Mucin-type O-linked Glycosylation Research Articles

Comprehensive pan-cancer analysis reveals the versatile role of GALNT7 in epigenetic alterations and immune modulation in cancer

Cancer is a leading cause of mortality globally, characterized by intricate molecular alterations, including epigenetic changes such as glycosylation. This study presents a comprehensive pan-cancer analysis of Polypeptide N-Acetylgalactosaminyltransferase 7 (GALNT7), an enzyme involved in mucin-type O-linked protein glycosylation. GALNT7 has previously been linked to various cancers, but a unified analysis across cancer types is lacking.Leveraging data from TCGA, GTEx, and other sources, we scrutinized GALNT7's expression, prognostic relevance, links to immune-related genes, immune cell infiltration, and its involvement in tumor genetic heterogeneity across 33 cancer types. GALNT7 exhibited diverse expression patterns across cancer types, showcasing its potential as an oncogenic factor, with its expression levels linked to both positive and negative prognoses, highlighting the context-specific nature of its role in cancer progression.We delved into the intricate interplay between GALNT7 and immune genes, unveiling positive and negative correlations, underscoring complex interactions in the tumor microenvironment. GALNT7 was found to impact immune cell infiltration, which could have implications for treatment strategies. Additionally, GALNT7 displayed associations with genetic tumor aspects, encompassing genomic instability, DNA repair issues, and genetic mutations, hinting at its pivotal role in shaping the genetic landscape of diverse cancers.Enrichment analysis uncovered potential functions of GALNT7 beyond glycosylation, such as its participation in signaling pathways and its association with various diseases, notably cancer.This comprehensive analysis elucidates the multifaceted role of GALNT7 in cancer biology, underlining its potential as a therapeutic target and biomarker across various cancer types. These findings provide valuable insights for future research and the development of personalized cancer treatment strategies.

Altered O-linked glycosylation in benign and malignant meningiomas.

Changes in protein glycosylation have been reported in various diseases, including cancer; however, the consequences of altered glycosylation in meningiomas remains undefined. We established two benign meningioma cell lines-SUT-MG12 and SUT-MG14, WHO grade I-and demonstrated the glycan and glycosyltransferase profiles of the mucin-type O-linked glycosylation in the primary benign meningioma cells compared with two malignant meningioma cell lines-HKBMM and IOMM-Lee, WHO grade III. Changes in O-linked glycosylation profiles in malignant meningiomas were proposed. Primary culture technique, morphological analysis, and immunocytochemistry were used to establish and characterize two benign meningioma cell lines. The glycan profiles of the primary benign and malignant meningiomas cell lines were then analyzed using lectin cytochemistry. The gene expression of O-linked glycosyltransferases, mucins, sialyltransferases, and fucosyltransferases were analyzed in benign and malignant meningioma using the GEO database (GEO series GSE16581) and quantitative-PCR (qPCR). Lectin cytochemistry revealed that the terminal galactose (Gal) and N-acetyl galactosamine (GalNAc) were highly expressed in primary benign meningioma cells (WHO grade I) compared to malignant meningioma cell lines (WHO grade III). The expression profile of mucin types O-glycosyltransferases in meningiomas were observed through the GEO database and gene expression experiment in meningioma cell lines. In the GEO database, C1GALT1-specific chaperone (COSMC) and mucin 1 (MUC1) were significantly increased in malignant meningiomas (Grade II and III) compared with benign meningiomas (Grade I). Meanwhile, in the cell lines, Core 2 β1,6-N-acetylglucosaminyltransferase-2 (C2GNT2) was highly expressed in malignant meningiomas. We then investigated the complex mucin-type O-glycans structures by determination of sialyltransferases and fucosyltransferases. We found ST3 β-galactoside α-2,3-sialyltransferase 4 (ST3GAL4) was significantly decreased in the GEO database, while ST3GAL1, ST3GAL3, α1,3 fucosyltransferases 1 and 8 (FUT1 and FUT8) were highly expressed in malignant meningioma cell lines-(HKBMM)-compared to primary benign meningioma cells-(SUT-MG12 and SUT-MG14). Our findings are the first to demonstrate the potential glycosylation changes in the O-linked glycans of malignant meningiomas compared with benign meningiomas, which may play an essential role in the progression, tumorigenesis, and malignancy of meningiomas.

