Sialyl Lewis X decorated integrin α3 on small extracellular vesicles promotes metastasis of bladder cancer via enhancing vascular permeability.

The permeability of blood vessels plays a crucial role in the spread of cancer cells, facilitating their metastasis at distant sites. Small extracellular vesicles (sEVs) are known to contribute to the metastasis of various cancers by crossing the blood vessel wall. However, the role of abnormal glycoconjugates on sEVs in tumor blood vessels remains unclear. Our study found elevated levels of fucosyltransferase VII (FUT7) and its product sialyl Lewis X (sLeX) in muscle-invasive bladder cancer (BLCA), with high levels of sLeX promoting the growth and invasion of BLCA cells. Further investigation revealed that sLeX was enriched in sEVs derived from BLCA. sLeX-decorated sEVs increased blood vessel permeability by disrupting the tight junctions of human umbilical vein endothelial cells (HUVECs). Using the glycoproteomics approach, we identified integrin α3 (ITGA3) as a sLeX-bearing glycoprotein in BLCA cells and their sEVs. Mechanically, sLeX modification stabilized ITGA3 by preventing its degradation in lysosomes. sEVs carrying sLeX-modified ITGA3 can be effectively internalized by HUVECs, leading to a decrease in the expression of tight junction protein. Conversely, silencing ITGA3 in sLeX-decorated sEVs restored tight junction proteins and reduced blood vessel permeability by inhibiting the MAPK pathway. Moreover, sLeX-modification of ITGA3 at Asn 265 in HUVECs promoted occludin dephosphorylation at Ser/Thr residues, followed by inducing its importin α1-mediated nuclear translocation, which resulted in the disruption of tight junctions. Our findings suggest a potential strategy for disrupting the formation of a metastatic microenvironment and preventing the spread of malignant bladder cancer.

Sialyl LewisX glycomimetics bearing an extended anionic chain targeting E- and P- selectin binding sites.

Neutrophil binding to vascular P- and E-selectin is the rate-limiting step in the recruitment of immune cells to sites of inflammation. Many diseases, including sickle cell anemia, post-myocardial infarction reperfusion injury, and acute respiratory distress syndrome are characterized by dysregulated inflammation. We have recently reported sialyl Lewisx analogues as potent antagonists of P- and E-selectin and demonstrated their in vivo immunosuppressive activity. A key component of these molecules is a tartrate diester that serves as an acyclic tether to orient the fucoside and the galactoside moiety in the required gauche conformation for optimal binding. The next stage of our study involved attaching an extended carbon chain onto one of the esters. This chain could be utilized to tether other pharmacophores, lipids, and contrast agents in the context of enhancing pharmacological applications through the sialyl Lewisx / receptor-mediated mechanism. Herein, we report our preliminary studies to generate a small library of tartrate based sialyl Lewisx analogues bearing extended carbon chains. Anionic charged chemical entities are attached to take advantage of proximal charged amino acids in the carbohydrate recognition domain of the selectin receptors. Starting with a common azido intermediate, synthesized using copper-catalyzed Huisgen 1,3-dipolar cycloadditions, these molecules demonstrate E- and P-selectin binding properties.

The Sialyl Lewis X Glycan Receptor Facilitates Infection of Subtype H7 Avian Influenza A Viruses.

