Development of Lamprey-derived Antibodies Against Human Blood Group Antigens.

A major challenge in the glycosciences is the scarcity of sensitive and specific glycan-binding reagents, such as monoclonal antibodies, for detecting and isolating glycans. Here we report the development and characterization of new monoclonal antibodies (mAbs) that bind carbohydrate-based red blood cell (RBC) antigens including the ABO(H) antigens. This approach exploits the immune system of the sea lamprey (Petromyzon marinus), which strongly responds to human glycans to enable the generation of high affinity antibodies.

Unraveling the glyco-immunity nexus in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive disease, and standard of care therapies have failed to yield significant clinical benefit, with invasive surgery being the only curative treatment for patients with early-stage disease. Tumor-associated glycans in pancreatic cancer have direct effects on the survival and propagation of the tumor proper and contribute to an immunosuppressed tumor microenvironment. The existence of a “tumor glycocode” in PDAC and the role of hypersialylation in this cancer have been hugely underscored.

Parallel Synthesis of Glycopeptide Libraries Using Glyco-SPOT Synthesis.

Synthesis of glycopeptide libraries is often limited by the cost and availability of the glycoamino acids. To circumvent this, we have devised a method of using SPOT synthesis for production of such libraries using cellulose filter paper as the solid support. We termed this method Glyco-SPOT synthesis, and it can be used to produce libraries of glycopeptides in microgram quantities in a parallel manner using microgram amounts of glycoamino acids.

-Glycopedia: Libraries for Native -glycan Structural Analysis.

Studies on glycans of glycoproteins are hampered by the lack of standards that reflect the wide diversity in structure typically observed. To this end we have exploited a large library of -glycan standards comprised of a unique collection of 226 -glycans including oligomannose, hybrid, and complex-type. We generated a method employing porous graphitized carbon (PGC) and liquid chromatography mass spectrometry (PGC-LC-MS), which can provide a high degree of resolution of underivatized -glycan structures.

A glycan atlas of the mammalian intestine through ontogeny and inflammation.

The muco-epithelial interface in the mammalian gut is composed of a mucus and epithelial lining fundamental to barrier function, microbe-host interactions, and intestinal homeostasis. This barrier is heavily glycosylated by O-linked sugars covalently linked to mucin glycoproteins, and N-linked sugars that coat epithelial surface proteins. Gut O- and N-glycans are thought to play central roles in barrier function, host defense, nutrition and attachment for commensals and pathogens, immunoregulation and cell-cell interactions.

Blood group O expression in normal tissues and tumors.

The H antigen (O blood group), the precursor to A and B blood groups, is expressed on human erythrocytes and other cells (e.g., endothelial cells).

Novel Click Coupling Chemistry to Explore Glycan Recognition.

Specific recognition of glycans by proteins is important in many biological processes and immune responses. Here we present a general approach for derivatizing free glycans with a novel linker MTZ (3-(methoxyamino)-propylamine added to a bioorthogonal-functional tetrazine tag) that exploits click chemistry to generate multiple platforms of glycan coupling. This derivatization preserves glycan integrity, is reversible and quantifiable, and incorporates a bioorthogonal tetrazine tag for click coupling.

Antibody-Fab and -Fc features promote Mycobacterium tuberculosis restriction.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), a leading cause of death by an infectious disease globally, has no efficacious vaccine. Antibodies are implicated in M. tuberculosis control, but the mechanisms of action remain poorly understood.

Endothelial Glycocalyx Turnover in Vascular Health and Disease: Rethinking Endothelial Dysfunction.

The endothelial glycocalyx, a glycan-rich layer on the luminal surface of endothelial cells lining blood and lymphatic vessels, plays a crucial role in vascular homeostasis by regulating vascular permeability, immune cell trafficking, and vascular tone. Dysregulated endothelial glycocalyx turnover-whether through altered synthesis, intracellular degradation, or shedding-contributes to endothelial dysfunction in conditions such as sepsis, ischemic events, and chronic inflammatory disorders including diabetes and atherosclerosis. In this review, we examine the structure, function, and turnover of the endothelial glycocalyx, emphasizing how pathological changes in its turnover drive vascular dysfunction.

