Depletion of b-series ganglioside prevents limb length discrepancy after growth plate injury.

INTRODUCTION: Growth plate damage in long bones often results in progressive skeletal growth imbalance and deformity, leading to significant physical problems. Gangliosides, key glycosphingolipids in cartilage, are notably abundant in articular cartilage and regulate chondrocyte homeostasis. This suggests their significant roles in regulating growth plate cartilage repair. METHODS: Chondrocytes from 3 to 5 day-old C57BL/6 mice underwent glycoblotting and mass spectrometry. Based on the results of the glycoblotting analysis, we employed GD3 synthase knockout mice (GD3-/-), which lack b-series gangliosides. In 3-week-old mice, physeal injuries were induced in the left tibiae, with right tibiae sham operated. Tibiae were analyzed at 5 weeks postoperatively for length and micro-CT for growth plate height and bone volume at injury sites. Tibial shortening ratio and bone mineral density were measured by micro-CT. RESULTS: Glycoblotting analysis indicated that b-series gangliosides were the most prevalent in physeal chondrocytes among ganglioside series. At 3 weeks, GD3-/- exhibited reduced tibial shortening (14.7 ± 0.2 mm) compared to WT (15.0 ± 0.1 mm, P = 0.03). By 5 weeks, the tibial lengths in GD3-/- (16.0 ± 0.4 mm) closely aligned with sham-operated lengths (P = 0.70). Micro-CT showed delayed physeal bridge formation in GD3-/-, with bone volume measuring 168.9 ± 5.8 HU at 3 weeks (WT: 180.2 ± 3.2 HU, P = 0.09), but normalizing by 5 weeks. CONCLUSION: This study highlights that GD3 synthase knockout mice inhibit physeal bridge formation after growth plate injury, proposing a new non-invasive approach for treating skeletal growth disorders.

Identification and validation of sialyltransferase ST3Gal5 in bladder cancer through bioinformatics and experimental analysis.

BACKGROUND: Bladder cancer (BLCA) is one of the top ten most common cancers in the world. Aberrant sialylation is a common feature in tumorigenesis and tumor immunity. This study seeks to explore the potential impact of sialyltransferase ST3Gal5 on BLCA. METHODS: Initially, glycosyltransferase-related DEGs (GRDEGs) were identified using multiple bioinformatics approaches in TCGA-BLCA cohort and validated using GEO databases. Clinical prognosis integration facilitated the determination of ST3Gal5 as an independent prognostic factor in BLCA, employing univariate and multivariate Cox regression analyses. Immune cell infiltration was assessed via CIBERSORT and ssGSEA analyses, while HLA and immune checkpoint genes' levels, along with drug sensitivity, were evaluated in low- and high-ST3Gal5 groups. The TIDE and IPS scores were used to gauge the immune checkpoint blockade (ICB) response. Furthermore, functional experiments, both in vivo and in vitro, were conducted to elucidate the biological roles of ST3Gal5. RESULTS: In agreement with bioinformatics findings, ST3Gal5 expression was down-regulated in BLCA tissues and cells, correlating with poorer prognostic outcomes. The StromalScore, ImmuneScore, and ESTIMATEScore were significantly elevated in low-ST3Gal5 group. Moreover, the levels of HLA and immune checkpoint genes were upregulated in low-ST3Gal5 group. Down-regulated ST3Gal5 promoted the proliferation, migration, and invasion of BLCA cells in vivo and in vitro. CONCLUSION: Our findings demonstrated that low ST3Gal5 level promoted tumorigenesis and progression of BLCA, implying its potential as a predictive biomarker and therapeutic target.

IgG sialylation occurs in B cells pre antibody secretion.

