Pseudomonas aeruginosa AlgF is a protein-protein interaction mediator required for acetylation of the alginate exopolysaccharide.

Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.

Absolute Affinities from Quantitative Shotgun Glycomics Using Concentration-Independent (COIN) Native Mass Spectrometry.

Native mass spectrometry (nMS) screening of natural glycan libraries against glycan-binding proteins (GBPs) is a powerful tool for ligand discovery. However, as the glycan concentrations are unknown, affinities cannot be measured directly from natural libraries. Here, we introduce Concentration-Independent (COIN)-nMS, which enables quantitative screening of natural glycan libraries by exploiting slow mixing of solutions inside a nanoflow electrospray ionization emitter. The affinities (Kd) of detected GBP-glycan interactions are determined, simultaneously, from nMS analysis of their time-dependent relative abundance changes. We establish the reliability of COIN-nMS using interactions between purified glycans and GBPs with known Kd values. We also demonstrate the implementation of COIN-nMS using the catch-and-release (CaR)-nMS assay for glycosylated GBPs. The COIN-CaR-nMS results obtained for plant, fungal, viral, and human lectins with natural libraries containing hundreds of N-glycans and glycopeptides highlight the assay's versatility for discovering new ligands, precisely measuring their affinities, and uncovering "fine" specificities. Notably, the COIN-CaR-nMS results clarify the sialoglycan binding properties of the SARS-CoV-2 receptor binding domain and establish the recognition of monosialylated hybrid and biantennary N-glycans. Moreover, pharmacological depletion of host complex N-glycans reduces both pseudotyped virions and SARS-CoV-2 cell entry, suggesting that complex N-glycans may serve as attachment factors.

Native Mass Spectrometry Quantitation of α2-3-Linked N-Acetylneuraminic Acid Content of Prostate-Specific Antigen: An Accurate Liquid Biopsy for Clinically Significant Prostate Cancer.

Application of the prostate-specific antigen (PSA) test, which measures PSA levels in blood, is standard in prostate cancer (PCa) screening. However, because PSA levels may be elevated for reasons other than PCa, it leads to high rates of misdiagnosis and overtreatment. Recently, alteration in the N-glycan sialylation of PSA, specifically increased levels of α2-3-linked N-acetylneuraminic acid (α2-3-Neu5Ac or α2-3-sialic acid), was identified as a potential biomarker for clinically significant PCa. Here, we introduce a robust top-down native mass spectrometry (MS) approach, performed using a combination of α2-3-Neu5Ac-specific and nonspecific neuraminidases and employing center-of-mass monitoring (CoMMon), for quantifying the levels of α2-3-Neu5Ac as a fraction of total N-linked Neu5Ac present on PSA extracted from blood serum. To illustrate the potential of the assay for clinical diagnosis and disease staging of PCa, the percentages of α2-3-Neu5Ac on PSA (%α23PSA) in the serum of low-grade (International Society of Urological Pathology Grade Group/GG1), intermediate-grade (GG2), and high-grade (GG3,4,5) PCa individuals were measured. We observed a high sensitivity (85.5%) and specificity (84.6%) for discrimination of GG1 from clinically significant GG2-5 patients when using a %α23PSA test cut-off of 28.0%. Our results establish that the %α23PSA in blood serum PSA, which can be precisely measured in a non-invasive manner with our dual neuraminidase native MS/CoMMon assay, can discriminate between clinically significant PCa (GG2-5) and low-grade PCa (GG1). Such discrimination has not been previously achieved and represents an important clinical need. This assay could greatly improve the standard PSA test and serve as a valuable PCa diagnostic tool.

Siglec-6 mediates the uptake of extracellular vesicles through a noncanonical glycolipid binding pocket.