The bacterial virulence factor NleA undergoes host-mediated O-linked glycosylation.

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are gastrointestinal pathogens responsible for severe diarrheal illness. EHEC and EPEC form "attaching and effacing" lesions during colonization and, upon adherence, inject proteins directly into host intestinal cells via the type III secretion system (T3SS). Injected bacterial proteins have a variety of functions but generally alter host cell biology to favor survival and/or replication of the pathogen. Non-LEE-encoded effector A (NleA) is a T3SS-injected effector of EHEC, EPEC, and the related mouse pathogen Citrobacter rodentium. Studies in mouse models indicate that NleA has an important role in bacterial virulence. However, the mechanism by which NleA contributes to disease remains unknown. We have determined that the following translocation into host cells, a serine and threonine-rich region of NleA is modified by host-mediated mucin-type O-linked glycosylation. Surprisingly, this region was not present in several clinical EHEC isolates. When expressed in C. rodentium, a non-modifiable variant of NleA was indistinguishable from wildtype NleA in an acute mortality model but conferred a modest increase in persistence over the course of infection in mixed infections in C57BL/6J mice. This is the first known example of a bacterial effector being modified by host-mediated O-linked glycosylation. Our data also suggests that this modification may confer a selective disadvantage to the bacteria during in vivo infection.

Galnt17 loss-of-function leads to developmental delay and abnormal coordination, activity, and social interactions with cerebellar vermis pathology

GALNT17 encodes a N-acetylgalactosaminyltransferase (GalNAc-T) protein specifically involved in mucin-type O-linked glycosylation of target proteins, a process important for cell adhesion, cell signaling, neurotransmitter activity, neurite outgrowth, and neurite sensing. GALNT17, also known as WBSCR17, is located at the edge of the Williams-Beuren Syndrome (WBS) critical region and adjacent to the AUTS2 locus, genomic regions associated with neurodevelopmental phenotypes that are thought to be co-regulated. Although previous data have implicated Galnt17 in neurodevelopment, the in vivo functions of this gene have not been investigated. In this study, we have analyzed behavioral, brain pathology, and molecular phenotypes exhibited by Galnt17 knockout (Galnt17−/−) mice. We show that Galnt17−/− mutants exhibit developmental neuropathology within the cerebellar vermis, along with abnormal activity, coordination, and social interaction deficits. Transcriptomic and protein analysis revealed reductions in both mucin type O-glycosylation and heparan sulfate synthesis in the developing mutant cerebellum along with disruption of pathways central to neuron differentiation, axon pathfinding, and synaptic signaling, consistent with the mutant neuropathology. These brain and behavioral phenotypes and molecular data confirm a specific role for Galnt17 in brain development and suggest new clues to factors that could contribute to phenotypes in certain WBS and AUTS2 syndrome patients.

GALNTs: master regulators of metastasis-associated epithelial-mesenchymal transition (EMT)?

In humans, the UDP-N-α-D galactosamine:polypeptide N-acetylgalactosaminyltransferases family (ppGalNAc-Ts, GalNAc-Ts or GALNTs) comprises 20 isoenzymes. They are responsible for the initial synthesis of α-GalNAc1,3-O-Ser/Thr, or Tn antigen, at initiation of mucin type O-linked glycosylation. This structure is normally extended by the further sequential action of glycosytransferases to build more complex linear or branched O-linked structures, but in cancers it is frequently left unelaborated, and its presence is often associated with poor patient prognosis. Altered levels of GALNT expression or distribution have also been extensively reported in a wide range of cancers. These changes would be predicted to result in marked alterations in GalNAc O-linked glycosylation, including altered levels of site specific O-linked glycosylation and changes in the glycan structures formed, including, potentially, exposure of truncated O-glycans such as Tn antigen. Many reports have demonstrated that altered levels of specific GALNTs have prognostic significance in cancers, or shown that they are associated with changes in cell behaviour, including proliferation, migration, invasion or growth and metastasis in animal models. We have previously reviewed how deregulation of GALNTs in several epithelial cancers is a feature of different stages metastasis. Here we consider evidence that changes in GALNT expression, and therefore consequent alterations in GalNAc O-linked glycosylation, may directly influence molecules implicated in aspects of epithelial-mesenchymal transition (EMT), a fundamental aspect of cancer metastasis, during which epithelial cancer cells lose their cell-cell junctions, apical-basal polarity and adhesive interactions with basement membrane and become mesenchymal, with a spindle-shaped morphology and increased migratory capacity.

