Impact of Glycan Depletion, Glycan Debranching and Increased Glycan Charge on HIV-1 Neutralization Sensitivity and Immunogenicity.

Broadly neutralizing antibodies (bNAbs) isolated from HIV-1 infected donors are vaccine paradigms. These bNAbs recognize envelope glycoprotein trimers that carry 75-90 oligomannose and complex-type glycans. Although bNAbs and their precursors must navigate past glycans, they usually also make some glycan contacts. Glycan-modified vaccines may therefore be useful to initiate and guide bNAb development. Here, we describe two ways to modify Env glycans for possible vaccine use: 1) using a cocktail of glycosidases (termed "NGAF3" (Neuraminidase, ß-Galactosidase, N-Acetylglucosaminidase, endoglycosidase F3 (endo F3)) to deplete complex glycans to try to minimize bNAb-glycan clashes and 2) co-expressing ß-1,4-galactosyltransferase 1 (B4G) and ß-galactoside α-2,6 sialyltransferase 1 (ST6) during Env biosynthesis, creating bNAb-preferred glycan structures. Mass spectrometry revealed that NGAF3 removed glycan heads at 3/7 sites occupied by complex glycans. B4G overexpression resulted in hybrid glycan development whenever complex glycans were closely spaced. The glycan at position 611 in of Env's gp41 transmembrane subunit was uniquely isolated from the effects of both endo F3 and B4G. B4G and ST6 co-expression increased hybrid and sialylated glycan abundance, reducing glycan complexity. In rabbit vaccinations, B4G + ST6 virus-like particles (VLPs) induced less frequent, weaker titer NAbs, implying that ST6-mediated increased Env charge dampens vaccine antibodies. In some cases, vaccine sera preferentially neutralized B4G + ST6-modified pseudovirus. HIV-1+ donor plasma NAbs were generally more effective against B4G + ST6 modified pseudovirus, suggesting a preference for less complex and/or α-2,6 sialylated Env trimers. Collectively, our data suggest that B4G and ST6 Env modifications are best suited for intermediate or late vaccine shots.

Possible involvement of sialidase and sialyltransferase activities in a stage-dependent recycling of sialic acid in some organs of type 1 and type 2 diabetic rats.

Background: Type 1 (T1D) and type 2 (T2D) diabetes lead to an aberrant metabolism of sialoglycoconjugates and elevated free serum sialic acid (FSSA) level. The present study evaluated sialidase and sialyltranferase activities in serum and some organs relevant to diabetes at early and late stages of T1D and T2D. Methods: Sialic acid level with sialidase and sialyltransferase activities were monitored in the serum, liver, pancreas, skeletal muscle and kidney of diabetic animals at early and late stages of the diseases. Results: The FSSA and activity of sialidase in the serum were significantly increased at late stage of both T1D and T2D while sialic acid level in the liver was significantly decreased in the early and late stages of T1D and T2D, respectively. Furthermore, the activity of sialidase was significantly elevated in most of the diabetes-relevant organs while the activity of sialyltransferase remained largely unchanged. A multiple regression analysis revealed the contribution of the liver to the FSSA while pancreas and kidney contributed to the activity of sialidase in the serum. Conclusions: We concluded that the release of hepatic sialic acid in addition to pancreatic and renal sialidase might (in)directly contribute to the increased FSSA during both types of diabetes mellitus.

Specific sialylation of N-glycans and its novel regulatory mechanism.

Altered glycosylation is a common feature of cancer cells. Some subsets of glycans are found to be frequently enriched on the tumor cell surface and implicated in different tumor phenotypes. Among these, changes in sialylation have long been associated with metastatic cell behaviors such as invasion and enhanced cell survival. Sialylation typically exists in three prominent linkages: α2,3, α2,6, and α2,8, catalyzed by a group of sialyltransferases. The aberrant expression of all three linkages has been related to cancer progression. The increased α2,6 sialylation on N-glycans catalyzed by ß-galactoside α2,6 sialyltransferase 1 (ST6Gal1) is frequently observed in many cancers. In contrast, functions of α2,3 sialylation on N-glycans catalyzed by at least three ß-galactoside α2,3-sialyltransferases, ST3Gal3, ST3Gal4, and ST3Gal6 remain elusive due to a possibility of compensating for one another. In this minireview, we briefly describe functions of sialylation and recent findings that different α2,3 sialyltransferases specifically modify target proteins, as well as sialylation regulatory mechanisms vis a complex formation among integrin α3ß1, Golgi phosphoprotein 3 (GOLPH3), phosphatidylinositol 4-kinase IIα (PI4KIIα), focal adhesion kinase (FAK) and sialyltransferase, which suggests a new concept for the regulation of glycosylation in cell biology.

