Sialylation of EGFR by ST6GAL1 induces receptor activation and modulates trafficking dynamics.

Aberrant glycosylation is a hallmark of a cancer cell. One prevalent alteration is an enrichment in α2,6-linked sialylation of N-glycosylated proteins, a modification directed by the ST6GAL1 sialyltransferase. ST6GAL1 is upregulated in many malignancies including ovarian cancer. Prior studies have shown that the addition of α2,6 sialic acid to the epidermal growth factor receptor (EGFR) activates this receptor, although the mechanism was largely unknown. To investigate the role of ST6GAL1 in EGFR activation, ST6GAL1 was overexpressed in the OV4 ovarian cancer line, which lacks endogenous ST6GAL1, or knocked-down in the OVCAR-3 and OVCAR-5 ovarian cancer lines, which have robust ST6GAL1 expression. Cells with high expression of ST6GAL1 displayed increased activation of EGFR and its downstream signaling targets, AKT and NFκB. Using biochemical and microscopy approaches, including total internal reflection fluorescence microscopy, we determined that the α2,6 sialylation of EGFR promoted its dimerization and higher order oligomerization. Additionally, ST6GAL1 activity was found to modulate EGFR trafficking dynamics following EGF-induced receptor activation. Specifically, EGFR sialylation enhanced receptor recycling to the cell surface following activation while simultaneously inhibiting lysosomal degradation. 3D widefield deconvolution microscopy confirmed that in cells with high ST6GAL1 expression, EGFR exhibited greater colocalization with Rab11 recycling endosomes and reduced colocalization with LAMP1-positive lysosomes. Collectively, our findings highlight a novel mechanism by which α2,6 sialylation promotes EGFR signaling by facilitating receptor oligomerization and recycling.

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.

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.

A Rapid and Sensitive MicroPlate Assay (MPSA) Using an Alkyne-Modified CMP-Sialic Acid Donor to Evaluate Human Sialyltransferase Specificity.

Human sialyltransferases primarily utilize CMP-Sias, especially transferring Neu5Ac from CMP-Neu5Ac to various acceptors. Advances in chemical biology have led to the synthesis of novel CMP-Sia donors suitable for bioorthogonal reactions in cell-based assays. However, the compatibility of these donors with all human enzymes remains uncertain. We synthesized a non-natural CMP-Sia donor with an alkyne modification on the N-acyl group of Neu5Ac, which was effectively used by human ST6Gal I and ST3Gal I. A sensitive MicroPlate Sialyltransferase Assay (MPSA) was developed and expanded to a panel of 13 human STs acting on glycoproteins. All assayed enzymes tolerated CMP-SiaNAl, allowing for the determination of kinetic parameters and turnover numbers. This study enhances the biochemical characterization of human sialyltransferases and opens new avenues for developing sialyltransferase inhibitors.

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.

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.

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.

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.

Probing the binding specificities of human Siglecs by cell-based glycan arrays.

Siglecs are a family of sialic acid-binding receptors expressed by cells of the immune system and a few other cell types capable of modulating immune cell functions upon recognition of sialoglycan ligands. While human Siglecs primarily bind to sialic acid residues on diverse types of glycoproteins and glycolipids that constitute the sialome, their fine binding specificities for elaborated complex glycan structures and the contribution of the glycoconjugate and protein context for recognition of sialoglycans at the cell surface are not fully elucidated. Here, we generated a library of isogenic human HEK293 cells with combinatorial loss/gain of individual sialyltransferase genes and the introduction of sulfotransferases for display of the human sialome and to dissect Siglec interactions in the natural context of glycoconjugates at the cell surface. We found that Siglec-4/7/15 all have distinct binding preferences for sialylated GalNAc-type O-glycans but exhibit selectivity for patterns of O-glycans as presented on distinct protein sequences. We discovered that the sulfotransferase CHST1 drives sialoglycan binding of Siglec-3/8/7/15 and that sulfation can impact the preferences for binding to O-glycan patterns. In particular, the branched Neu5Acα2-3(6-O-sulfo)Galß1-4GlcNAc (6'-Su-SLacNAc) epitope was discovered as the binding epitope for Siglec-3 (CD33) implicated in late-onset Alzheimer's disease. The cell-based display of the human sialome provides a versatile discovery platform that enables dissection of the genetic and biosynthetic basis for the Siglec glycan interactome and other sialic acid-binding proteins.

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.

Sialyltransferase ST6GAL-1 mediates resistance to chemoradiation in rectal cancer.