Efficient inhibition of O-glycan biosynthesis using the hexosamine analog Ac5GalNTGc

There is a critical need to develop small-molecule inhibitors of mucin-type O-linked glycosylation. The best-known reagent currently is benzyl-GalNAc, but it is effective only at millimolar concentrations. This article demonstrates that Ac5GalNTGc, a peracetylated C-2 sulfhydryl-substituted GalNAc, fulfills this unmet need. When added to cultured leukocytes, breast cells, and prostate cells, Ac5GalNTGc increased cell-surface VVA binding by ∼10-fold, indicating truncation of O-glycan biosynthesis. Cytometry, mass spectrometry, and western blotanalysis of HL-60 promyelocytes demonstrated that 50-80μM Ac5GalNTGc prevented elaboration of 30%-60% of the O-glycans beyond the Tn-antigen (GalNAcα1-Ser/Thr) stage. The effect of the compound on N-glycans and glycosphingolipids was small. Glycan inhibition induced by Ac5GalNTGc resulted in 50%-80% reduction in leukocyte sialyl-Lewis X expression and L-/P-selectin-mediated rolling under flow conditions. Ac5GalNTGc was pharmacologically active in mouse. It reduced neutrophil infiltration to sites of inflammation by ∼60%. Overall, Ac5GalNTGc may find diverse applications as a potent inhibitor of O-glycosylation.

O-linked mucin-type glycosylation regulates the transcriptional programme downstream of EGFR.

Aberrant mucin-type O-linked glycosylation is a common occurrence in cancer where the upregulation of sialyltransferases is often seen leading to the early termination of O-glycan chains. Mucin-type O-linked glycosylation is not limited to mucins and occurs on many cell surface glycoproteins including EGFR, where the number of sites can be limited. Upon EGF ligation, EGFR induces a signaling cascade and may also translocate to the nucleus where it directly regulates gene transcription, a process modulated by Galectin-3 and MUC1 in some cancers. Here, we show that upon EGF binding, breast cancer cells carrying different O-glycans respond by transcribing different gene expression signatures. MMP10, the principal gene upregulated when cells carrying sialylated core 1 glycans were stimulated with EGF, is also upregulated in ER-positive breast carcinoma reported to express high levels of ST3Gal1 and hence mainly core 1 sialylated O-glycans. In contrast, isogenic cells engineered to carry core 2 glycans upregulate CX3CL1 and FGFBP1 and these genes are upregulated in ER-negative breast carcinomas, also known to express longer core 2 O-glycans. Changes in O-glycosylation did not significantly alter signal transduction downstream of EGFR in core 1 or core 2 O-glycan expressing cells. However, striking changes were observed in the formation of an EGFR/galectin-3/MUC1/β-catenin complex at the cell surface that is present in cells carrying short core 1-based O-glycans but absent in core 2 carrying cells.

Cosmc-dependent mucin-type O-linked glycosylation is essential for podocyte function.

Mucin-type O-linked glycosylation, a posttranslational modification affecting the stability and biophysical characteristics of proteins, requires C1GalT1 (T synthase) and its obligate, X-linked chaperone Cosmc. Hypomorphic C1GalT1 mutations cause renal failure via not yet established mechanisms. We hypothesize that impaired Cosmc-dependent O-glycosylation in podocytes is sufficient to cause disease. Podocyte-specific Cosmc knockout mice were generated and phenotyped to test this hypothesis. Female heterozygous mice displaying mosaic inactivation of Cosmc in podocytes due to random X-linked inactivation were also examined. Mice with podocyte-specific Cosmc deletion develop profound albuminuria, foot process effacement, glomerular sclerosis, progressive renal failure, and impaired survival. Glomerular transcriptome analysis reveals early changes in cell adhesion, extracellular matrix organization, and chemokine-mediated signaling pathways, coupled with podocyte loss. Expression of the O-glycoprotein podoplanin was lost, while Tn antigen, representing immature O-glycans, was most abundantly found on podocalyxin. In contrast to hemizygous male and homozygous female animals, heterozygous female mosaic animals developed only mild albuminuria, focal foot process effacement, and nonprogressive kidney disease. Ultrastructurally, Cosmc-deficient podocytes formed Tn antigen-positive foot processes interdigitating with those of normal podocytes but not with other Cosmc-deficient cells. This suggests a cell nonautonomous mechanism for mucin-type O-glycoproteins in maintaining podocyte function. In summary, our findings demonstrated an essential and likely cell nonautonomous role for mucin-type O-glycosylation for podocyte function.

