Expression Of O-glycans Research Articles

O-Glycome Profiling of Breast Cancer Cell Lines to Understand Breast Cancer Brain Metastasis.

Breast cancer is the second leading cause of cancer-related death among women and a major source of brain metastases. Despite the increasing incidence of brain metastasis from breast cancer, the underlying mechanisms remain poorly understood. Altered glycosylation is known to play a role in various diseases including cancer metastasis. However, profiling studies of O-glycans and their isomers in breast cancer brain metastasis (BCBM) are scarce. This study analyzed the expression of O-glycans and their isomers in human breast cancer cell lines (MDA-MB-231, MDA-MB-361, HTB131, and HTB22), a brain cancer cell line (CRL-1620), and a brain metastatic breast cancer cell line (MDA-MB-231BR) using nanoLC-MS/MS, identifying 27 O-glycan compositions. We observed significant upregulation in the expression of HexNAc1Hex1NeuAc2 and HexNAc2Hex3, whereas the expression of HexNAc1Hex1NeuAc1 was downregulated in MDA-MB-231BR compared to other cell lines. In our isomeric analysis, we observed notable alterations in the isomeric forms of the O-glycan structure HexNAc1Hex1NeuAc1 in a comparison of different cell lines. Our analysis of O-glycans and their isomers in cancer cells demonstrated that changes in their distribution can be related to the metastatic process. We believe that our investigation will contribute to an enhanced comprehension of the significance of O-glycans and their isomers in BCBM.

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.

CRISPR-screen identifies ZIP9 and dysregulated Zn2+ homeostasis as a cause of cancer-associated changes in glycosylation.

In epithelial cancers, truncated O-glycans, such as the Thomson-nouveau antigen (Tn) and its sialylated form (STn), are upregulated on the cell surface and associated with poor prognosis and immunological escape. Recent studies have shown that these carbohydrate epitopes facilitate cancer development and can be targeted therapeutically; however, the mechanism underpinning their expression remains unclear. To identify genes directly influencing the expression of cancer-associated O-glycans, we conducted an unbiased, positive-selection, whole-genome CRISPR knockout-screen using monoclonal antibodies against Tn and STn. We show that knockout of the Zn2+-transporter SLC39A9 (ZIP9), alongside the well-described targets C1GALT1 (C1GalT1) and its molecular chaperone, C1GALT1C1 (COSMC), results in surface-expression of cancer-associated O-glycans. No other gene perturbations were found to reliably induce O-glycan truncation. We furthermore show that ZIP9 knockout affects N-linked glycosylation, resulting in upregulation of oligo-mannose, hybrid-type, and α2,6-sialylated structures as well as downregulation of tri- and tetra-antennary structures. Finally, we demonstrate that accumulation of Zn2+ in the secretory pathway coincides with cell-surface presentation of truncated O-glycans in cancer tissue, and that over-expression of COSMC mitigates such changes. Collectively, the findings show that dysregulation of ZIP9 and Zn2+ induces cancer-like glycosylation on the cell surface by affecting the glycosylation machinery.

O-Glycosylation Changes in Serum Immunoglobulin G Are Associated with Inflammation Development in Advanced Endometriosis.

Endometriosis is a gynecological disease, the pathogenesis of which seems to be directly related to inflammatory processes with an immune basis. Our study aimed to analyze the O-glycosylation of native serum IgG and IgG isolated from sera of women with advanced endometriosis, without endometriosis but with benign gynecological diseases, and from a control group of healthy women, in the context of its utility for differentiation of advanced endometriosis from the other two groups of women studied. For the analysis of serum IgG O-glycosylation and the expression of multi-antennary N-glycans, lectin-ELISA with lectins specific to O-glycans (MPL, VVL, and Jacalin) and highly branched N-glycans (PHA-L) was used. The relative reactivities of isolated serum IgG O-linked glycans with specific lectins as well as the MPL/VVL O-glycosylation ratio were significantly higher in patients with advanced endometriosis and those with other gynecological diseases when compared to the control group of healthy women. We also showed significantly higher expression of PHA-L-reactive multi-antennary N-glycans in isolated IgG in the advanced endometriosis and the non-endometriosis groups in comparison to the control group. Additionally, significantly higher expression of Jacalin-reactive O-glycans in isolated IgG was observed in the non-endometriosis than in the advanced endometriosis group. The results of the ROC curve and cluster analysis additionally confirmed that the lectin-based analysis of isolated serum IgG O-glycosylation and the expression of highly branched N-glycans may help distinguish women with advanced endometriosis from healthy women. Moreover, the analysis of the expression of Jacalin-reactive i-IgG O-glycans may be helpful in differentiation between women with advanced endometriosis and patients with other gynecological diseases with an inflammatory background. In the case of non-endometriosis patients, the observed differences were most probably caused by increased expression of core 3 type O-glycans.