Locally advanced rectal cancer is typically treated with chemoradiotherapy followed by surgery. Most patients do not display a complete response to chemoradiotherapy, but resistance mechanisms are poorly understood. ST6GAL-1 is a sialyltransferase that adds the negatively charged sugar, sialic acid (Sia), to cell surface proteins in the Golgi, altering their function. We therefore hypothesized that ST6GAL-1 could mediate resistance to chemoradiation in rectal cancer by inhibiting apoptosis. Patient-derived xenograft and organoid models of rectal cancer and rectal cancer cell lines were assessed for ST6GAL-1 protein with and without chemoradiation treatment. ST6GAL-1 mRNA was assessed in untreated human rectal adenocarcinoma by PCR assays. Samples were further assessed by Western blotting, Caspase-Glo apoptosis assays, and colony formation assays. The presence of functional ST6GAL-1 was assessed via flow cytometry using the Sambucus nigra lectin, which specifically binds cell surface α2,6-linked Sia, and via lectin precipitation. In patient-derived xenograft models of rectal cancer, we found that ST6GAL-1 protein was increased after chemoradiation in a subset of samples. Rectal cancer cell lines demonstrated increased ST6GAL-1 protein and cell surface Sia after chemoradiation. ST6GAL-1 was also increased in rectal cancer organoids after treatment. ST6GAL-1 knockdown in rectal cancer cell lines resulted in increased apoptosis and decreased survival after treatment. We concluded that ST6GAL-1 promotes resistance to chemoradiotherapy by inhibiting apoptosis in rectal cancer cell lines. More research will be needed to further elucidate the importance and mechanism of ST6GAL-1-mediated resistance.

ST6Gal-I-mediated sialylation of the epidermal growth factor receptor modulates cell mechanics and enhances invasion.

Heterogeneity within the glycocalyx influences cell adhesion mechanics and signaling. However, the role of specific glycosylation subtypes in influencing cell mechanics via alterations of receptor function remains unexplored. It has been shown that the addition of sialic acid to terminal glycans impacts growth, development, and cancer progression. In addition, the sialyltransferase ST6Gal-I promotes epidermal growth factor receptor (EGFR) activity, and we have shown EGFR is an 'allosteric mechano-organizer' of integrin tension. Here, we investigated the impact of ST6Gal-I on cell mechanics. Using DNA-based tension gauge tether probes of variable thresholds, we found that high ST6Gal-I activity promotes increased integrin forces and spreading in Cos-7 and OVCAR3, OVCAR5, and OV4 cancer cells. Further, employing inhibitors and function-blocking antibodies against ß1, ß3, and ß5 integrins and ST6Gal-I targets EGFR, tumor necrosis factor receptor, and Fas cell surface death receptor, we validated that the observed phenotypes are EGFR-specific. We found that while tension, contractility, and adhesion are extracellular-signal-regulated kinase pathway-dependent, spreading, proliferation, and invasion are phosphoinositide 3-kinase-Akt serine/threonine kinase dependent. Using total internal reflection fluorescence microscopy and flow cytometry, we also show that high ST6Gal-I activity leads to sustained EGFR membrane retention, making it a key regulator of cell mechanics. Our findings suggest a novel sialylation-dependent mechanism orchestrating cellular mechanics and enhancing cell motility via EGFR signaling.

Human brain sialoglycan ligand for CD33, a microglial inhibitory Siglec implicated in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by accumulation of misfolded proteins. Genetic studies implicate microglia, brain-resident phagocytic immune cells, in AD pathogenesis. As positive effectors, microglia clear toxic proteins, whereas as negative effectors, they release proinflammatory mediators. An imbalance of these functions contributes to AD progression. Polymorphisms of human CD33, an inhibitory microglial receptor, are linked to AD susceptibility; higher CD33 expression correlates with increased AD risk. CD33, also called Siglec-3, is a member of the sialic acid-binding immunoglobulin-type lectin (Siglec) family of immune regulatory receptors. Siglec-mediated inhibition is initiated by binding to complementary sialoglycan ligands in the tissue environment. Here, we identify a single sialoglycoprotein in human cerebral cortex that binds CD33 as well as Siglec-8, the most abundant Siglec on human microglia. The ligand, which we term receptor protein tyrosine phosphatase zeta (RPTPζ)S3L, is composed of sialylated keratan sulfate chains carried on a minor isoform/glycoform of RPTPζ (phosphacan) and is found in the extracellular milieu of the human brain parenchyma. Brains from human AD donors had twofold higher levels of RPTPζS3L than age-matched control donors, raising the possibility that RPTPζS3L overexpression limits misfolded protein clearance contributing to AD pathology. Mice express the same structure, a sialylated keratan sulfate RPTPζ isoform, that binds mouse Siglec-F and crossreacts with human CD33 and Siglec-8. Brains from mice engineered to lack RPTPζ, the sialyltransferase St3gal4, or the keratan sulfate sulfotransferase Chst1 lacked Siglec binding, establishing the ligand structure. The unique CD33 and Siglec-8 ligand, RPTPζS3L, may contribute to AD progression.

