Oxidative Release of Natural Glycans: Unraveling the Mechanism for Rapid N-Glycan Glycomics Analysis.

N-glycosylation is a critical post-translational modification involved in various biosynthetic pathways and disease mechanisms. In this study, we present an optimized oxidative release of natural glycans (ORNG) method using household bleach that enables the rapid and efficient release of N-glycans from biological samples. We thoroughly investigated the ORNG mechanism, identifying key intermediates and side products and providing valuable insights into the oxidative release process.

A Versatile One-Step Enzymatic Strategy for Efficient Imaging and Mapping of Tumor-Associated Tn Antigen.

Tn antigen (CD175), recognized as the precursor monosaccharide (α-GalNAc) of mucin O-glycan, is a well-known tumor-associated carbohydrate antigen (TACA). It has emerged as a potential biomarker for cancer diagnosis and prognosis. However, the role it plays in cancer biology remains elusive due to the absence of a sensitive and selective detection method.

Integrated analysis of natural glycans using a versatile pyrazolone-type heterobifunctional tag ANPMP.

Glycans mediate various biological processes through carbohydrate-protein interactions, and glycan microarrays have become indispensable tools for understanding these mechanisms. However, advances in functional glycomics are hindered by the absence of convenient and universal methods for obtaining natural glycan libraries with diverse structures from glycoconjugates. To address this challenge, we have developed an integrative approach that enables one-pot release and simultaneously capture, separation, structural characterization, and functional analysis of N/O-glycans.

Serum glycomic profile as a predictive biomarker of recurrence in patients with differentiated thyroid cancer.

Purpose: Thyroid cancer recurrence following curative thyroidectomy is associated with increased morbidity and mortality, but current surveillance strategies are inadequate for early detection. Prior studies indicate that tissue glycosylation is altered in thyroid cancer, but the utility of serum glycosylation in thyroid cancer surveillance remains unexplored. We therefore assessed the potential utility of altered serum glycomic profile as a tumor-specific target for disease surveillance in recurrent thyroid cancer.

A clade C HIV-1 vaccine protects against heterologous SHIV infection by modulating IgG glycosylation and T helper response in macaques.

The rising global HIV-1 burden urgently requires vaccines capable of providing heterologous protection. Here, we developed a clade C HIV-1 vaccine consisting of priming with modified vaccinia Ankara (MVA) and boosting with cyclically permuted trimeric gp120 (CycP-gp120) protein, delivered either orally using a needle-free injector or through parenteral injection. We tested protective efficacy of the vaccine against intrarectal challenges with a pathogenic heterologous clade C SHIV infection in rhesus macaques.

Regeneration of Free Reducing Glycans from Reductive Amination-Tagged Glycans by Oxone.

Glycans are usually fluorescently tagged by reductive amination for analytic tools. However, free reducing glycan regeneration is sometimes important and necessary for further structural or functional studies. Here, we introduce a new method for efficiently removing fluorescent tags from glycoconjugates by a simple treatment with Oxone.

SARS-CoV-2 and other coronaviruses bind to phosphorylated glycans from the human lung.

SARS-CoV, MERS-CoV, and potentially SARS-CoV-2 emerged as novel human coronaviruses following cross-species transmission from animal hosts. Although the receptor binding characteristics of human coronaviruses are well documented, the role of carbohydrate binding in addition to recognition of proteinaceous receptors has not been fully explored. Using natural glycan microarray technology, we identified N-glycans in the human lung that are recognized by various human and animal coronaviruses.

Cellular O-Glycome Reporter/Amplification (CORA): Analytical and Preparative Tools to Study Mucin-Type O-Glycans of Living Cells.

Mucin-type O-glycosylation (O-glycans, O-glycome) is among the most biologically important post-translational modification in glycoproteins but O-glycan structural diversity and expression are poorly understood due to the inadequacy of current analytical methods. We recently developed a new tool termed cellular O-glycome reporter/amplification (CORA), which uses O-glycan precursors, benzyl-α-GalNAc (Bn-α-GalNAc) or azido-Bn-α-GalNAc (N -Bn-α-GalNAc), as surrogates of protein O-glycosylation. Living cells metabolically convert these precursors to all types of O-GalNAc glycans representative of the cells' capabilities.

Targeting of Mammalian Glycans Enhances Phage Predation in the Gastrointestinal Tract.

