Sequential glycosylations at the multibasic cleavage site of SARS-CoV-2 spike protein regulate viral activity.

The multibasic furin cleavage site at the S1/S2 boundary of the spike protein is a hallmark of SARS-CoV-2 and plays a crucial role in viral infection. However, the mechanism underlying furin activation and its regulation remain poorly understood. Here, we show that GalNAc-T3 and T7 jointly initiate clustered O-glycosylations in the furin cleavage site of the SARS-CoV-2 spike protein, which inhibit furin processing, suppress the incorporation of the spike protein into virus-like-particles and affect viral infection. Mechanistic analysis reveals that the assembly of the spike protein into virus-like particles relies on interactions between the furin-cleaved spike protein and the membrane protein of SARS-CoV-2, suggesting a possible mechanism for furin activation. Interestingly, mutations in the spike protein of the alpha and delta variants of the virus confer resistance against glycosylation by GalNAc-T3 and T7. In the omicron variant, additional mutations reverse this resistance, making the spike protein susceptible to glycosylation in vitro and sensitive to GalNAc-T3 and T7 expression in human lung cells. Our findings highlight the role of glycosylation as a defense mechanism employed by host cells against SARS-CoV-2 and shed light on the evolutionary interplay between the host and the virus.

Previously reported CCDC26 risk variant and novel germline variants in GALNT13, AR, and MYO10 associated with familial glioma in Finland.

Predisposing factors underlying familial aggregation of non-syndromic gliomas are still to be uncovered. Whole-exome sequencing was performed in four Finnish families with brain tumors to identify rare predisposing variants. A total of 417 detected exome variants and 102 previously reported glioma-related variants were further genotyped in 19 Finnish families with brain tumors using targeted sequencing. Rare damaging variants in GALNT13, MYO10 and AR were identified. Two families carried either c.553C>T (R185C) or c.1214T>A (L405Q) on GALNT13. Variant c.553C>T is located on the substrate-binding site of GALNT13. AR c.2180G>T (R727L), which is located on a ligand-binding domain of AR, was detected in two families, one of which also carried a GALNT13 variant. MYO10 c.4448A>G (N1483S) was detected in two families and c.1511C>T (A504V) variant was detected in one family. Both variants are located on functional domains related to MYO10 activity in filopodia formation. In addition, affected cases in six families carried a known glioma risk variant rs55705857 in CCDC26 and low-risk glioma variants. These novel findings indicate polygenic inheritance of familial glioma in Finland and increase our understanding of the genetic contribution to familial glioma susceptibility.

GALNT14 in association with GDF-15 promotes stemness and drug resistance through ß-catenin signalling pathway in breast cancer.

BACKGROUND: Altered glycosylation plays a role in carcinogenesis. GALNT14 promotes cancer stem-like properties and drug resistance. GDF-15 is known to induces drug resistance and stemness markers for maintenance of breast cancer (BC) stem-like cell state. Currently there is lack of data on association of GDF-15 and GALNTs. In this study, the expression and interaction of GALNT14 and GDF-15 with stemness (OCT4 and SOX2) and drug resistance (ABCC5) markers were evaluated in BC. METHODS: We investigated tumour tissue from 30 BC patients and adjacent non-tumour tissues. Expression of serum GALNT14 from BC patients and matched healthy controls was evaluated. Expression of GALNT14, GDF-15, OCT4, SOX2, ABCC5, and ß-catenin in BC tissue was determined by RT-PCR. Knockdown of GALNT14 and GDF-15 in the MCF-7 cell line was done through siRNA, gene expression and protein expression of ß-catenin by western blot were determined. RESULTS: A significant increase in the expression of GALNT14, GDF-15, OCT4, SOX2, ABCC5, and ß-catenin was observed in BC tumour tissues compared to adjacent non-tumour tissues. The serum level of GALNT14 was significantly high in BC patients (80.7 ± 65.3 pg/ml) compared to healthy controls (12.2 ± 9.12 pg/ml) (p < 0.000). To further analyse the signalling pathway involved in BC stemness and drug resistance, GALNT14 and GDF-15 were knocked down in the MCF-7 cell line, and it was observed that after knockdown, the expression level of OCT4, SOX2, ABCC5, and ß-catenin was decreased, and co-knockdown with GALNT14 and GDF-15 further decreased the expression of genes. CONCLUSION: It can be concluded that GALNT14, in association with GDF-15, promotes stemness and intrinsic drug resistance in BC, possibly through the ß-catenin signalling pathway.