Forced expression of α2,3-sialyltransferase IV rescues impaired heart development in α2,6-sialyltransferase I-deficient medaka.

Sialic acids (Sias) are often linked to galactose (Gal) residues by α2,6- and α2,3-linkages in glycans of glycoproteins. Sias are indispensable for vertebrate development, because organisms deficient in some enzymes in the Sia synthetic pathway are lethal during the development. However, it remains unknown if the difference of Siaα2,6Gal or α2,3Gal linkage has a critical meaning. To find a clue to understand significance of the linkage difference at the organism level, medaka was used as a vertebrate model. In embryos, Siaα2,6Gal epitopes recognized by Sambucus nigra lectin (SNA) and Siaα2,3Gal epitopes recognized by Maackia amurensis lectin (MAA) were enriched in the blastodisc and the yolk sphere, respectively. When these lectins were injected in the perivitelline space, SNA, but not MAA, impaired embryo body formation at 1 day post-fertilization (dpf). Most Siaα2,6Gal epitopes occurred on N-glycans owing to their sensitivity to peptide:N-glycanase. Of knockout-medaka (KO) for either of two ß-galactoside:α2,6-sialyltransferase genes, ST6Gal I and ST6Gal II, only ST6Gal I-KO showed severe cardiac abnormalities at 7-16 dpf, leading to lethality at 14-18 dpf. Interestingly, however, these cardiac abnormalities of ST6Gal I-KO were rescued not only by forced expression of ST6Gal I, but also by that of ST6Gal II and the ß-galactoside:α2,3-sialyltransferase IV gene (ST3Gal IV). Taken together, the Siaα2,6Gal linkage synthesized by ST6Gal I are critical in heart development; however, it can be replaced by the linkages synthesized by ST6Gal II and ST3Gal IV. These data suggest that sialylation itself is more important than its particular linkage for the heart development.

Glycomic Analysis Reveals That Sialyltransferase Inhibition Is Involved in the Antiviral Effects of Arbidol.

Due to the high mutation rate of influenza virus and the rapid increase of drug resistance, it is imperative to discover host-targeting antiviral agents with broad-spectrum antiviral activity. Considering the discrepancy between the urgent demand of antiviral drugs during an influenza pandemic and the long-term process of drug discovery and development, it is feasible to explore host-based antiviral agents and strategies from antiviral drugs on the market. In the current study, the antiviral mechanism of arbidol (ARB), a broad-spectrum antiviral drug with potent activity at early stages of viral replication, was investigated from the aspect of hemagglutinin (HA) receptors of host cells. N-glycans that act as the potential binding receptors of HA on 16-human bronchial epithelial (16-HBE) cells were comprehensively profiled for the first time by using an in-depth glycomic approach based on TiO2-PGC chip-Q-TOF MS. Their relative levels upon the treatment of ARB and virus were carefully examined by employing an ultra-high sensitive qualitative method based on Chip LC-QQQ MS, showing that ARB treatment led to significant and extensive decrease of sialic acid (SA)-linked N-glycans (SA receptors), and thereby impaired the virus utilization on SA receptors for rolling and entry. The SA-decreasing effect of ARB was demonstrated to result from its inhibitory effect on sialyltransferases (ST), ST3GAL4 and ST6GAL1 of 16-HBE cells. Silence of STs, natural ST inhibitors, as well as sialidase treatment of 16-HBE cells, resulted in similar potent antiviral activity, whereas ST-inducing agent led to the diminished antiviral effect of ARB. These observations collectively suggesting the involvement of ST inhibition in the antiviral effect of ARB. IMPORTANCE This study revealed, for the first time, that ST inhibition and the resulted destruction of SA receptors of host cells may be an underlying mechanism for the antiviral activity of ARB. ST inhibition has been proposed as a novel host-targeting antiviral approach recently and several compounds are currently under exploration. ARB is the first antiviral drug on the market that was found to possess ST inhibiting function. This will provide crucial evidence for the clinical usages of ARB, such as in combination with neuraminidase (NA) inhibitors to exert optimized antiviral effect, etc. More importantly, as an agent that can inhibit the expression of STs, ARB can serve as a novel lead compound for the discovery and development of host-targeting antiviral drugs.

The vertebrate sialylation machinery: structure-function and molecular evolution of GT-29 sialyltransferases.

Every eukaryotic cell is covered with a thick layer of complex carbohydrates with essential roles in their social life. In Deuterostoma, sialic acids present at the outermost positions of glycans of glycoconjugates are known to be key players in cellular interactions including host-pathogen interactions. Their negative charge and hydrophilic properties enable their roles in various normal and pathological states and their expression is altered in many diseases including cancers. Sialylation of glycoproteins and glycolipids is orchestrated by the regulated expression of twenty sialyltransferases in human tissues with distinct enzymatic characteristics and preferences for substrates and linkages formed. However, still very little is known on the functional organization of sialyltransferases in the Golgi apparatus and how the sialylation machinery is finely regulated to provide the ad hoc sialome to the cell. This review summarizes current knowledge on sialyltransferases, their structure-function relationships, molecular evolution, and their implications in human biology.