Mendelian Randomization Analysis Reveals a Causal Influence of Circulating Sclerostin Levels on Bone Mineral Density and Fractures.

ABSTRACTIn bone, sclerostin is mainly osteocyte‐derived and plays an important local role in adaptive responses to mechanical loading. Whether circulating levels of sclerostin also play a functional role is currently unclear, which we aimed to examine by two‐sample Mendelian randomization (MR). A genetic instrument for circulating sclerostin, derived from a genomewide association study (GWAS) meta‐analysis of serum sclerostin in 10,584 European‐descent individuals, was examined in relation to femoral neck bone mineral density (BMD; n = 32,744) in GEFOS and estimated bone mineral density (eBMD) by heel ultrasound (n = 426,824) and fracture risk (n = 426,795) in UK Biobank. Our GWAS identified two novel serum sclerostin loci, B4GALNT3 (standard deviation [SD]) change in sclerostin per A allele (β = 0.20, p = 4.6 × 10−49) and GALNT1 (β = 0.11 per G allele, p = 4.4 × 10−11). B4GALNT3 is an N‐acetyl‐galactosaminyltransferase, adding a terminal LacdiNAc disaccharide to target glycocoproteins, found to be predominantly expressed in kidney, whereas GALNT1 is an enzyme causing mucin‐type O‐linked glycosylation. Using these two single‐nucleotide polymorphisms (SNPs) as genetic instruments, MR revealed an inverse causal relationship between serum sclerostin and femoral neck BMD (β = –0.12, 95% confidence interval [CI] –0.20 to –0.05) and eBMD (β = –0.12, 95% CI –0.14 to –0.10), and a positive relationship with fracture risk (β = 0.11, 95% CI 0.01 to 0.21). Colocalization analysis demonstrated common genetic signals within the B4GALNT3 locus for higher sclerostin, lower eBMD, and greater B4GALNT3 expression in arterial tissue (probability >99%). Our findings suggest that higher sclerostin levels are causally related to lower BMD and greater fracture risk. Hence, strategies for reducing circulating sclerostin, for example by targeting glycosylation enzymes as suggested by our GWAS results, may prove valuable in treating osteoporosis. © 2019 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc.

Regulation of AβPP Glycosylation Modification and Roles of Glycosylation on AβPP Cleavage in Alzheimer's Disease.

The presence of senile plaques in the gray matter of the brain is one of the major pathologic features of Alzheimer's disease (AD), and amyloid-β (Aβ) is the main component of extracellular deposits of the senile plaques. Aβ derives from amyloid-β precursor protein (AβPP) cleaved by β-secretase (BACE1) and γ-secretase, and the abnormal cleavage of AβPP is an important event leading to overproduction and aggregation of Aβ species. After translation, AβPP undergoes post-translational modifications (PTMs) including glycosylation and phosphorylation in the endoplasmic reticulum (ER) and Golgi apparatus, and these modifications play an important role in regulating the cleavage of this protein. In this Review, we summarize research progress on the modification of glycosylation, especially O-GlcNAcylation and mucin-type O-linked glycosylation (also known as O-GalNAcylation), on the regulation of AβPP cleavage and on the influence of AβPP's glycosylation in the pathogenesis of AD.

O-linked mucin-type glycosylation in breast cancer.