Phosphate-sensing mechanisms and functions of phosphate as a first messenger.

Bone secrets the hormone, fibroblast growth factor 23 (FGF23), as an endocrine organ to regulate blood phosphate level. Phosphate is an essential mineral for the human body, and around 85% of phosphate is present in bone as a constituent of hydroxyapatite, Ca10(PO4)6(OH)2. Because hypophosphatemia induces rickets/osteomalacia, and hyperphosphatemia results in ectopic calcification, blood phosphate (inorganic form) level must be regulated in a narrow range (2.5 mg/dL to 4.5 me/dL in adults). However, as yet it is unknown how bone senses changes in blood phosphate level, and how bone regulates the production of FGF23. Our previous data indicated that high extracellular phosphate phosphorylates FGF receptor 1 (FGFR1) in an unliganded manner, and its downstream intracellular signaling pathway regulates the expression of GALNT3. Furthermore, the post-translational modification of FGF23 protein via a gene product of GALNT3 is the main regulatory mechanism of enhanced FGF23 production due to high dietary phosphate. Therefore, our research group proposes that FGFR1 works as a phosphate-sensing receptor at least in the regulation of FGF23 production and blood phosphate level, and phosphate behaves as a first messenger. Phosphate is involved in various effects, such as stimulation of parathyroid hormone (PTH) synthesis, vascular calcification, and renal dysfunction. Several of these responses to phosphate are considered as phosphate toxicity. However, it is not clear whether FGFR1 is involved in these responses to phosphate. The elucidation of phosphate-sensing mechanisms may lead to the identification of treatment strategies for patients with abnormal phosphate metabolism.

Deficiency of polypeptide N-acetylgalactosamine transferase 9 contributes to a risk for Parkinson's disease via mitochondrial dysfunctions.

Polypeptide N-acetylgalactosamine transferase 9 (GALNT9) catalyzes the initial step of mucin-type O-glycosylation via linking N-acetylgalactosamine (GalNAc) to serine/threonine in a protein. To unravel the association of GALNT9 with Parkinson's disease (PD), a progressive neurodegenerative disorder, GALNT9 levels were evaluated in the patients with PD and mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and statistically analyzed based on the GEO datasets of GSE114918 and GSE216281. Glycoproteins with exposing GalNAc were purified using lectin affinity chromatography and identified by LC-MS/MS. The influence of GALNT9 on cells was evaluated via introducing a GALNT9-specific siRNA into SH-SY5Y cells. Consequently, GALNT9 deficiency was found to occur under PD conditions. GALNT9 silencing contributed to a causative factor in PD pathogenesis via reducing the levels of intracellular dopamine, tyrosine hydroxylase and soluble α-synuclein, and promoting α-synuclein aggregates. MS identification revealed 14 glycoproteins. 5 glycoproteins, including ACO2, ATP5B, CKB, CKMT1A, ALDOC, were associated with energy metabolism. GALNT9 silencing resulted in mitochondrial dysfunctions via increasing ROS accumulation, mitochondrial membrane depolarization, mPTPs opening, Ca2+ releasing and activation of the CytC-related apoptotic pathway. The dysfunctional mitochondria then triggered mitophagy, possibly intermediated by adenine nucleotide translocase 1. Our study suggests that GALNT9 is potentially developed into an auxiliary diagnostic index and therapeutic target of PD.

GALNT12 suppresses the bone-specific prostate cancer metastasis by activating BMP pathway via the O-glycosylation of BMPR1A.

Bone metastasis caused the majority death of prostate cancer (PCa) but the mechanism remains poorly understood. In this present study, we show that polypeptide N-acetylgalactosaminyltransferase 12 (GALNT12) suppresses bone-specific metastasis of PCa. GALNT12 suppresses proliferation, migration, invasion and cell division ability of PCa cells by activating the BMP pathway. Mechanistic investigations showed that GALNT12 augments the O-glycosylation of BMPR1A then actives the BMP pathway. Activated BMP signaling inhibits the expression of integrin αVß3 to reduce the bone-specific seeding of PCa cells. Furthermore, activated BMP signaling remolds the immune microenvironment by suppressing the STAT3 pathway. Our results of this study illustrate the role and mechanism of GALNT12 in the process of bone metastasis of PCa and identify GALNT12 as a potential therapeutic target for metastatic PCa.

