Top-Down Chemoenzymatic Approach to Synthesizing Diverse High-Mannose N-Glycans and Related Neoglycoproteins for Carbohydrate Microarray Analysis.

High-mannose-type N-glycans are an important component of neutralizing epitopes on HIV-1 envelope glycoprotein gp120. They also serve as signals for protein folding, trafficking, and degradation in protein quality control. A number of lectins and antibodies recognize high-mannose-type N-glycans, and glycan array technology has provided an avenue to probe these oligomannose-specific proteins. We describe in this paper a top-down chemoenzymatic approach to synthesize a library of high-mannose N-glycans and related neoglycoproteins for glycan microarray analysis. The method involves the sequential enzymatic trimming of two readily available natural N-glycans, the Man9GlcNAc2Asn prepared from soybean flour and the sialoglycopeptide (SGP) isolated from chicken egg yolks, coupled with chromatographic separation to obtain a collection of a full range of natural high-mannose N-glycans. The Asn-linked N-glycans were conjugated to bovine serum albumin (BSA) to provide neoglycoproteins containing the oligomannose moieties. The glycoepitopes displayed were characterized using an array of glycan-binding proteins, including the broadly virus-neutralizing agents, glycan-specific antibody 2G12, Galanthus nivalis lectin (GNA), and Narcissus pseudonarcissus lectin (NPA).

Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry.

Glucans are polymers of d-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure-function studies and their exploitation. We describe construction of a "glucome" microarray, the first sequence-defined glycome-scale microarray, using a "designer" approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear "homo" and "hetero" and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or ß-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.

Glycan Markers as Potential Immunological Targets in Circulating Tumor Cells.

We present here an experimental approach for exploring a new class of tumor biomarkers that are overexpressed by circulating tumor cells (CTCs) and are likely targetable in immunotherapy against tumor metastasis. Using carbohydrate microarrays, anti-tumor monoclonal antibodies (mAbs) were scanned against a large panel of carbohydrate antigens to identify potential tumor glycan markers. Subsequently, flow cytometry and fiber-optic array scanning technology (FAST) were applied to determine whether the identified targets are tumor-specific cell-surface markers and are, therefore, likely suitable for targeted immunotherapy. Finally, the tumor glycan-specific antibodies identified were validated using cancer patients' blood samples for their performance in CTC-detection and immunotyping analysis. In this article, identifying breast CTC-specific glycan markers and targeting mAbs serve as examples to illustrate this tumor biomarker discovery strategy.

Targeting N-glycan cryptic sugar moieties for broad-spectrum virus neutralization: progress in identifying conserved molecular targets in viruses of distinct phylogenetic origins.

Identifying molecular targets for eliciting broadly virus-neutralizing antibodies is one of the key steps toward development of vaccines against emerging viral pathogens. Owing to genomic and somatic diversities among viral species, identifying protein targets for broad-spectrum virus neutralization is highly challenging even for the same virus, such as HIV-1. However, viruses rely on host glycosylation machineries to synthesize and express glycans and, thereby, may display common carbohydrate moieties. Thus, exploring glycan-binding profiles of broad-spectrum virus-neutralizing agents may provide key information to uncover the carbohydrate-based virus-neutralizing epitopes. In this study, we characterized two broadly HIV-neutralizing agents, human monoclonal antibody 2G12 and Galanthus nivalis lectin (GNA), for their viral targeting activities. Although these agents were known to be specific for oligomannosyl antigens, they differ strikingly in virus-binding activities. The former is HIV-1 specific; the latter is broadly reactive and is able to neutralize viruses of distinct phylogenetic origins, such as HIV-1, severe acute respiratory syndrome coronavirus (SARS-CoV), and human cytomegalovirus (HCMV). In carbohydrate microarray analyses, we explored the molecular basis underlying the striking differences in the spectrum of anti-virus activities of the two probes. Unlike 2G12, which is strictly specific for the high-density Man9GlcNAc2Asn (Man9)-clusters, GNA recognizes a number of N-glycan cryptic sugar moieties. These include not only the known oligomannosyl antigens but also previously unrecognized tri-antennary or multi-valent GlcNAc-terminating N-glycan epitopes (Tri/m-Gn). These findings highlight the potential of N-glycan cryptic sugar moieties as conserved targets for broad-spectrum virus neutralization and suggest the GNA-model of glycan-binding warrants focused investigation.