Functional analysis of GCNT3 for cell migration and EMT of castration-resistant prostate cancer cells.

Castration-resistant prostate cancer (CRPC) is a malignant tumor that is resistant to androgen deprivation therapy. Treatments for CRPC are limited, and no diagnostic markers are currently available. O-glycans are known to play an important role in cell proliferation, migration, invasion, and metastasis of cancer cells. However, the differences in the O-glycan expression profiles for normal prostate cancer (PCa) cells compared with CRPC cells have not yet been investigated. In this study, the saccharide primer method was employed to analyze the O-glycans expressed in CRPC cells. Expression levels of core 4-type O-glycans were significantly increased in CRPC cells. Furthermore, the expression level of N-Acetylglucosaminyltransferase 3 (GCNT3), a core 4-type O-glycan synthase gene, was increased in CRPC cells. The expression of core 4-type O-glycans and GCNT3 was presumed to be regulated by androgen deprivation. GCNT3 knockdown induced cell migration and epithelial-mesenchymal transition. These observations elucidate the mechanism of acquisition of castration resistance in PCa and offer new possibilities for the development of diagnostic markers and therapeutic targets in the treatment of PCa.

Mucin-Type O-Glycosylation in the Drosophila Nervous System.

Mucin-type O-glycosylation, a predominant type of O-glycosylation, is an evolutionarily conserved posttranslational modification in animals. Mucin-type O-glycans are often found on mucins in the mucous membranes of the digestive tract. These glycan structures are also expressed in other cell types, such as blood cells and nephrocytes, and have crucial physiological functions. Altered expression of mucin-type O-glycans is known to be associated with several human disorders, including Tn syndrome and cancer; however, the physiological roles of mucin-type O-glycans in the mammalian brain remains largely unknown. The functions of mucin-type O-glycans have been studied in the fruit fly, Drosophila melanogaster. The basic structures of mucin-type O-glycans, including Tn antigen (GalNAcα1-Ser/Thr) and T antigen (Galβ1–3GalNAcα1-Ser/Thr), as well as the glycosyltransferases that synthesize them, are conserved between Drosophila and mammals. These mucin-type O-glycans are expressed in the Drosophila nervous system, including the central nervous system (CNS) and neuromuscular junctions (NMJs). In primary cultured neurons of Drosophila, mucin-type O-glycans show a characteristic localization pattern in axons. Phenotypic analyses using mutants of glycosyltransferase genes have revealed that mucin-type O-glycans are required for CNS development, NMJ morphogenesis, and synaptic functions of NMJs in Drosophila. In this review, we describe the roles of mucin-type O-glycans in the Drosophila nervous system. These findings will provide insight into the functions of mucin-type O-glycans in the mammalian brain.

Cellular O-Glycome Reporter/Amplification (CORA): Analytical and Preparative Tools to Study Mucin-Type O-Glycans of Living Cells.

