CRISPR screens and lectin microarrays identify high mannose N-glycan regulators.

Glycans play critical roles in cellular signaling and function. Unlike proteins, glycan structures are not templated from genetic sequences but synthesized by the concerted activity of many genes, making them historically challenging to study. Here, we present a strategy that utilizes CRISPR screens and lectin microarrays to uncover and characterize regulators of glycosylation.

The liquid lectin array detects compositional glycocalyx differences using multivalent DNA-encoded lectins on phage.

Selective detection of disease-associated changes in the glycocalyx is an emerging field in modern targeted therapies. Detecting minor glycan changes on the cell surface is a challenge exacerbated by the lack of correspondence between cellular DNA/RNA and glycan structures. We demonstrate that multivalent displays of lectins on DNA-barcoded phages-liquid lectin array (LiLA)-detect subtle differences in density of glycans on cells.

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.

An enantioselective and modular platform for C4'-modified nucleoside analogue synthesis enabled by intramolecular trans-acetalizations.

C4'-modified nucleoside analogues continue to attract global attention for their use in antiviral drug development and oligonucleotide-based therapeutics. However, current approaches to C4'-modified nucleoside analogues still involve lengthy (9-16 steps), non-modular routes that are unamenable to library synthesis. Towards addressing the challenges associated with their syntheses, we report a modular 5-step process to a diverse collection of C4'-modified nucleoside analogues through a sequence of intramolecular trans-acetalizations of readily assembled polyhydroxylated frameworks.

HIV-1 interaction with an -glycan-specific bacterial lectin enhances virus infectivity and resistance to neutralizing antibodies.

Bacteria dysbiosis and its accompanying inflammation or compromised mucosal integrity is associated with an increased risk of HIV-1 transmission. However, HIV-1 may also bind bacteria or bacterial products to impact infectivity and transmissibility. This study evaluated HIV-1 interactions with bacteria through glycan-binding lectins.

Patient subtyping analysis of baseline multi-omic data reveals distinct pre-immune states associated with antibody response to seasonal influenza vaccination.

Understanding the molecular mechanisms underpinning diverse vaccination responses is critical for developing efficient vaccines. Molecular subtyping can offer insights into heterogeneous nature of responses and aid in vaccine design. We analyzed multi-omic data from 62 haemagglutinin seasonal influenza vaccine recipients (2019-2020), including transcriptomics, proteomics, glycomics, and metabolomics data collected pre-vaccination.

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.

Infection and the Glycome─New Insights into Host Response.

Glycans play critical roles in the host-pathogen interactions leading to infection. However, we still understand very little about the dynamic nature of glycosylation in response to infection and its function in modulating host immunity. Many of the host proteins involved in immune defense are glycoproteins.

Integrated functional screens and multi-omics analyses identify α-2,3-sialylation as essential for melanoma maintenance.

Glycosylation is a hallmark of cancer biology, and altered glycosylation influences multiple facets of melanoma growth and progression. To identify glycosyltransferases, glycans, and glycoproteins essential for melanoma maintenance, we conducted an growth screen with a pooled shRNA library of glycosyltransferases, lectin microarray profiling of benign nevi and melanoma patient samples, and mass spectrometry-based glycoproteomics. We found that α-2,3 sialyltransferases ST3GAL1 and ST3GAL2 and corresponding α-2,3-linked sialosides are upregulated in melanoma compared to nevi and are essential for melanoma growth and .

CRISPR screens and lectin microarrays identify novel high mannose N-glycan regulators.

Glycans play critical roles in cellular signaling and function. Unlike proteins, glycan structures are not templated from genes but the concerted activity of many genes, making them historically challenging to study. Here, we present a strategy that utilizes pooled CRISPR screens and lectin microarrays to uncover and characterize regulators of cell surface glycosylation.

The microenvironment dictates glycocalyx construction and immune surveillance.

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with models that do not match the microenvironmental characteristics of human tissues. Using models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium.