Immunomodulatory Siglecs are controlled by their glycoprotein and glycolipid ligands. Siglec-glycolipid interactions are often studied outside the context of a lipid bilayer, missing the complex behaviors of glycolipids in a membrane. Through optimizing a liposomal formulation to dissect Siglec-glycolipid interactions, it is shown that Siglec-6 can recognize glycolipids independent of its canonical binding pocket, suggesting that Siglec-6 possesses a secondary binding pocket tailored for recognizing glycolipids in a bilayer. A panel of synthetic neoglycolipids is used to probe the specificity of this glycolipid binding pocket on Siglec-6, leading to the development of a neoglycolipid with higher avidity for Siglec-6 compared to natural glycolipids. This neoglycolipid facilitates the delivery of liposomes to Siglec-6 on human mast cells, memory B-cells and placental syncytiotrophoblasts. A physiological relevance for glycolipid recognition by Siglec-6 is revealed for the binding and internalization of extracellular vesicles. These results demonstrate a unique and physiologically relevant ability of Siglec-6 to recognize glycolipids in a membrane.

Structure of the AlgKX modification and secretion complex required for alginate production and biofilm attachment in Pseudomonas aeruginosa.

Synthase-dependent secretion systems are a conserved mechanism for producing exopolysaccharides in Gram-negative bacteria. Although widely studied, it is not well understood how these systems are organized to coordinate polymer biosynthesis, modification, and export across both membranes and the peptidoglycan. To investigate how synthase-dependent secretion systems produce polymer at a molecular level, we determined the crystal structure of the AlgK-AlgX (AlgKX) complex involved in Pseudomonas aeruginosa alginate exopolysaccharide acetylation and export. We demonstrate that AlgKX directly binds alginate oligosaccharides and that formation of the complex is vital for polymer production and biofilm attachment. Finally, we propose a structural model for the AlgEKX outer membrane modification and secretion complex. Together, our study provides insight into how alginate biosynthesis proteins coordinate production of a key exopolysaccharide involved in establishing persistent Pseudomonas lung infections.

How Choice of Model Membrane Affects Protein-Glycosphingolipid Interactions: Insights from Native Mass Spectrometry.

Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) are involved in numerous physiological and pathophysiological processes. Many model membrane systems are available for studying GBP-GSL interactions, but a systematic investigation has not been carried out on how the nature of the model membrane affects binding. In this work, we use electrospray ionization mass spectrometry (ESI-MS), both direct and competitive assays, to measure the binding of cholera toxin B subunit homopentamer (CTB5) to GM1 ganglioside in liposomes, bilayer islands [styrene maleic acid lipid particles (SMALPs), nanodiscs (NDs), and picodiscs (PDs)], and micelles. We find that direct ESI-MS analysis of CTB5 binding to GM1 is unreliable due to non-uniform response factors, incomplete extraction of bound GM1 in the gas phase, and nonspecific CTB5-GM1 interactions. Conversely, indirect proxy ligand ESI-MS measurements show that the intrinsic (per binding site) association constants of CTB5 for PDs, NDs, and SMALPs are similar and comparable to the affinity of soluble GM1 pentasaccharide (GM1os). The observed affinity decreases with increasing GM1 content due to molecular crowding stemming from GM1 clustering. Unlike the smaller model membranes, the observed affinity of CTB5 toward GM1 liposomes is ∼10-fold weaker than GM1os and relatively insensitive to the GM1 content. GM1 glycomicelles exhibit the lowest affinity, ∼35-fold weaker than GM1os. Together, the results highlight experimental design considerations for quantitative GBP-GSL binding studies involving multisubunit GBPs and factors to consider when comparing results obtained with different membrane systems. Notably, they suggest that bilayer islands with a low percentage of GSL, wherein clustering is minimized, are ideal for assessing intrinsic strength of GBP-GSL interactions in a membrane environment, while binding to liposomes, which is sub-optimal due to extensive clustering, may be more representative of authentic cellular environments.

The retaining ß-Kdo glycosyltransferase WbbB uses a double-displacement mechanism with an intermediate adduct rearrangement step.

