Deciphering Pathways and Thermodynamics of Protein Assembly Using Native Mass Spectrometry.

Protein oligomerization regulates many critical physiological processes, and its dysregulation can contribute to dysfunction and diseases. Elucidating the assembly pathways and quantifying their underlying thermodynamic and kinetic parameters are crucial for a comprehensive understanding of biological processes and for advancing therapeutics targeting abnormal protein oligomerization. Established binding assays, with limited mass precision, often rely on simplified models for data interpretation.

Elusive Protein-Glycosphingolipid Interactions Revealed by Membrane Anchor-Assisted Native Mass Spectrometry.

Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) present in cell membranes are implicated in a wide range of biological processes. However, studying GSL binding is hindered by the paucity of purified GSLs and the weak affinities typical of monovalent GBP-GSL interactions. Native mass spectrometry (nMS) performed using soluble model membranes is a promising approach for the discovery of GBP ligands, but the detection of weak interactions remains challenging.

Dissecting the abilities of murine Siglecs to interact with gangliosides.

Siglecs are cell surface receptors whose functions are tied to the binding of their sialoglycan ligands. Recently, we developed an optimized liposome formulation and used it to investigate the binding of human Siglecs (hSiglec) against a panel of gangliosides. Animal models, more specifically murine models, are used to understand human biology; however, species-specific differences can complicate the interpretation of the results.

Role of N343 glycosylation on the SARS-CoV-2 S RBD structure and co-receptor binding across variants of concern.

Glycosylation of the SARS-CoV-2 spike (S) protein represents a key target for viral evolution because it affects both viral evasion and fitness. Successful variations in the glycan shield are difficult to achieve though, as protein glycosylation is also critical to folding and structural stability. Within this framework, the identification of glycosylation sites that are structurally dispensable can provide insight into the evolutionary mechanisms of the shield and inform immune surveillance.

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.

Chemoenzymatic synthesis of genetically-encoded multivalent liquid N-glycan arrays.

Cellular glycosylation is characterized by chemical complexity and heterogeneity, which is challenging to reproduce synthetically. Here we show chemoenzymatic synthesis on phage to produce a genetically-encoded liquid glycan array (LiGA) of complex type N-glycans. Implementing the approach involved by ligating an azide-containing sialylglycosyl-asparagine to phage functionalized with 50-1000 copies of dibenzocyclooctyne.

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 ncentration-dependent (COIN)-nMS, which enables quantitative screening of natural glycan libraries by exploiting slow mixing of solutions inside a nanoflow electrospray ionization emitter.

Native Mass Spectrometry Quantitation of α2-3-Linked -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 -glycan sialylation of PSA, specifically increased levels of α2-3-linked -acetylneuraminic acid (α2-3-Neu5Ac or α2-3-sialic acid), was identified as a potential biomarker for clinically significant PCa.

The αTSR Domain of Circumsporozoite Protein Bound Heparan Sulfates and Elicited High Titers of Sporozoite Binding Antibody After Displayed by Nanoparticles.

Introduction: Malaria is a devastating infectious illness caused by protozoan parasites. The circumsporozoite protein (CSP) on sporozoites binds heparan sulfate proteoglycan (HSPG) receptors for liver invasion, a critical step for prophylactic and therapeutic interventions.

Methods: In this study, we characterized the αTSR domain that covers region III and the thrombospondin type-I repeat (TSR) of the CSP using various biochemical, glycobiological, bioengineering, and immunological approaches.

SARS-CoV-2 Spike N-Terminal Domain Engages 9--Acetylated α2-8-Linked Sialic Acids.

SARS-CoV-2 viruses engage ACE2 as a functional receptor with their spike protein. The S1 domain of the spike protein contains a C-terminal receptor binding domain (RBD) and an N-terminal domain (NTD). The NTD of other coronaviruses includes a glycan binding cleft.

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.

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.

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 (CTB) to GM1 ganglioside in liposomes, bilayer islands [styrene maleic acid lipid particles (SMALPs), nanodiscs (NDs), and picodiscs (PDs)], and micelles.

The retaining β-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 β-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-β-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.

The SARS-CoV-2 spike N-terminal domain engages 9- -acetylated α2-8-linked sialic acids.

Unlabelled: SARS-CoV-2 viruses engage ACE2 as a functional receptor with their spike protein. The S1 domain of the spike protein contains a C-terminal receptor-binding domain (RBD) and an N-terminal domain (NTD). The NTD of other coronaviruses includes a glycan-binding cleft.

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.

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.

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 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).

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.

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.

Fucosylated Human Milk Oligosaccharide Foraging within the Species Bifidobacterium pseudocatenulatum Is Driven by Glycosyl Hydrolase Content and Specificity.

Human milk enriches members of the genus in the infant gut. One species, Bifidobacterium pseudocatenulatum, is found in the gastrointestinal tracts of adults and breastfed infants. In this study, B.

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.

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.