Native mass spectrometry (nMS) is a powerful label-free method for detecting biomolecular complexes, resolving stoichiometry, and quantifying affinity (Kd). However, signal overlap in heterogeneous systems often limits its accuracy. Charge detection (CD)-nMS, which independently measures the mass-to-charge ratio and charge of individual ions, overcomes this challenge, enabling quantitative analysis of polydisperse and high molecular weight complexes with unresolved charge states. Here, we systematically validate CD-nMS for precise Kd determination using soluble protein-ligand complexes of known Kd and extend its application to quantify glycan ligand binding by a highly glycosylated immune lectin. We then demonstrate the implementation of slow mixing mode (SLOMO), a time-resolved mixing method that corrects for nonuniform response factors of interacting species, using CD-nMS to enable robust quantification of protein-protein interactions. Finally, we apply SLOMO-CD-nMS to directly detect and quantify bacterial toxin binding to glycolipids embedded in membrane-like assemblies, a capability not accessible with conventional nMS. These measurements uncovered previously unrecognized assembly pathways and demonstrate, for the first time, that SLOMO-CD-nMS can resolve and quantify multivalent lectin engagement with glycolipids in a native-like membrane context. Collectively, these results establish CD-nMS, alone or in combination with SLOMO, as a broadly applicable assay for quantitative characterization of complex biomolecular interactions across soluble, glycosylated, and membrane-associated systems.

N-Glycans play essential roles in diverse biological processes, and enzymes capable of precisely modifying them offer powerful means to modulate their function for therapeutic applications. However, the discovery of such enzymes is hindered by the limited availability of sensitive, fully representative substrates that accurately mimic native N-glycan structures. Here, we report a fluorescence-quenched (FQ) N-glycan probe, chemoenzymatically synthesized from egg-derived sialoglycopeptide (SGP), that addresses this limitation. The probe remains nonfluorescent ("OFF") until enzymatic cleavage of a linkage within the intact complex type N-glycan substrate, which in turn triggers a fluorescent signal ('ON'). Incubation of the FQ N-glycan probe with a panel of glycoside hydrolases (GHs) reported to target specific linkages within complex type N-glycans, results in an increase in fluorescence, which can be readily monitored. Furthermore, we show that this probe is compatible with single-emulsion droplet-based screening and confirm its utility through a trial screen that can recover N-glycan degrading clones from mixed microbial populations using fluorescence activated single-droplet dispensing (FASD). While demonstrated for egg-derived SGP, this chemoenzymatic strategy may be extended to diverse glycans and glycopeptide substrates, providing a strategy to generate fully representative glycan probes for ultrahigh-throughput screening to advance both glycoengineering and fundamental glycomics research.

Glycans constitute a structurally diverse and immunologically instructive layer that shapes how transplanted tissues are interpreted by the host immune system. Although glycoengineering approaches and glycocalyx-focused strategies have gained momentum, the mechanistic pathways through which immune cells decode glycan information remain underexplored in transplantation biology. This hybrid Perspective integrates selected mechanistic foundations with a broader conceptual framework that positions glycans as upstream immune checkpoints governing graft recognition and early innate–adaptive integration. We synthesize advances across four major axes of glycan-regulated immunity: Siglec (Sialic acid-binding immunoglobulin-type lectin)-mediated inhibitory circuits that calibrate macrophage, neutrophil, and NK-cell activation; C-type lectin receptor pathways that program antigen-presenting cells and govern antigen routing; NK-cell glycan-sensing mechanisms shaped by sialylation density, glycan topology, and ischemia–reperfusion–induced glycocalyx collapse; and complement regulation through Factor H, which interprets sialic acid motifs to restrain alternative pathway amplification. We further examine how these innate pathways intersect with glycan-dependent modulation of direct, indirect, and semi-direct allorecognition, including effects on MHC stability, exosomal transfer, antigen uptake, and T-cell intrinsic glycan checkpoints. Together, these mechanisms reveal that glycans function as a pre-recognition code that precedes and conditions classical protein-centric checkpoints by initiating, amplifying and sustaining the classical pathways, and influencing whether grafts are classified as self-like, stressed, or foreign. By consolidating these pathways into a unified model, this Perspective highlights glycan composition and architecture as a foundational design parameter for next-generation immune-compatible organ modifications and outlines mechanistic priorities for advancing glycan-informed strategies in transplantation.

