Uncovering the Role of N-Glycan Occupancy on the Cooperative Assembly of Spike and Angiotensin Converting Enzyme 2 Complexes: Insights from Glycoengineering and Native Mass Spectrometry.

Interactions between the SARS-CoV-2 Spike protein and ACE2 are one of the most scrutinized reactions of our time. Yet, questions remain as to the impact of glycans on mediating ACE2 dimerization and downstream interactions with Spike. Here, we address these unanswered questions by combining a glycoengineering strategy with high-resolution native mass spectrometry (MS) to investigate the impact of N-glycan occupancy on the assembly of multiple Spike-ACE2 complexes. We confirmed that intact Spike trimers have all 66 N-linked sites occupied. For monomeric ACE2, all seven N-linked glycan sites are occupied to various degrees; six sites have >90% occupancy, while the seventh site (Asn690) is only partially occupied (∼30%). By resolving the glycoforms on ACE2, we deciphered the influence of each N-glycan on ACE2 dimerization. Unexpectedly, we found that Asn432 plays a role in mediating dimerization, a result confirmed by site-directed mutagenesis. We also found that glycosylated dimeric ACE2 and Spike trimers form complexes with multiple stoichiometries (Spike-ACE2 and Spike2-ACE2) with dissociation constants (Kds) of ∼500 and <100 nM, respectively. Comparing these values indicates that positive cooperativity may drive ACE2 dimers to complex with multiple Spike trimers. Overall, our results show that occupancy has a key regulatory role in mediating interactions between ACE2 dimers and Spike trimers. More generally, since soluble ACE2 (sACE2) retains an intact SARS-CoV-2 interaction site, the importance of glycosylation in ACE2 dimerization and the propensity for Spike and ACE2 to assemble into higher oligomers are molecular details important for developing strategies for neutralizing the virus.

Pathogen-sugar interactions revealed by universal saturation transfer analysis.

Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily modified pathogen proteins can be confounded by overlapping sugar signals and/or compounded with known experimental constraints. Universal saturation transfer analysis (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin-lineage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike trimer binds sialoside sugars in an "end-on" manner. uSTA-guided modeling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar binding in SARS-CoV-2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins deep in the human lung as potentially relevant to virulence and/or zoonosis.

Surgical tracheostomies in COVID-19 patients: A multidisciplinary approach and lessons learned.

Surgical tracheostomies have a role in the weaning process of COVID-19 patients treated in intensive care units. A multidisciplinary team approach (MDT) is required for decision making. This process is augmented by specific standard operating practices implemented by senior clinicians. Here, we report on our early experience and outcomes with open tracheostomies in a cohort of COVID-19 patients. We outline the criteria that guide decision making and explore the challenges faced by our intensive care colleagues in the management of these patients. The cohort was 100% male with 90% of them having a raised Body Mass Index (BMI) and other comorbidities (hypertension and diabetes). 60% have been decannulated and have been stepped down the intensive care unit. We recorded no surgical complications or adverse events. The service to date has been shown to be effective, safe, largely reproducible and reflective.