Uncovering cryptic glycan markers in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE).

Using an integrated antigen microarray approach, we observed epitope-spreading of autoantibody responses to a variety of antigenic structures in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) and in the serum of mice with experimental autoimmune encephalomyelitis (EAE). These included previously described protein- and lipid-based antigenic targets and newly discovered autoimmunogenic sugar moieties, notably, autoantibodies specific for the oligomannoses in both MS patient CSF and the sera of mice with EAE. These glycans are often masked by other sugar moieties and belong to a class of cryptic autoantigens. We further determined that these targets are highly expressed on multiple cell types in MS and EAE lesions. Co-immunization of SJL/J mice with a Man9-KLH conjugate at the time of EAE induction elicited highly significant levels of anti-Man9-cluster autoantibodies. Nevertheless, this anti-glycan autoantibody response was associated with a significantly reduced clinical severity of EAE. The potential of these cryptic glycan markers and targeting antibodies for diagnostic and therapeutic interventions of neurological disorders has yet to be explored.

Anti-Oligomannose Antibodies as Potential Serum Biomarkers of Aggressive Prostate Cancer.

[Table: see text] This study bridges a carbohydrate microarray discovery and a large-scale serological validation of anti-oligomannose antibodies as novel serum biomarkers of aggressive prostate cancer (PCa). Experimentally, a Man9-cluster-specific enzyme-linked immunosorbent assay was established to enable sensitive detection of anti-Man9 antibodies in human sera. A large-cohort of men with PCa or benign prostatic hyperplasia (BPH) whose sera were banked at Stanford University was characterized using this assay. Subjects included patients with 100% Gleason grade 3 cancer (n = 84), with Gleason grades 4 and/or 5 cancer (n = 204), and BPH controls (n = 135). Radical prostatectomy Gleason grades and biochemical (PSA) recurrence served as key parameters for serum biomarker evaluation. It was found that IgGMan9 and IgMMan9 were widely present in the sera of men with BPH, as well as those with cancer. However, these antibody reactivities were significantly increased in the subjects with the largest volumes of high grade cancer. Detection of serum IgGMan9 and IgMMan9 significantly predicted the clinical outcome of PCa post-radical prostatectomy. Given these results, we suggest that IgGMan9 and IgMMan9 are novel serum biomarkers for monitoring aggressive progression of PCa. The potential of oligomannosyl antigens as targets for PCa subtyping and targeted immunotherapy is yet to be explored.

Assessment of Immunologically Potent Carbohydrates.

Carbohydrates have a great potential in generating structural and immunological diversities. Microbial pathogens often decorate their outmost surfaces with specific carbohydrate signatures. Carbohydrate antigens also differ significantly from protein antigens in physiochemical properties, especially in surface display of antigenic determinants in aqueous solutions. Technical optimization or modifications are often needed when we apply standard procedures for protein-based enzyme-linked immunosorbent assay (ELISA) to assess immunologically potent carbohydrates. We present here our laboratory protocols for performing carbohydrate ELISA and discuss several assay platforms that may be applied complementarily to explore the carbohydrate moieties that are critical for host immune recognition and induction of glycan-specific antibody responses.

Targeting the stage-specific embryonic antigen (SSEA)-0 tumor neoantigen.

Recognition of abnormal glycosylation in virtually any cancer type has raised a great interest in the glycan-based tumor biomarkers. Our team explored carbohydrate microarrays as a broad-spectrum immunoassay to probe the immunologically potent tumor glycan targets. This effort has led to the identification of a blood group precursor antigen SSEA-0 as a conserved breast cancer (BCA) marker. Since this immunogenic O-core glycan is normally hidden as a cryptic antigen but becomes overexpressed and surface-exposed by metastatic breast cancer cells (MBCA), its potential as a novel immunological target for precision immunotherapy against tumor metastasis warrants a focused investigation.