Changes in mucin-type O-linked glycosylation are seen in over 90% of breast cancers where increased sialylation is often observed and a change from branched glycans to linear glycans is often seen. There are many mechanisms involved including increased/altered expression of glycosyltransferases and relocalisation to the endoplasmic reticulum of the enzymes responsible for the addition of the first sugar, N-acetyl-d-galactosamine. It is now becoming clear that these changes can contribute to tumour growth and progression by modulating the micro-environment through glycan-sensing lectins expressed on immune cells, by modulating interactions with tumour surface receptors and by binding to selectins. The understanding of how changes in mucin-type O-linked glycosylation influence tumour growth and progression reveals new potential targets for therapeutic intervention in the treatment of breast cancer.

HEK293T cell lines defective for O-linked glycosylation.

Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE), galactokinase 1 (GALK1), and galactokinase 2 (GALK2) genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc) to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2), O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.

Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication.

Influenza A virus (IAV) affects the upper and lower respiratory tracts and rapidly induces the expression of mucins, which are common O-glycosylated proteins, on the epithelial surfaces of the respiratory tract. Although mucin production is associated with the inhibition of virus transmission as well as characteristic clinical symptoms, little is known regarding how mucins are produced on the surfaces of respiratory epithelial cells and how they affect IAV replication. In this study, we found that two microRNAs (miRNAs), miR-17-3p and miR-221, which target GalNAc transferase 3 (GALNT3) mRNA, are rapidly downregulated in human alveolar basal epithelial cells during the early stage of IAV infection. We demonstrated that the expression of GALNT3 mRNA is upregulated in an IAV replication-dependent fashion and leads to mucin production in bronchial epithelial cells. A lectin microarray analysis revealed that the stable expression of GALNT3 by human alveolar basal epithelial cells induces mucin-type O-glycosylation modifications similar to those present in IAV-infected cells, suggesting that GALNT3 promotes mucin-type O-linked glycosylation in IAV-infected cells. Notably, analyses using short interfering RNAs and miRNA mimics showed that GALNT3 knockdown significantly reduces IAV replication. Furthermore, IAV replication was markedly decreased in embryonic fibroblast cells obtained from galnt3-knockout mice. Interestingly, IAV-infected galnt3-knockout mice exhibited high mortality and severe pathological alterations in the lungs compared to those of wild-type mice. Our results demonstrate not only the molecular mechanism underlying rapid mucin production during IAV infection but also the contribution of O-linked glycosylation to the replication and propagation of IAV in lung cells. Viral infections that affect the upper or lower respiratory tracts, such as IAV, rapidly induce mucin production on the epithelial surfaces of respiratory cells. However, the details of how mucin-type O-linked glycosylation is initiated by IAV infection and how mucin production affects viral replication have not yet been elucidated. In this study, we show that levels of two miRNAs that target the UDP-GalNAc transferase GALNT3 are markedly decreased during the early stage of IAV infection, resulting in the upregulation of GALNT3 mRNA. We also demonstrate that the expression of GALNT3 initiates mucin production and affects IAV replication in infected cells. This is the first report demonstrating the mechanism underlying the miRNA-mediated initiation of mucin-type O-glycosylation in IAV-infected cells and its role in viral replication. Our results have broad implications for understanding IAV replication and suggest a strategy for the development of novel anti-influenza approaches.

GalNAc-T4 putatively modulates the estrogen regulatory network through FOXA1 glycosylation in human breast cancer cells.

GALNT4 belongs to a family of N-acetylgalactosaminyltransferases, which catalyze the transfer of GalNAc to Serine or Threonine residues in the initial step of mucin-type O-linked protein glycosylation. This glycosylation type is the most complex post-translational modification of proteins, playing important roles during cellular differentiation and in pathological disorders. Most of the breast cancer subtypes are estrogen receptor positive, and hence, the estrogen pathway represents a key regulatory network. We investigated the expression of GalNAc-T4 in a panel of mammary epithelial cell lines and found its expression is associated with the estrogen status of the cells. FOXA1, a key transcription factor, functions to promote estrogen responsive gene expression by acting as a cofactor to estrogen receptor alpha (ERα), but all the aspects of this regulatory mechanism are not fully explored. This study found that knockdown of GALNT4 expression in human breast cancer cells attenuated the protein expression of ERα, FOXA1, and Cyclin D1. Further, our immunoprecipitation assays depicted the possibility of FOXA1 to undergo O-GalNAc modifications with a decrease of GalNAc residues in the GALNT4 knockdown cells and also impairment in the FOXA1-ERα association. Rescuing GALNT4 expression could restore the interaction as well as the glycosylation of FOXA1. Together, these findings suggest a key role for GalNAc-T4 in the estrogen pathway through FOXA1 glycosylation.