Sialic acids as terminal sugar residues on cell surface or secreted proteins have many functional roles. In particular, the presence or absence of α2,6-linked sialic acid residues at the immunoglobulin G (IgG) Fc fragment can switch IgG effector functions from pro- to anti-inflammatory activity. IgG glycosylation is considered to take place inside the plasma blast/plasma cell while the molecule travels through the endoplasmic reticulum and Golgi apparatus before being secreted. However, more recent studies have suggested that IgG sialylation may occur predominantly post-antibody secretion. To what extent this extracellular IgG sialylation process contributes to overall IgG sialylation remains unclear, however. By generating bone marrow chimeric mice with a B cell-specific deletion of ST6Gal1, the key enzyme required for IgG sialylation, we now show that sialylation of the IgG Fc fragment exclusively occurs within B cells pre-IgG secretion. We further demonstrate that B cells expressing ST6Gal1 have a developmental advantage over B cells lacking ST6Gal1 expression and thus dominate the plasma cell pool and the resulting serum IgG population in mouse models in which both ST6Gal1-sufficient and -deficient B cells are present.

Blockade of Sialylation with Decrease in Polysialic Acid Levels Counteracts Transforming Growth Factor ß1-Induced Skin Fibroblast-to-Myofibroblast Transition.

Aberrant sialylation with overexpression of the homopolymeric glycan polysialic acid (polySia) was recently reported in fibroblasts from fibrotic skin lesions. Yet, whether such a rise in polySia levels or sialylation in general may be functionally implicated in profibrotic activation of fibroblasts and their transition to myofibroblasts remains unknown. Therefore, we herein explored whether inhibition of sialylation could interfere with the process of skin fibroblast-to-myofibroblast transition induced by the master profibrotic mediator transforming growth factor ß1 (TGFß1). Adult human skin fibroblasts were pretreated with the competitive pan-sialyltransferase inhibitor 3-Fax-peracetyl-Neu5Ac (3-Fax) before stimulation with recombinant human TGFß1, and then analyzed for polySia expression, cell viability, proliferation, migratory ability, and acquisition of myofibroblast-like morphofunctional features. Skin fibroblast stimulation with TGFß1 resulted in overexpression of polySia, which was effectively blunted by 3-Fax pre-administration. Pretreatment with 3-Fax efficiently lessened TGFß1-induced skin fibroblast proliferation, migration, changes in cell morphology, and phenotypic and functional differentiation into myofibroblasts, as testified by a significant reduction in FAP, ACTA2, COL1A1, COL1A2, and FN1 gene expression, and α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein levels, as well as a reduced contractile capability. Moreover, skin fibroblasts pre-administered with 3-Fax displayed a significant decrease in Smad3-dependent canonical TGFß1 signaling. Collectively, our in vitro findings demonstrate for the first time that aberrant sialylation with increased polySia levels has a functional role in skin fibroblast-to-myofibroblast transition and suggest that competitive sialyltransferase inhibition might offer new therapeutic opportunities against skin fibrosis.

Digitoxin inhibits ICC cell properties via the NF­κB/ST6GAL1 signaling pathway.

Intrahepatic cholangiocarcinoma (ICC) is a type of liver cancer associated with poor prognosis and increased mortality; the limited treatment strategy highlights the urgent need for investigation. Traditional Chinese Medicine (TCM), used alone or in combination with other treatments, can enhance therapeutic efficacy, improve life quality of patients and extend overall survival. In total, two rounds of screening of a TCM library of 2,538 active compounds were conducted using a Cell Counting Kit­8 assay and ICC cell lines. Cell proliferation and migration abilities were assessed through colony formation, 5­ethynyl­2'­deoxyuridine, would healing and Transwell assays. The impact of digitoxin (DT) on signaling pathways was initially investigated using RNA sequencing and further validated using reverse transcription­quantitative PCR, western blotting, lectin blotting and flow cytometry. ICC cells stably overexpressing ST6 ß­galactoside α­2,6­sialyltransferase 1 (ST6GAL1) were generated through lentiviral transfection. It was shown that DT emerged as a highly effective anti­ICC candidate from two rounds high­throughput library screening. DT could inhibit the proliferation and migration of ICC cells by suppressing NF­κB activation and reducing nuclear phosphorylated­NF­κB levels, along with diminishing ST6GAL1 mRNA and protein expression. The aforementioned biological effects and signal pathways of DT could be counteracted by overexpressing ST6GAL1 in ICC cells. In conclusion, DT suppressed ICC cell proliferation and migration by targeting the NF­κB/ST6GAL1 signaling axis. The findings of the present study indicated the promising therapeutic effects of DT in managing ICC, offering new avenues for treatment strategies.