WbbB, a lipopolysaccharide O-antigen synthesis enzyme from Raoultella terrigena, contains an N-terminal glycosyltransferase domain with a highly modified architecture that adds a terminal ß-Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) residue to the O-antigen saccharide, with retention of stereochemistry. We show, using mass spectrometry, that WbbB forms a covalent adduct between the catalytic nucleophile, Asp232, and Kdo. We also determine X-ray structures for the CMP-ß-Kdo donor complex, for Kdo-adducts with D232N and D232C WbbB variants, for a synthetic disaccharide acceptor complex, and for a ternary complex with both a Kdo-adduct and the acceptor. Together, these structures show that the enzyme-linked Asp232-Kdo adduct rotates to reposition the Kdo into a second sub-site, which then transfers Kdo to the acceptor. Retaining glycosyltransferases were thought to use only the front-side SNi substitution mechanism; here we show that retaining glycosyltransferases can also potentially use double-displacement mechanisms, but incorporating an additional catalytic subsite requires rearrangement of the protein's architecture.

Mass spectrometry-based shotgun glycomics for discovery of natural ligands of glycan-binding proteins.

The non-covalent associations of complex carbohydrates (glycans) with glycan-binding proteins mediate many important physiological and pathophysiological processes. Identifying these interactions is essential to understanding their diverse biological functions and enables the development of new disease treatments and diagnostics. Knowledge of the repertoire of glycans recognized by most glycan-binding proteins and their affinities is incomplete. Mass spectrometry-based screening of natural glycan libraries has emerged as a promising approach to defining the glycan interactome of glycan-binding proteins. Here, we review recent advances in mass spectrometry-based natural library screening that have led to the discovery of glycan ligands of endogenous and exogenous proteins and illuminated their binding specificities.

The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing.

The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.

Quantifying Biomolecular Interactions Using Slow Mixing Mode (SLOMO) Nanoflow ESI-MS.

Electrospray ionization mass spectrometry (ESI-MS) is a powerful label-free assay for detecting noncovalent biomolecular complexes in vitro and is increasingly used to quantify binding thermochemistry. A common assumption made in ESI-MS affinity measurements is that the relative ion signals of free and bound species quantitatively reflect their relative concentrations in solution. However, this is valid only when the interacting species and their complexes have similar ESI-MS response factors (RFs). For many biomolecular complexes, such as protein-protein interactions, this condition is not satisfied. Existing strategies to correct for nonuniform RFs are generally incompatible with static nanoflow ESI (nanoESI) sources, which are typically used for biomolecular interaction studies, thereby significantly limiting the utility of ESI-MS. Here, we introduce slow mixing mode (SLOMO) nanoESI-MS, a direct technique that allows both the RF and affinity (K d) for a biomolecular interaction to be determined from a single measurement using static nanoESI. The approach relies on the continuous monitoring of interacting species and their complexes under nonhomogeneous solution conditions. Changes in ion signals of free and bound species as the system approaches or moves away from a steady-state condition allow the relative RFs of the free and bound species to be determined. Combining the relative RF and the relative abundances measured under equilibrium conditions enables the K d to be calculated. The reliability of SLOMO and its ease of use is demonstrated through affinity measurements performed on peptide-antibiotic, protease-protein inhibitor, and protein oligomerization systems. Finally, affinities measured for the binding of human and bacterial lectins to a nanobody, a viral glycoprotein, and glycolipids displayed within a model membrane highlight the tremendous power and versatility of SLOMO for accurately quantifying a wide range of biomolecular interactions important to human health and disease.

CRISPR-Click Enables Dual-Gene Editing with Modular Synthetic sgRNAs.