The "Janus-like" RNA-editing machinery in innate antiviral immunity.

Our innate immune systems are evolved to provide the first line of immune defense against microbial infections. A key effector component is the adenosine deaminase acting on the RNA-1 (ADAR-1)/interferon (IFN) pathway of the innate cytoplasmic immunity that mounts rapid responses to many viral pathogens. As an RNA-editing enzyme, ADAR-1 targets viral RNA intermediates in the cytoplasmic compartment to interfere with the infection. However, ADAR-1 may also edit characteristic RNA structures of certain host genes, notably, the 5-hydroxytryptamine (serotonin) receptor 2C (5-HT2CR). Dysfunction of 5-HT2CR has been linked to the pathology of several human mental conditions, such as Schizophrenia, anxiety, bipolar disorder, major depression, and the mental illnesses of substance use disorders (SUD). Thus, the ADAR-1-mediated RNA editing may be either beneficial or harmful; these effects need to be tightly modulated to sustain innate antiviral immunity while restricting undesired off-target self-reactivity. In this communication, we discuss ideas and tools to identify the orphan drug candidates, including small molecules and biologics that may serve as effective modulators of the ADAR-1/IFN innate immunity and are thereby promising for use in treating or preventing SUD- and/or viral infection-associated mental illnesses.

The human epithelial carcinoma antigen recognized by monoclonal antibody AE3 is expressed on a sulfoglycolipid in addition to neoplastic mucins.

The term human epithelial carcinoma antigen (HCA) has been applied collectively to mucin-type high molecular weight (>1000kDa) glycoproteins that are over-expressed in epithelial cancers. Since the 1990s, over 40 monoclonal antibodies have been raised that recognize HCA. There has been evidence that the antigenic determinants are mostly carbohydrates, but details have been elusive. Here we have carried out carbohydrate microarray analyses of one of the monoclonal antibodies, AE3, that has been regarded the 'most carcinoma specific' in respect to its ability to detect HCA in sera of patients with epithelial cancers. The microarrays encompassed a series of 492 sequence-defined glycan probes in the form of glycolipids and neoglycolipids. We have thus established that the antigen recognized by antibody AE3 is a carbohydrate sequence distinct from the A, B, H, Lewis(a/b), Lewis(x/y) and T antigens, but that it is strongly expressed on the monosulfated tetra-glycosyl ceramide, SM1a, Galß1-3GalNAcß1-4(3-O-sulfate)Galß1-4GlcCer. This is the first report of an anti-HCA to be characterized with respect to its recognition sequence and of the occurrence of the antigen on a glycolipid as well as on glycoproteins. Knowledge of a discrete glycan sequence as target antigen now opens the way to its exploration as a serologic cancer biomarker, namely to determine if the antigen elicits an autoantibody response in early non-metastatic cancer, or if it is shed and immunochemically detectable in more advanced disease.

CTLA-4 expression by B-1a B cells is essential for immune tolerance.

CTLA-4 is an important regulator of T-cell function. Here, we report that expression of this immune-regulator in mouse B-1a cells has a critical function in maintaining self-tolerance by regulating these early-developing B cells that express a repertoire enriched for auto-reactivity. Selective deletion of CTLA-4 from B cells results in mice that spontaneously develop autoantibodies, T follicular helper (Tfh) cells and germinal centers (GCs) in the spleen, and autoimmune pathology later in life. This impaired immune homeostasis results from B-1a cell dysfunction upon loss of CTLA-4. Therefore, CTLA-4-deficient B-1a cells up-regulate epigenetic and transcriptional activation programs and show increased self-replenishment. These activated cells further internalize surface IgM, differentiate into antigen-presenting cells and, when reconstituted in normal IgH-allotype congenic recipient mice, induce GCs and Tfh cells expressing a highly selected repertoire. These findings show that CTLA-4 regulation of B-1a cells is a crucial immune-regulatory mechanism.