The human gastrointestinal mucosal surface consists of a eukaryotic epithelium, a prokaryotic microbiota, and a carbohydrate-rich interface that separates them. In the gastrointestinal tract, the interaction of bacteriophages (phages) and their prokaryotic hosts influences the health of the mammalian host, especially colonization with invasive pathobionts. Antibiotics may be used, but they also kill protective commensals.

Enteroaggregative E. coli Adherence to Human Heparan Sulfate Proteoglycans Drives Segment and Host Specific Responses to Infection.

Enteroaggregative Escherichia coli (EAEC) is a significant cause of acute and chronic diarrhea, foodborne outbreaks, infections of the immunocompromised, and growth stunting in children in developing nations. There is no vaccine and resistance to antibiotics is rising. Unlike related E.

Unique repertoire of anti-carbohydrate antibodies in individual human serum.

Humoral immunity to pathogens and other environmental challenges is paramount to maintain normal health, and individuals lacking or unable to make antibodies are at risk. Recent studies indicate that many human protective antibodies are against carbohydrate antigens; however, little is known about repertoires and individual variation of anti-carbohydrate antibodies in healthy individuals. Here we analyzed anti-carbohydrate antibody repertoires (ACARs) of 105 healthy individual adult donors, aged 20-60 from different ethnic backgrounds to explore variations in antibodies, as defined by binding to glycan microarrays and by affinity purification.

Preparation of Complex Glycans From Natural Sources for Functional Study.

One major barrier in glycoscience is the lack of diverse and biomedically relevant complex glycans in sufficient quantities for functional study. Complex glycans from natural sources serve as an important source of these glycans and an alternative to challenging chemoenzymatic synthesis. This review discusses preparation of complex glycans from several classes of glycoconjugates using both enzymatic and chemical release approaches.

Strategy for Isolation, Preparation, and Structural Analysis of Chondroitin Sulfate Oligosaccharides from Natural Sources.

The structure of chondroitin sulfate oligosaccharides (CSOs), especially their sulfation pattern, has been found to be closely related with many biological pathways and diseases. However, detailed functional analysis such as their interaction with glycan binding proteins (GBPs) has been lagging, presumably due to the unavailability of well-defined, diverse structures. Besides challenging chemical and enzymatic synthesis, this is also due to the challenges in their purification at the isomer level and structural analysis owing to their instability, structural complexity, and low mass spectrometry detection sensitivity.

Amplification and Preparation of Cellular O-Glycomes for Functional Glycomics.

Mucin-type O-glycans play key roles in many cellular processes, and they are often altered in human diseases. A major challenge in studying the role of O-glycans through functional O-glycomics is the absence of a complete repertoire of the glycans that comprise the human O-glycome. Here we describe a cellular O-glycome preparation strategy, Preparative Cellular O-Glycome Reporter/Amplification (pCORA), that introduces 4-N-Bn-GalNAc(Ac) as a novel precursor in large-scale cell cultures to generate usable amounts of O-glycans as a potential O-glycome factory.

Preparative scale purification of natural glycans by closed-loop recycle HPLC.

While glycoscience has become well recognized as an indispensable area in biomedical research, studies on the function of individual glycans remains a great challenge due to the lack of tools and methods. One of the greatest impediments to progress in this area is the lack of biomedically relevant complex glycans in sufficient quantity and purity for structural and functional analysis. Despite recent advances in chemoenzymatic synthesis of complex glycans, generating significant amounts of pure glycans is limited to laboratories with specialized expertise.

-Benzylhydroxylamine (BHA) as a Cleavable Tag for Isolation and Purification of Reducing Glycans.

Glycoscience has been recognized as an important area in biomedical research. Currently, a major obstacle for glycoscience study is the lack of diverse, biomedically relevant, and complex glycans in quantities sufficient for exploring their structural and functional aspects. Complementary to chemoenzymatic synthesis, natural glycans could serve as a great source of biomedically relevant glycans if they are available in sufficient quantities.

Synthesis and Characterization of Versatile O-Glycan Precursors for Cellular O-Glycomics.