Establishment of a humanized ST6GAL1 mouse model for influenza research.

BACKGROUND: This study aimed to construct and characterize a humanized influenza mouse model expressing hST6GAL1. METHODS: Humanized fragments, consisting of the endothelial cell-specific K18 promoter, human ST6GAL1-encoding gene, and luciferase gene, were microinjected into the fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice. The offspring were identified using PCR. Mice exhibiting elevated expression of the hST6GAL1 gene were selectively bred for propagation, and in vivo analysis was performed for screening. Expression of the humanized gene was tested by performing immunohistochemical (IHC) analysis. Hematologic and biochemical analyses using the whole blood and serum of humanized hST6GAL1 mice were performed. RESULTS: Successful integration of the human ST6GAL1 gene into the mouse genome led to the overexpression of human SiaT ST6GAL1. Seven mice were identified as carrying copies of the humanized gene, and the in vivo analysis indicated that hST6GAL1 gene expression in positive mice mirrored influenza virus infection characteristics. The IHC results revealed that hST6GAL1 was expressed in the lungs of humanized mice. Moreover, the hematologic and biochemical parameters of the positive mice were within the normal range. CONCLUSION: A humanized influenza mouse model expressing the hST6GAL1 gene was successfully established and characterized.

GD2 and its biosynthetic enzyme GD3 synthase promote tumorigenesis in prostate cancer by regulating cancer stem cell behavior.

While better management of loco-regional prostate cancer (PC) has greatly improved survival, advanced PC remains a major cause of cancer deaths. Identification of novel targetable pathways that contribute to tumor progression in PC could open new therapeutic options. The di-ganglioside GD2 is a target of FDA-approved antibody therapies in neuroblastoma, but the role of GD2 in PC is unexplored. Here, we show that GD2 is expressed in a small subpopulation of PC cells in a subset of patients and a higher proportion of metastatic tumors. Variable levels of cell surface GD2 expression were seen on many PC cell lines, and the expression was highly upregulated by experimental induction of lineage progression or enzalutamide resistance in CRPC cell models. GD2high cell fraction was enriched upon growth of PC cells as tumorspheres and GD2high fraction was enriched in tumorsphere-forming ability. CRISPR-Cas9 knockout (KO) of the rate-limiting GD2 biosynthetic enzyme GD3 Synthase (GD3S) in GD2high CRPC cell models markedly impaired the in vitro oncogenic traits and growth as bone-implanted xenograft tumors and reduced the cancer stem cell and epithelial-mesenchymal transition marker expression. Our results support the potential role of GD3S and its product GD2 in promoting PC tumorigenesis by maintaining cancer stem cells and suggest the potential for GD2 targeting in advanced PC.

Novel genetically glycoengineered human dendritic cell model reveals regulatory roles of α2,6-linked sialic acids in DC activation of CD4+ T cells and response to TNFα.

