Heparin-Like Properties of Sulfated Alginates with Defined Sequences and Sulfation Degrees

Sulfated glycosaminoglycans have a vast range of protein interactions relevant to the development of new biomaterials and pharmaceuticals, but their characterization and application is complicated mainly due to a high structural variability and the relative difficulty to isolate large quantities of structurally homogeneous samples. Functional and versatile analogues of heparin/heparan sulfate can potentially be created from sulfated alginates, which offer structure customizability through targeted enzymatic epimerization and precise tuning of the sulfation degree. Alginates are linear polysaccharides consisting of β-d-mannuronic acid (M) and α-l-guluronic acid (G), derived from brown algae and certain bacteria. The M/G ratio and distribution of blocks are critical parameters for the physical properties of alginates and can be modified in vitro using mannuronic-C5-epimerases to introduce sequence patterns not found in nature. Alginates with homogeneous sequences (poly-M, poly-MG, and poly-G) and similar molecular weights were chemically sulfated and structurally characterized by the use of NMR and elemental analysis. These sulfated alginates were shown to bind and displace HGF from the surface of myeloma cells in a manner similar to heparin. We observed dependence on the sulfation degree (DS) as well as variation in efficacy based on the alginate monosaccharide sequence, relating to relative flexibility and charge density in the polysaccharide chains. Co-incubation with human plasma showed complement compatibility of the alginates and lowering of soluble terminal complement complex levels by sulfated alginates. The sulfated polyalternating (poly-MG) alginate proved to be the most reproducible in terms of precise sulfation degrees and showed the greatest relative degree of complement inhibition and HGF interaction, maintaining high activity at low DS values

Comparison of mean force (a) and force variance (b) versus force loading rate for the alginate-epimerase pairwise interactions.

<p>Comparison of mean force (a) and force variance (b) versus force loading rate for the alginate-epimerase pairwise interactions.</p

The epimerization process, epimerases and prevailing alginate residue sequences of the various epimerase substrates and resulting products.

<p><i>(a)</i> The mannuronan C-5 epimerases possess the ability to epimerize β-d-mannuronate residues (M) to its epimer form; α-l-guluronate residue (G). <i>(b)</i> The naturally occurring epimerases are known to form long stretches of systematically epimerized alginates. While AlgE4 can produce polyalternating structures from polymannuronic alginates, AlgE6 can epimerize both polymannuronic and polyalternating structures to form polyguluronic alginates.</p

Average values of energy landscape parameters obtained for the constant-force rupture-rate analysis.

<p>The estimates were obtained using multiple regressions of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.e001" target="_blank">Eq 1</a> (<i>v</i> = 1/2) after conversion to constant-force rupture-rate (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.e006" target="_blank">Eq 6</a>) to mean rates with corresponding mean forces. The number of force loading rate intervals employed and procedure are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.t002" target="_blank">Table 2</a>. The resulting master curves for the poly-M and the poly-MG complexes are shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.g004" target="_blank">4B, 4D, 4F</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.g005" target="_blank">5B, 5D and 5F</a>, respectively.</p

Gallery of rupture events of the various AlgE-poly-M <i>(a</i>,<i>b</i>,<i>c)</i> and AlgE-poly-MG <i>(d</i>,<i>e</i>,<i>f)</i> molecular pairs.

<p>The red curves are recorded with AFM, while the blue curves represent forced ruptures recorded with OT. The displacement scale corresponds to the z-piezo translation distance, and bead separation for the data collected employing AFM and OT, respectively. Some of the AFM curves display interactions at short displacement distances (~ 0–50 nm) that may reflect non-specific interaction between the AFM-tip and the mica slide (e.g. red curves in a,b, and c). Bead-bead interactions are in some recordings present at the adhesion region of the OT experiments, such as the ones in <i>(c)</i> and <i>(d)</i>. Interactions in the adhesion region either due to AFM-tip mica slide contact in the AFM experiments or due to strong bead-bead interactions in the OT were not included in the analysis as single-bond rupture events.</p

Interactions of AlgE–poly-MG complexes determined by direct unbinding.

