Functions Of O-glycans Research Articles

O-Glycosylation of a male seminal fluid protein influences sperm binding and female postmating behavior.

Glycoproteins are abundant within the human reproductive system and alterations in glycosylation lead to reproductive disorders, suggesting that glycans play an important role in reproductive function. In this study, we used the Drosophila reproductive system as a model to investigate the biological functions of O-glycosylation. We found that O-glycosylation in the male accessory glands, an organ responsible for secreting seminal fluid proteins, plays important roles in female postmating behavior. The loss of one O-glycosyltransferase, PGANT9, in the male reproductive system resulted in decreased egg production in mated females. We identified one substrate of PGANT9, lectin-46Ca (CG1656), which is known to affect female postmating responses. We further show that the loss of lectin-46Ca O-glycosylation affects its ability to associate with sperm tails, resulting in reduced transfer within the female reproductive system. Our results provide the first example that O-glycosylation of a seminal fluid protein affects its ability to associate with sperm in vivo. These studies may shed light on the biological function of O-glycans in mammalian reproduction.

The sialyl-Tn antigen synthase genes regulates migration-proliferation dichotomy in prostate cancer cells under hypoxia.

A low-oxygen (hypoxia) tumor microenvironment can facilitate chemotherapy and radiation therapy resistance in tumors and is associated with a poor prognosis. Hypoxia also affects PCa (prostate cancer) phenotype transformation and causes therapeutic resistance. Although O-glycans are known to be involved in the malignancy of various cancers under hypoxia, the expression and function of O-glycans in PCa are not well understood. In this study, the saccharide primer method was employed to analyze O-glycan expression in PCa cells. Results showed that the expression of sTn antigens was increased in PCa cells under hypoxia. Furthermore, it was found that ST6GalNAc1, the sTn antigen synthase gene, was involved in the migration-proliferation dichotomy and drug resistance in PCa cells under hypoxia. The results of this study will contribute to the development of novel diagnostic markers and drug targets for PCa under hypoxia.

Global functions of O-glycosylation: promises and challenges in O-glycobiology.

Mucin type O-glycosylation is one of the most diverse types of glycosylation, playing essential roles in tissue development and homeostasis. In complex organisms, O-GalNAc glycans comprise a substantial proportion of the glycocalyx, with defined functions in hemostatic, gastrointestinal, and respiratory systems. Furthermore, O-GalNAc glycans are important players in host-microbe interactions, and changes in O-glycan composition are associated with certain diseases and metabolic conditions, which in some instances can be used for diagnosis or therapeutic intervention. Breakthroughs in O-glycobiology have gone hand in hand with the development of new technologies, such as advancements in mass spectrometry, as well as facilitation of genetic engineering in mammalian cell lines. High-throughput O-glycoproteomics have enabled us to draw a comprehensive map of O-glycosylation, and mining this information has supported the definition and confirmation of functions related to site-specific O-glycans. This includes protection from proteolytic cleavage, as well as modulation of binding affinity or receptor function. Yet, there is still much to discover, and among the important next challenges will be to define the context-dependent functions of O-glycans in different stages of cellular differentiation, cellular metabolism, host-microbiome interactions, and in disease. In this review, we present the achievements and the promises in O-GalNAc glycobiology driven by technological advances in analytical methods, genetic engineering, and systems biology.

Site-Specific O-Glycosylation Analysis of SARS-CoV-2 Spike Protein Produced in Insect and Human Cells.

Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence is difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S—insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.

Mucin-Type O-GalNAc Glycosylation in Health and Disease.

Mucin-type GalNAc O-glycosylation is one of the most abundant and unique post-translational modifications. The combination of proteome-wide mapping of GalNAc O-glycosylation sites and genetic studies with knockout animals and genome-wide analyses in humans have been instrumental in our understanding of GalNAc O-glycosylation. Combined, such studies have revealed well-defined functions of O-glycans at single sites in proteins, including the regulation of pro-protein processing and proteolytic cleavage, as well as modulation of receptor functions and ligand binding. In addition to isolated O-glycans, multiple clustered O-glycans have an important function in mammalian biology by providing structural support and stability of mucins essential for protecting our inner epithelial surfaces, especially in the airways and gastrointestinal tract. Here the many O-glycans also provide binding sites for both endogenous and pathogen-derived carbohydrate-binding proteins regulating critical developmental programs and helping maintain epithelial homeostasis with commensal organisms. Finally, O-glycan changes have been identified in several diseases, most notably in cancer and inflammation, where the disease-specific changes can be used for glycan-targeted therapies. This chapter will review the biosynthesis, the biology, and the translational perspectives of GalNAc O-glycans.

