Metal-Mediated Il-8 Binding to Heparan Sulfate Evaluated by Electrochemical Impedance Spectroscopy

Abstract

Heparan sulfate (HS) interactions with interleukin 8 (IL-8) are crucial for immune system response. The structural features of the HS and the environmental entities, such as metal ions, can regulate these interactions. However, it is challenging to evaluate the effect of each parameter on the interactions because of low accessibility to well-defined saccharides and the lack of characteristic features to be determined by analytical tools. We evaluated the effect of the HS structural features on IL-8 binding affinity utilizing electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS). We showed that the metal ions Ca²⁺ and Mg²⁺ dissimilarly mediate the interactions of HS and IL-8 in structure-dependent manner of the HS. We showed that in all glycans, a positive synergistic effect on IL-8 binding was observed. For several glycans, the presence of ions resulted in a dramatic increase in the affinity to IL-8, while for other glycans, a milder effect was observed. This demonstrated that both structural motifs and environmental features are crucial for maintaining the interactions between the HS and IL-8.

Keywords: EIS; X‐ray photoelectron spectroscopy; heparan sulfate; interleukin 8; metal ions.

Scheme 1

The effect of the glycosaminoglycans' structural features on metal ions and IL‐8‐related interactions independently and synergistically. Left: the effect of sulfation on metal ion‐mediated IL‐8 binding. Right: the combined effect of sulfation pattern and uronic acid stereochemistry on metal ion binding preference. Bottom: the combined effect of sulfation pattern and uronic acid stereochemistry on metal ion‐mediated IL‐8 binding presented in this work.

Figure 1

Structure of the HS tetrasaccharide used in the study where G‐6S comprises Glucuronic acid (GlcA) and 6S N‐acetyl glucosamine (GlcNAc) a), I‐6S comprises iduronic acid (IdoA) and 6S GlcNAc b), G‐3S comprises GlcA and 3S GlcNAc, and their electrografting process to give GC modified with the tetrasaccharides G‐6S‐GC a), I‐6S‐GC b), and G‐3S‐GC c).

Figure 2

The effect of metal ions, proteins, and their combination on the tetrasaccharide monolayer is evaluated by EIS via three routes independently. a) Metal ion‐free G‐6S‐GC exposure to IL‐8 (route 1), Mg²⁺ ion‐mediated exposure to IL‐8 (route 2), and Ca²⁺ ion‐mediated exposure to IL‐8 (route 3). b) Nyquist plots represent the EIS response recorded for each route. The green curve represents the electrode before the exposure to ions or IL‐8, red after exposure to Mg²⁺, blue after exposure to Ca²⁺, and purple after exposure to IL‐8.

Figure 3

a) The individual and synergistic effect of metal ions and protein on the tetrasaccharides. Normalized R_CT for the response of the tetrasaccharides to metal ions and IL‐8 in the presence or absence of metal ions. The error bars are based on the standard deviation of five independent electrodes. b) The saccharide‐specific synergistic effect. The relative increase represents the difference between exposure to metal ions alone and the combination of IL‐8 and metal ions. The relative value was calculated by dividing the response after exposure to the IL‐8‐metal ion combination by the response to metal ions alone. The dashed line represents the threshold for response significance.

Figure 4

XPS analyses of G‐6S‐GC after exposure to IL‐8 in the presence of Ca²⁺ (blue Route 3 step ii), or Mg²⁺ (red Route 2 step ii), or absence of metal ions (purple Route 1 step i) and without exposure to IL‐8 (black), where a) is the spectra of Ca2p, b) is the spectra of Mg2p, c) is the spectra of N1s, and d) is the spectra of S2p, where the insert is the zoom on the disulfide bonds.

Figure 5

Schematic representation of the IL‐8 interaction with the HS, with the factors that affect the binding affinity. Addition of Mg²⁺ to the system increases the interaction, while addition of Ca²⁺ has a stronger effect. Change in sulfation pattern from 6‐O to 3‐O or glucuronic acid to iduronic acid decreases the interactions where the effect of sulfation pattern is higher.