Dendritic cells (DCs) are central for the initiation and regulation of appropriate immune responses. While several studies suggest important regulatory roles of sialoglycans in DC biology, our understanding is still inadequate primarily due to a lack of appropriate models. Previous approaches based on enzymatic- or metabolic-glycoengineering and primary cell isolation from genetically modified mice have limitations related to specificity, stability, and species differences. This study addresses these challenges by introducing a workflow to genetically glycoengineer the human DC precursor cell line MUTZ-3, described to differentiate and maturate into fully functional dendritic cells, using CRISPR-Cas9, thereby providing and validating the first isogenic cell model for investigating glycan alteration on human DC differentiation, maturation, and activity. By knocking out (KO) the ST6GAL1 gene, we generated isogenic cells devoid of ST6GAL1-mediated α(2,6)-linked sialylation, allowing for a comprehensive investigation into its impact on DC function. Glycan profiling using lectin binding assay and functional studies revealed that ST6GAL1 KO increased the expression of important antigen presenting and co-stimulatory surface receptors and a specifically increased activation of allogenic human CD4 + T cells. Additionally, ST6GAL1 KO induces significant changes in surface marker expression and cytokine response to TNFα-induced maturation, and it affects migration and the endocytic capacity. These results indicate that genetic glycoengineering of the isogenic MUTZ-3 cellular model offers a valuable tool to study how specific glycan structures influence human DC biology, contributing to our understanding of glycoimmunology.

Decreasing ganglioside synthesis delays motor and cognitive symptom onset in Spg11 knockout mice.

Biallelic variants in the SPG11 gene account for the most common form of autosomal recessive hereditary spastic paraplegia characterized by motor and cognitive impairment, with currently no therapeutic option. We previously observed in a Spg11 knockout mouse that neurodegeneration is associated with accumulation of gangliosides in lysosomes. To test whether a substrate reduction therapy could be a therapeutic option, we downregulated the key enzyme involved in ganglioside biosynthesis using an AAV-PHP.eB viral vector expressing a miRNA targeting St3gal5. Downregulation of St3gal5 in Spg11 knockout mice prevented the accumulation of gangliosides, delayed the onset of motor and cognitive symptoms, and prevented the upregulation of serum levels of neurofilament light chain, a biomarker widely used in neurodegenerative diseases. Importantly, similar results were observed when Spg11 knockout mice were administrated venglustat, a pharmacological inhibitor of glucosylceramide synthase expected to decrease ganglioside synthesis. Downregulation of St3gal5 or venglustat administration in Spg11 knockout mice strongly decreased the formation of axonal spheroids, previously associated with impaired trafficking. Venglustat had similar effect on cultured human SPG11 neurons. In conclusion, this work identifies the first disease-modifying therapeutic strategy in SPG11, and provides data supporting its relevance for therapeutic testing in SPG11 patients.

Sialyltransferase ST3GAL6 silencing reduces α2,3-sialylated glycans to regulate autophagy by decreasing HSPB8-BAG3 in the brain with hepatic encephalopathy. / å”¾æ¶²é ¸ç³–åŸºè½¬ç§»é ¶ST3GAL6的沉默可通过减少α2,3-å”¾æ¶²é ¸åŒ–èšç³–é™ä½ŽHSPB8-BAG3è¡¨è¾¾ä»Žè€Œè°ƒæŽ§è‚æ€§è„‘ç— å°é¼ å¤§è„‘ä¸­çš„è‡ªå™¬.

End-stage liver diseases, such as cirrhosis and liver cancer caused by hepatitis B, are often combined with hepatic encephalopathy (HE); ammonia poisoning is posited as one of its main pathogenesis mechanisms. Ammonia is closely related to autophagy, but the molecular mechanism of ammonia's regulatory effect on autophagy in HE remains unclear. Sialylation is an essential form of glycosylation. In the nervous system, abnormal sialylation affects various physiological processes, such as neural development and synapse formation. ST3 ß|-galactoside α2,|3-sialyltransferase 6 (ST3GAL6) is one of the significant glycosyltransferases responsible for adding α2,3-linked sialic acid to substrates and generating glycan structures. We found that the expression of ST3GAL6 was upregulated in the brains of mice with HE and in astrocytes after ammonia induction, and the expression levels of α2,3-sialylated glycans and autophagy-related proteins microtubule-associated protein light chain 3 (LC3) and Beclin-1 were upregulated in ammonia-induced astrocytes. These findings suggest that ST3GAL6 is related to autophagy in HE. Therefore, we aimed to determine the regulatory relationship between ST3GAL6 and autophagy. We found that silencing ST3GAL6 and blocking or degrading α2,3-sialylated glycans by way of Maackia amurensis lectin-II (MAL-II) and neuraminidase can inhibit autophagy. In addition, silencing the expression of ST3GAL6 can downregulate the expression of heat shock protein ß8 (HSPB8) and Bcl2-associated athanogene 3 (BAG3). Notably, the overexpression of HSPB8 partially restored the reduced autophagy levels caused by silencing ST3GAL6 expression. Our results indicate that ST3GAL6 regulates autophagy through the HSPB8-BAG3 complex.