The glycosylation of proteins endows them with distinct biophysical properties and allows them to play fundamental roles in cellular communication. Much of our understanding of glycoproteins has derived from the ability to enzymatically manipulate glycan structures. In particular, selective cleavage of glycans from proteins simplifies the analysis of glycoproteins and the determination of structure-activity relationships. However, limited enzymatic tools are available for the study of mucin-type O-glycans. To address this, we carried out the directed evolution of a glycoside hydrolase to increase its ability to cleave the sialyl T-antigen, a ubiquitous O-glycan structure in humans. We employed ultrahigh-throughput droplet-based microfluidics to rapidly screen vast libraries of variants in pL-sized droplets, thus minimizing the quantities of complex substrate required. Furthermore, by use of fluorescent protein-fusion and ratiometric gating during droplet sorting we could account for varying expression levels and identify highly active hits that could have been overlooked due to lower expression levels. Within just two rounds of screening, we uncovered variants with 840-fold enhancements in activity and new specificities compared to those of the WT enzyme. This campaign highlights the versatility of glycoside hydrolases and provides a broadly applicable strategy to engineer enzymatic tools for glycomics through microfluidic screening.

ABO-incompatible kidney transplantation is widely used to meet the escalating need for organs. Current recipient-centric desensitization protocols involving antibody depletion through plasmapheresis increase the risk of infections, perioperative bleeding events and costs. Here we present a donor-centric desensitization protocol, converting type-A kidneys into enzyme-converted O kidneys during hypothermic perfusion to remove the A antigen from the kidneys. An ex vivo model resulted in no antibody-mediated injury. Encouraged by this, an enzyme-converted O kidney was transplanted into a type-O brain-dead recipient with a high titre of anti-A antibody, and no hyperacute rejection was observed. The graft was well tolerated with no evidence of antibody-mediated rejection for 2 days. Antibody-mediated lesions and complement deposition were found starting 3 days post-transplant, coinciding with A-antigen regeneration, and later higher Banff scores, suggesting an immune-mediated response. Single-cell sequencing confirms the elevated expression of accommodation-related genes, suggesting the potential for longer-term tolerance. This study provides a donor-centric organ engineering strategy and has the potential to broaden the reach of ABO-incompatible kidney transplantation, improving the fairness of and access to organ allocation. An ex vivo model and pre-clinical study in a brain-dead recipient provide enzyme-converted O organs to avoid hyperacute rejection in ABO-incompatible kidney transplant patients.

We challenge the conclusion that the β-glucosidase in question directly catalyses hydrolysis of the substrate ester linkage. Rather we propose that this enzyme performs a normal glucoside hydrolysis and that the released aglycone undergoes rearrangement with formation of a quinone methide-like species through spontaneous cleavage of the ester.

Sulfation is a common, but poorly understood, post-glycosylational modification (PGM) used to modulate biological function. To deepen our understanding of the roles of various sulfated glycoforms and their relevant binding proteins, we must expand our enzymatic toolkit for their synthesis. Here, we bypass the need for both sulfotransferases and glycosyltransferases by engineering a series of mutants of a 6-SulfoGlcNAcase, from Streptococcus pneumoniae, to directly and efficiently synthesize not only the ubiquitous 6S-GlcNAc-β-1,3-Gal linkage prevalent within host glycans, but also the 6S-GlcNAc-β-1,6-GalNAc commonly observed within core-6 O-glycans, and the more exotic 6S-GlcNAc-β-1,4-GalNAc linkage. We further elaborate these into complex sulfated N-glycan and O-glycan structures of biological relevance. By utilizing the cost-effective activated donor pNP-6S-GlcNAc in conjunction with mutant GH185 6-SulfoGlcNAcases we demonstrate a simple yet powerful in vitro method for generating well-defined sulfated oligosaccharides and glycoforms for use in a variety of applications including glycan arrays, glycan remodeling, and specificity studies with carbohydrate binding proteins such as lectins.

The most common blood type (O) is based upon the H-antigen, which in its most common form (Type II) is fucosyl α-1,2-LacNAc. We here describe a decagram scale synthesis of this key trisaccharide and its conversion to various aryl and alkyl glycosides. These can then be used as substrates for enzymatic elaboration to the A and B antigens for use in plate-based high throughput screens for enzymes to convert blood types. We further demonstrate the use of such substrates in the assay of fucosidases that are active on the H antigen.