The alteration and role of glycoconjugates in Alzheimer's disease.

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder characterized by abnormal protein deposition. With an alarming 30 million people affected worldwide, AD poses a significant public health concern. While inhibiting key enzymes such as ß-site amyloid precursor protein-cleaving enzyme 1 and γ-secretase or enhancing amyloid-ß clearance, has been considered the reasonable strategy for AD treatment, their efficacy has been compromised by ineffectiveness. Furthermore, our understanding of AD pathogenesis remains incomplete. Normal aging is associated with a decline in glucose uptake in the brain, a process exacerbated in patients with AD, leading to significant impairment of a critical post-translational modification: glycosylation. Glycosylation, a finely regulated mechanism of intracellular secondary protein processing, plays a pivotal role in regulating essential functions such as synaptogenesis, neurogenesis, axon guidance, as well as learning and memory within the central nervous system. Advanced glycomic analysis has unveiled that abnormal glycosylation of key AD-related proteins closely correlates with the onset and progression of the disease. In this context, we aimed to delve into the intricate role and underlying mechanisms of glycosylation in the etiopathology and pathogenesis of AD. By highlighting the potential of targeting glycosylation as a promising and alternative therapeutic avenue for managing AD, we strive to contribute to the advancement of treatment strategies for this debilitating condition.

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 Acids Blockade-Based Chemo-Immunotherapy Featuring Cancer Cell Chemosensitivity and Antitumor Immune Response Synergies.

Immune checkpoint blockade (ICB) has significantly improved the prognosis of patients with cancer, although the majority of such patients achieve low response rates; consequently, new therapeutic approaches are urgently needed. The upregulation of sialic acid-containing glycans is a common characteristic of cancer-related glycosylation, which drives disease progression and immune escape via numerous pathways. Herein, the development of self-assembled core-shell nanoscale coordination polymer nanoparticles loaded with a sialyltransferase inhibitor, referred to as NCP-STI which effectively stripped diverse sialoglycans from cancer cells, providing an antibody-independent pattern to disrupt the emerging Siglec-sialic acid glyco-immune checkpoint is reported. Furthermore, NCP-STI inhibits sialylation of the concentrated nucleoside transporter 1 (CNT1), promotes the intracellular accumulation of anticancer agent gemcitabine (Gem), and enhances Gem-induced immunogenic cell death (ICD). As a result, the combination of NCP-STI and Gem (NCP-STI/Gem) evokes a robust antitumor immune response and exhibits superior efficacy in restraining the growth of multiple murine tumors and pulmonary metastasis. Collectively, the findings demonstrate a novel form of small molecule-based chemo-immunotherapy approach which features sialic acids blockade that enables cooperative effects of cancer cell chemosensitivity and antitumor immune responses for cancer treatment.

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.

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.

Synthesis of α-Hydroxy-1,2,3-Triazole-linked Sialyltransferase Inhibitors and Evaluation of Selectivity Towards ST3GAL1, ST6GAL1 and ST8SIA2.

Tumour-derived sialoglycans, bearing the charged nonulosonic sugar sialic acid at their termini, play a critical role in tumour cell adhesion and invasion, as well as evading cell death and immune surveillance. Sialyltransferases (ST), the enzymes responsible for the biosynthesis of sialylated glycans, are highly upregulated in cancer, with tumour hypersialylation strongly correlated with tumour growth, metastasis and drug resistance. As a result, desialylation of the tumour cell surface using either targeted delivery of a pan-ST inhibitor (or sialidase) or systemic delivery of a non-toxic selective ST inhibitors are being pursued as potential new anti-metastatic strategies against multiple cancers including pancreatic, ovarian, breast, melanoma and lung cancer. Herein, we have employed molecular modelling to give insights into the selectivity observed in a series of selective ST inhibitors that incorporate a uridyl ring in place of the cytidine of the natural donor (CMP-Neu5Ac) and replace the charged phosphodiester linker of classical ST inhibitors with a neutral α-hydroxy-1,2,3-triazole linker. The inhibitory activities of the nascent compounds were determined against recombinant human ST enzymes (ST3GAL1, ST6GAL1, ST8SIA2) showing promising activity and selectivity towards specific ST sub-types. Our ST inhibitors are non-toxic and show improved synthetic accessibility and drug-likeness compared to earlier nucleoside-based ST inhibitors.

Novel regulatory mechanisms of N-glycan sialylation: Implication of integrin and focal adhesion kinase in the regulation.

BACKGROUND: Sialylation of glycoproteins, including integrins, is crucial in various cancers and diseases such as immune disorders. These modifications significantly impact cellular functions and are associated with cancer progression. Sialylation, catalyzed by specific sialyltransferases (STs), has traditionally been considered to be regulated at the mRNA level. SCOPE OF REVIEW: Recent research has expanded our understanding of sialylation, revealing ST activity changes beyond mRNA level variations. This includes insights into COPI vesicle formation and Golgi apparatus maintenance and identifying specific target proteins of STs that are not predictable through recombinant enzyme assays. MAJOR CONCLUSIONS: This review summarizes that Golgi-associated pathways largely influence the regulation of STs. GOLPH3, GORAB, PI4K, and FAK have become critical elements in sialylation regulation. Some STs have been revealed to possess specificity for specific target proteins, suggesting the presence of additional, enzyme-specific regulatory mechanisms. GENERAL SIGNIFICANCE: This study enhances our understanding of the molecular interplay in sialylation regulation, mainly focusing on the role of integrin and FAK. It proposes a bidirectional system where sialylations might influence integrins and vice versa. The diversity of STs and their specific linkages offer new perspectives in cancer research, potentially broadening our understanding of cellular mechanisms and opening avenues for new therapeutic approaches in targeting sialylation pathways.

Development of a Novel, Potent, and Selective Sialyltransferase Inhibitor for Suppressing Cancer Metastasis.

Sialyltransferase-catalyzed membrane protein and lipid glycosylation plays a vital role as one of the most abundant post-translational modifications and diversification reactions in eukaryotes. However, aberrant sialylation has been associated with cancer malignancy and metastasis. Sialyltransferases thus represent emerging targets for the development of small molecule cancer drugs. Herein, we report the inhibitory effects of a recently discovered lithocholic acid derivative FCW393 on sialyltransferase catalytic activity, integrin sialyation, cancer-associated signal transduction, MDA-MB-231 and B16F10 cell migration and invasion, and in in vivo studies, on tumor growth, metastasis, and angiogenesis. FCW393 showed effective and selective inhibition of the sialyltransferases ST6GAL1 (IC50 = 7.8 µM) and ST3GAL3 (IC50 = 9.45 µM) relative to ST3GAL1 (IC50 > 400 µM) and ST8SIA4 (IC50 > 100 µM). FCW393 reduced integrin sialylation in breast cancer and melanoma cells dose-dependently and downregulated proteins associated with the integrin-regulated FAK/paxillin and GEF/Rho/ROCK pathways, and with the VEGF-regulated Akt/NFκB/HIF-1α pathway. FCW393 inhibited cell migration (IC50 = 2.6 µM) and invasion in in vitro experiments, and in in vivo studies of tumor-bearing mice, FCW393 reduced tumor size, angiogenesis, and metastatic potential. Based on its demonstrated selectivity, cell permeability, relatively low cytotoxicity (IC50 = 55 µM), and high efficacy, FCW393 shows promising potential as a small molecule experimental tool compound and a lead for further development of a novel cancer therapeutic.

Biological evaluation of sulfonate and sulfate analogues of lithocholic acid: A bioisosterism-guided approach towards the discovery of potential sialyltransferase inhibitors for antimetastatic study.

The naturally occurring bile acid lithocholic acid (LCA) has been a crucial core structure for many non-sugar-containing sialyltranferase (ST) inhibitors documented in literature. With the aim of elucidating the impact of the terminal carboxyl acid substituent of LCA on its ST inhibition, in this present study, we report the (bio)isosteric replacement-based design and synthesis of sulfonate and sulfate analogues of LCA. Among these compounds, the sulfate analogue SPP-002 was found to selectively inhibit N-glycan sialylation by at least an order of magnitude, indicating a substantial improvement in both potency and selectivity when compared to the unmodified parent bile acid. Molecular docking analysis supported the stronger binding of the synthetic analogue in the enzyme active site. Treatment with SPP-002 also hampered the migration, adhesion, and invasion of MDA-MB-231 cells in vitro by suppressing the expression of signaling proteins involved in the cancer metastasis-associated integrin/FAK/paxillin pathway. In totality, these findings offer not only a novel structural scaffold but also valuable insights for the future development of more potent and selective ST inhibitors with potential therapeutic effects against tumor cancer metastasis.

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.

Sialylated glycoproteins and sialyltransferases in digestive cancers: Mechanisms, diagnostic biomarkers, and therapeutic targets.

Sialic acid (SA), as the ultimate epitope of polysaccharides, can act as a cap at the end of polysaccharide chains to prevent their overextension. Sialylation is the enzymatic process of transferring SA residues onto polysaccharides and is catalyzed by a group of enzymes known as sialyltransferases (SiaTs). It is noteworthy that the sialylation level of glycoproteins is significantly altered when digestive cancer occurs. And this alteration exhibits a close correlation with the progression of these cancers. In this review, from the perspective of altered SiaTs expression levels and changed glycoprotein sialylation patterns, we summarize the pathogenesis of gastric cancer (GC), colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), and hepatocellular carcinoma (HCC). Furthermore, we propose potential early diagnostic biomarkers and prognostic indicators for different digestive cancers. Finally, we summarize the therapeutic value of sialylation in digestive system cancers.

Sialic acid in the regulation of blood cell production, differentiation and turnover.

Sialic acid is a unique sugar moiety that resides in the distal and most accessible position of the glycans on mammalian cell surface and extracellular glycoproteins and glycolipids. The potential for sialic acid to obscure underlying structures has long been postulated, but the means by which such structural changes directly affect biological processes continues to be elucidated. Here, we appraise the growing body of literature detailing the importance of sialic acid for the generation, differentiation, function and death of haematopoietic cells. We conclude that sialylation is a critical post-translational modification utilized in haematopoiesis to meet the dynamic needs of the organism by enforcing rapid changes in availability of lineage-specific cell types. Though long thought to be generated only cell-autonomously within the intracellular ER-Golgi secretory apparatus, emerging data also demonstrate previously unexpected diversity in the mechanisms of sialylation. Emphasis is afforded to the mechanism of extrinsic sialylation, whereby extracellular enzymes remodel cell surface and extracellular glycans, supported by charged sugar donor molecules from activated platelets.

CCCTC-binding factor: the specific transcription factor of ß-galactoside α-2,6-sialyltransferase 1 that upregulates the sialylation of anti-citrullinated protein antibodies in rheumatoid arthritis.

OBJECTIVE: Sialylation of the crystallizable fragment (Fc) of ACPAs, which is catalysed by ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) could attenuate inflammation of RA. In this study, we screened the transcription factor of ST6GAL1 and elucidated the mechanism of transcriptionally upregulating sialylation of ACPAs in B cells to explore its role in the progression of RA. METHODS: Transcription factors interacting with the P2 promoter of ST6GAL1 were screened by DNA pull-down and liquid chromatography with tandem mass spectrometry (LC-MS/MS), and verified by chromatin immunoprecipitation (ChIP), dual luciferase reporter assay and electrophoretic mobility shift assay (EMSA). The function of the CCCTC-binding factor (CTCF) on the expression of ST6GAL1 and the inflammatory effect of ACPAs were verified by knocking down and overexpressing CTCF in B cells. The CIA model was constructed from B cell-specific CTCF knockout mice to explore the effect of CTCF on arthritis progression. RESULTS: We observed that the levels of ST6GAL1 and ACPAs sialylation decreased in the serum of RA patients and were negatively correlated with DAS28 scores. Subsequently, CTCF was screened and verified as the transcription factor interacting with the P2 promoter of ST6GAL1, which enhances the sialylation of ACPAs, thus weakening the inflammatory activity of ACPAs. Furthermore, the above results were also verified in the CIA model constructed from B cell-specific CTCF knockout mice. CONCLUSION: CCCTC-binding factor is the specific transcription factor of ß-galactoside α-2,6-sialyltransferase 1 in B cells that upregulates the sialylation of ACPAs in RA and attenuates the disease progression.

Pax3 induces target-specific reinnervation through axon collateral expression of PSA-NCAM.

Damaged or dysfunctional neural circuits can be replaced after a lesion by axon sprouting and collateral growth from undamaged neurons. Unfortunately, these new connections are often disorganized and rarely produce clinical improvement. Here we investigate how to promote post-lesion axonal collateral growth, while retaining correct cellular targeting. In the mouse olivocerebellar path, brain-derived neurotrophic factor (BDNF) induces correctly-targeted post-lesion cerebellar reinnervation by remaining intact inferior olivary axons (climbing fibers). In this study we identified cellular processes through which BDNF induces this repair. BDNF injection into the denervated cerebellum upregulates the transcription factor Pax3 in inferior olivary neurons and induces rapid climbing fiber sprouting. Pax3 in turn increases polysialic acid-neural cell adhesion molecule (PSA-NCAM) in the sprouting climbing fiber path, facilitating collateral outgrowth and pathfinding to reinnervate the correct targets, cerebellar Purkinje cells. BDNF-induced reinnervation can be reproduced by olivary Pax3 overexpression, and abolished by olivary Pax3 knockdown, suggesting that Pax3 promotes axon growth and guidance through upregulating PSA-NCAM, probably on the axon's growth cone. These data indicate that restricting growth-promotion to potential reinnervating afferent neurons, as opposed to stimulating the whole circuit or the injury site, allows axon growth and appropriate guidance, thus accurately rebuilding a neural circuit.

