The trispecific DARPin ensovibep inhibits diverse SARS-CoV-2 variants.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.

Characterization and immunogenicity of a Shigella flexneri 2a O-antigen bioconjugate vaccine candidate.

Shigellosis remains a major cause of diarrheal disease in developing countries and causes substantial morbidity and mortality in children. Vaccination represents a promising preventive measure to fight the burden of the disease, but despite enormous efforts, an efficacious vaccine is not available to date. The use of an innovative biosynthetic Escherichia coli glycosylation system substantially simplifies the production of a multivalent conjugate vaccine to prevent shigellosis.

Supercharging Reagent for Enhanced Liquid Chromatographic Separation and Charging of Sialylated and High-Molecular-Weight Glycopeptides for NanoHPLC-ESI-MS/MS Analysis.

Recent developments in proteomic techniques have led to the development of mass spectrometry (MS)-based methods to characterize site-specific glycosylation of proteins. However, appropriate analytical tools to characterize acidic and high-molecular-weight (hMW) glycopeptides are still lacking. In this study, we demonstrate that the addition of supercharging reagent, m-nitrobenzyl alcohol (m-NBA), into mobile phases greatly facilitates the analysis of acidic and hMW glycopeptides.

Immunogenicity and safety of a tetravalent E. coli O-antigen bioconjugate vaccine in animal models.

Background: Extra-intestinal pathogenic Escherichia coli (ExPEC) are major human pathogens; however, no protective vaccine is currently available. We assessed in animal models the immunogenicity and safety of a 4-valent E. coli conjugate vaccine (ExPEC-4V, serotypes O1, O2, O6 and O25 conjugated to Exotoxin A from Pseudomonas aeruginosa (EPA)) produced using a novel in vivo bioconjugation method.

Purification and characterization of a Shigella conjugate vaccine, produced by glycoengineering Escherichia coli.

Shigellosis remains a major cause of diarrheal disease in developing countries and causes substantial morbidity and mortality in children. Glycoconjugate vaccines consisting of bacterial surface polysaccharides conjugated to carrier proteins are the most effective vaccines for controlling invasive bacterial infections. Nevertheless, the development of a multivalent conjugate vaccine to prevent Shigellosis has been hampered by the complex manufacturing process as the surface polysaccharide for each strain requires extraction, hydrolysis, chemical activation and conjugation to a carrier protein.

Improvement of dolichol-linked oligosaccharide biosynthesis by the squalene synthase inhibitor zaragozic acid.

The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG.

Congenital disorders of glycosylation: an update on defects affecting the biosynthesis of dolichol-linked oligosaccharides.

Defects in the biosynthesis of the oligosaccharide precursor for N-glycosylation lead to decreased occupancy of glycosylation sites and thereby to diseases known as congenital disorders of glycosylation (CDG). In the last 20 years, approximately 1,000 CDG patients have been identified presenting with multiple organ dysfunctions. This review sets the state of the art by listing all mutations identified in the 15 genes (PMM2, MPI, DPAGT1, ALG1, ALG2, ALG3, ALG9, ALG12, ALG6, ALG8, DOLK, DPM1, DPM3, MPDU1, and RFT1) that yield a deficiency of dolichol-linked oligosaccharide biosynthesis.

HPLC and mass spectrometry analysis of dolichol-phosphates at the cell culture scale.

Dolichols (Dol) are polyprenol lipids that are essential structural components of eukaryotic membranes. In addition, the phosphorylated derivatives of Dol function as lipid anchors of mono- and oligosaccharide precursors involved in protein glycosylation. The biological importance of Dol phosphates (Dol-P) is illustrated by the severe outcome of human disorders linked to Dol biosynthetic defects, such as Dol-kinase deficiency.

RFT1 deficiency in three novel CDG patients.

The medical significance of N-glycosylation is underlined by a group of inherited human disorders called Congenital Disorders of Glycosylation (CDG). One key step in the biosynthesis of the Glc(3)Man(9)GlcNAc(2)-PP-dolichol precursor, essential for N-glycosylation, is the translocation of Man(5)GlcNAc(2)-PP-dolichol across the endoplasmic reticulum membrane. This step is facilitated by the RFT1 protein.

Human RFT1 deficiency leads to a disorder of N-linked glycosylation.

N-linked glycosylation is an essential posttranslational modification of proteins in eukaryotes. The substrate of N-linked glycosylation, dolichol pyrophosphate (DolPP)-GlcNAc(2)Man(9)Glc(3), is assembled through a complex series of ordered reactions requiring the translocation of the intermediate DolPP-GlcNAc(2)Man(5) structure across the endoplasmic-reticulum membrane. A young patient diagnosed with a congenital disorder of glycosylation characterized by an intracellular accumulation of DolPP-GlcNAc(2)Man(5) was found to carry a homozygous point mutation in the RFT1 gene.