SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2.

We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ACE2-binding site. Both ACE2 and heparin can bind independently to spike protein in vitro, and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities.

Proteomics-based screening of the endothelial heparan sulfate interactome reveals that C-type lectin 14a (CLEC14A) is a heparin-binding protein.

Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.

Alteration of Neuropilin-1 and Heparan Sulfate Interaction Impairs Murine B16 Tumor Growth.

Neuropilin-1 acts as a coreceptor with vascular endothelial growth factor receptors to facilitate binding of its ligand, vascular endothelial growth factor. Neuropilin-1 also binds to heparan sulfate, but the functional significance of this interaction has not been established. A combinatorial library screening using heparin oligosaccharides followed by molecular dynamics simulations of a heparin tetradecasaccharide suggested a highly conserved binding site composed of amino acid residues extending across the b1 and b2 domains of murine neuropilin-1. Mutagenesis studies established the importance of arginine513 and lysine514 for binding of heparin to a recombinant form of Nrp1 composed of the a1, a2, b1, and b2 domains. Recombinant Nrp1 protein bearing R513A,K514A mutations showed a significant loss of heparin-binding, heparin-induced dimerization, and heparin-dependent thermal stabilization. Isothermal calorimetry experiments suggested a 1:2 complex of heparin tetradecasaccharide:Nrp1. To study the impact of altered heparin binding in vivo, a mutant allele of Nrp1 bearing the R513A,K514A mutations was created in mice (Nrp1D) and crossbred to Nrp1+/- mice to examine the impact of altered heparan sulfate binding. Analysis of tumor formation showed variable effects on tumor growth in Nrp1D/D mice, resulting in a frank reduction in tumor growth in Nrp1D/- mice. Expression of mutant Nrp1D protein was normal in tissues, suggesting that the reduction in tumor growth was due to the altered binding of heparin/heparan sulfate to neuropilin-1. These findings suggest that the interaction of neuropilin-1 with heparan sulfate modulates its stability and its role in tumor formation and growth.

The human milk oligosaccharide 3'sialyllactose reduces low-grade inflammation and atherosclerosis development in mice.

Macrophages contribute to the induction and resolution of inflammation and play a central role in chronic low-grade inflammation in cardiovascular diseases caused by atherosclerosis. Human milk oligosaccharides (HMOs) are complex unconjugated glycans unique to human milk that benefit infant health and act as innate immune modulators. Here, we identify the HMO 3'sialyllactose (3'SL) as a natural inhibitor of Toll-Like Receptor (TLR) 4-induced low-grade inflammation in macrophages and endothelium. Transcriptome analysis in macrophages revealed that 3'SL attenuates mRNA levels of a selected set of inflammatory genes and promotes the activity of Liver X Receptor (LXR) and Sterol Regulatory Element-binding Protein-1 (SREBP). These acute anti-inflammatory effects of 3'SL were associated with reduced histone H3K27 acetylation at a subset of lipopolysaccharide (LPS)-inducible enhancers distinguished by preferential enrichment for CCCTC-binding factor (CTCF), Interferon Regulatory Factor 2 (IRF2), B-cell lymphoma 6 (BCL6), and other transcription factor recognition motifs. In a murine atherosclerosis model, both subcutaneous and oral administration of 3'SL significantly reduced atherosclerosis development and the associated inflammation. This study provides evidence that 3'SL attenuates inflammation by a transcriptional mechanism to reduce atherosclerosis development in the context of cardiovascular disease.

Vascular Proteome Responses Precede Organ Dysfunction in a Murine Model of Staphylococcus aureus Bacteremia.

Vascular dysfunction and organ failure are two distinct, albeit highly interconnected, clinical outcomes linked to morbidity and mortality in human sepsis. The mechanisms driving vascular and parenchymal damage are dynamic and display significant molecular cross talk between organs and tissues. Therefore, assessing their individual contribution to disease progression is technically challenging. Here, we hypothesize that dysregulated vascular responses predispose the organism to organ failure. To address this hypothesis, we have evaluated four major organs in a murine model of Staphylococcus aureus sepsis by combining in vivo labeling of the endothelial cell surface proteome, data-independent acquisition (DIA) mass spectrometry, and an integrative computational pipeline. The data reveal, with unprecedented depth and throughput, that a septic insult evokes organ-specific proteome responses that are highly compartmentalized, synchronously coordinated, and significantly correlated with the progression of the disease. These responses include abundant vascular shedding, dysregulation of the intrinsic pathway of coagulation, compartmentalization of the acute phase response, and abundant upregulation of glycocalyx components. Vascular cell surface proteome changes were also found to precede bacterial invasion and leukocyte infiltration into the organs, as well as to precede changes in various well-established cellular and biochemical correlates of systemic coagulopathy and tissue dysfunction. Importantly, our data suggest a potential role for the vascular proteome as a determinant of the susceptibility of the organs to undergo failure during sepsis. IMPORTANCE Sepsis is a life-threatening response to infection that results in immune dysregulation, vascular dysfunction, and organ failure. New methods are needed for the identification of diagnostic and therapeutic targets. Here, we took a systems-wide approach using data-independent acquisition (DIA) mass spectrometry to track the progression of bacterial sepsis in the vasculature leading to organ failure. Using a murine model of S. aureus sepsis, we were able to quantify thousands of proteins across the plasma and parenchymal and vascular compartments of multiple organs in a time-resolved fashion. We showcase the profound proteome remodeling triggered by sepsis over time and across these compartments. Importantly, many vascular proteome alterations precede changes in traditional correlates of organ dysfunction, opening a molecular window for the discovery of early markers of sepsis progression.

Dissecting structure-function of 3-O-sulfated heparin and engineered heparan sulfates.

Heparan sulfate (HS) polysaccharides are master regulators of diverse biological processes via sulfated motifs that can recruit specific proteins. 3-O-sulfation of HS/heparin is crucial for anticoagulant activity, but despite emerging evidence for roles in many other functions, a lack of tools for deciphering structure-function relationships has hampered advances. Here, we describe an approach integrating synthesis of 3-O-sulfated standards, comprehensive HS disaccharide profiling, and cell engineering to address this deficiency. Its application revealed previously unseen differences in 3-O-sulfated profiles of clinical heparins and 3-O-sulfotransferase (HS3ST)­specific variations in cell surface HS profiles. The latter correlated with functional differences in anticoagulant activity and binding to platelet factor 4 (PF4), which underlies heparin-induced thrombocytopenia, a known side effect of heparin. Unexpectedly, cells expressing the HS3ST4 isoenzyme generated HS with potent anticoagulant activity but weak PF4 binding. The data provide new insights into 3-O-sulfate structure-function and demonstrate proof of concept for tailored cell-based synthesis of next-generation heparins.

SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2.

We show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities.