Comparative structural analysis of the glycosylation of salivary and buccal cell proteins: Innate protection against infection by Candida albicans

Mucosal epithelial surfaces, such as line the oral cavity, are common sites of microbial colonization by bacteria, yeast and fungi. The microbial interactions involve adherence between the glycans on the host cells and the carbohydrate-binding proteins of the pathogen. Saliva constantly bathes the buccal cells of the epithelial surface of the mouth and we postulate that the sugars on the salivary glycoproteins provide an innate host immune mechanism against infection by competitively inhibiting pathogen binding to the cell membranes. The structures of the N-and O-linked oligosaccharides on the glycoproteins of saliva and buccal cell membranes were analyzed using capillary carbon liquid chromatography-electrospray ionization MS/MS. The 190 glycan structures that were characterized were qualitatively similar, but differed quantitatively, between saliva and epithelial buccal cell membrane proteins. The similar relative abundance of the terminal glycan epitope structures (e.g. ABO(H) blood group, sialylation and Lewis-type antigens) on saliva and buccal cell membrane glycoproteins indicated that the terminal N-and O-linked glycan substructures in saliva could be acting as decoy-binding receptors to competitively inhibit the attachment of pathogens to the surface of the oral mucosa. A flow cytometry-based binding assay quantified the interaction between buccal cells and the commensal oral pathogen Candida albicans. Whole saliva and released glycans from salivary proteins inhibited the interaction of C. albicans with buccal epithelial cells, confirming the protective role of the glycans on salivary glycoproteins against pathogen infection. © 2012 The Author

Specific Sialoforms Required for the Immune Suppressive Activity of Human Soluble CD52

Human CD52 is a small glycopeptide (12 amino acid residues) with one N-linked glycosylation site at asparagine 3 (Asn3) and several potential O-glycosylation serine/threonine sites. Soluble CD52 is released from the surface of activated T cells and mediates immune suppression via its glycan moiety. In suppressing activated T cells, it first sequesters the pro-inflammatory high mobility group Box 1 (HMGB1) protein, which facilitates its binding to the inhibitory sialic acid-binding immunoglobulin-like lectin-10 (Siglec-10) receptor. We aimed to identify the features of CD52 glycan that underlie its bioactivity. Analysis of native CD52 purified from human spleen revealed extensive heterogeneity in N-glycosylation and multi-antennary sialylated N-glycans with abundant polyLacNAc extensions, together with mainly di-sialylated O-glycosylation type structures. Glycomic (porous graphitized carbon-ESI-MS/MS) and glycopeptide (C8-LC-ESI-MS) analysis of recombinant soluble human CD52-immunoglobulin Fc fusion proteins revealed that CD52 bioactivity was correlated with a high abundance of tetra-antennary α-2,3/6 sialylated N-glycans. Removal of α-2,3 sialylation abolished bioactivity, which was restored by re-sialylation with α-2,3 sialyltransferases. When glycoforms of CD52-Fc were fractionated by anion exchange MonoQ-GL chromatography, bioactive fractions displayed mainly tetra-antennary, α-2,3 sialylated N-glycan structures and a lower relative abundance of bisecting GlcNAc structures compared to non-bioactive fractions. In addition, O-glycan core type-2 di-sialylated structures at Ser12 were more abundant in bioactive CD52 fractions. Understanding the structural features of CD52 glycan required for its bioactivity will aid its development as an immunotherapeutic agent

Targeting cancer glycosylation repolarizes tumor-associated macrophages allowing effective immune checkpoint blockade.

Immune checkpoint blockade (ICB) has substantially improved the prognosis of patients with cancer, but the majority experiences limited benefit, supporting the need for new therapeutic approaches. Up-regulation of sialic acid-containing glycans, termed hypersialylation, is a common feature of cancer-associated glycosylation, driving disease progression and immune escape through the engagement of Siglec receptors on tumor-infiltrating immune cells. Here, we show that tumor sialylation correlates with distinct immune states and reduced survival in human cancers. The targeted removal of Siglec ligands in the tumor microenvironment, using an antibody-sialidase conjugate, enhanced antitumor immunity and halted tumor progression in several murine models. Using single-cell RNA sequencing, we revealed that desialylation repolarized tumor-associated macrophages (TAMs). We also identified Siglec-E as the main receptor for hypersialylation on TAMs. Last, we found that genetic and therapeutic desialylation, as well as loss of Siglec-E, enhanced the efficacy of ICB. Thus, therapeutic desialylation represents an immunotherapeutic approach to reshape macrophage phenotypes and augment the adaptive antitumor immune response