Single Molecule Force Spectroscopy at Cell Surfaces to Study Physical Properties of Heparan Sulfate Chains and Protein‐Heparan Sulfate Interactions

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ABSTRACTHeparins and heparan sulfates (HS) are highly sulfated glycosaminoglycans that contribute to various physiological and pathophysiological processes such as coagulation, signal transduction, and extracellular matrix organization. In addition to its use as an anticoagulant, HS is therefore considered a promising compound for various biomedical applications such as tissue engineering. The wide range of different biological activities is facilitated by the large structural diversity of HS. At the molecular level, the biological properties of HS are linked to the interaction with HS‐binding proteins, which is controlled by a complex code of sulfation. A comprehensive understanding of the HS structure and associated biological properties is essential for the effective exploitation of HS in biomedical applications. In the present study, we used super‐resolution microscopy in combination with single‐molecule force spectroscopy (SMFS) to characterize HS on cell surfaces. Following our previous studies, we used melanoma cells as a model system. We found punctate accumulations of HS on the surface of melanoma cells in colocalisation with nanometric cellular protrusions. These protrusions were less prominent on surfaces of HS‐deficient melanoma cells. Applying SMFS experiments, we characterized the apparent length and flexibility of single HS chains and discussed the impact of HS‐binding proteins on the structural configuration of HS. Melanoma cells with an altered sulfation after knockout of the HS 6‐O sulfotransferase 1, were used as reference. Our data analysis suggests that the apparent median length of single HS chains was 137 nm, whereas chain segments of approximately 50 nm were involved in HS‐protein interactions. In conclusion, we have successfully applied super‐resolution microscopy and SMFS to study the distribution and structural configuration of HS on the surface of mammalian cells. In the future, genetic engineering of mammalian cells may allow the production of defined HS tailored for specific biomedical applications, such as anticoagulation or tissue regeneration.