Sialic acid blockade inhibits the metastatic spread of prostate cancer to bone.

BACKGROUND: Bone metastasis is a common consequence of advanced prostate cancer. Bisphosphonates can be used to manage symptoms, but there are currently no curative treatments available. Altered tumour cell glycosylation is a hallmark of cancer and is an important driver of a malignant phenotype. In prostate cancer, the sialyltransferase ST6GAL1 is upregulated, and studies show ST6GAL1-mediated aberrant sialylation of N-glycans promotes prostate tumour growth and disease progression. METHODS: Here, we monitor ST6GAL1 in tumour and serum samples from men with aggressive prostate cancer and using in vitro and in vivo models we investigate the role of ST6GAL1 in prostate cancer bone metastasis. FINDINGS: ST6GAL1 is upregulated in patients with prostate cancer with tumours that have spread to the bone and can promote prostate cancer bone metastasis in vivo. The mechanisms involved are multi-faceted and involve modification of the pre-metastatic niche towards bone resorption to promote the vicious cycle, promoting the development of M2 like macrophages, and the regulation of immunosuppressive sialoglycans. Furthermore, using syngeneic mouse models, we show that inhibiting sialylation can block the spread of prostate tumours to bone. INTERPRETATION: Our study identifies an important role for ST6GAL1 and α2-6 sialylated N-glycans in prostate cancer bone metastasis, provides proof-of-concept data to show that inhibiting sialylation can suppress the spread of prostate tumours to bone, and highlights sialic acid blockade as an exciting new strategy to develop new therapies for patients with advanced prostate cancer. FUNDING: Prostate Cancer Research and the Mark Foundation For Cancer Research, the Medical Research Council and Prostate Cancer UK.

Highly efficient biosynthesis of 3'-sialyllactose in engineered Escherichia coli.

3'-Sialyllactose (3'-SL), one of the abundant and important sialylated human milk oligosaccharides, is an emerging food ingredient used in infant formula milk. We previously developed an efficient route for 3'-SL biosynthesis in metabolically engineered Escherichia coli BL21(DE3). Here, several promising α2,3-sialyltransferases were re-evaluated from the byproduct synthesis perspective. The α2,3-sialyltransferase from Neisseria meningitidis MC58 (NST) with great potential and the least byproducts was selected for subsequent molecular modification. Computer-assisted mutation sites combined with a semi-rational modification were designed and performed. A combination of two mutation sites (P120H/N113D) of NST was finally confirmed as the best one, which significantly improved 3'-SL biosynthesis, with extracellular titers of 24.5 g/L at 5-L fed-batch cultivations. When NST-P120H/N113D was additionally integrated into the genome of host EZAK (E. coli BL21(DE3)ΔlacZΔnanAΔnanT), the final strain generated 32.1 g/L of extracellular 3'-SL in a 5-L fed-batch fermentation. Overall, we underscored the existence of by-products and improved 3'-SL production by engineering N. meningitidis α2,3-sialyltransferase.

Generation of a human induced pluripotent stem cell line from a patient with GM3 synthase deficiency using self-replicating RNA vector.