Mucin-type O-glycosylation (O-glycans, O-glycome) is among the most biologically important post-translational modification in glycoproteins but O-glycan structural diversity and expression are poorly understood due to the inadequacy of current analytical methods. We recently developed a new tool termed cellular O-glycome reporter/amplification (CORA), which uses O-glycan precursors, benzyl-α-GalNAc (Bn-α-GalNAc) or azido-Bn-α-GalNAc (N3 -Bn-α-GalNAc), as surrogates of protein O-glycosylation. Living cells metabolically convert these precursors to all types of O-GalNAc glycans representative of the cells' capabilities. The amplification and secretion of the O-glycome products greatly facilitates their analysis and functional studies. Here we describe protocols for analytical and preparative applications. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Cellular O-glycome reporter/amplification for the analysis of mucin-type O-glycans from living cells Basic Protocol 2: Preparation of cellular O-glycans from living cells for functional glycomics and glycan microarrays Basic Protocol 3: Conjugation of cellular O-glycans with a bifunctional fluorescent tag Basic Protocol 4: 2D-HPLC purification and MALDI-TOF/MS identification of individual PYAB-Bn-O-glycan.

Placental Glycoredox Dysregulation Associated with Disease Progression in an Animal Model of Superimposed Preeclampsia.

Pregnancies carried by women with chronic hypertension are at increased risk of superimposed preeclampsia, but the placental pathways involved in disease progression remain poorly understood. In this study, we used the stroke-prone spontaneously hypertensive rat (SHRSP) model to investigate the placental mechanisms promoting superimposed preeclampsia, with focus on cellular stress and its influence on galectin–glycan circuits. Our analysis revealed that SHRSP placentas are characterized by a sustained activation of the cellular stress response, displaying significantly increased levels of markers of lipid peroxidation (i.e., thiobarbituric acid reactive substances (TBARS)) and protein nitration and defective antioxidant enzyme expression as early as gestation day 14 (which marks disease onset). Further, lectin profiling showed that such redox imbalance was associated with marked alterations of the placental glycocode, including a prominent decrease of core 1 O-glycan expression in trophoblasts and increased decidual levels of sialylation in SHRSP placentas. We also observed significant changes in the expression of galectins 1, 3 and 9 with pregnancy progression, highlighting the important role of the galectin signature as dynamic interpreters of placental microenvironmental challenges. Collectively, our findings uncover a new role for the glycoredox balance in the pathogenesis of superimposed preeclampsia representing a promising target for interventions in hypertensive disorders of pregnancy.

Core 2 β1,6-N-acetylglucosaminyltransferases accelerate the escape of choriocarcinoma from natural killer cell immunity

Hyperglycosylated human chorionic gonadotropin (H-hCG) is secreted from choriocarcinoma and contains a core2 O-glycan formed by core2 β1,6-N-acetylglucosaminyl transferase (C2GnT). Choriocarcinoma is considered immunogenic as it is gestational and contains paternal chromosomal components. Here we examined the function of C2GnT in the evasion of choriocarcinoma cells from natural killer (NK) cell-mediating killing. We determined that C2GnT is highly expressed in malignant gestational trophoblastic neoplasms. C2GnT KO downregulates core2 O-glycan expression in choriocarcinoma cells, which are more efficiently killed by NK cells than control cells. C2GnT KO cell containing tumor necrosis factor-related apoptosis inducing ligand have lower viability than control cells. Additionally, poly-N-acetyllactosamine in core2 branched oligosaccharides on MHC class I-related chain A (MICA) and mucin1 (MUC1) is significantly reduced in C2GnT KO cells. Meanwhile, the cumulative survival rate of nude mice inoculated with C2GnT KO tumors was higher than that of the control group. These findings suggest that choriocarcinoma cells may escape NK cell-mediated killing via glycosylation of MICA and MUC1.

Clinicopathological significance of core 3 O-glycan synthetic enzyme, β1,3-N-acetylglucosaminyltransferase 6 in pancreatic ductal adenocarcinoma.