Loss of intestinal O-glycans promotes spontaneous duodenal tumors.

Mucin-type O-glycans, primarily core 1- and core 3-derived O-glycans, are the major mucus barrier components throughout the gastrointestinal tract. Previous reports identified the biological role of O-glycans in the stomach and colon. However, the biological function of O-glycans in the small intestine remains unknown. Using mice lacking intestinal core 1- and core 3-derived O-glycans [intestinal epithelial cell C1galt1(-/-);C3GnT(-/-) or double knockout (DKO)], we found that loss of O-glycans predisposes DKO mice to spontaneous duodenal tumorigenesis by ∼1 yr of age. Tumor incidence did not increase with age; however, tumors advanced in aggressiveness by 20 mo. O-glycan deficiency was associated with reduced luminal mucus in DKO mice before tumor development. Altered intestinal epithelial homeostasis with enhanced baseline crypt proliferation characterizes these phenotypes as assayed by Ki67 staining. In addition, fluorescence in situ hybridization analysis reveals a significantly lower bacterial burden in the duodenum compared with the large intestine. This phenotype is not reduced with antibiotic treatment, implying O-glycosylation defects, rather than bacterial-induced inflammation, which causes spontaneous duodenal tumorigenesis. Moreover, inflammatory responses in DKO duodenal mucosa are mild as assayed with histology, quantitative PCR for inflammation-associated cytokines, and immunostaining for immune cells. Importantly, inducible deletion of intestinal O-glycans in adult mice leads to analogous spontaneous duodenal tumors, although with higher incidence and heightened severity compared with mice with O-glycans constitutive deletion. In conclusion, these studies reveal O-glycans within the small intestine are critical determinants of duodenal cancer risk. Future studies will provide insights into the pathogenesis in the general population and those at risk for this rare but deadly cancer.

Inhibition of Mucin-Type O-Glycosylation through Metabolic Processing and Incorporation of N-Thioglycolyl-d-galactosamine Peracetate (Ac5GalNTGc)

Mucin-type O-glycans form one of the most abundant and complex post-translational modifications (PTM) on cell surface proteins that govern adhesion, migration, and trafficking of hematopoietic cells. Development of targeted approaches to probe functions of O-glycans is at an early stage. Among several approaches, small molecules with unique chemical functional groups that could modulate glycan biosynthesis form a critical tool. Herein, we show that metabolism of peracetyl N-acyl-D-galactosamine derivatives carrying an N-thioglycolyl (Ac5GalNTGc, 1) moiety-but not N-glycolyl (Ac5GalNGc, 2) and N-acetyl (Ac4GalNAc, 3)-through the N-acetyl-D-galactosamine (GalNAc) salvage pathway induced abrogation of MAL-II and PNA epitopes in Jurkat cells. Mass spectrometry of permethylated O-glycans from Jurkat cells confirmed the presence of significant amounts of elaborated O-glycans (sialyl-T and disialyl-T) which were inhibited upon treatment with 1. O-Glycosylation of CD43, a cell surface antigen rich in O-glycans, was drastically reduced by 1 in a thiol-dependent manner. By contrast, only mild effects were observed for CD45 glycoforms. Direct metabolic incorporation of 1 was confirmed by thiol-selective Michael addition reaction of immunoprecipitated CD43-myc/FLAG. Mechanistically, CD43 glycoforms were unperturbed by peracetylated N-(3-acetylthiopropanoyl) (4), N-(4-acetylthiobutanoyl) (5), and N-methylthioacetyl (6) galactosamine derivatives, N-thioglycolyl-D-glucosamine (7, C-4 epimer of 1), and α-O-benzyl 2-acetamido-2-deoxy-D-galactopyranoside (8), confirming the critical requirement of both free sulfhydryl and galactosamine moieties for inhibition of mucin-type O-glycans. Similar, yet differential, effects of 1 were observed for CD43 glycoforms in multiple hematopoietic cells. Development of small molecules that could alter glycan patterns in an antigen-selective and cell-type selective manner might provide avenues for understanding biological functions of glycans.