A sialyltransferases-related gene signature serves as a potential predictor of prognosis and therapeutic response for bladder cancer.

BACKGROUND: Aberrant glycosylation, catalyzed by the specific glycosyltransferase, is one of the dominant features of cancers. Among the glycosyltransferase subfamilies, sialyltransferases (SiaTs) are an essential part which has close linkages with tumor-associated events, such as tumor growth, metastasis and angiogenesis. Considering the relationship between SiaTs and cancer, the current study attempted to establish an effective prognostic model with SiaTs-related genes (SRGs) to predict patients' outcome and therapeutic responsiveness of bladder cancer. METHODS: RNA-seq data, clinical information and genomic mutation data were downloaded (TCGA-BLCA and GSE13507 datasets). The comprehensive landscape of the 20 SiaTs was analyzed, and the differentially expressed SiaTs-related genes were screened with "DESeq2" R package. ConsensusClusterPlus was applied for clustering, following with survival analysis with Kaplan-Meier curve. The overall survival related SRGs were determined with univariate Cox proportional hazards regression analysis, and the least absolute shrinkage and selection operator (LASSO) regression analysis was performed to generate a SRGs-related prognostic model. The predictive value was estimated with Kaplan-Meier plot and the receiver operating characteristic (ROC) curve, which was further validated with the constructed nomogram and decision curve. RESULTS: In bladder cancer tissues, 17 out of the 20 SiaTs were differentially expressed with CNV changes and somatic mutations. Two SiaTs_Clusters were determined based on the expression of the 20 SiaTs, and two gene_Clusters were identified based on the expression of differentially expressed genes between SiaTs_Clusters. The SRGs-related prognostic model was generated with 7 key genes (CD109, TEAD4, FN1, TM4SF1, CDCA7L, ATOH8 and GZMA), and the accuracy for outcome prediction was validated with ROC curve and a constructed nomogram. The SRGs-related prognostic signature could separate patients into high- and low-risk group, where the high-risk group showed poorer outcome, more abundant immune infiltration, and higher expression of immune checkpoint genes. In addition, the risk score derived from the SRGs-related prognostic model could be utilized as a predictor to evaluate the responsiveness of patients to the medical therapies. CONCLUSIONS: The SRGs-related prognostic signature could potentially aid in the prediction of the survival outcome and therapy response for patients with bladder cancer, contributing to the development of personalized treatment and appropriate medical decisions.

Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase.

BACKGROUND: In whole-cell bio-catalysis, the biosystems engineering paradigm shifts from the global reconfiguration of cellular metabolism as in fermentation to a more focused, and more easily modularized, optimization of comparably short cascade reactions. Human milk oligosaccharides (HMO) constitute an important field for the synthetic application of cascade bio-catalysis in resting or non-living cells. Here, we analyzed the central catalytic module for synthesis of HMO-type sialo-oligosaccharides, comprised of CMP-sialic acid synthetase (CSS) and sialyltransferase (SiaT), with the specific aim of coordinated enzyme co-expression in E. coli for reaction flux optimization in whole cell conversions producing 3'-sialyllactose (3SL). RESULTS: Difference in enzyme specific activity (CSS from Neisseria meningitidis: 36 U/mg; α2,3-SiaT from Pasteurella dagmatis: 5.7 U/mg) was compensated by differential protein co-expression from tailored plasmid constructs, giving balance between the individual activities at a high level of both (α2,3-SiaT: 9.4 × 102 U/g cell dry mass; CSS: 3.4 × 102 U/g cell dry mass). Finally, plasmid selection was guided by kinetic modeling of the coupled CSS-SiaT reactions in combination with comprehensive analytical tracking of the multistep conversion (lactose, N-acetyl neuraminic acid (Neu5Ac), cytidine 5'-triphosphate; each up to 100 mM). The half-life of SiaT in permeabilized cells (≤ 4 h) determined the efficiency of 3SL production at 37 °C. Reaction at 25 °C gave 3SL (40 ± 4 g/L) in ∼ 70% yield within 3 h, reaching a cell dry mass-specific productivity of ∼ 3 g/(g h) and avoiding intermediary CMP-Neu5Ac accumulation. CONCLUSIONS: Collectively, balanced co-expression of CSS and SiaT yields an efficient (high-flux) sialylation module to support flexible development of E. coli whole-cell catalysts for sialo-oligosaccharide production.