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 might be involved in host cell entry of SARS-CoV-2. Using a surface plasmon resonance direct binding assay, we found that both monomeric and trimeric SARS-CoV-2 spike more tightly bind to immobilized heparin (K _D = 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. Our study augments our knowledge in SARS-CoV-2 pathogenesis and advances carbohydrate-based COVID-19 therapeutic development.

We performed next-generation sequencing in patients with familial steroid-sensitive nephrotic syndrome (SSNS) and identified a homozygous segregating variant (p.H310Y) in the gene encoding clavesin-1 ( CLVS1 ) in a consanguineous family with 3 affected individuals. Knockdown of the clavesin gene in zebrafish (clvs2) produced edema phenotypes due to disruption of podocyte structure and loss of glomerular filtration barrier integrity that could be rescued by WT CLVS1 but not the p.H310Y variant. Analysis of cultured human podocytes with CRISPR/Cas9-mediated CLVS1 knockout or homozygous H310Y knockin revealed deficits in clathrin-mediated endocytosis and increased susceptibility to apoptosis that could be rescued with corticosteroid treatment, mimicking the steroid responsiveness observed in patients with SSNS. The p.H310Y variant also disrupted binding of clavesin-1 to α -tocopherol transfer protein, resulting in increased reactive oxygen species (ROS) accumulation in CLVS1 -deficient podocytes. Treatment of CLVS1 -knockout or homozygous H310Y-knockin podocytes with pharmacological ROS inhibitors restored viability to control levels. Taken together, these data identify CLVS1 as a candidate gene for SSNS, provide insight into therapeutic effects of corticosteroids on podocyte cellular dynamics, and add to the growing evidence of the importance of endocytosis and oxidative stress regulation to podocyte function.

Brucellosis continues to be a significant global zoonotic infection, with diagnosis largely relying on the detection of antibodies against the Brucella O-polysaccharide (O-PS) A and M antigens. In this study, computational methods, including homology modeling, molecular docking, and molecular dynamics simulations, were applied to investigate the interaction of the four murine monoclonal antibodies (mAbs) YsT9.1, YsT9.2, Bm10, and Bm28 with hexasaccharide fragments of the A and M epitopes. Through stringent stability criteria, based on interaction energies and mobility of the antigens, high-affinity binding of A antigen with YsT9.1 antibody and M antigen with Bm10 antibody was predicted. In both the complexes hydrophobic interactions dominate the antigen–antibody binding. These findings align well with experimental epitope mapping, indicating YsT9.1’s preference for internal sequences of the A epitope and Bm10’s preference for internal elements of the M epitope. Interestingly, no stable complexes were identified for YsT9.2 or Bm28 interacting with A or M antigen. This study provides valuable insights into the mechanism of molecular recognition of Brucella O-antigens that can be applied for the development of improved diagnostics, synthetic glycomimetics, and improved vaccine strategies.

The adhesion of HIV gp120 antigen to human cells is modulated in part by interactions with heparan sulfate. The HXB2 strain of gp120 has been shown to interact with heparin primarily through the V3 loop, although other domains including the C-terminal domain were also implicated. However, the JR-FL strain (representative of CCR5-interacting strains that make up newest infections) was shown to have a drastically lowered affinity to heparin due to the loss of several basic residues in the V3 loop, and deletion of the V3 loop in JR-FL gp120 was shown to abrogate some, but not all, heparin binding. Here, we use high resolution hydroxyl radical protein footprinting to measure the changes in protein surface oxidation levels that result from the binding of a model heparin fragment (fondaparinux). Protection in both the V3 loop and the N-terminus of JR-FP gp120 is observed. The well-defined composition of fondaparinux allowed us to perform docking simulations, which showed two clusters of fondaparinux binding: the V3 loop, and a domain consisting of the N- and C-termini. Together, the experimental and theoretical results indicate the heparin/heparan sulfate binding sites on JR-FL gp120 and the efficient interaction of fondaparinux, a widely exploited therapeutic carbohydrate, on gp120.

Introduction: Nephrotic syndrome (NS), a common glomerular disease in children, is classified based on response to corticosteroid therapy as either steroid-sensitive nephrotic syndrome (SSNS), or steroid-resistant nephrotic syndrome (SRNS). However, there are currently no reliable predictors of therapy response at initial clinical presentation. Methods: We conducted genome-wide association studies, developed polygenic risk scores (PRS) for therapy response and analyzed classical HLA alleles in 1,997 (994 discovery and 1,003 replication/validation cohorts) previously unstudied children with NS and 3,558 ancestry-matched controls. Results: A significant association with HLA loci defined by variants in HLA-DQB1, HLA-DRB1 , and HLA-DQA1 were found for SSNS (but not SRNS), along with a second immune-related SSNS locus: CLEC16A . A PRS that discriminates between SSNS and SRNS was validated in two independent cohorts. The HLA haplotype HLA-DRB1*07:01∼DQA1*02:01∼DQB1*02:02 was associated with ∼4 times the risk of developing SSNS. A model incorporating HLA haplotype, PRS score, and age at onset of the disease was the best predictor of steroid responsiveness with an AUC of 0.68-0.70 and an overall classification accuracy of SSNS versus SRNS of 67-71%. Conclusions: Our findings confirm that SSNS (unlike SRNS) is an immune-mediated HLA-associated disorder. The PRS for therapy response and HLA haplotype can serve as biomarkers and provide a foundation for more accurate diagnoses and tailored and individualized treatment. Translational statement: To identify biomarkers of pattern of steroid responsiveness in childhood-onset nephrotic syndrome, we carried out a case-control study that included over 4,000 samples, including 994 patients with NS in the discovery phase and an additional 1,003 cases from two independent replication cohorts. We identified significant risk of steroid-sensitive NS (SSNS) at HLA class II genes and CLEC16A (a gene important in the regulation of T and B lymphocytes). The HLA haplotype DRB1*07:01∼DQA1*02:01∼DQB1*02:02 was associated with four-fold increased odds of SSNS. A model incorporating HLA haplotype, polygenic risk score, and age at onset of the disease was the best predictor of steroid responsiveness providing useful delineation of steroid sensitivity from steroid resistance. In conclusion, age at onset of disease, HLA class II variants and polygenic risk scores are useful biomarkers of corticosteroid response in childhood NS and may serve as useful clinical decision support tools to guide treatment. Abstract Figure: Graphical Abstract