Biosynthetic Machinery Involved in Aberrant Glycosylation: Promising Targets for Developing of Drugs Against Cancer

Cancer cells depend on altered metabolism and nutrient uptake to generate and keep the malignant phenotype. The hexosamine biosynthetic pathway is a branch of glucose metabolism that produces UDP-GlcNAc and its derivatives, UDP-GalNAc and CMP-Neu5Ac and donor substrates used in the production of glycoproteins and glycolipids. Growing evidence demonstrates that alteration of the pool of activated substrates might lead to different glycosylation and cell signaling. It is already well established that aberrant glycosylation can modulate tumor growth and malignant transformation in different cancer types. Therefore, biosynthetic machinery involved in the assembly of aberrant glycans are becoming prominent targets for anti-tumor drugs. This review describes three classes of glycosylation, O-GlcNAcylation, N-linked, and mucin type O-linked glycosylation, involved in tumor progression, their biosynthesis and highlights the available inhibitors as potential anti-tumor drugs.

The role of ppGalNAc-T family in breast cancer development and progression.

Glycosylation of proteins is an essential process in all eukaryotes. Mucin-type O-linked glycosylation is an evolutionarily conserved protein modification as a kind of glycosylation of proteins. The role of O-glycosylation was well documented in multiple cancers. While in breast cancer, the enzymes that catalyzed the initiation of O-glycosylation remained elusive. In this review, we briefly introduced the process of the initiation of O-glycosylation and summarized the roles of enzymes that catalyzed the initiation step of O-glycosylation in the breast cancer carcinogenesis, development, and progression. Finally, we summarized some attempts exploring the therapy against aberrant O-glycosylation.

Glycan imaging in intact rat hearts and glycoproteomic analysis reveal the upregulation of sialylation during cardiac hypertrophy.

In the heart, glycosylation is involved in a variety of physiological and pathological processes. Cardiac glycosylation is dynamically regulated, which remains challenging to monitor in vivo. Here we describe a chemical approach for analyzing the dynamic cardiac glycome by metabolically labeling the cardiac glycans with azidosugars in living rats. The azides, serving as a chemical reporter, are chemoselectively conjugated with fluorophores using copper-free click chemistry for glycan imaging; derivatizing azides with affinity tags allows enrichment and proteomic identification of glycosylated cardiac proteins. We demonstrated this methodology by visualization of the cardiac sialylated glycans in intact hearts and identification of more than 200 cardiac proteins modified with sialic acids. We further applied this methodology to investigate the sialylation in hypertrophic hearts. The imaging results revealed an increase of sialic acid biosynthesis upon the induction of cardiac hypertrophy. Quantitative proteomic analysis identified multiple sialylated proteins including neural cell adhesion molecule 1, T-kininogens, and α2-macroglobulin that were upregulated during hypertrophy. The methodology may be further extended to other types of glycosylation, as exemplified by the mucin-type O-linked glycosylation. Our results highlight the applications of metabolic glycan labeling coupled with bioorthogonal chemistry in probing the biosynthesis and function of cardiac glycome during pathophysiological responses.

Activation of Epidermal Growth Factor Receptor Mediates Mucin Production Stimulated by p40, a Lactobacillus rhamnosus GG-derived Protein