Gene-editing systems such as CRISPR-Cas9 readily enable individual gene phenotypes to be studied through loss of function. However, in certain instances, gene compensation can obfuscate the results of these studies, necessitating the editing of multiple genes to properly identify biological pathways and protein function. Performing multiple genetic modifications in cells remains difficult due to the requirement for multiple rounds of gene editing. While fluorescently labeled guide RNAs (gRNAs) are routinely used in laboratories for targeting CRISPR-Cas9 to disrupt individual loci, technical limitations in single gRNA (sgRNA) synthesis hinder the expansion of this approach to multicolor cell sorting. Here, we describe a modular strategy for synthesizing sgRNAs where each target sequence is conjugated to a unique fluorescent label, which enables fluorescence-activated cell sorting (FACS) to isolate cells that incorporate the desired combination of gene-editing constructs. We demonstrate that three short strands of RNA functionalized with strategically placed 5'-azide and 3'-alkyne terminal deoxyribonucleotides can be assembled in a one-step, template-assisted, copper-catalyzed alkyne-azide cycloaddition to generate fully functional, fluorophore-modified sgRNAs. Using these synthetic sgRNAs in combination with FACS, we achieved selective cleavage of two targeted genes, either separately as a single-color experiment or in combination as a dual-color experiment. These data indicate that our strategy for generating double-clicked sgRNA allows for Cas9 activity in cells. By minimizing the size of each RNA fragment to 41 nucleotides or less, this strategy is well suited for custom, scalable synthesis of sgRNAs.

Mass Spectrometry-Based Shotgun Glycomics Using Labeled Glycan Libraries.

Mass spectrometry-based shotgun glycomics (MS-SG) is a rapid, sensitive, label-, and immobilization-free approach for the discovery of natural ligands of glycan-binding proteins (GBPs). To perform MS-SG, natural libraries of glycans derived from glycoconjugates in cells or tissues are screened against a target GBP using catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS). Because glycan concentrations are challenging to determine, ligand affinities cannot be directly measured. In principle, relative affinities can be ranked by combining CaR-ESI-MS data with relative concentrations established by hydrophilic interaction liquid chromatography (HILIC) performed on the fluorophore-labeled glycan library. To validate this approach, as well as the feasibility of performing CaR-ESI-MS directly on labeled glycans, libraries of labeled N-glycans extracted from the human monocytic U937 cells or intestinal tissues were labeled with 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), or procainamide (proA). The libraries were screened against plant and human GBPs with known specificities for α2-3- and α2-6-linked sialosides and quantified by HILIC. Dramatic differences, in some cases, were found for affinity rankings obtained with libraries labeled with different fluorophores, as well as those produced using the combined unlabeled/labeled library approach. The origin of these differences could be explained by differential glycan labeling efficiencies, the impact of specific labels on glycan affinities for the GBPs, and the relative efficiency of release of ligands from GBPs in CaR-ESI-MS. Overall, the results of this study suggest that the 2-AB(CaR-ESI-MS)/2-AB(HILIC) combination provides the most reliable description of the binding specificities of GBPs for N-glycans and is recommended for MS-SG applications.

Sialic acid-containing glycolipids mediate binding and viral entry of SARS-CoV-2.

Emerging evidence suggests that host glycans influence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we reveal that the receptor-binding domain (RBD) of the spike (S) protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (Sia), with preference for monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind to the RBD. The monomeric affinities (Kd = 100-200 µM) of gangliosides for the RBD are similar to another negatively charged glycan ligand of the RBD proposed as a viral co-receptor, heparan sulfate (HS) dp2-dp6 oligosaccharides. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to angiotensin-converting enzyme 2 (ACE2)-expressing cells is decreased following depletion of cell surface Sia levels using three approaches: sialyltransferase (ST) inhibition, genetic knockout of Sia biosynthesis, or neuraminidase treatment. These effects on RBD binding and both pseudotyped and authentic SARS-CoV-2 viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

Quantifying Carbohydrate-Active Enzyme Activity with Glycoprotein Substrates Using Electrospray Ionization Mass Spectrometry and Center-of-Mass Monitoring.

