O-GalNAc glycans, also known as mucin-type O-glycans, are primary constituents of mucins on various mucosal sites of the body and also ubiquitously expressed on cell surface and secreted proteins. They have crucial roles in a wide range of physiological and pathological processes, including tumor growth and progression. In addition, altered expression of O-GalNAc glycans is frequently observed during different disease states. Research dedicated to unraveling the structure-function relationships of O-GalNAc glycans has led to the discovery of disease biomarkers and diagnostic tools and the development of O-glycopeptide-based cancer vaccines. Many of these efforts require amino acid-linked O-GalNAc core structures as building blocks to assemble complex O-glycans and glycopeptides. There are eight core structures (cores one to eight), from which all mucin-type O-glycans are derived. In this protocol, we describe the first divergent synthesis of all eight cores from a versatile precursor in practical scales. The protocol involves (i) chemical synthesis of the orthogonally protected precursor (3 days) from commercially available materials, (ii) chemical synthesis of five unique glycosyl donors (1-2 days for each donor) and (iii) selective deprotection of the precursor and assembly of the eight cores (2-4 days for each core). The procedure can be adopted to prepare O-GalNAc cores linked to serine, threonine and tyrosine, which can then be utilized directly for solid-phase glycopeptide synthesis or chemoenzymatic synthesis of complex O-glycans. The procedure empowers researchers with fundamental organic chemistry skills to prepare gram scales of any desired O-GalNAc core(s) or all eight cores concurrently.
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Protein glycosylation influences cellular recognition and regulates protein interactions, but how glycosylation functions alongside other common posttranslational modifications (PTMs), like tyrosine sulfation (sTyr), is unclear. We produced a library of 53 chemoenzymatically synthesized glycosulfopeptides representing N-terminal domains of human and murine P-selectin glycoprotein ligand-1 (PSGL-1), varying in sTyr and O-glycosylation (structure and site). Using these, we identified key roles of PSGL-1 O-glycosylation and sTyr in controlling interactions with specific chemokines. Results demonstrate that sTyr positively affects CCL19 and CCL21 binding to PSGL-1 N terminus, whereas O-glycan branching and sialylation reduced binding. For murine PSGL-1, interference between PTMs is greater, attributed to proximity between the two PTMs. Using fluorescence polarization, we found sTyr is a positive determinant for some chemokines. We showed that synthetic sulfopeptides are potent in decreasing chemotaxis of human dendritic cells toward CCL19 and CCL21. Our results provide new research avenues into the interplay of PTMs regulating leukocyte/chemokine interactions.
Copyright © 2023 Elsevier Ltd. All rights reserved.

The underlying pathology of immunoglobulin A (IgA) nephropathy (IgAN), the most common glomerulonephritis worldwide, is driven by the deposition of immune complexes containing galactose-deficient IgA1 [Tn(+)IgA1] in the glomerular mesangium. Here, we report that novel anti-Tn circulating immune complexes (anti-Tn CICs) contain predominantly IgM, representing large macromolecular complexes of ~1.2 megadaltons to several megadalton sizes together with Tn(+)IgA1 and some IgG. These complexes are significantly elevated in sera of patients with IgAN, which contains higher levels of complement C3, compared to healthy individuals. Anti-Tn CICs are bioactive and induce specific proliferation of human renal mesangial cells. We found that these anti-Tn CICs can be dissociated with small glycomimetic compounds, which mimic the Tn antigen of Tn(+)IgA1, releasing IgA1 from anti-Tn CICs. This glycomimetic compound can also significantly inhibit the proliferative activity of anti-Tn CICs of patients with IgAN. These findings could enhance both the diagnosis of IgAN and its treatment, as specific drug treatments are now unavailable.

Glycan recognition is typically studied using free glycans, but glycopeptide presentations represent more physiological conditions for glycoproteins. To facilitate studies of glycopeptide recognition, we developed Glyco-SPOT synthesis, which enables the parallel production of diverse glycopeptide libraries at microgram scales. The method uses a closed system for prolonged reactions required for coupling Fmoc-protected glycoamino acids, including O-, N-, and S-linked glycosides, and release conditions to prevent side reactions. To optimize reaction conditions and sample reaction progress, we devised a biopsy testing method. We demonstrate the efficient utilization of such microscale glycopeptide libraries to determine the specificity of glycan-recognizing antibodies (e.g., CTD110.6) using microarrays, enzyme specificity on-array and in-solution (e.g., ST6GalNAc1, GCNT1, and T-synthase), and binding kinetics using fluorescence polarization. We demonstrated that the glycosylation on these peptides can be expanded using glycosyltransferases both in-solution and on-array. This technology will promote the discovery of biological functions of peptide modifications by glycans.Copyright © 2020 Elsevier Ltd. All rights reserved.

In cancers, increased fucosylation (attachment of fucose sugar residues) on cell-surface glycans, resulting from the abnormal upregulation of the expression of specific fucosyltransferase enzymes (FUTs), is one of the most important types of glycan modifications associated with malignancy. Fucosylated glycans on cell surfaces are involved in a multitude of cellular interactions and signal regulation in normal biological processes, as well as in disease. For example, sialyl LewisX is a fucosylated cell-surface glycan that is abnormally abundant in some cancers where it has been implicated in facilitating metastasis, allowing circulating tumor cells to bind to the epithelial tissue within blood vessels and invade into secondary sites by taking advantage of glycan-mediated interactions. To identify inhibitors of FUT enzymes as potential cancer therapeutics, we have developed a novel high-throughput assay that makes use of a fluorogenically labeled oligosaccharide as a probe of fucosylation. This probe, which consists of a 4-methylumbelliferyl glycoside, is recognized and hydrolyzed by specific glycoside hydrolase enzymes to release fluorescent 4-methylumbelliferone, yet when the probe is fucosylated prior to treatment with the glycoside hydrolases, hydrolysis does not occur and no fluorescent signal is produced. We have demonstrated that this assay can be used to measure the inhibition of FUT enzymes by small molecules, because blocking fucosylation will allow glycosidase-catalyzed hydrolysis of the labeled oligosaccharide to produce a fluorescent signal. Employing this assay, we have screened a focused library of small molecules for inhibitors of a human FUT enzyme involved in the synthesis of sialyl LewisX and demonstrated that our approach can be used to identify potent FUT inhibitors from compound libraries in microtiter plate format.