GM3 synthase deficiency (GM3SD) is caused by biallelic variants in the ST3GAL5 gene. Early clinical features of GM3SD include infantile onset of severe irritability and feeding difficulties, early intractable seizures, growth failure, hypotonia, sensorineural hearing impairment. We describe the generation and characterization the human induced pluripotent stem cell (hiPSC) line derived from fibroblasts of a 13-year-old girl with GM3 synthase deficiency resulted compound heterozygous for two new variants in the ST3GAL5 gene, c.1166A > G (p.His389Arg) and the c.1024G > A (p.Gly342Ser). The generated hiPSC line shows a normal karyotype, expresses pluripotency markers, and is able to differentiate into the three germ layers.

Limited impact of cancer-derived gangliosides on anti-tumor immunity in colorectal cancer.

Aberrant glycosylation is a key mechanism employed by cancer cells to evade immune surveillance, induce angiogenesis and metastasis, among other hallmarks of cancer. Sialic acids, distinctive terminal glycan structures located on glycoproteins or glycolipids, are prominently upregulated across various tumor types, including colorectal cancer (CRC). Sialylated glycans modulate anti-tumor immune responses through their interactions with Siglecs, a family of glycan-binding receptors with specificity for sialic acid-containing glycoconjugates, often resulting in immunosuppression. In this paper, we investigated the immunomodulatory function of ST3Gal5, a sialyltransferase that catalyzes the addition of α2-3 sialic acids to glycosphingolipids, since lower expression of ST3Gal5 is associated with better survival of CRC patients. We employed CRISPR/Cas9 to knock out the ST3Gal5 gene in two murine CRC cell lines MC38 and CT26. Glycomics analysis confirmed the removal of sialic acids on glycolipids, with no discernible impact on glycoprotein sialylation. Although knocking out ST3Gal5 in both cell lines did not affect in vivo tumor growth, we observed enhanced levels of regulatory T cells in CT26 tumors lacking ST3Gal5. Moreover, we demonstrate that the absence of ST3Gal5 affected size and blood vessel density only in MC38 tumors. In summary, we ascertain that sialylation of glycosphingolipids has a limited influence on the anti-tumor immune response in CRC, despite detecting alterations in the tumor microenvironment, possibly due to a shift in ganglioside abundance.

CCR3-dependent eosinophil recruitment is regulated by sialyltransferase ST3Gal-IV.

Eosinophil recruitment is a pathological hallmark of many allergic and helminthic diseases. Here, we investigated chemokine receptor CCR3-induced eosinophil recruitment in sialyltransferase St3gal4-/- mice. We found a marked decrease in eosinophil extravasation into CCL11-stimulated cremaster muscles and into the inflamed peritoneal cavity of St3gal4-/- mice. Ex vivo flow chamber assays uncovered reduced adhesion of St3gal4-/- compared to wild type eosinophils. Using flow cytometry, we show reduced binding of CCL11 to St3gal4-/- eosinophils. Further, we noted reduced binding of CCL11 to its chemokine receptor CCR3 isolated from St3gal4-/- eosinophils. This was accompanied by almost absent CCR3 internalization of CCL11-stimulated St3gal4-/- eosinophils. Applying an ovalbumin-induced allergic airway disease model, we found a dramatic reduction in eosinophil numbers in bronchoalveolar lavage fluid following intratracheal challenge with ovalbumin in St3gal4-deficient mice. Finally, we also investigated tissue-resident eosinophils under homeostatic conditions and found reduced resident eosinophil numbers in the thymus and adipose tissue in the absence of ST3Gal-IV. Taken together, our results demonstrate an important role of ST3Gal-IV in CCR3-induced eosinophil recruitment in vivo rendering this enzyme an attractive target in reducing unwanted eosinophil infiltration in various disorders including allergic diseases.

Human ST3Gal II and ST6GalNAc IV genes increase human serum-mediated cytotoxicity to xenogeneic cells.