The mucus layer coating the gastrointestinal tract serves as the first line of intestinal defense against infection and injury. Probiotics promote mucin production by goblet cells in the intestine. p40, a Lactobacillus rhamnosus GG-derived soluble protein, has been shown to transactivate the EGF receptor (EGFR) in intestinal epithelial cells, which is required for inhibition of apoptosis and preservation of barrier function in the colon, thereby ameliorating intestinal injury and colitis. Because activation of EGFR has been shown to up-regulate mucin production in goblet cells, the purpose of this study was to investigate the effects and mechanisms of p40 regulation of mucin production. p40 activated EGFR and its downstream target, Akt, in a concentration-dependent manner in LS174T cells. p40 stimulated Muc2 gene expression and mucin production in LS174T cells, which were abolished by inhibition of EGFR kinase activity, down-regulation of EGFR expression by EGFR siRNA transfection, or suppression of Akt activation. Treatment with p40 increased mucin production in the colonic epithelium, thus thickening the mucus layer in the colon of wild type, but not of Egfr(wa5) mice, which have a dominant negative mutation in the EGFR kinase domain. Furthermore, inhibition of mucin-type O-linked glycosylation suppressed the effect of p40 on increasing mucin production and protecting intestinal epithelial cells from TNF-induced apoptosis in colon organ culture. Thus, these results suggest that p40-stimulated activation of EGFR mediates up-regulation of mucin production, which may contribute to the mechanisms by which p40 protects the intestinal epithelium from injury.

Cyclooxygenase-2 Enzyme Induces the Expression of the -2,3-Sialyltransferase-3 (ST3Gal-I) in Breast Cancer

Aberrant glycosylation is a common feature of malignant change. Changes in mucin-type O-linked glycosylation in breast cancer can result in the expression of truncated core 1-based sialylated glycans rather than the core 2-based glycans observed in normal mammary epithelium cells. This has been shown, in part, to be due to changes in the expression of glycosyltransferases, including the up-regulation of some sialyltransferases. Using the breast cancer cell line T47D, we have shown that PGE2, one of the final products of the cyclooxygenase-2 (COX-2) pathway, can induce the mRNA expression of the sialyltransferase α-2,3-sialyltransferase-3 (ST3Gal-I), resulting in increased sialyltransferase activity, demonstrated by a reduction in PNA lectin staining. Induction of COX-2 in the MDA-MB-231 breast cancer cell line also results in the increased expression of ST3Gal-I, leading to increased sialylation of the substrate of ST3Gal-I, core 1 Galβ1,3GalNAc. This effect on sialylation could be reversed by the selective COX-2 inhibitor celecoxib. The use of siRNA to knock down COX-2 and overexpression of COX-2 in MDA-MD-231 cells confirmed the involvement of COX-2 in the up-regulation of ST3Gal-I. Moreover, analysis of the expression of ST3Gal-I and COX-2 by 74 primary breast cancers showed a significant correlation between the two enzymes. COX-2 expression has been associated with a number of tumors, including breast cancer, where its expression is associated with poor prognoses. Thus, these results suggest the intriguing possibility that some of the malignant characteristics associated with COX-2 expression may be via the influence that COX-2 exerts on the glycosylation of tumor cells.

Mass spectrometric analysis of hepatitis C viral envelope protein E2 reveals extended microheterogeneity of mucin-type O-linked glycosylation

The infectious liver disease hepatitis C is caused by the small, enveloped, positive single-strand RNA hepatitis C virus (HCV). The HCV genome encodes for a single polyprotein precursor of ∼3010 amino acid residues. Host and cellular proteases co- and posttranslational process the precursor creating six nonstructural (NS) proteins and four structural components. Properly folded forms of the envelope proteins E1 and E2 form the associated E1-E2 complex. This complex represents a significant antigenic component at the viral surface that can interact with several target cell receptors. Extent and type of glycosylation is an important factor for virulence and escape from the immune system. Detailed characterization of the glycosylated sites is helpful for the understanding of different phenotypes as well as for the development of E1/E2-related treatments of HCV infection. Here, we have investigated in detail the O-linked glycosylation of the HCV envelope protein E2 expressed in and isolated from human embryonic kidney (HEK 293) cells. Using nano-liquid chromatography and tandem mass spectrometry approaches, we clearly have identified six residues for O-linked glycosylation within isolated glycopeptides (Ser393, Thr396, Ser401, Ser404, Thr473 and Thr518), carrying mainly Core 1 and Core 2 mucin-type structures. Based on our data, Thr385 is probably glycosylated as well. In addition, we could show that Ser479 within the hyper variable region (HVR) I is not O-glycosylated. For most of these sites, different degrees of microheterogeneity could be verified. Concerning HCV E2, this is the first case of experimentally proven O-linked glycosylation in detail via mass spectrometry.