This review explores the evolving landscape of glycoengineering on the cell surface, a pivotal field in translational medicine with profound implications for immunomodulation and regenerative therapies. The cell membrane and glycocalyx, composed of proteins, lipids, and glycans, govern cellular interactions, immune recognition, and tissue regeneration. Given the dynamic nature and important biological roles of these structures, precise glycoengineering strategies are essential for modulating cell behavior without compromising function and safety. We delve into genetic and non-genetic approaches to manipulation of cell surface patterns, including targeted glycan and glycosaminoglycan modifications, metabolic glycoengineering, the use of synthetic glycopolymers, and bio-orthogonal glycan functionalization. Enzymatic strategies for glycan cleavage and addition further expand the toolkit for controlled cell surface remodeling. These advancements hold promise for cancer immunotherapy, organ transplantation, islet transplantation, and cardiovascular disease treatments. Despite the considerable potential of glycoengineering, a number of challenges, such as membrane instability and unintended cellular alterations remain, necessitating precise control over modifications. This review critically evaluates emerging strategies, discussing their limitations and future directions in biomedical and bioengineering applications. By integrating glycocalyx biology with advanced engineering techniques, we provide a roadmap for harnessing cell surface engineering to enhance therapeutic efficacy, immune modulation, biomaterial innovation, and regenerative medicine.

The vast majority of the glycosidases characterised so far follow one of the variations of the “Koshland” mechanisms to hydrolyse glycosidic bonds. Herein we describe a large-scale screen of a human gut microbiome metagenomic library using an assay that selectively identifies non-Koshland glycosidase activities. This screen led to identification of a commonly occurring cluster of enzymes with unprecedentedly broad substrate specificities that is thoroughly characterised, mechanistically and structurally. Not only do these enzymes break glycosidic linkages of both α and β stereochemistry and multiple connectivities, but also substrates that are not cleaved by standard glycosidases. These include thioglycosides such as glucosinolates and pseudo-glycosidic bonds of pharmaceuticals such as acarbose. This is achieved via a distinct mechanism of hydrolysis that involves stepwise oxidation, elimination and hydration steps, each catalysed by enzyme modules that are in many cases interchangeable between organisms and substrate classes. These appear to constitute a substantial alternative pathway for glycan degradation. ### Competing Interest Statement The authors have declared no competing interest.

Purpose: Our group reported an ex vivo ABO antigen removal technique that converts donor lung of ABO-A1 blood type to universal O type, which may allow safe ABO incompatible (ABOi) lung transplant (Wang A, Sci Transl Med 2022). The present study investigates the ABO-A1 genotype in its relation to the expression and regeneration of ABO antigen. We hypothesize that the heterozygous genotype is associated with lower antigen generating activity, thereby represent a lower risk patient group for future ABOi lung transplantation.

Enzymatic cleavage of the terminal alpha-N-acetylgalactosamine of the A-antigen of red blood cells by gut commensal organisms such as Flavonifractor plautii is achieved in a two-step process in which a specific deacetylase initially generates a terminal galactosamine residue, and then a second alpha-galactosaminidase cleaves the sugar to leave the H-antigen of O blood types. An interesting question concerns how such an enzyme avoids the electrostatic destabilization of the transition state that would be imposed by the protonated amine, leading us to a structural analysis. The three-dimensional structure of this GH36 enzyme, in conjunction with mechanistic studies, reveals that, while the enzyme adopts a very similar overall structure and double displacement mechanism to those of other members of glycoside hydrolase Clan-D, the active site now contains an essential cobalt ion, which coordinates the 2-position amine of the substrate. Moreover, two other metals (zinc and manganese) appear to play structural roles, while no other Clan-D glycosidases are known to show any metal dependence. Indeed, no other glycosidase is known for such a diverse metal ion binding requirement. Coordination of the substrate amine to cobalt will suppress its protonation at the operating pH of the enzyme, thereby bypassing the problem of electrostatic destabilization of the oxocarbenium ion-like transition state and allowing the reaction to proceed efficiently. This is an alternative solution to a mechanistic problem while maintaining an otherwise very similar active site.

Carbohydrate-active enzymes (CAZymes) are responsible for the biosynthesis, modification and degradation of all glycans in Nature. Advances in genomic and metagenomic methodologies, in conjunction with lower cost gene synthesis, have provided access to a steady stream of new CAZymes with both well-established and novel mechanisms. At the same time, increasing access to cryo-EM has resulted in exciting new structures, particularly of transmembrane glycosyltransferases of various sorts. This improved understanding has resulted in widespread progress in applications of CAZymes across diverse fields, including therapeutics, organ transplantation, foods, and biofuels. Herein, we highlight a few of the many important advances that have recently been made in the understanding and applications of CAZymes.