An unusual dual sugar-binding lectin domain controls the substrate specificity of a mucin-type O-glycosyltransferase.

N-acetylgalactosaminyl-transferases (GalNAc-Ts) initiate mucin-type O-glycosylation, an abundant and complex posttranslational modification that regulates host-microbe interactions, tissue development, and metabolism. GalNAc-Ts contain a lectin domain consisting of three homologous repeats (α, ß, and γ), where α and ß can potentially interact with O-GalNAc on substrates to enhance activity toward a nearby acceptor Thr/Ser. The ubiquitous isoenzyme GalNAc-T1 modulates heart development, immunity, and SARS-CoV-2 infectivity, but its substrates are largely unknown. Here, we show that both α and ß in GalNAc-T1 uniquely orchestrate the O-glycosylation of various glycopeptide substrates. The α repeat directs O-glycosylation to acceptor sites carboxyl-terminal to an existing GalNAc, while the ß repeat directs O-glycosylation to amino-terminal sites. In addition, GalNAc-T1 incorporates α and ß into various substrate binding modes to cooperatively increase the specificity toward an acceptor site located between two existing O-glycans. Our studies highlight a unique mechanism by which dual lectin repeats expand substrate specificity and provide crucial information for identifying the biological substrates of GalNAc-T1.

Runx2 Regulates Galnt3 and Fgf23 Expressions and Galnt3 Decelerates Osteoid Mineralization by Stabilizing Fgf23.

Runx2 (runt related transcription factor 2) is an essential transcription factor for osteoblast proliferation and differentiation. Uridine diphosphate (UDP)-N-acetylgalactosamine (GalNAc): polypeptide GalNAc-transferase 3 (Galnt3) prevents proteolytic processing of fibroblast growth factor 23 (Fgf23), which is a hormone that regulates the serum level of phosphorus. Runx2 and Galnt3 were expressed in osteoblasts and osteocytes, and Fgf23 expression was restricted to osteocytes in bone. Overexpression and knock-down of Runx2 upregulated and downregulated, respectively, the expressions of Galnt3 and Fgf23, and Runx2 directly regulated the transcriptional activity of Galnt3 in reporter assays. The expressions of Galnt3 and Fgf23 in osteoblast-specific Runx2 knockout (Runx2fl/flCre) mice were about half those in Runx2fl/fl mice. However, the serum levels of phosphorus and intact Fgf23 in Runx2fl/flCre mice were similar to those in Runx2fl/fl mice. The trabecular bone volume was increased during aging in both male and female Galnt3-/- mice, but the osteoid was reduced. The markers for bone formation and resorption in Galnt3-/- mice were similar to the control in both sexes. Galnt3-/- mice exhibited hyperphosphatemia and hypercalcemia, and the intact Fgf23 was about 40% that of wild-type mice. These findings indicated that Runx2 regulates the expressions of Galnt3 and Fgf23 and that Galnt3 decelerates the mineralization of osteoid by stabilizing Fgf23.

ISOGlyP: O-Glycosylation Site Prediction Using Peptide Sequences and GALNTs.

Mucin-type O-glycosylation is one of the most common posttranslational modifications of proteins. The abnormal expression of various polypeptide GalNAc-transferases (GALNTs) which initiate and define sites of O-glycosylation is linked to many cancers and other diseases. Many current O-glycosylation prediction programs utilize O-glycoproteomics data obtained without using the transferase isoform(s) responsible for the glycosylation. With 20 different GALNTs in humans, having the ability to predict and interpret O-glycosylation sites in terms of specific GALNT isoforms is invaluable.To fill this gap, ISOGlyP (isoform-specific O-glycosylation prediction) has been developed. Using position-specific enhancement values generated based on GalNAc-T isoform-specific amino acid preferences, ISOGlyP predicts the propensity that a site would be glycosylated by a specific transferase. ISOGlyP gave an overall prediction accuracy of 70% against in vivo data, which is comparable to that of the NetOGlyc4.0 predictor. Additionally, ISOGlyP can identify the known effects of long- and short-range prior glycosylation and can generate potential peptide sequences selectively glycosylated by specific isoforms. ISOGlyP is freely available for use at https://ISOGlyP.utep.edu . The code is also available on GitHub ( https://github.com/jonmohl/ISOGlyP ).