Statin effects on immunoglobulin-G glycomic architecture and the link to cardiovascular disease.

Background: Immunoglobulin G (IgG) plays a critical role in immune defense yet our understanding of its role in cardiovascular disease (CVD) is evolving. Observational studies have correlated statin use with changes in IgG N-glycan structures. However, statin effects on IgG N-glycan changes have not been tested in randomized controlled trials, and their direct association with CVD remains unclear.

O-GalNAc glycans are enriched in neuronal tracts and regulate nodes of Ranvier.

Protein O-glycosylation is a critical modification in the brain, as genetic variants in the pathway are associated with common and severe neuropsychiatric phenotypes. However, little is known about the most abundant O-glycans in the mammalian brain, which are N-acetylgalactosamine (O-GalNAc) linked. Here, we determined the spatial localization, protein carriers, and cellular function of O-GalNAc glycans in the mouse brain.

Swift Universal Glycan Acquisition (SUGA) Enables Quantitative Glycan Profiling across Diverse Sample Types.

The ability to rapidly analyze complex mixtures of glycans derived from glycoproteins is important, but techniques are often laborious and require multiple glycan derivatization steps. Here, we describe an approach termed Swift Universal Glycan Acquisition (SUGA) in which the total released, nonreduced -glycan samples are analyzed following direct injection and electrospray ionization in a mass spectrometer with a rapid 3 min run time for each sample. As electrospray ionization (ESI) can generate multiple charge states and adducts for the same glycan composition (MS1), deconvolution is performed to yield the relative intensity profile for each detected glycan composition; each annotated composition is supported by an annotated MS2 spectrum.

C1GALT1C1-Associated Mosaic Disorder of Glycosylation in a Female.

Cosmc, encoded by the X-linked C1GALT1C1, is a molecular chaperone in the endoplasmic reticulum and a master regulator of O-glycosylation of mammalian glycoproteins. Recently, we described a germline mutation in C1GALT1C1 in two male patients, giving rise to a congenital disorder of glycosylation-COSMC-CDG. Here, we have identified a female patient with a de novo mosaic variant in C1GALT1C1 (c.

Differential expression of CD175 and CA19-9 in pancreatic adenocarcinoma.

Alterations in protein glycosylation are observed in many solid tumor types leading to formation of tumor-associated carbohydrate antigens (TACAs). The most common TACA is the Tn antigen (CD175), which is a mucin-type O-GalNAc-Ser/Thr/Tyr glycan in membrane and secreted glycoproteins. In addition, two other TACAs are CA19-9 (sialyl-Lewis a), which is used as a prognostic serum marker for pancreatic cancer, and its isomer sialyl-Lewis x (SLex, CD15s), which is overexpressed in many cancer types and associated with metastasis.

Thyroid Carcinoma Glycoproteins Express Altered -Glycans with 3-O-Sulfated Galactose Residues.

Aberrant protein glycosylation is a hallmark alteration of cancer and is highly associated with cancer progression. Papillary thyroid cancer (PTC) is the most common type of thyroid cancer, but the -glycosylation of its glycoproteins has not been well characterized. In this work, we analyzed multiple freshly prepared PTC specimens along with paired normal tissue obtained from thyroidectomies.

Intact quantitation and evaluation of a PEG-glycosulfopeptide as a therapeutic P-selectin antagonist.

Peptide-based therapeutics are recognized as potent and selective molecules but are often limited by short circulating half-lives, instability towards enzymatic degradation, and immunogenicity. To address these limitations and improve their pharmacological properties, peptides are commonly modified by the covalent attachment of polyethylene glycol (PEG). However, the large molecular weight and polydispersity of PEG chains complicate the interpretation of the full structure of PEGylated peptide therapeutics using standard analytical techniques.

The restricted N-glycome of neurons is programmed during differentiation.

