified by heparin affinity chromatography. This observation therefore suggested that the specific heparin-induced conformational change in AIIt did not occur in other annexins.

Table IV. Heparin-induced conformational changes in several annexins

Results are expressed as means ± S.D. (n = 5). Percent change at Delta varepsilon 222 nm = Delta varepsilon 222 nm(AIIt) - Delta varepsilon 222 nm(AIIt + ligand)/Delta varepsilon 222 nm(AIIt) × 100. Annexins (0.2 mg/ml) were incubated in buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2 mM DTT, 1 mM CaCl2, and 5.0 or 0.5 µM heparin (AIIt). Annexins used in this study were isolated as described (17) and further purified by Ca2+-dependent binding to a heparin affinity column as described (15).

Annexin Heparin binding Change at Delta varepsilon 222 nm
N-Acetylglucosamine Heparin

%
Annexin I + 6.2  ± 1.7 1.0  ± 1.0
Annexin II + 6.2  ± 1.7 6.4  ± 3.8
Annexin II tetramer + 6.2  ± 1.7 41.0  ± 3.4
Annexin III + 4.5  ± 0.7 5.3  ± 2.1
Annexin IV + 10.0  ± 1.4 3.7  ± 2.6
Annexin V + 5.0  ± 0.5 3.6  ± 2.1
Annexin VI + 21.5  ± 9.1 17.8  ± 4.3
p11  - NDa 1.0  ± 0.1

a ND, not determined.

Affect of Heparin on Membrane-bound AIIt

AIIt is a phospholipid-binding protein that has been shown to be located on both the intracellular and extracellular surfaces of the plasma membrane (9). Since the binding of heparin to AIIt has such a profound effect on the conformation of the protein, it was important to investigate the possibility that the interaction of AIIt with heparin might disrupt the binding of AIIt with membranes. However, as shown in Fig. 9, heparin did not block the interaction of AIIt with phospholipid liposomes.


Fig. 9. Effect of heparin on the binding of AIIt to phospholipid vesicles. Phospholipid liposomes consisting of phosphatidylserine, phosphatidylethanolamine, and cholesterol were prepared as described under "Experimental Procedures" and then incubated at 20 °C with 30 mM Hepes, pH 7.5, 50 mM KCl, 2.0 mM MgCl2, 0.2 mM CaCl2, 1.0 µM heparin, and 0.56 µM AIIt. After 30 min, the reaction was centrifuged at 14,000 × g for 10 min, and the pellet was analyzed by SDS-polyacrylamide gel electrophoresis. Lane 1, AIIt standard; lane 2, liposomes and heparin incubated for 10 min before addition of Ca2+ and AIIt; lane 3, AIIt incubated with heparin and Ca2+ before addition to the liposomes; lane MWM, molecular weight markers.
[View Larger Version of this Image (38K GIF file)]


DISCUSSION

Previous work from our laboratory established that AIIt is a Ca2+-dependent heparin-binding protein (15). The interaction of AIIt with heparin was also shown to be inhibited by tyrosine phosphorylation of AIIt (15). Since the role that heparin binding plays in the structure or function of AIIt is unknown, the current study was aimed at the characterization of the interaction of heparin with AIIt. Analysis of the CD spectra of AIIt showed that the binding of heparin to AIIt resulted in a profound change in the conformation of AIIt (Fig. 1 and Table I). We also found that in the absence of Ca2+, a small change in the conformation of AIIt occurred upon heparin binding.

Animal carbohydrate-binding proteins can be broadly classified into seven major groups. These include the C-type or Ca2+-dependent lectins, the S-type or Gal-binding galectins, P-type mannose 6-phosphate receptors, the I-type lectins, the pentraxins, the hyaluronan-binding proteins, and the heparin-binding proteins (26). The C-type lectins bind several carbohydrates including mannose and galactose and require Ca2+ to form a coordination bond with the sugar ligand. The galectins bind only beta -galactoside, whereas the P-type proteins bind only mannose 6-phosphate. The I-type lectins bind only sialic acid, whereas the pentraxins bind several carbohydrates such as heparin and sialic acid as well as phosphorylcholine. The hyaluronan-binding proteins bind only hyaluronan. The heparin-binding proteins generally demonstrate Ca2+-independent binding of both heparin and heparan sulfate. Since AIIt binds heparin (Table II) but not phosphorylcholine (15), AIIt is most likely a member of the heparin-binding family of proteins. However, AIIt appears to be unique among heparin-binding protein members in that the binding of AIIt to heparin is stimulated by Ca2+. Furthermore, AIIt appears to be a unique member of the heparin-binding proteins because AIIt can discriminate between heparin and heparan sulfate ligands.

Several consensus sequences have been identified among members of the heparin-binding family of proteins. For example, the heparin-binding sequence of the C-terminal region of fibronectin has been identified as WQPPRARI (27). In contrast, a region of thrombospondin containing the sequence WSPW has been identified as the heparin-binding region of the protein (28, 29). Analysis of several heparin-binding proteins has suggested the potential existence of two consensus sequences referred to as Cardin-Weintraub heparin-binding consensus sequences (16, 30). Site-directed mutagenesis and binding studies with synthetic or isolated peptides from several of these proteins have confirmed that this consensus region is often involved in binding heparin (30-34, 36). Other studies have shown that the orientation of the Cardin-Weintraub consensus sequence within the protein is critical and may determine if the consensus sequence participates in heparin binding (33, 37). As shown in Table III, the p36 subunit of AIIt contains a Cardin-Weintraub heparin-binding consensus sequence. Furthermore, a peptide to this region of the p36 subunit of AIIt (300LKIRSEFKKKYGKSLYY316) undergoes a conformational change upon heparin binding (Fig. 8). These results therefore suggest that residues 300-316 of the p36 subunit of AIIt are involved in heparin binding.