Putative SNPs in Ovar-DRB1 and GALNTL6 Genes Conferring Susceptibility to Natural Infection of Haemonchus Contortus in Southern Indian Sheep.

AIM: To explore associations between phenotypic traits and polymorphisms in the DRB1 and GALNT6 gene in Nellore, Deccani and Kenguri sheep naturally infected with Haemonchus contortus. MATERIALS AND METHODS: Blood and faecal samples were collected to evaluate fecal worm egg counts (FEC), packed cell volume (PCV), hemoglobin (Hb), eosinophilia and for DNA isolation. RESULTS: Animals were grouped into susceptible and resistant groups based on EPG counts. FEC and circulating eosinophilia were higher in a susceptible group. Log FEC was negatively correlated (P < 0.01) with PCV, and Hb estimates. The second exon of DRB1 and intron variant of GALNTL6 genes were amplified from DNA samples of resistant and susceptible sheep. Characterization of Ovar-DRB1 amplicon by RFLP revealed two genotypes ('bb' and 'ab'). The genotype frequencies differed significantly between both groups (P < 0.05). The 'bb' genotypes had higher (P < 0.05) log FEC value than 'ab' genotypes and 'b' allele was linked with susceptibility to haemonchosis in sheep. The mean FEC of Nellore sheep was high indicating susceptibility of the breed and also in which the frequency of 'b' allele was more compared to the other two breeds. OVAR-DRB1 genotypes associated with FEC did not affect PCV and Hb. PCR-RFLP assay developed to determine the genotypes with respect to SNP rs424521894 of GALNTL6 revealed monomorphic nature at the locus in the breeds studied. CONCLUSION: MHC polymorphism could be used as a genetic marker for the selection of sheep resistant to H. contortus. However, a more intensive study, involving controlled infections and other GALNTL6 SNPs may be enforced to make any decisive assertion.

CircRNA Galntl6 sponges miR-335 to ameliorate stress-induced hypertension through upregulating Lig3 in rostral ventrolateral medulla.

Rostral ventrolateral medulla (RVLM) is thought to serve as a major vasomotor center that participates in controlling the progression of stress-induced hypertension (SIH). Circular RNAs (circRNAs) perform important functions in the regulation of diverse physiological and pathological processes. However, information concerning the functions of RVLM circRNAs on SIH remains limited. RNA sequencing was performed to profile circRNA expression in RVLMs from SIH rats, which were induced by electric foot shocks and noises. The functions of circRNA Galntl6 in reducing blood pressure (BP) and its potential molecular mechanisms on SIH were investigated via various experiments, such as Western blot and intra-RVLM microinjection. A total of 12,242 circRNA transcripts were identified, among which circRNA Galntl6 was dramatically downregulated in SIH rats. The upregulation of circRNA Galntl6 in RVLM effectively decreased the BP, sympathetic outflow, and neuronal excitability in SIH rats. Mechanistically, circRNA Galntl6 directly sponged microRNA-335 (miR-335) and restrained it to reduce oxidative stress. Reintroduction of miR-335 observably reversed the circRNA Galntl6-induced attenuation of oxidative stress. Furthermore, Lig3 can be a direct target of miR-335. MiR-335 inhibition substantially increased the expression of Lig3 and suppressed oxidative stress, and these favorable effects were blocked by Lig3 knockdown. CircRNA Galntl6 is a novel factor that impedes SIH development, and the circRNA Galntl6/miR-335/Lig3 axis represents one of the possible mechanisms. These findings demonstrated circRNA Galntl6 as a possibly useful target for the prevention of SIH.

Disordered testosterone transport in mice lacking the ganglioside GM2/GD2 synthase gene.