Coronaviruses' sugar shields as vaccine candidates.

A successful global healthcare response relies on versatile vaccines and production of broadly virus-neutralizing antibodies by the immune system to protect us from emerging infectious diseases. The present 2019 severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic highlights the urgent need for development of anti-viral biodefense. Due to the genetic and proteomic diversities of viral pathogens, establishing versatile anti-viral vaccines or therapeutic agents is highly challenging. Carbohydrate antigens represent an important class of immunological targets for vaccine development and immunotherapy against microbial infections. In this mini review, some concepts and strategies for exploring the potential of immunogenic sugar moieties as CoV vaccine candidates are presented.

Differential Expression of Blood Group Precursor Antigen in Human Breast Cancer Tissue.

There is a pressing need for biomarkers for targeted immunotherapy against breast cancer (BCA), the leading cause of cancer death in women. Previously, a blood group precursor O-core epitope gpCl was found to be highly expressed in breast circulating tumor cells (BCTCs) and BCA cell lines with cancer stem cell (BCSC) features. In this pilot study, the breast tissue distribution of gpC1 was examined using tissue microarrays (TMAs). Notably, gpC1 positive cells were detected in the major histological types of neoplastic breast tissues. Conversely, none of the breast tissues derived from subjects without BCA were gpC1 positive. Thus, gpC1 expression seems to be tumor-specific but not histological type-dependent, reflecting perhaps its characteristics as a conserved epitope of oncofetal blood group precursor antigens.

Carbohydrate cluster microarrays fabricated on three-dimensional dendrimeric platforms for functional glycomics exploration.

We reported here a novel, ready-to-use bioarray platform and methodology for construction of sensitive carbohydrate cluster microarrays. This technology utilizes a three-dimensional (3-D) poly(amidoamine) starburst dendrimer monolayer assembled on glass surface, which is functionalized with terminal aminooxy and hydrazide groups for site-specific coupling of carbohydrates. A wide range of saccharides, including monosaccharides, oligosaccharides and polysaccharides of diverse structures, are applicable for the 3-D bioarray platform without prior chemical derivatization. The process of carbohydrate coupling is effectively accelerated by microwave radiation energy. The carbohydrate concentration required for microarray fabrication is substantially reduced using this technology. Importantly, this bioarray platform presents sugar chains in defined orientation and cluster configurations. It is, thus, uniquely useful for exploration of the structural and conformational diversities of glyco-epitope and their functional properties.

Chemoenzymatic synthesis and lectin array characterization of a class of N-glycan clusters.

N-Glycans are major components of many glycoproteins. These sugar moieties are frequently involved in important physiological and disease processes via their interactions with a variety of glycan-binding proteins (GBP). Clustering effect is an important feature in many glycan-lectin interactions. We describe in this paper a chemoenzymatic synthesis of novel N-glycan clusters using a tandem endoglycosidase-catalyzed transglycosylation. It was found that the internal beta-1,2-linked GlcNAc moieties in the N-glycan core, once exposed in the nonreducing terminus, was able to serve as acceptors for transglycosylation catalyzed by Endo-A and EndoM-N175A. This efficient chemoenzymatic method allows a quick extension of the sugar chains to form a class of glycan clusters in which sugar residues are all connected by native glycosidic linkages found in natural N-glycans. In addition, a discriminative enzymatic reaction at the two GlcNAc residues could be fulfilled to afford novel hybrid clusters. Lectin microarray studies revealed unusual properties in glyco-epitope expression by this panel of structurally well-defined synthetic N-glycans. These new compounds are likely valuable for functional glycomics studies to unveil new functions of both glycans and carbohydrate-binding proteins.

Liquid Biopsy in the OMICS Era of Tumor Medicine.

