Sequential in vitro enzymatic N-glycoprotein modification reveals site-specific rates of glycoenzyme processing (preprint)

N-glycosylation is an essential eukaryotic post-translational modification that affects various glycoprotein properties, including folding, solubility, protein-protein interactions, and half-life. N-glycans are processed in the secretory pathway to form varied ensembles of structures, and diversity at a single site on a glycoprotein is termed microheterogeneity. To understand the factors that influence glycan microheterogeneity, we hypothesized that local steric and electrostatic factors surrounding each site influences glycan availability to enzymatic modification. We tested this hypothesis by expression of a panel of reporter N-linked glycoproteins in MGAT1-null HEK293 cells to produce immature Man5GlcNAc2 glycoforms (38 glycan sites total). These glycoproteins were then sequentially modified in vitro from high-mannose to hybrid and on to biantennary, core fucosylated, complex structures by a panel of N-glycosylation enzymes and each reaction time-course was quantified by LC-MS/MS. Substantial differences in rates of in vitro enzymatic modification were observed between glycan sites on the same protein and differences in modification rates varied depending on the glycoenzyme being evaluated. By comparison, proteolytic digestion of the reporters prior to N-glycan processing eliminated differences in in vitro enzymatic modification. Comparison of in vitro rates of enzymatic modification with the glycan structures found on the mature reporters expressed in wild type cells correlate well with the enzymatic bottlenecks found in vitro. These data suggest that higher-order local structures surrounding each glycosylation site contribute to the efficiency of modification both in vitro and in vivo to establish the spectrum of site-specific microheterogeneity found on N-linked glycoproteins.

Integrated Chemoenzymatic Approach to Streamline the Assembly of Complex Glycopeptides in the Liquid Phase.

Glycosylation of proteins is a complicated post-translational modification. Despite the significant progress in glycoproteomics, accurate functions of glycoproteins are still ambiguous owing to the difficulty in obtaining homogeneous glycopeptides or glycoproteins. Here, we describe a streamlined chemoenzymatic method to prepare complex glycopeptides by integrating hydrophobic tag-supported chemical synthesis and enzymatic glycosylations. The hydrophobic tag is utilized to facilitate peptide chain elongation in the liquid phase and expeditious product separation. After removal of the tag, a series of glycans are installed on the peptides via efficient glycosyltransferase-catalyzed reactions. The general applicability and robustness of this approach are exemplified by efficient preparation of 16 well-defined SARS-CoV-2 O-glycopeptides, 4 complex MUC1 glycopeptides, and a 31-mer glycosylated glucagon-like peptide-1. Our developed approach will open up a new range of easy access to various complex glycopeptides of biological importance.

Melville’s Anatomies Two Decades Later: A Forum

Melville scholars gathered virtually as part of the 2021 American Literature Association Conference to celebrate and re-assess the contribution made by a landmark volume in late twentieth-century scholarship on Melville, Samuel Otter’s Melville’s Anatomies (1999). The roundtable had initially been planned as a face-to-face contribution to the 2020 ALA Conference, which was cancelled due to COVID, and so the scholars who had initially meant to discuss Otter’s book near the occasion of the twentieth anniversary of its publication found that their conversations had to be delayed by a year. We offer the full reflections of the six scholars who discussed the volume in hopes that this can serve as a model for future reflections on landmarks of Melville scholarship and major new contributions to the field that can appear in this venue. © 2022 The Melville Society and Johns Hopkins University Press