<p><i>(a</i>, <i>c</i>, <i>e)</i> Constant-speed rupture-force representation of the interactions i.e. mean forces versus mean loading rates (symbols) and fits of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.e003" target="_blank">Eq 3</a> with <i>ν</i> = 1/2(lines) to the experimental data. The estimates of the molecular interaction parameters are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.t002" target="_blank">Table 2</a>. The light grey data points are forced ruptures recorded with the OT, while the dark grey data points are forced ruptures recorded with the AFM. By combining the two techniques we can access a larger range of loading rates as can be seen in <i>(a</i>, <i>c</i>, <i>e)</i>. The inserts show two selected histograms of the unbinding forces, within the low and high loading rate regions, respectively. The histogram plots, one for each energy barrier, exhibit the distribution of unbinding forces for which <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.e002" target="_blank">Eq 2</a> with <i>ν</i> = 1/2(lines) was fitted. <i>(b</i>, <i>d</i>, <i>f)</i> Constant-force rupture-rate representation with <i>ν</i> = 1/2 of the interactions for the poly-M-complexes. The data are presented as mean rates versus mean forces obtained analytically from the constant-speed rupture-force experiments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.e006" target="_blank">Eq 6</a>). The resulting estimates of the molecular interaction parameters are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.t003" target="_blank">Table 3</a>.</p

Interactions of AlgE–poly-M complexes determined by direct unbinding.

<p><i>(a</i>, <i>c</i>, <i>e)</i> Constant-speed rupture-force representation of the interactions i.e. mean forces versus mean loading rates (symbols) and fits of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.e003" target="_blank">Eq 3</a> using <i>ν</i> = 1/2 (lines) to the experimental data. The estimates of the molecular interaction parameters are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.t002" target="_blank">Table 2</a>. The light grey data points are forced ruptures recorded with the OT, while the dark grey data points are forced ruptures recorded with the AFM. By combining the two techniques we can access a larger range of loading rates as can be seen in <i>(a</i>, <i>c</i>, <i>e)</i>. The inserts show two selected histograms of the unbinding forces, within the low and high loading rate regions, respectively, indicate typical distributions of the data within the loading rate intervals. The distribution of unbinding force P(f) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.e002" target="_blank">Eq 2</a>) for <i>ν</i> = 1/2 (lines) is included on top of the histograms. <i>(b</i>, <i>d</i>, <i>f)</i> Constant-force rupture-rate representation with fits using <i>ν</i> = 1/2 of the interactions for the poly-M-complexes. The data are presented as mean rates versus mean forces obtained analytically from the constant-speed rupture-force experiments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.e006" target="_blank">Eq 6</a>). The resulting estimates of the molecular interaction parameters are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.t003" target="_blank">Table 3</a>.</p

Schematic illustration of reconstructed energy landscapes for the AlgE4-poly-M and AlgE4 –poly-MG pairwise interactions based on the obtained parameters (Table 1).

<p>Schematic illustration of reconstructed energy landscapes for the AlgE4-poly-M and AlgE4 –poly-MG pairwise interactions based on the obtained parameters (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.t001" target="_blank">Table 1</a>).</p

Averages of energy landscape parameters for epimerase–alginate interactions.

<p>The parameters <i>x</i>_<i>β</i>, <i>τ</i>^<i>0</i> and <i>ΔG</i>^<i>#</i> were estimated using the constant-speed rupture-force analysis after multiple regressions of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.e003" target="_blank">Eq 3</a> with <i>v</i> = 1/2 to observed mean forces for all force loading rate intervals. The number of force loading rate intervals was varied from 20 and up to 30 yielding 20–30 estimates of the most probable unbinding force at the corresponding mean loading rates. The number of mean force estimates was about the same for the inner and outer barriers. The resulting master curves for the poly-M and the poly-MG complexes are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.g004" target="_blank">Fig 4A, 4C and 4E</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141237#pone.0141237.g005" target="_blank">Fig 5A, 5C and 5E</a>, respectively.</p