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.

Influenza Defective Interfering Virus Promotes Multiciliated Cell Differentiation and Reduces the Inflammatory Response in Mice.

Influenza defective interfering (DI) viruses have long been considered promising antiviral candidates because of their ability to interfere with replication-competent viruses and induce antiviral immunity. However, the mechanisms underlying DI-mediated antiviral immunity have not been extensively explored. Here, we demonstrated the interferon (IFN)-independent protection conferred by the influenza DI virus against homologous virus infection in mice deficient in type I and III IFN signaling.

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.

HIV-1 interaction with an -glycan-specific bacterial lectin enhances virus infectivity and resistance to neutralization by antibodies.

Bacteria dysbiosis has been associated with an increased risk of HIV-1 transmission and acquisition. The prevalent idea is that bacteria dysbiosis compromises mucosal integrity and promotes inflammatory conditions to cause recruitment and activation of immune cells that harbor or are targeted by HIV-1. However, it is also possible that HIV-1 directly binds bacteria or bacterial products to impact virus infectivity and transmissibility.

High-Throughput Analysis Reveals miRNA Upregulating α-2,6-Sialic Acid through Direct miRNA-mRNA Interactions.

Chemical biology has revealed the importance of sialic acids as a major signal in physiology and disease. The terminal modification α-2,6-sialic acid is controlled by the enzymes ST6GAL1 and ST6GAL2. Dysregulation of this glycan impacts immunological recognition and cancer development.

α2,6-Sialylation Is Upregulated in Severe COVID-19, Implicating the Complement Cascade.

Better understanding of the molecular mechanisms underlying COVID-19 severity is desperately needed in current times. Although hyper-inflammation drives severe COVID-19, precise mechanisms triggering this cascade and what role glycosylation might play therein are unknown. Here we report the first high-throughput glycomic analysis of COVID-19 plasma samples and autopsy tissues.

Deep learning explains the biology of branched glycans from single-cell sequencing data.

Glycosylation is ubiquitous and often dysregulated in disease. However, the regulation and functional significance of various types of glycosylation at cellular levels is hard to unravel experimentally. Multi-omics, single-cell measurements such as SUGAR-seq, which quantifies transcriptomes and cell surface glycans, facilitate addressing this issue.

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.

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).

SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation.

Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a mechanism by which the SARS-CoV-2 virus coopts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-β. We reveal that the SARS-CoV-2 encoded nonstructural protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein.

Prevaccination Glycan Markers of Response to an Influenza Vaccine Implicate the Complement Pathway.

A key to improving vaccine design and vaccination strategy is to understand the mechanism behind the variation of vaccine response with host factors. Glycosylation, a critical modulator of immunity, has no clear role in determining vaccine responses. To gain insight into the association between glycosylation and vaccine-induced antibody levels, we profiled the pre- and postvaccination serum protein glycomes of 160 Caucasian adults receiving the FLUZONE influenza vaccine during the 2019-2020 influenza season using lectin microarray technology.

α2,6-Sialylation is Upregulated in Severe COVID-19 Implicating the Complement Cascade.

Better understanding of the mechanisms of COVID-19 severity is desperately needed in current times. Although hyper-inflammation drives severe COVID-19, precise mechanisms triggering this cascade and what role glycosylation might play therein is unknown. Here we report the first high-throughput glycomic analysis of COVID-19 plasma samples and autopsy tissues.

Glycomic Analysis Reveals a Conserved Response to Bacterial Sepsis Induced by Different Bacterial Pathogens.

Sepsis is an extreme inflammatory response to infection that occurs in the bloodstream and causes damage throughout the body. Glycosylation is known to play a role in immunity and inflammation, but the role of glycans in sepsis is not well-defined. Herein, we profiled the serum glycomes of experimental mouse sepsis models to identify changes induced by 4 different clinical bacterial pathogens (Gram-positive: and , Gram-negative: and Typhimurium) using our lectin microarray technology.