Mucin-type O-glycans are involved in cancer initiation and progression, although details of their biological and clinicopathological roles remain unclear. The aim of this study was to investigate the clinicopathological significance of β1,3-N-acetylglucosaminyltransferase 6 (β3Gn-T6), an essential enzyme for the synthesis of core 3 O-glycan and several other O-glycans in pancreatic ductal adenocarcinoma (PDAC). We performed immunohistochemical and lectin-histochemical analyses to detect the expression of β3Gn-T6 and several O-glycans in 156 cases of PDAC with pancreatic intraepithelial neoplasias (PanINs) and corresponding normal tissue samples. The T antigen, Tn antigen, sialyl Lewis X (sLeX) antigen, and sLeX on core 2 O-glycan were more highly expressed in PDAC cells than in normal pancreatic duct epithelial cells (NPDEs). Conversely, the expression of 6-sulfo N-acetyllactosamine on extended core 1 O-glycan was found in NPDEs and was low in PDAC cells. These glycan expression levels were not associated with patient outcomes. β3Gn-T6 was expressed in ~20% of PDAC cases and 30–40% of PanINs but not in NPDEs. Higher expression of β3Gn-T6 was found in PDAC cells in more differentiated adenocarcinoma cases showing significantly longer disease-free survival in both univariate and multivariate analyses. In addition, the expression of β3Gn-T6 in PDAC cells and PanINs significantly correlated with the expression of MUC5AC in these cells, suggesting that β3Gn-T6 expression is related to cellular differentiation status of the gastric foveolar phenotype. Thus, it is likely that β3Gn-T6 expression in PDAC cells is a favorable prognostic factor in PDAC patients, and that the expression of β3Gn-T6 correlates with the gastric foveolar phenotype in pancreatic carcinogenesis.

Dual-Probe Approach for Mass Spectrometric Quantification of MUC1-Specific Terminal Gal/GalNAc In Situ.

Protein glycosylation is a prevalent post-translational modification that mediates a variety of cellular processes. For membrane proteins, glycosylation at their terminal motif is usually more functional. Among the various glycosylation types found in membrane proteins, O-glycosylation is the most common and is closely correlated with a variety of cancer types, including breast cancer. Slightly aberrant expression of certain O-glycans can significantly affect cancer progression, especially at the cancer-related membrane protein level. To collect biological information on protein-specific glycosylation and further explore clinical applications, quantitative detection of glycosylation is essential. However, few assays have been reported for the in situ detection of protein-specific glycosylation to date. Herein, we developed a dual-probe approach for mass spectrometric quantification of protein-specific glycosylation using the terminal galactose/N-acetylgalactosamine (Gal/GalNAc) of MUC1 as a model. The dual-probe (i.e., protein probe and glycan probe) system was first designed and built. The protein probe contained an aptamer for MUC1 protein recognition and a capture DNA sequence. Correspondingly, the glycan probe had a DNA sequence complementary to that of the capture DNA, a substrate peptide containing a reporter peptide, and a tryptic cleavage site, and could be covalently linked with the terminal Gal/GalNAc. Exonuclease III enabled recycling of the hybridization-dehybridization process in a restricted space. Finally, the reporter peptide was tryptically released and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The mass response of the reporter peptide represented the amount of MUC1-specific terminal Gal/GalNAc. This dual-probe approach was applied for in situ detection of MUC1-specific terminal Gal/GalNAc in three human breast cancer cell lines and 32 pairs of matched breast cancer tissue samples. The relationship between MUC1-specific terminal Gal/GalNAc expression and breast cancer diagnosis/prognosis was also assessed.

O-glycan recognition and function in mice and human cancers.

Protein glycosylation represents a nearly ubiquitous post-translational modification, and altered glycosylation can result in clinically significant pathological consequences. Here we focus on O-glycosylation in tumor cells of mice and humans. O-glycans are those linked to serine and threonine (Ser/Thr) residues via N-acetylgalactosamine (GalNAc), which are oligosaccharides that occur widely in glycoproteins, such as those expressed on the surfaces and in secretions of all cell types. The structure and expression of O-glycans are dependent on the cell type and disease state of the cells. There is a great interest in O-glycosylation of tumor cells, as they typically express many altered types of O-glycans compared with untransformed cells. Such altered expression of glycans, quantitatively and/or qualitatively on different glycoproteins, is used as circulating tumor biomarkers, such as CA19-9 and CA-125. Other tumor-associated carbohydrate antigens (TACAs), such as the Tn antigen and sialyl-Tn antigen (STn), are truncated O-glycans commonly expressed by carcinomas on multiple glycoproteins; they contribute to tumor development and serve as potential biomarkers for tumor presence and stage, both in immunohistochemistry and in serum diagnostics. Here we discuss O-glycosylation in murine and human cells with a focus on colorectal, breast, and pancreatic cancers, centering on the structure, function and recognition of O-glycans. There are enormous opportunities to exploit our knowledge of O-glycosylation in tumor cells to develop new diagnostics and therapeutics.