Cosmc Is Essential for O-Glycosylation of Platelet Glycoproteins and Platelet Function

AbstractAbstract 383Platelets express a large number of glycoproteins involved in adhesion and signaling events, but the specific roles of the Asn-linked (N-glycans) and Ser/Thr-linked (O-glycans) pathways are not well understood. While O-glycans are known to play important roles as selectin ligands important in leukocyte recruiting and lymphocyte homing, little is known about the functions of O-glycans in platelet biology. O-glycan biosynthesis requires sequential actions of glycosyltransferases in the Golgi apparatus. Key among these is the T-synthase, which converts the Tn antigen (GalNAcα1-Ser/Thr) to the core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr. Core 1 O-glycans are the common precursor for complex O-glycans on secreted and membrane glycoproteins on hematopoietic cells. Biosynthesis of an active T-synthase requires a unique and T-synthase-specific molecular chaperone termed Cosmc. In mice, disruption of either Cosmc or T-synthase is embryonic lethal due in part to impaired angiogenesis. In humans, somatic mutations in Cosmc on Xq24 result in Tn syndrome. Tn syndrome, which is characterized by the Tn antigen expression on blood cells of all lineages, is associated with thrombocytopenia, indicating a potential role of Cosmc and complex O-glycans in platelet biology and hemostasis.To further explore the roles of O-glycans in hemostasis we generated mice with deletions of Cosmc in hematopoietic and endothelial cells (EHC-Cosmc-/y) by breeding floxed (Cosmcflox/flox) females with Tie2-Cre transgenic male mice. Any EHC-Cosmc-/y mice are null, since the Cosmc gene is X-linked. Cre-mediated excision of the Cosmc gene was effective as demonstrated by the almost complete absence of Cosmc transcript, T-synthase activity, and the expression of Tn antigen on EHC-Cosmc-/y platelets. A high incidence of post-natal lethality (89%) due to hemorrhage was observed in EHC-Cosmc-/y mice along with a smaller body size, and the few survivors died within 3–5 months as a result of either pulmonary or GI hemorrhage. Tail-bleeding times were markedly prolonged in the surviving EHC-Cosmc-/y mice in comparison to both littermate controls and heterozygous females.Consistent with the human phenotype observed in patients with Tn syndrome, EHC Cosmc-/y mice were markedly macrothrombocytopenic (166±57 103/mm3) compared with their littermate controls (787±124 103/mm3). EHC Cosmc-/y platelets were more than twice as large as controls and appeared circular in contrast with the normal discoid shape of control platelets. The severity of the bleeding phenotype led us to more closely examine the platelet phenotype and function. In EHC Cosmc-/y platelet extracts both GPIbα and integrinαIIb appeared as proteolytically cleaved fragments, and platelets from EHC Cosmc-/y mouse had low expression of GPIbα on their cell surface as measured by flow cytometry. Levels of transcripts encoding GPIbα, GPIIb, GPV, and P-selectin in platelets were normal compared to those from control mice. EHC Cosmc-/y platelets have remarkably reduced adhesion and spreading on Von Willebrand factor coated plates, consistent with the low levels of GPIbα on the platelet surface. A similar defect in adhesion and binding was observed to a fibrinogen surface. Additionally, EHC Cosmc-/y platelets had significantly impaired activation in response to thrombin with decreased binding of antibodies to P-selectin and the activation-specific integrin αIIbβ3 antibody JON/A following stimulation. Female heterozygotes (Cosmc-/+) appeared normal on gross examination, however occasionally dysfunctional megathrombocytes were observed in peripheral blood consistent with lyonization. This study is the first to demonstrate the essential role of Cosmc in regulating correct platelet O-glycosylation, and to indicate the importance of complex O-glycans in platelet morphogenesis, biogenesis, and function. Disclosures:No relevant conflicts of interest to declare.

Biosynthesis and functions of O-glycans and regulation of mucin antigen expression in cancer.

Biosynthesis and functions of O-glycans and regulation of mucin antigen expression in cancer.