Genetic disruption of glycosyltransferases has provided clear information on the roles of their reaction products in the body. Our group has studied the function of glycosphingolipids by genetic engineering of glycosyltransferases in cell culture and in mice, which has demonstrated both expected and unexpected results. Among these findings, aspermatogenesis in ganglioside GM2/GD2 synthase knockout mice was one of the most surprising and intriguing results. There were no sperms in testis, and multinuclear giant cells were detected instead of spermatids. Although serum levels of testosterone in the male mice were extremely low, testosterone accumulated in the interstitial tissues, including Leydig cells, and seemed not to be transferred into the seminiferous tubules or vascular cavity from Leydig cells. This was considered to be the cause of aspermatogenesis and low serum levels of testosterone. Patients with a mutant GM2/GD2 synthase gene (SPG26) showed similar clinical signs, not only in terms of the neurological aspects, but also in the male reproductive system. The mechanisms for testosterone transport by gangliosides are discussed here based on our own results and reports from other laboratories.

N-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sda Synthase B4GALNT2.

The Sda carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human B4GALNT2 gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and SF-B4GALNT2) sharing identical transmembrane and luminal domains. Both isoforms are trans-Golgi proteins and the LF-B4GALNT2 also localizes to post-Golgi vesicles thanks to its extended cytoplasmic tail. Control mechanisms underpinning Sda and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual N-glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type N-glycan. We explored the influence of this N-glycan using site-directed mutagenesis and showed that each mutant had a slightly decreased expression level, impaired stability, and reduced enzyme activity. Furthermore, we observed that the mutant SF-B4GALNT2 was partially mislocalized in the endoplasmic reticulum, whereas the mutant LF-B4GALNT2 was still localized in the Golgi and post-Golgi vesicles. Lastly, we showed that the formation of homodimers was drastically impaired in the two mutated isoforms. An AlphaFold2 model of the LF-B4GALNT2 dimer with an N-glycan on each monomer corroborated these findings and suggested that N-glycosylation of each B4GALNT2 isoform controlled their biological activity.

Bump-and-hole engineering of human polypeptide N-acetylgalactosamine transferases to dissect their protein substrates and glycosylation sites in cells.

Despite the known disease relevance of glycans, the biological function and substrate specificities of individual glycosyltransferases are often ill-defined. Here, we describe a protocol to develop chemical, bioorthogonal reporters for the activity of the GalNAc-T family of glycosyltransferases using a tactic termed bump-and-hole engineering. This allows identification of the protein substrates and glycosylation sites of single GalNAc-Ts. Despite requiring transfection of cells with the engineered transferases and enzymes for biosynthesis of bioorthogonal substrates, the tactic complements methods in molecular biology. For complete details on the use and execution of this protocol, please refer to Schumann et al. (2020)1, Cioce et al. (2021)2, and Cioce et al. (2022)3.

GALNT2 promotes invasiveness of colorectal cancer cells partly through AXL.

GalNAc-type O-glycosylation and its initiating GalNAc transferases (GALNTs) play crucial roles in a wide range of cellular behaviors. Among 20 GALNT members, GALNT2 is consistently associated with poor survival of patients with colorectal cancer in public databases. However, its clinicopathological significance in colorectal cancer remains unclear. In this study, immunohistochemistry showed that GALNT2 was overexpressed in colorectal tumors compared with the adjacent nontumor tissues. GALNT2 overexpression was associated with poor survival of colorectal cancer patients. Forced expression of GALNT2 promoted migration and invasion as well as peritoneal metastasis of colorectal cancer cells. In contrast, GALNT2 knockdown with siRNAs or knockout with CRISPR/Cas9 system suppressed these malignant properties. Interestingly, we found that GALNT2 modified O-glycans on AXL and determined AXL levels via the proteasome-dependent pathway. In addition, the GALNT2-promoted invasiveness was significantly reversed by AXL siRNAs. These findings suggest that GALNT2 promotes colorectal cancer invasion at least partly through AXL.

Ganglioside Microdomains on Cellular and Intracellular Membranes Regulate Neuronal Cell Fate Determination.

