High-resolution probing heparan sulfate-antithrombin interaction on a single endothelial cell surface: single-molecule AFM studies

Abstract

Heparan sulfate (HS) plays diverse functions in multiple biological processes by interacting with a wide range of important protein ligands, such as the key anticoagulant factor, antithrombin (AT). The specific interaction of HS with a protein ligand is determined mainly by the sulfation patterns on the HS chain. Here, we reported the probing single-molecule interaction of AT and HS (both wild type and mutated) expressed on the endothelial cell surface under near-physiological conditions by atomic force microscopy (AFM). Functional AFM imaging revealed the uneven distribution of HS on the endothelial cell surface though they are highly expressed. Force spectroscopy measurements using an AT-functionalized AFM tip revealed that AT interacts with endothelial HS on the cell surface through multiple binding sites. The interaction essentially requires HS to be N-, 2-O- and/or 6-O-sulfated. This work provides a new tool to probe the HS-protein ligand interaction at a single-molecular level on the cell surface to elucidate the functional roles of HS.

Fig. 1

AFM Images of Ndst1^f/f cells. (A) Topography images of Ndst1^f/f cells. (B) Enlarged area marked with black square in (A). (C) and (D) are corresponding amplitude images of (A) and (B). Circle and straight line in (D) indicate the features of granule and cytoskeleton on cell surface, respectively. The images were obtained by Top MAC mode with a magnetic AFM tip.

Fig. 2

Functional imaging of endothelial cell surface with AT-modified AFM tip. (A) Schematics for cell lines with different levels of HS expression (Ext1^f/f, Ext1^−/−, Ndst1^f/f, and Ndst1^−/−) as well as an AT-functionalized AFM tip. (B) Topographical image for part of a Ndst1^f/f cell surface as an example. (C) Corresponding recognition image of (B).

Fig. 3

Force spectroscopy analysis on Ext1^f/f and Ext1^−/− cell surfaces. (A) Representative force-distance curves between an AT-modified tip and HS interaction on Ext1^f/f (wildtype) and Ext1^−/− endothelial cell surfaces. (B) Comparison of rupture forces between Ext1^f/f and Ext1^−/− cells. The loading rate is 39 nN/s, and more than 1000 rupture forces were measured from the force-distance curve for each genotypic cell.

Fig. 4

Force spectroscopy analysis on Ndst1^f/f and Ndst1^−/− endothelial cell surfaces. (A) Representative force-distance curves between an AT-modified tip and HS interaction on Ndst1^f/f and Ndst1^−/− endothelial cell surfaces. (B) Comparison of rupture forces between Ndst1^f/f and Ndst1^−/− cells. (C) Schematic for treatment of Ndst1^f/f cell with heparinase, and subsequent rupture force determination. (D) Distribution of rupture force after heparinase treatment. The histogram indicates the force decreased to 25.77 ± 8.30 pN. The loading rate was 39 nN/s and more than 1000 rupture forces were measured from the force-distance curve for each genotypic cell.