Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing an unprecedented global pandemic demanding the urgent development of therapeutic strategies. Microarray binding experiments, using an extensive heparan sulfate (HS) oligosaccharide library, showed that the receptor binding domain (RBD) of the spike of SARS-CoV-2 can bind HS in a length- and sequence-dependent manner. A hexasaccharide composed of IdoA2S-GlcNS6S repeating units was identified as the minimal binding epitope. Surface plasmon resonance showed the SARS-CoV-2 spike protein binds with a much higher affinity to heparin (K_D = 55 nM) compared to the RBD (K_D = 1 μM) alone. It was also found that heparin does not interfere in angiotensin-converting enzyme 2 (ACE2) binding or proteolytic processing of the spike. However, exogenous administered heparin or a highly sulfated HS oligosaccharide inhibited RBD binding to cells. Furthermore, an enzymatic removal of HS proteoglycan from physiological relevant tissue resulted in a loss of RBD binding. The data support a model in which HS functions as the point of initial attachment allowing the virus to travel through the glycocalyx by low-affinity high-avidity interactions to reach the cell membrane, where it can engage with ACE2 for cell entry. Microarray binding experiments showed that ACE2 and HS can simultaneously engage with the RBD, and it is likely no dissociation between HS and RBD is required for binding to ACE2. The results highlight the potential of using HS oligosaccharides as a starting material for therapeutic agent development.

Significance: Although heparan sulfate (HS) modified by a 3- O -sulfate (3-OS) has been implicated in many biological processes, there is only a rudimentary understanding of ligand requirements of HS-binding proteins requiring this type of modification. We developed a modular synthetic approach that provided a structurally diverse collection of HS oligosaccharides with and without 3-OS. The compounds were printed as a glycan microarray to determine ligand requirements of HS-binding proteins, which revealed specific HS sequons for binding, and led to the discovery of proteins needing 3-OS for binding. The compounds made it possible to determine receptor requirements of herpes simplex virus, and substrate specificities of heparin lyases. The platform provides an important means to examine the biology of 3-OS−modified HS.

This study provides proof of concept for cell-based heparin without heparin-induced thrombocytopenia side effects.

Despite evident regulatory roles of heparan sulfate (HS) saccharides in numerous biological processes, definitive information on the bioactive sequences of these polymers is lacking, with only a handful of natural structures sequenced to date. Here, we develop a “Shotgun” Ion Mobility Mass Spectrometry Sequencing (SIMMS²) method in which intact HS saccharides are dissociated in an ion mobility mass spectrometer and collision cross section values of fragments measured. Matching of data for intact and fragment ions against known values for 36 fully defined HS saccharide structures (from di- to decasaccharides) permits unambiguous sequence determination of validated standards and unknown natural saccharides, notably including variants with 3O-sulfate groups. SIMMS² analysis of two fibroblast growth factor-inhibiting hexasaccharides identified from a HS oligosaccharide library screen demonstrates that the approach allows elucidation of structure-activity relationships. SIMMS² thus overcomes the bottleneck for decoding the informational content of functional HS motifs which is crucial for their future biomedical exploitation.