Differential distribution of N- and O-Glycans and variable expression of sialyl-T antigen on HeLa cells-Revealed by direct fluorescent glycan imaging.

Cells are covered with glycans. The expression and distribution of specific glycans on the surface of a cell are important for various cellular functions. Imaging these glycans is essential to aid elucidation of their biological roles. Here, utilizing methods of direct fluorescent glycan imaging, in which fluorescent sialic acids are directly incorporated into substrate glycans via recombinant sialyltranferases, we report the differential distribution of N- and O-glycans and variable expression of sialyl-T antigen on HeLa cells. While the expression of N-glycans tends to be more peripheral at positions where cell-cell interaction occurs, O-glycan expression is more granular but relatively evenly distributed on positive cells. While N-glycans are expressed on all cells, sialyl-T antigen expression exhibits a wide spectrum of variation with some cells being strongly positive and some cells being almost completely negative. The differential distribution of N- and O-glycans on cell surface reflects their distinctive roles in cell biology.

Mucins and Truncated O-Glycans Unveil Phenotypic Discrepancies between Serous Ovarian Cancer Cell Lines and Primary Tumours.

Optimal research results rely on the selection of cellular models capable of recapitulating the characteristics of primary tumours from which they originate. The expression of mucins (MUC16 and MUC1) and truncated O-glycans (Tn, STn and T) represents a characteristic footprint of serous ovarian carcinomas (SOCs). Therefore, selecting ovarian cancer (OVCA) cell lines that reflect this phenotype is crucial to explore the putative biological role of these biomarkers in the SOC setting. Here, we investigated a panel of OVCA cell lines commonly used as SOC models, and tested whether, when cultured in 2D and 3D conditions, these recapitulate the mucin and O-glycan expression profiles of SOCs. We further explored the role of truncating the O-glycosylation capacity in OVCAR3 cells through knockout of the COSMC chaperone, using in vitro and in vivo assays. We found that the majority of OVCA cell lines of serous origin do not share the mucin and truncated O-glycan footprint of SOCs, although 3D cultures showed a higher resemblance. We also found that genetic truncation of the O-glycosylation capacity of OVCAR3 cells did not enhance oncogenic features either in vitro or in vivo. This study underscores the importance of well-characterized cellular models to study specific features of ovarian cancer.

Gastric gland mucin-specific O-glycan expression decreases as tumor cells progress from lobular endocervical gland hyperplasia to cervical mucinous carcinoma, gastric type.

Gastric gland mucin-specific O-glycans are unique in having α1,4-linked N-acetylglucosamine (αGlcNAc) attached to MUC6. We previously reported decreased expression of αGlcNAc relative to MUC6 in gastric and pancreatic neoplasms, but its significance in cervical glandular lesions remained unclear. Here, we analyzed MUC5AC, MUC6, αGlcNAc, and p16 expression in 9 lesions of mucinous carcinoma, gastric type with minimal deviation adenocarcinoma (GAS-MDA), 5 of GAS with nonMDA (GAS-nonMDA), 14 of typical lobular endocervical gland hyperplasia (LEGH), and 5 of atypical LEGH (33 total lesions). All 33 were MUC5AC-positive. Moreover, all 14 typical LEGH, 5 atypical LEGH, 8 of 9 GAS-MDA, and 3 of 5 GAS-nonMDA were MUC6-positive. All 14 typical LEGH, 2 of 5 atypical LEGH, 3 of 9 GAS-MDA, and 1 of 5 GAS-nonMDA were αGlcNAc-positive. The proportion of αGlcNAc-positive atypical LEGH or GAS-MDA or GAS-nonMDA lesions was significantly smaller than that seen in typical LEGH lesions (P< 0.001 and P< 0.01, respectively). Of 33 lesions, 32 were p16-negative. Furthermore, when we evaluated MUC6 and αGlcNAc immunoreactivity semi-quantitatively in all 33 lesions, in typical LEGH and GAS-MDA, the immunohistochemical score for αGlcNAc was significantly lower than that for MUC6 (P< 0.01). We did not observe significantly decreased αGlcNAc expression relative to MUC6 in typical LEGH lesions. These studies suggest that αGlcNAc expression decreases as typical LEGH progresses to GAS. Given the difficulty in distinguishing MDA and atypical LEGH from typical LEGH in H.E. staining, we propose that immunohistochemical analysis of αGlcNAc and MUC6 could be useful.