Gangliosides are sialylated glycosphingolipids (GSLs) with essential but enigmatic functions in brain activities and neural stem cell (NSC) maintenance. Our group has pioneered research on the importance of gangliosides for growth factor receptor signaling and epigenetic regulation of NSC activity and differentiation. The primary localization of gangliosides is on cell-surface microdomains and the drastic dose and composition changes during neural differentiation strongly suggest that they are not only important as biomarkers, but also are involved in modulating NSC fate determination. Ganglioside GD3 is the predominant species in NSCs and GD3-synthase knockout (GD3S-KO) revealed reduction of postnatal NSC pools with severe behavioral deficits. Exogenous administration of GD3 significantly restored the NSC pools and enhanced the stemness of NSCs with multipotency and self-renewal. Since morphological changes during neurogenesis require a huge amount of energy, mitochondrial functions are vital for neurogenesis. We discovered that a mitochondrial fission protein, the dynamin-related protein-1 (Drp1), as a novel GD3-binding protein, and GD3 regulates mitochondrial dynamics. Furthermore, we discovered that GM1 ganglioside promotes neuronal differentiation by an epigenetic regulatory mechanism. Nuclear GM1 binds with acetylated histones on the promoters of N-acetylgalactosaminyltransferase (GalNAcT; GM2 synthase) as well as on the NeuroD1 genes in differentiated neurons. In addition, epigenetic activation of the GalNAcT gene was detected as accompanied by an apparent induction of neuronal differentiation in NSCs responding to an exogenous supplement of GM1. GM1 is indeed localized in the nucleus where it can interact with transcriptionally active histones. Interestingly, GM1 could induce epigenetic activation of the tyrosine hydroxylase (TH) gene, with recruitment of nuclear receptor related 1 (Nurr1, also known as NR4A2), a dopaminergic neuron-associated transcription factor, to the TH promoter region. In this way, GM1 epigenetically regulates dopaminergic neuron specific gene expression. GM1 interacts with active chromatin via acetylated histones to recruit transcription factors at the nuclear periphery, resulting in changes in gene expression for neuronal differentiation. The significance is that multifunctional gangliosides modulate lipid microdomains to regulate functions of important molecules on multiple sites: the plasma membrane, mitochondrial membrane, and nuclear membrane. Versatile gangliosides could regulate functional neurons as well as sustain NSC functions via modulating protein and gene activities on ganglioside microdomains.

GALNT14-mediated O-glycosylation on PHB2 serine-161 enhances cell growth, migration and drug resistance by activating IGF1R cascade in hepatoma cells.

The single nucleotide polymorphism (SNP) rs9679162 located on GALNT14 gene predicts therapeutic outcomes in patients with intermediate and advanced hepatocellular carcinoma (HCC), but the molecular mechanism remains unclear. Here, the associations between SNP genotypes, GALNT14 expression, and downstream molecular events were determined. A higher GALNT14 cancerous/noncancerous ratio was associated with the rs9679162-GG genotype, leading to an unfavorable postoperative prognosis. A novel exon-6-skipped GALNT14 mRNA variant was identified in patients carrying the rs9679162-TT genotype, which was associated with lower GALNT14 expression and favorable prognosis. Cell-based experiments showed that elevated levels of GALNT14 promoted HCC growth, migration, and resistance to anticancer drugs. Using a comparative lectin-capture glycoproteomic approach, PHB2 was identified as a substrate for GALNT14-mediated O-glycosylation. Site-directed mutagenesis experiments revealed that serine-161 (Ser161) was the O-glycosylation site. Further analysis showed that O-glycosylation of PHB2-Ser161 was required for the GALNT14-mediated growth-promoting phenotype. O-glycosylation of PHB2 was positively correlated with GALNT14 expression in HCC, resulting in increased interaction between PHB2 and IGFBP6, which in turn led to the activation of IGF1R-mediated signaling. In conclusion, the GALNT14-rs9679162 genotype was associated with differential expression levels of GALNT14 and the generation of a novel exon-6-skipped GALNT14 mRNA variant, which was associated with a favorable prognosis in HCC. The GALNT14/PHB2/IGF1R cascade modulated the growth, migration, and anticancer drug resistance of HCC cells, thereby opening the possibility of identifying new therapeutic targets against HCC.

Global mapping of GalNAc-T isoform-specificities and O-glycosylation site-occupancy in a tissue-forming human cell line.