Carbohydrate-active enzymes (CAZymes) play critical roles in diverse physiological and pathophysiological processes and are important for a wide range of biotechnology applications. Kinetic measurements offer insight into the activity and substrate specificity of CAZymes, information that is of fundamental interest and supports diverse applications. However, robust and versatile kinetic assays for monitoring the kinetics of intact glycoprotein and glycolipid substrates are lacking. Here, we introduce a simple but quantitative electrospray ionization mass spectrometry (ESI-MS) method for measuring the kinetics of CAZyme reactions involving glycoprotein substrates. The assay, referred to as center-of-mass (CoM) monitoring (CoMMon), relies on continuous (real-time) monitoring of the CoM of an ensemble of glycoprotein substrates and their corresponding CAZyme products. Notably, there is no requirement for calibration curves, internal standards, labeling, or mass spectrum deconvolution. To demonstrate the reliability of CoMMon, we applied the method to the neuraminidase-catalyzed cleavage of N-acetylneuraminic acid (Neu5Ac) residues from a series of glycoproteins of varying molecular weights and degrees of glycosylation. Reaction progress curves and initial rates determined with CoMMon are in good agreement (initial rates within ≤5%) with results obtained, simultaneously, using an isotopically labeled Neu5Ac internal standard, which enabled the time-dependent concentration of released Neu5Ac to be precisely measured. To illustrate the applicability of CoMMon to glycosyltransferase reactions, the assay was used to measure the kinetics of sialylation of a series of asialo-glycoproteins by a human sialyltransferase. Finally, we show how combining CoMMon and the competitive universal proxy receptor assay enables the relative reactivity of glycoprotein substrates to be quantitatively established.

Submicron Emitters Enable Reliable Quantification of Weak Protein-Glycan Interactions by ESI-MS.

Interactions between carbohydrates (glycans) and glycan-binding proteins (GBPs) regulate a wide variety of important biological processes. However, the affinities of most monovalent glycan-GBP complexes are typically weak (dissociation constant (Kd) > µM) and difficult to reliably measure with conventional assays; consequently, the glycan specificities of most GBPs are not well established. Here, we demonstrate how electrospray ionization mass spectrometry (ESI-MS), implemented with nanoflow ESI emitters with inner diameters of ∼50 nm, allows for the facile quantification of low-affinity glycan-GBP interactions. The small size of the droplets produced from these submicron emitters effectively eliminates the formation of nonspecific glycan-GBP binding (false positives) during the ESI process up to ∼mM glycan concentrations. Thus, interactions with affinities as low as ∼5 mM can be measured directly from the mass spectrum. The general suppression of nonspecific adducts (including nonvolatile buffers and salts) achieved with these tips enables ESI-MS glycan affinity measurements to be performed on C-type lectins, a class of GBPs that bind glycans in a calcium-dependent manner and are important regulators of immune response. At physiologically relevant calcium ion concentrations (2-3 mM), the extent of Ca2+ nonspecific adduct formation observed using the submicron emitters is dramatically suppressed, allowing glycan affinities, and the influence of Ca2+ thereon, to be measured. Finally, we show how the use of submicron emitters and suppression of nonspecific binding enable the quantification of labile (prone to in-source dissociation) glycan-GBP interactions.

A versatile soluble siglec scaffold for sensitive and quantitative detection of glycan ligands.

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are immunomodulatory receptors that are regulated by their glycan ligands. The connections between Siglecs and human disease motivate improved methods to detect Siglec ligands. Here, we describe a new versatile set of Siglec-Fc proteins for glycan ligand detection. Enhanced sensitivity and selectivity are enabled through multimerization and avoiding Fc receptors, respectively. Using these Siglec-Fc proteins, Siglec ligands are systematically profiled on healthy and cancerous cells and tissues, revealing many unique patterns. Additional features enable the production of small, homogenous Siglec fragments and development of a quantitative ligand-binding mass spectrometry assay. Using this assay, the ligand specificities of several Siglecs are clarified. For CD33 (Siglec-3), we demonstrate that it recognizes both α2-3 and α2-6 sialosides in solution and on cells, which has implications for its link to Alzheimer's disease susceptibility. These soluble Siglecs reveal the abundance of their glycan ligands on host cells as self-associated molecular patterns.