Although the monomeric annexins I-VI bind to a heparin affinity column in the presence of Ca2+, a heparin-dependent conformational change was not observed for these proteins (Table IV). The p36 subunit of AIIt can exist as a monomer or as a heterotetramer. Heterotetrameric AIIt is composed of two p36 subunits and two p11 subunits. Considering that the p36 subunit (annexin II) binds to a heparin affinity column and contains the Cardin-Weintraub consensus sequence, it was surprising that the p36 subunit did not undergo a conformational change upon heparin binding. This suggests that the heparin-binding site of the p36 subunit and other monomeric annexins is preformed and does not require the recruitment of residues from other regions of the protein. This is consistent with the observation that carbohydrate-binding proteins undergo few if any changes in conformation upon carbohydrate binding (26). The p11 subunit of AIIt does not bind heparin and does not contain any heparin-binding consensus sequences. It is therefore unlikely that the heparin-dependent conformational change in AIIt was due to the coordinated binding of heparin by both the p36 and p11 subunits of AIIt. We cannot, however, rule out the possibility that the binding of the p36 subunit to the p11 subunit induces a conformational change in the p11 subunit that results in exposure of a novel heparin-binding domain. The simplest explanation for the large conformational change in AIIt upon heparin binding is that the orientation of the p36 subunits in AIIt is not optimal for heparin binding. Therefore, the binding of heparin to AIIt results in the realignment of the p36 subunits.

Of particular interest was our observation that the Ca2+-dependent conformational change in AIIt was induced by heparin, but not by other negatively charged glycosaminoglycans such as heparan sulfate, chondroitin sulfate, and dextran sulfate. Heparan sulfates are structurally related glycosaminoglycans that are found on cell surfaces and in the extracellular matrix, where they form the chains of heparan sulfate proteoglycans and bear only short stretches of dense sulfation. In contrast, heparin is the glycosaminoglycan that is secreted by mast cells and other hematopoietic cells and therefore may serve as a signaling molecule (38, 39). To date, a heparin-binding protein capable of distinguishing between heparin and heparan sulfate has not been described. Recently, annexin IV was shown to bind heparin, but the binding of heparin to this protein was inhibited by a variety of carbohydrates including glucose, N-acetylneuraminic acid, heparan sulfate, and chondroitin sulfate (40). In contrast, we have found that heparan sulfate or other glycosaminoglycans do not induce a conformational change in AIIt (Table II). Furthermore, high concentrations of heparan sulfate (50 µM) do not inhibit the conformational change in AIIt elicited by 0.5 µM heparin (Table II), therefore suggesting that heparan sulfate does not bind to AIIt. However, considering the heterogeneity of the cell-surface heparan sulfate proteoglycan (38), it is possible that AIIt may interact with other heparan sulfate proteoglycans.

We also observed that AIIt formed a large complex with heparin and that this complex was pelleted by centrifugation at 400,000 × g. Analysis of the binding isotherm suggested that AIIt bound heparin with an apparent Kd of 32 ± 6 nM (mean ± S.D., n = 3) and a stoichiometry of 11 ± 0.9 mol of AIIt/mol of heparin (mean ± S.D., n = 3). This Kd for the binding of heparin to AIIt is slightly lower than the Kd reported for the binding of heparin to heparinase (60 nM), acidic fibroblast growth factor (50-140 nM), or fibronectin (34 nM), but higher than that reported for the binding of heparin to basic fibroblast growth factor (2.2 nM) or antithrombin III (11 nM) (32, 41, 42). AIIt does not bind to disaccharides of heparin, but does bind to 3-kDa heparin that contains ~10 monosaccharides (Table II). The binding of ~11 molecules of AIIt to a single 17-kDa heparin strand that contains ~50 monosaccharide units (Fig. 5) suggests that AIIt requires ~4-5 monosaccharide units for binding.

The physiological significance of the binding of heparin to AIIt is unclear. Heparin has been shown to interact with enzymes of the clotting and fibrinolysis systems (24), protect proteins from inactivation, play an essential role in the interaction of growth factors with their receptors, directly activate growth factor receptors, and serve as an essential cofactor in cell-cell recognition and cell-matrix adhesion processes (27, 35, 43-47). AIIt is the major cellular receptor for tenascin-C and plasminogen (8, 14). It is therefore possible that heparin might be involved in the regulation of the interaction of AIIt with these ligands.


FOOTNOTES

*   This work was supported by a grant from the Medical Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Medical Biochemistry, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. N. W., Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-3022; Fax: 403-283-4841; E-mail: waisman{at}acs.ucalgary.ca.
1   The abbreviations used are: AIIt, annexin II tetramer; DTT, dithiothreitol.

ACKNOWLEDGEMENTS

We thank Dr. Robert W. Woody for the generous gift of the SELCON computer program and Dr. Narasimha Sreerama (Colorado State University) for helpful discussions concerning interpretation of CD data using the SELCON computer program.


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