Abstract Introduction Recently, enzymatic blood group conversion has been used to convert human kidneys from blood group A to universal blood group O using bacterial enzymes from Flavonifractor plautii. However, little is known about the functional consequences of antigen removal when a treated kidney is transplanted in an ABO-incompatible (ABOi) recipient. Here, we investigated classical complement activation during ABOi conditions in enzyme treated (ABOe) vs control human type A kidneys. Method Three biological pairs of type A human kidneys rejected for transplantation and offered for research were used in this study. One kidney per pair was treated with 1mg/L of FpGalNAc deacetylase and FpGalactosaminidase during 6hrs of hypothermic machine perfusion while the contralateral kidney was perfused without enzymes. Both kidneys were subsequently perfused for 4hrs in ABOi conditions using normothermic machine perfusion (10% AB serum; mouse anti-A IgM titre 1:128; type O red blood cells). Tissue-bound components of classical complement activation were assessed in pre- and post-perfusion biopsies. Results Following ABOi perfusion, control kidneys showed peritubular deposition of the classical complement component C1qA and further complement activation components C4d and C5b-9. The staining directly co-localised with regions of anti-A staining. In ABOe kidneys, no anti-A staining was observed, with no C1qA, C4d, or C5b-9 staining found in post-perfusion biopsies. Conclusions We have shown for the first time that ABOe kidneys do not activate the classical complement pathway in ABOi conditions. This work shows the potential of the enzymatic blood group conversion to evade hyperacute antibody-mediated rejection during renal transplantation.

Building upon a previously established (2+3)-cycloaddition strategy, a series of N,N-dialkylated aminocyclopentanes was synthesized using a partially protected eno-furanose as the starting point. The resulting N-methylisoxazolidine was subsequently transformed into the corresponding aminocyclopentane, which was further N-alkylated, yielding a collection of compounds with potential as inhibitors and pharmacological chaperones of beta-d-glucocerebrosidase. A comprehensive screening involving a range of biologically relevant glycosidases unveiled that these compounds exhibit remarkable potency and selectivity as inhibitors of human lysosomal beta-d-glucocerebrosidase. However, none of these compounds exhibit significant activity enhancement of Morbus Gaucher related p.N409S/p.L483P mutant beta-d-glucocerebrosidase.

Carbohydrate-active enzymes (CAZymes) constitute a diverse set of enzymes that catalyze the assembly, degradation, and modification of carbohydrates. These enzymes have been fashioned into potent, selective catalysts by millennia of evolution, and yet are also highly adaptable and readily evolved in the laboratory. To identify and engineer CAZymes for different purposes, (ultra)high-throughput screening campaigns have been frequently utilized with great success. This review provides an overview of the different approaches taken in screening for CAZymes and how mechanistic understandings of CAZymes can enable new approaches to screening. Within, we also cover how cutting-edge techniques such as microfluidics, advances in computational approaches and synthetic biology, as well as novel assay designs are leading the field towards more informative and effective screening approaches.

ABO blood group compatibility restrictions present the first barrier to donor-recipient matching in kidney transplantation. Here, we present the use of two enzymes, FpGalNAc deacetylase and FpGalactosaminidase, from the bacterium Flavonifractor plautii to enzymatically convert blood group A antigens from the renal vasculature of human kidneys to 'universal' O-type. Using normothermic machine perfusion (NMP) and hypothermic machine perfusion (HMP) strategies, we demonstrate blood group A antigen loss of approximately 80% in as little as 2 h NMP and HMP. Furthermore, we show that treated kidneys do not bind circulating anti-A antibodies in an ex vivo model of ABO-incompatible transplantation and do not activate the classical complement pathway. This strategy presents a solution to the donor organ shortage crisis with the potential for direct clinical translation to reduce waiting times for patients with end stage renal disease. ABO blood group compatibility restrictions limit the availability of organs for patients awaiting transplantation. Here, the authors show the rapid enzymatic removal of blood group A antigens from the vasculature of human kidneys using normothermic and hypothermic machine perfusion technologies to make universal blood group O organs for transplantation.

In search of efficient alpha-galactosidases that can convert B red blood cells (RBCs) to universal type RBCs, we have developed a simple and robust system for ultrahigh-throughput droplet-based microfluidic screening. Here, a multienzyme coupled assay with a fluorogenic B antigen tetrasaccharide substrate is encapsulated within single emulsion water-in-oil droplets alongside single cells from metagenomic libraries. The resulting fluorescent droplets containing candidate B antigen cleaving enzymes are sorted using a commercially available, walk-up droplet sorting instrument before validation, cloning, and characterization of the hits. Using this approach, we identified and characterized an alpha-1,3-galactosidase (PvGH110) from the human gut microbiome capable of converting B-to-O-type RBCs. The simplicity, efficiency, and accessibility of our microfluidic-based system make it suitable for nonspecialist laboratories and offer a promising tool to discover enzymes that enable the generation of a universal O blood type.