Mucin-type-O-glycosylation on proteins is integrally involved in human health and disease and is coordinated by an enzyme family of 20 N-acetylgalactosaminyltransferases (GalNAc-Ts). Detailed knowledge on the biological effects of site-specific O-glycosylation is limited due to lack of information on specific glycosylation enzyme activities and O-glycosylation site-occupancies. Here we present a systematic analysis of the isoform-specific targets of all GalNAc-Ts expressed within a tissue-forming human skin cell line, and demonstrate biologically significant effects of O-glycan initiation on epithelial formation. We find over 300 unique glycosylation sites across a diverse set of proteins specifically regulated by one of the GalNAc-T isoforms, consistent with their impact on the tissue phenotypes. Notably, we discover a high variability in the O-glycosylation site-occupancy of 70 glycosylated regions of secreted proteins. These findings revisit the relevance of individual O-glycosylation sites in the proteome, and provide an approach to establish which sites drive biological functions.

O-Glycosylating Enzyme GALNT2 Predicts Worse Prognosis in Cervical Cancer.

Identification of novel biomarkers is helpful for the diagnosis and treatment of cervical cancer. Mucin glycosylating enzyme GALNT2 modulates mucin O-glycosylation, and has been revealed as a regulator of tumorigenesis in various cancers. However, the expression pattern of GALNT2 in cervical cancer is still unclear. In this study, we demonstrated that the mRNA expression and protein level of GALNT2 were increased in cervical high-grade intraepithelial neoplasia and tumor tissues compared with normal cervix tissues. Kaplan-Meier plotter showed that overexpression of GALNT2 was associated with worse overall survival in TCGA cohort (p < 0.001, HR = 2.65, 95% CI = 1.62-4.34) and poor disease free survival in GSE44001 cohort (p = 0.0218, HR = 2.15, 95% CI = 1.14-4.06). In addition, GSEA analysis showed that various immune-related pathways were closely related to the expression of GALNT2 in cervical cancer. Moreover, co-expression of GALNT2 and IL1A, IL1B, IL11, CXCL1, CXCL2, CXCL5, CXCL6, CXCR1, or CCR3 predicted poor overall survival, and the expression of GALNT2 also affected the prognostic value of CD47, CD274, CD276, CSF1R, TNFSF9, and TNFSF11 in cervical cancer patients. These findings suggest that GALNT2 might be used as a prognostic biomarker in cervical cancer.

GALNT3 protects against phosphate-induced calcification in vascular smooth muscle cells by enhancing active FGF23 and inhibiting the wnt/ß-catenin signaling pathway.

Vascular calcification (VC) acts as a notable risk factor in the cardiovascular system. Disorder of phosphorus (Pi) metabolism promotes VC. Recent findings show that polypeptide N-acetylgalactosaminyltransferase 3(GALNT3) is Pi responsive and with potent effects on Pi homeostasis. However, whether GALNT3 is involved in high Pi-induced VC remains unclear. The present study investigated the potential role of GALNT3 as a novel regulator of VC. In vitro, human aortic smooth muscle cells (HASMCs) calcification was induced by inorganic Pi, while in vivo, C57BL/6 J mice were used to determine the effects of GALNT3 on Vitamin D3-induced medial arterial calcification. Alizarin red staining, Von Kossa staining, calcium and alkaline phosphatase (ALP) activity were performed to test VC. We showed that expression of GALNT3 was increased in the calcified HASMCs and aortas of the calcified mice.In vitro, overexpression of GALNT3 increased the levels of active full-length FGF23, accompanied by suppression of the osteoblast-related factors (Runx2 and BMP2), and further inhibited the formation of calcified nodules. Moreover, the protein levels of Wnt3a and active ß-catenin were determined and it was found that GALNT3 significantly inhibited their expression. LiCl, a Wnt/ß-catenin signaling activator, was observed to reverse the protective effect of GALNT3 overexpression. The opposite results were observed in the GALNT3 knockdown cells. In vivo, overexpression of GALNT3 by adeno-associated virus decreased the serum Pi and slowed the formation of aortic calcification in the calcified mice. In conclusion, our results indicate that GALNT3 counteracts high Pi-induced osteoblastic differentiation of VSMCs and protects against the initiation and progression of VC by inhibiting the Wnt/ß-catenin signaling pathway.