Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and continues to spread around the globe at an unprecedented rate. To date, no effective therapeutic is available to fight its associated disease, COVID-19. Our discovery of a novel insertion of glycosaminoglycan (GAG)-binding motif at S1/S2 proteolytic cleavage site (681-686 (PRRARS)) and two other GAG-binding-like motifs within SARS-CoV-2 spike glycoprotein (SGP) led us to hypothesize that host cell surface GAGs may interact SARS-CoV-2 SGPs to facilitate host cell entry. Using a surface plasmon resonance direct binding assay, we found that both monomeric and trimeric SARS-CoV-2 SGP bind more tightly to immobilized heparin (K D ¼ 40 pM and 73 pM, respectively) than the SARS-CoV and MERS-CoV SGPs (500 nM and 1 nM, respectively). In competitive binding studies, the IC 50 of heparin, tri-sulfated non-anticoagulant heparan sulfate, and non-anticoagulant low molecular weight heparin against SARS-CoV-2 SGP binding to immobilized heparin were 0.056 μM, 0.12 μM, and 26.4 μM, respectively. Finally, unbiased computational ligand docking indicates that heparan sulfate interacts with the GAG-binding motif at the S1/S2 site on each monomer interface in the trimeric SARS-CoV-2 SGP, and at another site (453-459 (YRLFRKS)) when the receptor-binding domain is in an open conformation. The current study serves a foundation to further investigate biological roles of GAGs in SARS-CoV-2 pathogenesis. Furthermore, our findings may provide additional basis for further heparin-based interventions for COVID-19 patients exhibiting thrombotic complications.
Background: Endothelial glycocalyx degradation contributes to the pathogenesis of critical illness. Results: Mechanically ventilated subjects exhibited plasma glycocalyx breakdown signatures (glycosaminoglycan fragments) characteristic of direct versus indirect etiologies of respiratory failure. Conclusion: Circulating glycosaminoglycans provide insight into respiratory failure pathophysiology. Significance: This is the first study to characterize circulating glycosaminoglycans during critical illness, offering insight into the mechanisms underlying respiratory failure.
The ability to fine tune gene expression has created the field of metabolic pathway optimization and balancing where a variety of factors affecting flux balance are carefully modulated to improve product titers, yields, and productivity. Using a library of isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible mutant T7 promoters of varied strength a combinatorial method was developed for transcriptional balancing of the violacein pathway. Violacein biosynthesis involves a complex five-gene pathway that is an excellent model for exploratory metabolic engineering efforts into pathway regulation and control due to many colorful intermediates and side products allowing for easy analysis and strain comparison. Upon screening approximately 4% of the total initial library, several high-titer mutants were discovered that resulted in up to a 63-fold improvement over the control strain. With further fermentation optimization, titers were improved to 1829 ± 46 mg/L; a 2.6-fold improvement in titer and a 30-fold improvement in productivity from previous literature reports.
Although most pharmaceutical heparin used today is obtained from porcine intestine, heparin has historically been prepared from bovine lung and ovine intestine. There is some regulatory concern about establishing the species origin of heparin. This concern began with the outbreak of mad cow disease in the 1990s and was exacerbated during the heparin shortage in the 2000s and the heparin contamination crisis of 2007–2008. Three heparins from porcine, ovine, and bovine were characterized through state-of-the-art carbohydrate analysis methods with a view profiling their physicochemical properties. Differences in molecular weight, monosaccharide and disaccharide composition, oligosaccharide sequence, and antithrombin III-binding affinity were observed. These data provide some insight into the variability of heparins obtained from these three species and suggest some analytical approaches that may be useful in confirming the species origin of a heparin active pharmaceutical ingredient.
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