Search Results (6)

A key function of naturally occurring antibodies is to control pathologically altered cells, such as those with aberrant glycosylation. Age-related diminution in the pool of B cells producing these immunoglobulins is linked to impaired anti-tumor immunity. In this study, the levels of antibodies against tumor-associated carbohydrate antigens (TACAs)—common in gastric cancer (GC) and other malignancies—were analyzed in 235 treatment-naïve GC patients (stages I–IV) and 76 healthy donors using a printed glycan array (PGA). We found that anti-glycan IgM levels, but not IgG, reduced with age in both patients and donors. Crucially, IgM levels against most glycans were significantly lower in the GC cohort compared with healthy donors, a trend that remained after age adjustment. Furthermore, an immunohistochemical analysis revealed that human anti-GalNAcα (Tn) antibodies—a well-characterized TACA in gastrointestinal cancers—bound to tumor cells and exhibited perinuclear and membrane staining in non-tumor surface cells within the same organ. These data support the hypothesis that gastric cancer patients have reduced levels of anti-glycan IgMs, which are responsible for the early recognition of transformed cells. This specific immunodeficiency may contribute to a permissive environment for tumor development. Full article

Background. Naturally occurring human antibodies against glycans recognize and quickly eliminate infectious bacteria, viruses and aberrantly glycosylated neoplastic malignant cells, and they often initiate processes that involve the complement system. Methods. Using a printed glycan array (PGA) containing 605 glycoligands (oligo- and polysaccharides, glycopeptides), we examined which of the glycan-binding antibodies are able to activate the complement system. Using this PGA, the specificities of antibodies of the IgM and IgG classes were determined in the blood serum of healthy donors (suggested as mostly natural), and, then, using the same array, it was determined which types of the bound immunoglobulins were also showing C3 deposition. Results. It was found that about 30% of anti-glycan antibodies in human serum detected by the PGA did not activate the complement. They were mostly IgGs and directed to bacterial O-antigens; no apparent common structural motif within their target polysaccharides was found. Antibodies to blood group systems ABO and Forssman, xeno-antigens, a number of polysaccharides from various strains of S. enterica, E. coli and P. alcalifaciens, as well as small fragments of bacterial polysaccharides were recognized by complement-activating antibodies as expected. A complement-activating antibody was affinity-isolated on glycan-Sepharose from human serum, and, in the presence of the complement, it lysed red blood cells coated with the same glycan (kodecytes, where glycans expressed on biological membranes), while an isolated complement non-activating antibody did not, which confirms the validity of the solid-phase PGA results. Conclusions. Thus, ~30% of human anti-glycan antibodies lack the ability to activate the complement system. The function of the widely represented immunoglobulins that do not cause C3 deposition remains unclear. Full article

Protein–carbohydrate interactions happen to be a crucial facet of biology, discharging a myriad of functions. Microarrays have become a premier choice to discern the selectivity, sensitivity and breadth of these interactions in a high-throughput manner. The precise recognition of target glycan ligands among the plethora of others is central for any glycan-targeting probe being tested by microarray analyses. Ever since the introduction of the microarray as an elemental tool for high-throughput glycoprofiling, numerous distinct array platforms possessing different customizations and assemblies have been developed. Accompanying these customizations are various factors ushering variances across array platforms. In this primer, we investigate the influence of various extrinsic factors, namely printing parameters, incubation procedures, analyses and array storage conditions on the protein–carbohydrate interactions and evaluate these factors for the optimal performance of microarray glycomics analysis. We hereby propose a 4D approach (Design–Dispense–Detect–Deduce) to minimize the effect of these extrinsic factors on glycomics microarray analyses and thereby streamline cross-platform analyses and comparisons. This work will aid in optimizing microarray analyses for glycomics, minimize cross-platform disparities and bolster the further development of this technology. Full article

Microarrays are one of the trailblazing technologies of the last two decades and have displayed their importance in all the associated fields of biology. They are widely explored to screen, identify, and gain insights on the characteristics traits of biomolecules (individually or in complex solutions). A wide variety of biomolecule-based microarrays (DNA microarrays, protein microarrays, glycan microarrays, antibody microarrays, peptide microarrays, and aptamer microarrays) are either commercially available or fabricated in-house by researchers to explore diverse substrates, surface coating, immobilization techniques, and detection strategies. The aim of this review is to explore the development of biomolecule-based microarray applications since 2018 onwards. Here, we have covered a different array of printing strategies, substrate surface modification, biomolecule immobilization strategies, detection techniques, and biomolecule-based microarray applications. The period of 2018–2022 focused on using biomolecule-based microarrays for the identification of biomarkers, detection of viruses, differentiation of multiple pathogens, etc. A few potential future applications of microarrays could be for personalized medicine, vaccine candidate screening, toxin screening, pathogen identification, and posttranslational modifications. Full article

Galectins bind various pathogens through recognition of distinct carbohydrate structures. In this work, we examined the binding of four human galectins to the Gram-negative bacteria Klebsiella pneumoniae (Kpn) and non-typeable Haemophilus influenzae (NTHi), which display different surface glycans. In particular, Kpn cells are covered by a polysaccharide capsule and display an O-chain-containing lipopolysaccharide (LPS), whereas NTHi is not capsulated and its LPS, termed lipooligosacccharide (LOS), does not contain O-chain. Binding assays to microarray-printed bacteria revealed that galectins-3, -4, and -8, but not galectin-1, bind to Kpn and NTHi cells, and confocal microscopy attested binding to bacterial cells in suspension. The three galectins bound to array-printed Kpn LPS. Moreover, analysis of galectin binding to mutant Kpn cells evidenced that the O-chain is the docking point for galectins on wild type Kpn. Galectins-3, -4, and -8 also bound the NTHi LOS. Microarray-assisted comparison of the binding to full-length and truncated LOSs, as well as to wild type and mutant cells, supported LOS involvement in galectin binding to NTHi. However, deletion of the entire LOS oligosaccharide chain actually increased binding to NTHi cells, indicating the availability of other ligands on the bacterial surface, as similarly inferred for Kpn cells devoid of both O-chain and capsule. Altogether, the results illustrate galectins’ versatility for recognizing different bacterial structures, and point out the occurrence of so far overlooked galectin ligands on bacterial surfaces. Full article

The level of human natural antibodies of immunoglobulin M isotype against Le^C in patients with breast cancer is lower than in healthy women. The epitope specificity of these antibodies has been characterized using a printed glycan array and enzyme-linked immunosorbent assay (ELISA), the antibodies being isolated from donors’ blood using Le^C-Sepharose (Le^C is Galβ1-3GlcNAcβ). The isolated antibodies recognize the disaccharide but do not bind to glycans terminated with Le^C, which implies the impossibility of binding to regular glycoproteins of non-malignant cells. The avidity (as dissociation constant value) of antibodies probed with a multivalent disaccharide is 10⁻⁹ M; the nanomolar level indicates that the concentration is sufficient for physiological binding to the cognate antigen. Testing of several breast cancer cell lines showed the strongest binding to ZR 75-1. Interestingly, only 7% of the cells were positive in a monolayer with a low density, increasing up to 96% at highest density. The enhanced interaction (instead of the expected inhibition) of antibodies with ZR 75-1 cells in the presence of Galβ1-3GlcNAcβ disaccharide, indicates that the target epitope of anti-Le^C antibodies is a molecular pattern with a carbohydrate constituent rather than a glycan. Full article