Carbohydrate-antigens widely existed on glycoproteins and glycosphingolipids of all mammalian cells play a crucial role in self-defense and immunity. Xeno-reactive antibodies included in natural human sera play a protecting role in an acute phase-rejection of xenotransplantation. In this study, we investigated the effect of an alteration of glycosylation-pattern, caused by human sialyltransferases such as hST3Gal II or hST6GalNAc IV, on human serum mediated cytotoxicity in pig kidney PK15 cells. From LDH cytotoxicity assay, cytotoxicity to human serum was significantly increased in hST3Gal II and hST6GalNAc IV-transfected PK15 cells, as compared to the control. In the hST6Gal I-carrying cells, the cytotoxicity to human serum was rather decreased. Moreover, flow cytometry analysis revealed that an alteration of pig glycosylation-pattern by hST3Gal II or hST6GalNAc IV influences on a binding of human IgM or IgG, respectively, in pig kidney cells, regardless of Gal antigen alteration. Finally, we found that hST6GalNAc IV contributed to increase of terminal disialylated tetrasaccharide structure, disialyl T antigen, as evidenced by increase of the MAL II lectin binding capacity in the hST6GalNAc IV-transfected PK15 cells, compared with control. Therefore, our results suggest that carbohydrate antigens, such as disialyl T antigen, newly synthesized by the ST3Gal II- and ST6GalNAc IV are potentially believed to be new xeno-reactive elements.

Characterization and application of active human α2,6-sialyltransferases ST6GalNAc V and ST6GalNAc VI recombined in Escherichia coli.

Eukaryotic sialyltransferases play key roles in many physiological and pathological events. The expression of active human recombinant sialyltransferases in bacteria is still challenging. In the current study, the genes encoding human N-acetylgalactosaminide α2,6-sialyltransferase V (hST6GalNAc V) and N-acetylgalactosaminide α2,6-sialyltransferase VI (hST6GalNAc VI) lacking the N-terminal transmembrane domains were cloned into the expression vectors, pET-32a and pET-22b, respectively. Soluble and active forms of recombinant hST6GalNAc V and hST6GalNAc VI when coexpressed with the chaperone plasmid pGro7 were successfully achieved in Escherichia coli. Further, lactose (Lac), Lacto-N-triose II (LNT II), lacto-N-tetraose (LNT), and sialyllacto-N-tetraose a (LSTa) were used as acceptor substrates to investigate their activities and substrate specificities. Unexpectedly, both can transfer sialic acid onto all those substrates. Compared with hST6GalNAc V expressed in the mammalian cells, the recombinant two α2,6-sialyltransferases in bacteria displayed flexible substrate specificities and lower enzymatic efficiency. In addition, an important human milk oligosaccharide disialyllacto-N-tetraose (DSLNT) can be synthesized by both human α2,6-sialyltransferases expressed in E. coli using LSTa as an acceptor substrate. To the best of our knowledge, these two active human α2,6-sialyltransferases enzymes were expressed in bacteria for the first time. They showed a high potential to be applied in biotechnology and investigating the molecular mechanisms of biological and pathological interactions related to sialylated glycoconjugates.

N-linked α2,6-sialylation of integrin ß1 by the sialyltransferase ST6Gal1 promotes cell proliferation and stemness in gestational trophoblastic disease.

