Glycosylation occurs at all major types of biomolecules, including proteins, lipids, and RNAs to form glycoproteins, glycolipids, and glycoRNAs in mammalian cells, respectively. The carbohydrate moiety, known as glycans on glycoproteins and glycolipids, is diverse in their compositions and structures. Normal cells have their unique array of glycans or glycome which play pivotal roles in many biological processes. The glycan structures in cancer cells, however, are often altered, some having unique structures which are termed as tumor-associated carbohydrate antigens (TACAs). TACAs as tumor biomarkers are glycan epitopes themselves, or glycoconjugates. Some of those TACAs serve as tumor glyco-biomarkers in clinical practice, while others are the immune therapeutic targets for treatment of cancers. A monoclonal antibody (mAb) to GD2, an intermediate of sialic-acid containing glycosphingolipids, is an example of FDA-approved immune therapy for neuroblastoma indication in young adults and many others. Strategies for targeting the aberrant glycans are currently under development, and some have proceeded to clinical trials. In this review, we summarize the currently established and most promising aberrant glycosylation as therapeutic targets for solid tumors.

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.

Altered protein glycosylation is typically associated with cognitive defects and other phenotypes, but there is a lack of knowledge about the brain glycoproteome. Here, we used the newly available O-glycoprotease IMPa from Pseudomonas aeruginosa for comprehensive O-glycoproteomic analyses of the mouse brain. In this approach, total tryptic glycopeptides were prepared, extracted, purified, and conjugated to a solid support before an enzymatic cleavage by IMPa. O-glycopeptides were analyzed by electron-transfer/higher-energy collision dissociation (EThcD), which permits site-specific and global analysis of all types of O-glycans. We developed two complementary approaches for the analysis of the total O-glycoproteome using HEK293 cells and derivatives. The results demonstrated that IMPa and EThcD facilitate the confident localization of O-glycans on glycopeptides. We then applied these approaches to characterize the O-glycoproteome of the mouse brain, which revealed the high frequency of various sialylated O-glycans along with the unusual presence of the Tn antigen. Unexpectedly, the results demonstrated that glycoproteins in the brain O-glycoproteome only partly overlap with those reported for the brain N-glycoproteome. These approaches will aid in identifying the novel O-glycoproteomes of different cells and tissues and foster clinical and translational insights into the functions of protein O-glycosylation in the brain and other organs.

Mutations in genes encoding molecular chaperones can lead to chaperonopathies, but none have so far been identified causing congenital disorders of glycosylation. Here we identified two maternal half-brothers with a novel chaperonopathy, causing impaired protein O-glycosylation. The patients have a decreased activity of T-synthase ( C1GALT1 ), an enzyme that exclusively synthesizes the T-antigen, a ubiquitous O-glycan core structure and precursor for all extended O-glycans. The T-synthase function is dependent on its specific molecular chaperone Cosmc, which is encoded by X-chromosomal C1GALT1C1 . Both patients carry the hemizygous variant c.59C>A (p.Ala20Asp; A20D-Cosmc) in C1GALT1C1 . They exhibit developmental delay, immunodeficiency, short stature, thrombocytopenia, and acute kidney injury (AKI) resembling atypical hemolytic uremic syndrome. Their heterozygous mother and maternal grandmother show an attenuated phenotype with skewed X-inactivation in blood. AKI in the male patients proved fully responsive to treatment with the complement inhibitor Eculizumab. This germline variant occurs within the transmembrane domain of Cosmc, resulting in dramatically reduced expression of the Cosmc protein. Although A20D-Cosmc is functional, its decreased expression, though in a cell or tissue-specific manner, causes a large reduction of T-synthase protein and activity, which accordingly leads to expression of varied amounts of pathological Tn-antigen (GalNAcα1-O-Ser/Thr/Tyr) on multiple glycoproteins. Transient transfection of patient lymphoblastoid cells with wild-type C1GALT1C1 partially rescued the T-synthase and glycosylation defect. Interestingly, all four affected individuals have high levels of galactose-deficient IgA1 in sera. These results demonstrate that the A20D-Cosmc mutation defines a novel O-glycan chaperonopathy and causes the altered O-glycosylation status in these patients.

Mucin-type O-glycans on glycoproteins are pivotal for biology and impact the quality of biotherapeutics. Furthermore, glycans on host cells serve as ligands for lectins/adhesins on bacteria for bacterium-host interactions in the colonization or attachment/invasion of bacteria. Defining the structure-function relationship of O-glycans is hindered by a lack of enzyme(s) to release sialylated O-glycans from glycoproteins. Here we show identification of endo-α-N-acetylgalactosaminidases (O-glycanases, GH101) with broad substrate specificities, termed Peptide:O-Glycosidase (POGase). In 5 POGase orthologs identified, we characterize one that releases sialylated O-glycans from glycopeptides, glycoproteins and biotherapeutics. Three peptide motifs differentiate the POGase existing in phylum Actinomycetota from known O-glycanases in other bacteria. While the GH101 domain classifies POGases, other domains confer the efficient enzyme activity and binding to major glycans decorating epithelial cells. The dual functional POGases encompassing broader O-glycanase and adhesin activities will facilitate the study of O-glycomics, quality assessment of biotherapeutics, and development of microbiology and medicine.

Tumor-associated carbohydrate antigens (TACAs) are expressed in a majority of carcinoma-type malignancies, but not in normal tissue, and they play an important role in driving oncogenic transformation. A highly relevant TACA is the Tn antigen, which is ubiquitously expressed in many human carcinomas, and which likely represents a promising diagnostic biomarker and candidate for molecular-targeted therapy. Over the last several decades, a myriad of immunotherapies and vaccination therapies against the Tn antigen have been developed; however, these have not shown sufficient anti-tumor efficacy. This review presents a novel methodology for generating a defined anti-Tn monoclonal antibody (mAb) and presents the latest cancer immunotherapies with anti-Tn mAb (targeted therapy, CAR-T cell therapy, and antibody-drug conjugates) within the hostile tumor microenvironment.