The protein glycome of individual cell types in the brain is unexplored, despite the critical function of these modifications in development and disease. In aggregate, the most abundant asparagine (N-) linked glycans in the adult brain are high mannose structures, and specifically ManGlcNAc (Man-5), which normally exits the ER for further processing in the Golgi. Mannose structures are uncommon in other organs and often overlooked or excluded in most studies.

Antibody-Fab and -Fc features promote restriction.

the causative agent of tuberculosis (TB), is a leading cause of death by an infectious disease globally, with no efficacious vaccine. Antibodies are implicated in control, but the mechanisms of antibody action remain poorly understood. We assembled a library of TB monoclonal antibodies (mAb) and screened for the ability to restrict in mice, identifying protective antibodies targeting known and novel antigens.

Unique Glycans in Synaptic Glycoproteins in Mouse Brain.

The synapse is an essential connection between neuronal cells in which the membrane and secreted glycoproteins regulate neurotransmission. The post-translational modifications of glycoproteins with carbohydrates, although essential for their functions as well as their specific localization, are not well understood. Oddly, whereas galactose addition to glycoproteins is required for neuronal functions, galactosylation is severely restricted for Asn-linked on N-glycans in the brain, and genetic evidence highlights the important roles of galactose in brain functions and development.

Insights Into Glycobiology and the Protein-Glycan Interactome Using Glycan Microarray Technologies.

Glycans linked to proteins and lipids and also occurring in free forms have many functions, and these are partly elicited through specific interactions with glycan-binding proteins (GBPs). These include lectins, adhesins, toxins, hemagglutinins, growth factors, and enzymes, but antibodies can also bind glycans. While humans and other animals generate a vast repertoire of GBPs and different glycans in their glycomes, other organisms, including phage, microbes, protozoans, fungi, and plants also express glycans and GBPs, and these can also interact with their host glycans.

Cosmc regulates O-glycan extension in murine hepatocytes.

Hepatocytes synthesize a vast number of glycoproteins found in their membranes and secretions, many of which contain O-glycans linked to Ser/Thr residues. As the functions and distribution of O-glycans on hepatocyte-derived membrane glycoproteins and blood glycoproteins are not well understood, we generated mice with a targeted deletion of Cosmc (C1Galt1c1) in hepatocytes. Liver glycoproteins in WT mice express typical sialylated core 1 O-glycans (T antigen/CD176) (Galβ1-3GalNAcα1-O-Ser/Thr), whereas the Cosmc knockout hepatocytes (HEP-Cosmc-KO) lack extended O-glycans and express the Tn antigen (CD175) (GalNAcα1-O-Ser/Thr).

Plant-Produced Therapeutic Crizanlizumab Monoclonal Antibody Binds P-Selectin to Alleviate Vaso-occlusive Pain Crises in Sickle Cell Disease.

Sickle Cell Disease (SCD) is a severe genetic disorder causing vascular occlusion and pain by upregulating the adhesion molecule P-selectin on endothelial cells and platelets. It primarily affects infants and children, causing chronic pain, circulatory problems, organ damage, and complications. Thus, effective treatment and management are crucial to reduce SCD-related risks.

Galectin-4 Antimicrobial Activity Primarily Occurs Through its C-Terminal Domain.

Although immune tolerance evolved to reduce reactivity with self, it creates a gap in the adaptive immune response against microbes that decorate themselves in self-like antigens. This is particularly apparent with carbohydrate-based blood group antigens, wherein microbes can envelope themselves in blood group structures similar to human cells. In this study, we demonstrate that the innate immune lectin, galectin-4 (Gal-4), exhibits strain-specific binding and killing behavior towards microbes that display blood group-like antigens.

N-Glycosylation Profiles of Immunoglobulin G and Future Cardiovascular Events.

Background: Posttranslational glycosylation of IgG can modulate its inflammatory capacity through structural variations. We examined the association of baseline IgG N-glycans and an IgG glycan score with incident cardiovascular disease (CVD).

Methods: IgG N-glycans were measured in 2 nested CVD case-control studies: JUPITER (Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin; NCT00239681; primary prevention; discovery; Npairs=162); and TNT trial (Treating to New Targets; NCT00327691; secondary prevention; validation; Npairs=397).