Mass Spectrometry-Based Shotgun Glycomics for Discovery of Natural Ligands of Glycan-Binding Proteins.

Glycans attached to lipids and membrane-bound and secreted proteins and peptides mediate many important physiological and pathophysiological processes through interactions with glycan-binding proteins (GBPs). However, uncovering functional glycan ligands is challenging due to the large number of naturally occurring glycan structures, the limited availability of glycans in their purified form, the low affinities of GBP-glycan interactions, and limitations in existing binding assays. This work explores the application of catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) for screening libraries of N-glycans derived from natural sources. The assay was tested by screening a small-defined library of complex N-glycans at equimolar concentrations against plant and human GBPs with known specificities for either α2-3- or α2-6-linked sialosides, with affinities in the millimolar to micromolar range. Validation experiments, performed in negative ion mode, revealed that bound N-glycan ligands are readily released, as intact deprotonated ions, from GBPs in the gas phase using collision-induced dissociation. Moreover, the relative abundances of the released ligands closely match their solution affinities. The results obtained for a natural N-glycan library produced from cultured immune cells serve to highlight the ease with which CaR-ESI-MS can screen complex mixtures of N-glycans for interactions. Additionally, scaling the relative abundances of released glycan ligands according to their relative abundances in solution, as determined by hydrophilic interaction-ultrahigh-performance liquid chromatography of the fluorescently labeled library, allows the relative affinities of glycan ligands to be ranked.

Neoglycolipids as Glycosphingolipid Surrogates for Protein Binding Studies Using Nanodiscs and Native Mass Spectrometry.

Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) in the membranes of cells are implicated in a wide variety of normal and pathophysiological processes. Despite the critical biological roles these interactions play, the GSL ligands of most GBPs have not yet been identified. The limited availability of purified GSLs represents a significant challenge to the discovery and characterization of biologically relevant GBP-GSL interactions. The present work investigates the use of neoglycolipids (NGLs) as surrogates for GSLs for catch-and-release-electrospray ionization mass spectrometry (CaR-ESI-MS)-based screening, implemented with nanodiscs, for the discovery of GSL ligands. Three pairs of NGLs based on the blood group type A and B trisaccharides, with three different lipid head groups but all with "ring-closed" monosaccharide residue at the reducing end, were synthesized. The incorporation efficiencies (into nanodiscs) of the NGLs and their affinities for a fragment of family 51 carbohydrate-binding module (CBM) identified an amide-linked 1,3-di-O-hexadecyl-glycerol moiety as the optimal lipid structure. Binding measurements performed on cholera toxin B subunit homopentamer (CTB5) and nanodiscs containing an NGL consisting of the optimal lipid moiety and the GM1 ganglioside pentasaccharide yielded affinities similar, within a factor of 2, to those of native GM1. Finally, nanodiscs containing the optimal A and B trisaccharide NGLs, as well as the corresponding NGLs of lactose, A type 2 tetrasaccharide, and the GM1 and GD2 pentasaccharides were screened against the family 51 CBM, human galectin-7, and CTB5 to illustrate the potential of NGLs to accelerate the discovery of GSL ligands of GBPs.

Structural and biochemical characterization of the exopolysaccharide deacetylase Agd3 required for Aspergillus fumigatus biofilm formation.

The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Deletion of a gene encoding a putative deacetylase, Agd3, leads to defects in GAG deacetylation, biofilm formation, and virulence. Here, we show that Agd3 deacetylates GAG in a metal-dependent manner, and is the founding member of carbohydrate esterase family CE18. The active site is formed by four catalytic motifs that are essential for activity. The structure of Agd3 includes an elongated substrate-binding cleft formed by a carbohydrate binding module (CBM) that is the founding member of CBM family 87. Agd3 homologues are encoded in previously unidentified putative bacterial exopolysaccharide biosynthetic operons and in other fungal genomes.