Liquid biopsy uses noninvasive blood test to assess tumor heterogeneity and evolution in real time. It looks for tumor components in the blood circulation, such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), to provide tumor-specific information. By detecting multiplex tumor biomarkers, including nucleic acids, proteins, carbohydrates, and other tumor-derived substances, liquid biopsy helps with early tumor diagnosis, tumor evolution monitoring, and prognosis prediction. With the development of high-throughput OMICS tools like carbohydrate microarray and high-speed fiber-optic array scanning technology (FAST scan), it is now practical to identify glycan markers of CTCs and cancer stem cells (CSCs), especially those that are cell-surface exposed and readily accessible for immune recognition and targeting. Potential of this class of biomarkers in tumor subtyping and targeted immunotherapy is yet to be explored.

Programmed cell removal by calreticulin in tissue homeostasis and cancer.

Macrophage-mediated programmed cell removal (PrCR) is a process essential for the clearance of unwanted (damaged, dysfunctional, aged, or harmful) cells. The detection and recognition of appropriate target cells by macrophages is a critical step for successful PrCR, but its molecular mechanisms have not been delineated. Here using the models of tissue turnover, cancer immunosurveillance, and hematopoietic stem cells, we show that unwanted cells such as aging neutrophils and living cancer cells are susceptible to "labeling" by secreted calreticulin (CRT) from macrophages, enabling their clearance through PrCR. Importantly, we identified asialoglycans on the target cells to which CRT binds to regulate PrCR, and the availability of such CRT-binding sites on cancer cells correlated with the prognosis of patients in various malignancies. Our study reveals a general mechanism of target cell recognition by macrophages, which is the key for the removal of unwanted cells by PrCR in physiological and pathophysiological processes.

Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells.

We describe here the development of a carbohydrate-based microarray to extend the scope of biomedical research on carbohydrate-mediated molecular recognition and anti-infection responses. We have demonstrated that microbial polysaccharides can be immobilized on a surface-modified glass slide without chemical conjugation. With this procedure, a large repertoire of microbial antigens (approximately 20,000 spots) can be patterned on a single micro-glass slide, reaching the capacity to include most common pathogens. Glycoconjugates of different structural characteristics are shown here to be applicable for microarray fabrication, extending the repertoires of diversity and complexity of carbohydrate microarrays. The printed microarrays can be air-dried and stably stored at room temperature for long periods of time. In addition, the system is highly sensitive, allowing simultaneous detection of a broad spectrum of antibody specificities with as little as a few microliters of serum specimen. Finally, the potential of carbohydrate microarrays is demonstrated by the discovery of previously undescribed cellular markers, Dex-Ids.

A practical protocol for carbohydrate microarrays.

We have established a high-throughput biochip platform for constructing carbohydrate microarrays. Using this technology, carbohydrate-containing macromolecules of diverse structures, including polysaccharides, natural glycoconjugates, and mono- and oligosaccharides coupled to carrier molecules, can be stably immobilized on a glass chip without chemical modification. Here, we describe a practical protocol for this technology. We hope that anyone who has access to a standard cDNA microarray facility will be able to explore this technology for his or her own research interest. We also provide an example to illustrate that the carbohydrate microarray is also a discovery tool; this is particularly useful for identifying immunologic sugar moieties, including complex carbohydrates of cancer cells and sugar signatures of previously unrecognized microbial pathogens.

A Liposome-Based Approach to the Integrated Multi-Component Antigen Microarrays.

This report describes an experimental procedure for constructing integrated lipid, carbohydrate, and protein microarrays. In essence, it prints liposomes on nitrocellulose-coated micro-glass slides, a biochip substrate for spotting protein and carbohydrate microarrays, and the substances that can form liposomes (homo-liposomes) or can be incorporated into liposomes (hetero-liposomes) are suitable for microarray construction using existing microarray spotting devices. Importantly, this technology allows simultaneous detection of serum antibody activities among the three major classes of antigens, i.e., lipids, carbohydrates, and proteins. The potential of this technology is illustrated by its use in revealing a broad-spectrum of pre-existing anti-lipid antibodies in blood circulation and monitoring the epitope spreading of autoantibody reactivities among protein, carbohydrate, and lipid antigens in experimental autoimmune encephalomyelitis (EAE).