Turning universal O into rare Bombay type blood.

Red blood cell antigens play critical roles in blood transfusion since donor incompatibilities can be lethal. Recipients with the rare total deficiency in H antigen, the O Bombay phenotype, can only be transfused with group O blood to avoid serious transfusion reactions. We discover FucOB from the mucin-degrading bacteria Akkermansia muciniphila as an α-1,2-fucosidase able to hydrolyze Type I, Type II, Type III and Type V H antigens to obtain the afucosylated Bombay phenotype in vitro.

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis.

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs.

Mapping O-glycosylation Sites Using OpeRATOR and LC-MS.

O-glycosylation is a difficult posttranslational modification to analyze. O-glycans are labile and often cluster making their analysis by LC-MS very challenging. OpeRATOR is an O-glycan specific protease that cleaves the protein backbone N-terminally of glycosylated serine and threonine residues.

Fast Afucosylation Profiling of Glycoengineered Antibody Subunits by Middle-Up Mass Spectrometry.

Middle-up LC-MS antibody characterization workflows using reduction or IdeS digestion for a focused assessment of N-glycan profiling of three representative glycoengineered monoclonal antibodies (mAbs), namely, obinutuzumab (GlycomAb technology, Glycart/Roche), benralizumab (Potelligent Technology, BioWa, Kyowa Kirin) and mAb B (kifunensine) and compared to mAb A, produced in a common CHO cell line. In addition, EndoS or EndoS2 enzyme are used for quantitative determination of Fc-glycan core afucosylation and high mannose for these antibodies, as requested by health authorities for Fc-competent therapeutics mAbs critical quality attributes (CQAs).

Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA from Akkermansia muciniphila.

Akkermansia muciniphila is a mucin-degrading bacterium commonly found in the human gut that promotes a beneficial effect on health, likely based on the regulation of mucus thickness and gut barrier integrity, but also on the modulation of the immune system. In this work, we focus in OgpA from A. muciniphila, an O-glycopeptidase that exclusively hydrolyzes the peptide bond N-terminal to serine or threonine residues substituted with an O-glycan.

Optimization of GIG for -Glycopeptide Characterization with Sialic Acid Linkage Determination.

-Glycoprotein analysis has been historically challenging due, in part, to a dearth of available enzymes active in the release of -glycans. Moreover, chemical releasing methods, such as β-elimination/Michael addition, are not specific to -glycan release and can also eliminate phosphoryl substitutions. Both of these events leave behind deaminated serine and threonine and thus can lead to ambiguous structural conclusions.

Glycan-Mediated Technology for Obtaining Homogeneous Site-Specific Conjugated Antibody-Drug Conjugates: Synthesis and Analytical Characterization by Using Complementary Middle-up LC/HRMS Analysis.

Conventional antibody-drug conjugate (ADC) manufacturing methods are based on the nonselective bioconjugation of cytotoxic drugs to lysine and cysteine residues. This results in highly heterogeneous mixtures of different drug-antibody ratios (DAR) that can significantly affect the safety and efficacy of the ADC product. Recently, an innovative procedure named GlyCLICK was suggested, consisting of a two-step enzymatic procedure to transform Fc-glycans present on IgG mAbs into two site-specific anchor points for the conjugation of any alkyne-containing payload of choice.

Antibody Conjugations via Glycosyl Remodeling.

The antibody Fc-glycans are interesting targets for conjugation of cytotoxic compounds due to their localization and their chemical composition. In striving to obtain site-specific conjugates, the antibody Fc-glycans have been explored in numerous ways. Here we present a two-step enzymatic methodology coupled to click-chemistry for conjugation at the core GlcNAc of the Fc-glycan resulting in ADCs that are homogenous with a DAR 2.

On enzymatic remodeling of IgG glycosylation; unique tools with broad applications.

The importance of IgG glycosylation has been known for many years not only by scientists in glycobiology but also by human pathogens that have evolved specific enzymes to modify these glycans with fundamental impact on IgG function. The rise of IgG as a major therapeutic scaffold for many cancer and immunological indications combined with the availability of unique enzymes acting specifically on IgG Fc-glycans have spurred a range of applications to study this important post-translational modification on IgG. This review article introduces why the IgG glycans are of distinguished interest, gives a background on the unique enzymatic tools available to study the IgG glycans and finally presents an overview of applications utilizing these enzymes for various modifications of the IgG glycans.

Deciphering Protein O-Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment.