Vibrio-binding gangliosides in fish intestinal tracts.

It has been clarified that pathogens bind to glycosphingolipid (GSL) receptors in mammals, but there have been very few reports on pathogen-binding GSLs in fish. Vibrios are facultative anaerobic bacteria ubiquitous in marine and brackish environments. They are members of the normal intestinal microflora of healthy fish, but some species can cause a disease called vibriosis in fish and shellfish when the hosts are physiologically or immunologically weakened. The adherence of vibrios to host intestinal tracts is a significant event not only for survival and growth but also in terms of pathogenicity. We show in this mini-review that sialic acid-containing GSLs (gangliosides), GM4 and GM3, are receptors to which vibrios adhere to epithelial cells in the intestinal tract of fish. We also describe the enzymes responsible for synthesizing these Vibrio-binding gangliosides in fish.

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.

Rab11-mediated post-Golgi transport of the sialyltransferase ST3GAL4 suggests a new mechanism for regulating glycosylation.

Glycosylation, the most common posttranslational modification of proteins, is a stepwise process that relies on tight regulation of subcellular glycosyltransferase location to control the addition of each monosaccharide. Glycosyltransferases primarily reside and function in the endoplasmic reticulum (ER) and the Golgi apparatus; whether and how they traffic beyond the Golgi, how this trafficking is controlled, and how it impacts glycosylation remain unclear. Our previous work identified a connection between N-glycosylation and Rab11, a key player in the post-Golgi transport that connects recycling endosomes and other compartments. To learn more about the specific role of Rab11, we knocked down Rab11 in HeLa cells. Our findings indicate that Rab11 knockdown results in a dramatic enhancement in the sialylation of N-glycans. Structural analyses of glycans using lectins and LC-MS revealed that α2,3-sialylation is selectively enhanced, suggesting that an α2,3-sialyltransferase that catalyzes the sialyation of glycoproteins is activated or upregulated as the result of Rab11 knockdown. ST3GAL4 is the major α2,3-sialyltransferase that acts on N-glycans; we demonstrated that the localization of ST3GAL4, but not the levels of its mRNA, protein, or donor substrate, was altered by Rab11 depletion. In knockdown cells, ST3GAL4 is densely distributed in the trans-Golgi network, compared with the wider distribution in the Golgi and in other peripheral puncta in control cells, whereas the α2,6-sialyltransferase ST6GAL1 is predominantly localized to the Golgi regardless of Rab11 knockdown. This indicates that Rab11 may negatively regulate α2,3-sialylation by transporting ST3GAL4 to post-Golgi compartments (PGCs), which is a novel mechanism of glycosyltransferase regulation.

Extracellular ST6GAL1 regulates monocyte-macrophage development and survival.

Interaction of immune cells with the systemic environment is necessary for the coordinated development and execution of immune responses. Monocyte-macrophage lineage cells reside at the junction of innate and adaptive immunity. Previously we reported that the sialyltransferase ST6GAL1 in the extracellular milieu modulates B cell development and IgG production, granulocyte production, and attenuates acute airway inflammation to bacterial challenge in mouse models. Here, we report that extracellular ST6GAL1 also elicits profound responses in monocyte-macrophage lineage cells. We show that recombinant ST6GAL1 adheres to subsets of thioglycolate-elicited inflammatory cells in the mouse peritoneum and to cultured human monocyte THP-1 cells. Exposure of the inflammatory cells to recombinant ST6GAL1 elicited wholesale changes in the gene expression profile of primary mouse myeloid cells; most notable was the striking up-regulation of monocyte-macrophage and monocyte-derived dendritic cell development pathway signature genes and transcription factors PU.1, NFκB and their target genes, driving increased monocyte-macrophage population and survival ex vivo. In the cultured human monocyte cells, the essential cell surface receptor of the monocyte-macrophage lineage, the M-CSF receptor (M-CSF-R, Csfr1) was a target of extracellular ST6GAL1 catalytic activity. Extracellular ST6GAL1 activated the M-CSF-R and initiated intracellular signaling events, namely, the nuclear translocation of NFκB subunit p65, and phosphorylation of ERK 1/2 and AKT. The findings implicate extracellular ST6GAL1 in monocyte development by a mechanism initiated at the cell surface and support an emerging paradigm of an extracellular glycan-modifying enzyme as a central regulator coordinating immune hematopoietic cell development and function.