INTRODUCTION: Gestational trophoblastic disease (GTD) encompasses a spectrum of rare pre-malignant and malignant entities originating from trophoblastic tissue, including partial hydatidiform mole, complete hydatidiform mole and choriocarcinoma. ß-galactoside α2,6 sialyltransferase 1 (ST6Gal1), the primary sialyltransferase responsible for the addition of α2,6 sialic acids, is strongly associated with the occurrence and development of several tumor types. However, the role of ST6Gal1/α2,6 -sialylation of trophoblast cells in GTD is still not well understood. METHODS: The expression of ST6Gal1 was investigated in GTD and human immortalized trophoblastic HTR-8/SVneo cells and human gestational choriocarcinoma JAR cells. We evaluated the effect of ST6Gal1 on proliferation and stemness of trophoblastic cells. We also examined the effect of internal miR-199a-5p on ST6Gal1 expression. The role of ST6Gal1 in regulating α2,6-sialylated integrin ß1 and its significance in the activation of integrin ß1/focal adhesion kinase (FAK) signaling pathway were also explored. RESULTS: ST6Gal1 was observed to be highly expressed in GTD. Overexpression of ST6Gal1 promoted the proliferation and stemness of HTR-8/SVneo cells, whereas knockdown of ST6Gal1 suppressed the viability and stemness of JAR cells. MiR-199a-5p targeted and inhibited the expression of ST6Gal1 in trophoblastic cells. In addition, we revealed integrin ß1 was highly α2,6-sialylated in JAR cells. Inhibition of ST6Gal1 reduced α2,6-sialylation on integrin ß1 and suppressed the integrin ß1/FAK pathway in JAR cells, thereby affecting its biological functions. DISCUSSION: This study demonstrated that ST6Gal1 plays important roles in promoting proliferation and stemness through the integrin ß1 signaling pathway in GTD. Therefore, ST6Gal1 may have a potential role in the occurrence and development of GTD.

Sialyltransferase ST3GAL4 confers osimertinib resistance and offers strategies to overcome resistance in non-small cell lung cancer.

The third-generation EGFR-TKI osimertinib is widely used in EGFR-mutated positive non-small cell lung cancer (NSCLC) patients, but drug resistance is inevitable. The currently known mechanisms only explain resistance in a small proportion of patients. For most patients, the mechanism of osimertinib resistance is still unclear, especially for EGFR-independent resistance. Herein, we thoroughly investigated the novel mechanism of osimertinib resistance and treatment strategies. We identified that ST3GAL4, a sialyltransferase, catalyzes terminal glycan sialylation of receptor protein tyrosine kinases, which induces acquired resistance to osimertinib in vitro and in vivo. In addition, ST3GAL4 is generally overexpressed in osimertinib-resistant patients with unknown resistance mechanisms. ST3GAL4 modifies MET glycosylation on N785 with sialylation, which antagonizes K48-related ubiquitin-dependent MET degradation and subsequently activates MET and its downstream proliferation signaling pathways. Meanwhile, ST3GAL4 knockdown or inhibition by brigatinib resensitizes resistant non-small cell lung cancer cells to osimertinib in vitro and in vivo This study suggests that ST3GAL4 can induce acquired resistance to osimertinib, which may be an important EGFR-independent resistance mechanism Furthermore, targeting ST3GAL4 with brigatinib provides new strategies to overcome osimertinib resistance.

Functions of Sialyltransferases in gynecological malignancies: A systematic review.

INTRODUCTION: The biosynthesis of tumor-associated sialoglycans involves Sialyltransferases expressed in cancer cells differentially. The current review aspires to bridge the existing knowledge gaps by consolidating evidence regarding the role of Sialyltransferases in gynecological malignant tumors (ovarian, cervix, endometrial, and breast). METHODS: In this systematic review, we searched databases, including PubMed, Embase, Web of Science, Scopus and Cochrane Library. Twenty-two high-quality articles were selected out of 559 researched studies using radiomics quality score (RQS) tools. RESULTS: Our findings indicated that 7 articles were related to Sialyltransferases in ovarian cancer, in which 6 studies was examined only ST6Gal-I and one study examined the ST3Gal-I, ST3Gal-II, ST3Gal-III, ST3Gal-IV, ST3Gal-VI, and ST3Gal-6. In addition, 5 articles were related to Sialyltransferases in cervix cancer (ST6Gal-I), 3 articles to endometrial cancer (ST6Gal-I, ST3Gal-III, ST3Gal-IV, and ST3Gal-6), and 7 articles to breast cancer (ST6Gal-I gene in 5 studies, ST6GAL-II gene in one study, and ST8SIA1 and ST3GAL-V genes in one study). CONCLUSION: ST6Gal-I gene expression occurs at a high speed in ovarian, cervix, endometrial, and breast cancers, leading to metastasis to distant cells, cell destruction, cell invasion, and reduced patient survival.