Glycosylation plays a critical role in the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Over 50% of mammalian cellular proteins are typically glycosylated; this modification is involved in a wide range of biological functions such as barrier formation against intestinal microbes and serves as signaling molecules for selectins and galectins in the innate immune system. N-linked glycosylation analysis has been greatly facilitated owing to a range of specific enzymes available for their release.

Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp.

Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG.

EndoSd: an IgG glycan hydrolyzing enzyme in Streptococcus dysgalactiae subspecies dysgalactiae.

Aim: The aim of this study was to identify and characterize EndoS-like enzymes in Streptococcus dysgalactiae subspecies dysgalactiae (SDSD).

Materials & Methods: PCR, DNA sequencing, recombinant protein expression, lectin blot, ultra high performance liquid chromatography analysis and a chitinase assay were used to identify ndoS-like genes and characterize EndoSd.

Results: EndoSd were found in four SDSD strains.

Rapid and improved characterization of therapeutic antibodies and antibody related products using IdeS digestion and subunit analysis.

The immunoglobulin degrading enzyme from Streptococcus pyogenes, IdeS, was discovered as a mechanism by which pathogenic bacteria circumvent antibody mediated immune defense. IdeS was found to rapidly and specifically cleave IgG into F(ab')2 and Fc/2 fragments. The enzymatic specificity has led to a range of recent developments in the analytical strategies for characterization of monoclonal therapeutic antibodies and related products such as antibody-drug conjugates, bispecific antibodies, antibody mixtures and Fc-fusion proteins.

A novel mechanism for NETosis provides antimicrobial defense at the oral mucosa.

Neutrophils are essential for host defense at the oral mucosa and neutropenia or functional neutrophil defects lead to disordered oral homeostasis. We found that neutrophils from the oral mucosa harvested from morning saliva had released neutrophil extracellular traps (undergone NETosis) in vivo. The NETosis was mediated through intracellular signals elicited by binding of sialyl Lewis(X) present on salival mucins to l-selectin on neutrophils.

EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans.

Enzymes that affect glycoproteins of the human immune system, and thereby modulate defense responses, are abundant among bacterial pathogens. Two endoglycosidases from the human pathogen Streptococcus pyogenes, EndoS and EndoS2, have recently been shown to hydrolyze N-linked glycans of human immunoglobulin G. However, detailed characterization and comparison of the hydrolyzing activities have not been performed.

Bacterial glycosidases in pathogenesis and glycoengineering.

Glycosylation is a common post-translational protein modification and many key proteins of the immune system are glycosylated. As the true experts of our immune system, pathogenic bacteria produce enzymes that can modify the carbohydrates (glycans) of the defense mechanisms in order to favor bacterial survival and persistence. At the intersection between bacterial pathogenesis and glycobiology, there is an increased interest in studying the bacterial enzymes that modify the protein glycosylation of their colonized or infected hosts.

EndoE from Enterococcus faecalis hydrolyzes the glycans of the biofilm inhibiting protein lactoferrin and mediates growth.

Glycosidases are widespread among bacteria. The opportunistic human pathogen Enterococcus faecalis encodes several putative glycosidases but little is known about their functions. The identified endo-β-N-acetylglucosaminidase EndoE has activity on the N-linked glycans of the human immunoglobulin G (IgG).

EndoS2 is a unique and conserved enzyme of serotype M49 group A Streptococcus that hydrolyses N-linked glycans on IgG and α1-acid glycoprotein.

Many bacteria have evolved ways to interact with glycosylation functions of the immune system of their hosts. Streptococcus pyogenes [GAS (group A Streptococcus)] secretes the enzyme EndoS that cleaves glycans on human IgG and impairs the effector functions of the antibody. The ndoS gene, encoding EndoS, has, until now, been thought to be conserved throughout the serotypes.

Study of the IgG endoglycosidase EndoS in group A streptococcal phagocyte resistance and virulence.

Background: The secreted enzyme EndoS, an endoglycosidase from Streptococcus pyogenes, hydrolyzes the N-linked glycan of the constant region of immunoglobulin G (IgG) heavy chain and renders the antibody unable to interact with Fc receptors and elicit effector functions. In this study we couple targeted allelic replacement mutagenesis and heterologous expression to elucidate the contribution of EndoS to group A Streptococcus (GAS) phagocyte resistance and pathogenicity in vitro and in vivo.

Results: Knocking out the EndoS gene in GAS M1T1 background revealed no significant differences in bacterial survival in immune cell killing assays or in a systemic mouse model of infection.