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.

RFT1-CDG: deafness as a novel feature of congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are genetic diseases due to defects in the synthesis of glycans and in the attachment of glycans to lipids and proteins. Actually, some 42 CDG are known including defects in protein N-glycosylation, in protein O-glycosylation, in lipid glycosylation, and in multiple and other glycosylation pathways. Most CDG are multisystem diseases and a large number of signs and symptoms have already been reported in CDG.

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.

A new case of ALG8 deficiency (CDG Ih).

Unlabelled: Congenital disorders of glycosylation (CDG) represent an expanding group of inherited diseases. One of them, ALG8 deficiency (CDG Ih), leads to protein N-glycosylation defects caused by malfunction of glucosyltransferase 2 (Dol-P-Glc:Glc1-Man(9)-GlcNAc(2)-P-P-Dol glucosyltransferase) resulting in inefficient addition of the second glucose residue onto lipid-linked oligosaccharides. So far, only five patients have been described with ALG8 deficiency.

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.

Structural requirements for membrane assembly of proteins spanning the membrane several times.

We have investigated the structural requirements for the biogenesis of proteins spanning the membrane several times. Proteins containing various combinations of topological signals (signal anchor and stop transfer sequences) were synthesized in a cell-free translation system and their membrane topology was determined. Proteins spanning the membrane twice were obtained when a signal anchor sequence was followed by either a stop transfer sequence or a second signal anchor sequence.

A tripartite structure of the signals that determine protein insertion into the endoplasmic reticulum membrane.

Multilineage colony stimulating factor is a secretory protein with a cleavable signal sequence that is unusually long and hydrophobic. Using molecular cloning techniques we exchanged sequences NH2- or COOH-terminally flanking the hydrophobic signal sequence. Such modified fusion proteins still inserted into the membrane but their signal sequence was not cleaved.

Signal recognition particle arrests elongation of nascent secretory and membrane proteins at multiple sites in a transient manner.

The signal recognition particle (SRP) has been shown to target nascent secretory and membrane proteins to the endoplasmic reticulum. In the wheat germ cell-free system, SRP arrests the elongation of the nascent chains until the translational complex is docked to the endoplasmic reticulum membrane where the interaction between SRP and docking protein causes a release of the nascent chain arrest. For two secretory proteins, arrested peptides of 70 amino acids have been identified (Walter, P.

Translation arrest by oligodeoxynucleotides complementary to mRNA coding sequences yields polypeptides of predetermined length.

We investigated the arrest of mRNA translation at predetermined sites by oligodeoxynucleotides complementary to defined coding sequences within a mRNA. An in vitro transcription and a wheat germ cell-free translation system were used for the synthesis of mRNA and protein, respectively. Oligodeoxynucleotides (10-, 15- and 20-mer) arrested polypeptide synthesis in a concentration-dependent manner at the site of their hybridization to the mRNA, as judged by the size of the translation products.

Cell proliferation and milk protein gene expression in rabbit mammary cell cultures.

We analyzed the synthesis of DNA, the rate of cell proliferation, and the expression of milk protein genes in mammary cells grown as primary cultures on or in collagen gels in chemically defined media. We assessed DNA synthesis and cell growth, measured by [(3) H]- thymidine incorporation into acid-insoluble material, DNA content, and cell counts, in a progesterone- and prolactin-containing medium. In some experiments, cultures were pulsed for 1 h with [(3)H]thymidine and dissociated into individual cells which were cytocentrifuged and processed for immunocytochemistry and autoradiography.

Effect of cell shape change on the function and differentiation of rabbit mammary cells in culture.

We examined the role of cell shape, cytodifferentiation, and tissue topography on the induction and maintenance of functional differentiation in rabbit mammary cells grown as primary cultures on two-dimensional collagen surfaces or in three-dimensional collagen matrices. Mammary glands from mid-pregnant rabbits were dissociated into single cells, and epithelial cells were enriched by isopycnic centrifugation. Small spheroids of epithelial cells (approximately 50 cells) that formed on a rotary shaker were plated on or embedded in collagen gels.