De novo expression of human polypeptide N-acetylgalactosaminyltransferase 6 (GalNAc-T6) in colon adenocarcinoma inhibits the differentiation of colonic epithelium

Aberrant expression of O-glycans is a hallmark of epithelial cancers. Mucin-type O-glycosylation is initiated by a large family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) that target different proteins and are differentially expressed in cells and organs. Here, we investigated the expression patterns of all of the GalNAc-Ts in colon cancer by analyzing transcriptomic data. We found that GalNAc-T6 was highly up-regulated in colon adenocarcinomas but absent in normal-appearing adjacent colon tissue. These results were verified by immunohistochemistry, suggesting that GalNAc-T6 plays a role in colon carcinogenesis. To investigate the function of GalNAc-T6 in colon cancer, we used precise gene targeting to produce isogenic colon cancer cell lines with a knockout/rescue system for GALNT6 GalNAc-T6 expression was associated with a cancer-like, dysplastic growth pattern, whereas GALNT6 knockout cells showed a more normal differentiation pattern, reduced proliferation, normalized cell-cell adhesion, and formation of crypts in tissue cultures. O-Glycoproteomic analysis of the engineered cell lines identified a small set of GalNAc-T6-specific targets, suggesting that this isoform has unique cellular functions. In support of this notion, the genetically and functionally closely related GalNAc-T3 homolog did not show compensatory functionality for effects observed for GalNAc-T6. Taken together, these data strongly suggest that aberrant GalNAc-T6 expression and site-specific glycosylation is involved in oncogenic transformation.

Glycosylation Changes in Brain Cancer.

Protein glycosylation is a posttranslational modification that affects more than half of all known proteins. Glycans covalently bound to biomolecules modulate their functions by both direct interactions, such as the recognition of glycan structures by binding partners, and indirect mechanisms that contribute to the control of protein conformation, stability, and turnover. The focus of this Review is the discussion of aberrant glycosylation related to brain cancer. Altered sialylation and fucosylation of N- and O-glycans play a role in the development and progression of brain cancer. Additionally, aberrant O-glycan expression has been implicated in brain cancer. This Review also addresses the clinical potential and applications of aberrant glycosylation for the detection and treatment of brain cancer. The viable roles glycans may play in the development of brain cancer therapeutics are addressed as well as cancer-glycoproteomics and personalized medicine. Glycoprotein alterations are considered as a hallmark of cancer while high expression in body fluids represents an opportunity for cancer assessment.

Gastric gland mucin-specific O-glycan expression decreases with tumor progression from precursor lesions to pancreatic cancer.

Pancreatic cancer is lethal, as it is often detected late. Thus, novel biomarkers of precursor lesions are needed to devise timely therapies. Pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) are major precursors of pancreatic cancer. In normal gastric mucosa, gastric gland mucin‐specific O‐glycans are unique in having α1,4‐linked N‐acetylglucosamine (αGlcNAc) residues attached to MUC6. Recently we reported that αGlcNAc functions as a tumor suppressor for differentiated‐type gastric adenocarcinoma (Karasawa et al., J Clin Invest 122, 923, 2012). MUC6 is also expressed in pancreatic neoplasms, including PanIN and IPMN, but the role of αGlcNAc expression in pancreatic neoplasms remains unknown. Here, we analyze expression patterns of αGlcNAc, MUC6 and MUC5AC in pancreatic neoplasms and compare them with progression from PanIN to invasive ductal adenocarcinoma (IDAC) (the PanIN‐IDAC sequence; 20 cases) and from IPMN to IPMN with associated invasive carcinoma (IPMNAIC) (the IPMN‐IPMNAIC sequence; 20 cases). At both sequences, the frequency of MUC6‐positive and αGlcNAc‐positive lesions decreased with tumor progression. We then compared expression levels of αGlcNAc and MUC6 at each step of the progression. At the PanIN‐IDAC sequence, αGlcNAc expression significantly decreased relative to MUC6 in low‐grade PanIN (P = 0.021), high‐grade PanIN/intraductal spread of IDAC (P = 0.031) and IDAC (P = 0.013). At the IPMN‐IPMNAIC sequence, decreased αGlcNAc expression was also observed in low‐grade IPMN exhibiting gastric‐type morphology (P = 0.020). These results suggest that decreased expression of αGlcNAc relative to MUC6 occurs early and marks the initiation of tumor progression to pancreatic cancer.