Protein O-glycosylation is a universal post-translational modification and plays essential roles in many biological processes. Recently we reported a technology termed cellular O-glycome reporter/amplification (CORA) to amplify and profile mucin-type O-glycans of living cells growing in the presence of peracetylated Benzyl-α-GalNAc (AcGalNAc-α-Bn). However, the application and development of the CORA method are limited by the properties of the precursor benzyl aglycone, which is relatively inert to further chemical modifications.

Next-Generation Glycan Microarray Enabled by DNA-Coded Glycan Library and Next-Generation Sequencing Technology.

Interactions of glycans with proteins, cells, and microorganisms play important roles in cell-cell adhesion and host-pathogen interaction. Glycan microarray technology, in which multiple glycan structures are immobilized on a single glass slide and interrogated with glycan-binding proteins (GBPs), has become an indispensable tool in the study of protein-glycan interactions. Despite its great success, the current format of the glycan microarray requires expensive, specialized instrumentation and labor-intensive assay and image processing procedures, which limit automation and possibilities for high-throughput analyses.

History and future of shotgun glycomics.

Glycans in polysaccharides and glycoconjugates of the hydrophilic exterior of all animal cells participate in signal transduction, cellular adhesion, intercellular signaling, and sites for binding of pathogens largely through protein-glycan interactions. Microarrays of defined glycans have been used to study the binding specificities of biologically relevant glycan-binding proteins (GBP), but such arrays are limited by their lack of diversity or relevance to the GBP being investigated. Shotgun glycan microarrays are made up of structurally undefined glycans that were released from natural sources, labeled with bifunctional reagents so that they can be monitored during their purification using multidimensional chromatographic procedures, stored as a tagged glycan library (TGL) and subsequently printed onto microarrays at equal molar concentrations.

Sensitive and robust MALDI-TOF-MS glycomics analysis enabled by Girard's reagent T on-target derivatization (GTOD) of reducing glycans.

Sensitive glycomics analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is of great importance but significantly hampered by their low ionization efficiency and labile sialic acid moieties. Chemical derivatization offers a viable way to improve both the ionization efficiency and analytical sensitivity of the glycans in MS analysis by altering their hydrophobicity or charge property. Here we employed Girard's reagent T (GT) for on-target derivatization (GTOD) of reducing glycan under mild acid condition to form stable hydrazones at room temperature, allowing rapid and sensitive identification of neutral and sialylated glycans in positive-ion mode as only permanently positive charged molecular ions without multiple ion adducts by MALDI-TOF-MS.

Anthranilic Acid as a Versatile Fluorescent Tag and Linker for Functional Glycomics.

The advancement of glycoscience is critically dependent on the access to a large number of glycans for their functional study. Naturally occurring glycans are considered a viable source for diverse and biologically relevant glycan libraries. A mixture of free reducing glycans released from natural sources can be fluorescently tagged and separated by chromatography to produce a natural glycan library.

Reductive chemical release of N-glycans as 1-amino-alditols and subsequent 9-fluorenylmethyloxycarbonyl labeling for MS and LC/MS analysis.

Unlabelled: Glycoproteins play pivotal roles in a series of biological processes and their glycosylation patterns need to be structurally and functionally characterized. However, the lack of versatile methods to release N-glycans as functionalized forms has been undermining glycomics studies. Here a novel method is developed for dissociation of N-linked glycans from glycoproteins for analysis by MS and online LC/MS.

Large scale preparation of high mannose and paucimannose N-glycans from soybean proteins by oxidative release of natural glycans (ORNG).

Despite the important advances in chemical and chemoenzymatic synthesis of glycans, access to large quantities of complex natural glycans remains a major impediment to progress in Glycoscience. Here we report a large-scale preparation of N-glycans from a kilogram of commercial soy proteins using oxidative release of natural glycans (ORNG). The high mannose and paucimannose N-glycans were labeled with a fluorescent tag and purified by size exclusion and multidimensional preparative HPLC.

Simultaneous Release and Labeling of O- and N-Glycans Allowing for Rapid Glycomic Analysis by Online LC-UV-ESI-MS/MS.

Most glycoproteins and biological protein samples undergo both O- and N-glycosylation, making characterization of their structures very complicated and time-consuming. Nevertheless, to fully understand the biological functions of glycosylation, both the glycosylation forms need to be analyzed. Herein we report a versatile, convenient one-pot method in which O- and N-glycans are simultaneously released from glycoproteins and chromogenically labeled in situ and thus available for further characterization.