Remdesivir extraction by extracorporeal life support circuits

INTRODUCTION: Many patients with severe COVID-19 require extracorporeal membrane oxygenation (ECMO) and/or continuous renal replacement therapy (CRRT), both of which can alter drug disposition. Lipophilic, highly protein bound drugs can adsorb to circuit materials, while hydrophilic, minimally protein bound drugs are likely to be filtered. HYPOTHESIS: Remdesivir (RDV) is lipophilic and highly protein bound making it likely to be adsorbed by circuit components and minimally cleared by hemofiltration/dialysis. RDV's active metabolite GS-441524 is hydrophilic and minimally protein bound and should be minimally adsorbed but rapidly filtered. METHODS: We administered RDV and GS-441524 to blood-primed, closed loop, ex vivo ECMO and CRRT circuits and measured drug concentrations over time. Drugs were also administered to a separate control tube to determine drug degradation. Each experiment was performed in triplicate. Drug recovery (%) was calculated by dividing each concentration by the initial concentration. RESULTS: Mean (standard deviation) recovery of RDV in the ECMO circuits (n=3) was low at 33.3% (2.0) at 6 hours. Recovery in the control (n=3) at 6 hours was 29.3% (2.0) and not significantly different from recovery in the ECMO circuits at 6 hours (p=0.07). Substantial loss of RDV in the CRRT circuits (n=3) occurred within minutes. Recovery was 14.4% (5.7) at 5 minutes and 4.7% (1.0) at 3 hours and significantly different compared to control recovery at 3 hours (p=0.008). Recovery of GS-441524 was higher than RDV in the ECMO circuits (n=3). After 6 hours, recovery was at 75.8% (16.5). Mean recovery in the control (n=3) at 6 hours was 70.6% (6.2) and not significantly different from recovery in the ECMO circuit at 6 hours (p=0.7). In the CRRT circuits (n=3), GS-441524 recovery was low at 15.9% (3.0) at 30 minutes and 0% (0) at 3 hours and significantly different from the control (p=0.005). CONCLUSIONS: RDV is extracted by ECMO and CRRT primarily by drug adsorption to circuit materials and potentially by drug metabolism in the blood. GS-441524 was not substantially extracted by the ECMO circuit but rapidly cleared by hemodiafiltration in the CRRT circuit. The extent of loss for both molecules, especially in CRRT, suggests that in patients supported with ECMO and CRRT, dosing adjustments are needed.

Variable posttranslational modifications of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), started in 2019 in China and quickly spread into a global pandemic. Nucleocapsid protein (N protein) is highly conserved and is the most abundant protein in coronaviruses and is thus a potential target for both vaccine and point-of-care diagnostics. N Protein has been suggested in the literature as having posttranslational modifications (PTMs), and accurately defining these PTMs is critical for its potential use in medicine. Reports of phosphorylation of N protein have failed to provide detailed site-specific information. We have performed comprehensive glycomics, glycoproteomics and proteomics experiments on two different N protein preparations. Both were expressed in HEK293 cells; one was in-house expressed and purified without a signal peptide (SP) sequence, and the other was commercially produced with a SP channeling it through the secretory pathway. Our results show completely different PTMs on the two N protein preparations. The commercial product contained extensive N- and O-linked glycosylation as well as O-phosphorylation on site Thr393. Conversely, the native N Protein model had O-phosphorylation at Ser176 and no glycosylation, highlighting the importance of knowing the provenance of any commercial protein to be used for scientific or clinical studies. Recent studies have indicated that N protein can serve as an important diagnostic marker for COVID-19 and as a major immunogen by priming protective immune responses. Thus, detailed structural characterization of N protein may provide useful insights for understanding the roles of PTMs on viral pathogenesis, vaccine design and development of point-of-care diagnostics.

A flexible, pan-species, multi-antigen platform for the detection and monitoring of SARS-CoV-2-specific antibody responses (preprint)

The SARS-CoV-2 pandemic and the vaccination effort that is ongoing has created an unmet need for accessible, affordable, flexible and precise platforms for monitoring the induction, specificity and maintenance of virus-specific immune responses. Herein we validate a multiplex (Luminex-based) assay capable of detecting SARS-CoV-2-specific antibodies irrespective of host species, antibody isotype, and specimen type (e.g. plasma, serum, saliva or blood spots). The well-established precision of Luminex-based assays provides the ability to follow changes in antibody levels over time to many antigens, including multiple permutations of the most common SARS-CoV-2 antigens. This platform can easily measure antibodies known to correlate with neutralization activity as well as multiple non-SARS-CoV-2 antigens such as vaccines (e.g. Tetanus toxoid) or those from frequently encountered agents (influenza), which serve as stable reference points for quantifying the changing SARS-specific responses. All of the antigens utilized in our study can be made in-house, many in E. coli using readily available plasmids. Commercially sourced antigens may also be incorporated and newly available antigen variants can be rapidly produced and integrated, making the platform adaptable to the evolving viral strains in this pandemic.