C1GALT1 Seems to Promote In Vitro Disease Progression in Ovarian Cancer.

Aberrant glycosylation affects many cellular properties in cancers. The core 1 β1,3-galactosyltransferase (C1GALT1), an enzyme that controls the formation of mucin-type O-glycans, has been reported to regulate hepatocellular and mammary carcinogenesis. This study aimed to explore the role of C1GALT1 in ovarian cancer. C1GALT1 expression was assessed in a public database based on microarray data from 1287 ovarian cancer patients and ovarian cancerous tissues. Lectin blotting and flow cytometry analysis were conducted to detect changes in O-glycans on ovarian cancer cells. Effects of C1GALT1 on cell growth, migration, and sphere formation were analyzed in C1GALT1 knockdown or overexpressing ovarian cancer cells in vitro. Expression of cancer stemness-related genes was analyzed by quantitative reverse transcription polymerase chain reaction. High C1GALT1 expression shows a trend toward association with poor survival in ovarian cancer patients. C1GALT1 modifies O-glycan expression on surfaces and glycoproteins of ovarian cancer cells. Knockdown of C1GALT1 decreased cell growth, migration, and sphere formation of ES-2 and OVTW59-p4 cells. Conversely, overexpression of C1GALT1 promoted such malignant properties of SKOV3 cells. Furthermore, C1GALT1 regulated the expression of several cancer stemness-related genes, including CD133, CD24, Oct4, Nanog, and SNAI2, in ovarian cancer cells. C1GALT1 modifies O-glycan expression and enhances malignant behaviors in ovarian cancer cells, suggesting that C1GALT1 plays a role in the pathogenesis of ovarian cancer and targeting C1GALT1 could be a promising approach for ovarian cancer therapy.

Differential surface glycoprofile of buffalo bull spermatozoa during mating and non-mating periods

The buffalo has a seasonal reproduction activity with mating and non-mating periods occurring from late autumn to winter and from late spring to beginning of autumn, respectively. Sperm glycocalyx plays an important role in reproduction as it is the first interface between sperm and environment. Semen quality is poorer during non-mating periods, so we aimed to evaluate if there were also seasonal differences in the surface glycosylation pattern of mating period spermatozoa (MPS) compared with non-mating period spermatozoa (NMPS). The complexity of carbohydrate structures makes their analysis challenging, and recently the high-throughput microarray approach is now providing a new tool into the evaluation of cell glycosylation status. We adopted a novel procedure in which spermatozoa was spotted on microarray slides, incubated with a panel of 12 biotinylated lectins and Cy3-conjugated streptavidin, and then signal intensity was detected using a microarray scanner. Both MPS and NMPS microarrays reacted with all the lectins and revealed that the expression of (i) O-glycans with NeuNAcα2-3Galβ1,3(±NeuNAcα2-6)GalNAc, Galβ1,3GalNAc and GalNAcα1,3(l-Fucα1,2)Galβ1,3/4GlcNAcβ1 was not season dependent; (ii) O-linked glycans terminating with GalNAc, asialo N-linked glycans terminating with Galβ1,4GlcNAc, GlcNAc, as well as α1,6 and α1,2-linked fucosylated oligosaccharides was predominant in MPS; (iii) high mannose- and biantennary complex types N-glycans terminating with α2,6 sialic acids and terminal galactose were lower in MPS. Overall, this innovative cell microarray method was able to identify specific glycosylation changes that occur on buffalo bull sperm surface during the mating and non-mating periods.