Subtype H7 avian influenza A viruses (IAVs) are enzootic in wild aquatic birds and have caused sporadic spillovers into domestic poultry and humans. Here, we determined the distribution of fucosylated α2,3 sialoglycan (i.e., sialyl Lewis X [SLeX]) in chickens and five common dabbling duck species and the association between SLeX and cell/tissue/host tropisms of H7 IAVs. Receptor binding analyses showed that H7 IAVs bind to both α2,3-linked (SA2,3Gal) and α2,6-linked sialic acids (SA2,6Gal), but with a higher preference for SLeX; H7 IAVs replicated more efficiently in SLeX-overexpressed than SLeX-deficient MDCK cells. While chickens and all tested dabbling ducks expressed abundant SA2,3Gal and SA2,6Gal, SLeX was detected in both respiratory and gastrointestinal tissues of chickens and mallard ducks and in only the respiratory tissues of gadwall, green-wing teal, and northern shoveler but not in wood ducks. Viral-tissue binding assays showed that H7 IAVs bind to chicken colon crypt cells that express SLeX but fewer bind to mallard colon crypt cells, which do not express SLeX; H7 IAVs bind efficiently to epithelial cells of all tissues expressing SA2,3Gal. High viral replication was identified in both chickens and mallards infected with an H7 virus, regardless of SLeX expression, and viruses were detected in all cells to the same degree as viruses detected in the viral-tissue binding assays. In summary, this study suggests that SLeX facilitates infection of H7 viruses, but other types of SA2,3Gal glycan receptors shape the tissue/host tropisms of H7 IAVs. IMPORTANCE In addition to causing outbreaks in domestic poultry, subtype H7 IAVs can cause sporadic spillover infections in lower mammals and humans. In this study, we showed that SLeX expression varies among wild dabbling ducks. Although it facilitated virus binding and affected infection of H7 IAV in cells, SLeX expression is not the only determinant of viral replication at either the tissue or host level. This study suggested that access to heterologous SA2,3Gal glycan receptors, including fucosylated α2,3-linked sialoglycans, shape tissue and host tropism of H7 IAVs in aquatic wild birds.

An Introduction to the Relationship Between Lewis x and Malignancy Mainly Related to Breast Cancer and Head Neck Squamous Cell Carcinoma (HNSCC).

Lewis x functions as an adhesion molecule in glycolipids and glycoproteins since it mediates homophilic and heterophilic attachment of normal and tumoral cells. During malignancy, altered glycosylation is a frequent event; accumulating data support the expression of Lewis x in tumors although controversial results have been described including its relationship with patient survival. This report has been developed as an introduction to the relationship between Lewis x expression and breast cancer and head and neck squamous cell carcinoma (HNSCC). Results obtained in our laboratory are presented in the context of the literature.

Complementary Role of GlcNAc6ST2 and GlcNAc6ST3 in Synthesis of CL40-Reactive Sialylated and Sulfated Glycans in the Mouse Pleural Mesothelium.

Sialyl 6-sulfo Lewis X (6-sulfo sLeX) and its derivative sialyl 6-sulfo N-acetyllactosamine (LacNAc) are sialylated and sulfated glycans of sialomucins found in the high endothelial venules (HEVs) of secondary lymphoid organs. A component of 6-sulfo sLeX present in the core 1-extended O-linked glycans detected by the MECA-79 antibody was previously shown to exist in the lymphoid aggregate vasculature and bronchial mucosa of allergic and asthmatic lungs. The components of 6-sulfo sLeX in pulmonary tissues under physiological conditions remain to be analyzed. The CL40 antibody recognizes 6-sulfo sLeX and sialyl 6-sulfo LacNAc in O-linked and N-linked glycans, with absolute requirements for both GlcNAc-6-sulfation and sialylation. Immunostaining of normal mouse lungs with CL40 was performed and analyzed. The contribution of GlcNAc-6-O-sulfotransferases (GlcNAc6STs) to the synthesis of the CL40 epitope in the lungs was also elucidated. Here, we show that the expression of the CL40 epitope was specifically detected in the mesothelin-positive mesothelium of the pulmonary pleura. Moreover, GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5), but not GlcNAc6ST1 (encoded by Chst2) or GlcNAc6ST4 (encoded by Chst7), are required for the synthesis of CL40-positive glycans in the lung mesothelium. Furthermore, neither GlcNAc6ST2 nor GlcNAc6ST3 is sufficient for in vivo expression of the CL40 epitope in the lung mesothelium, as demonstrated by GlcNAc6ST1/3/4 triple-knock-out and GlcNAc6ST1/2/4 triple-knock-out mice. These results indicate that CL40-positive sialylated and sulfated glycans are abundant in the pleural mesothelium and are synthesized complementarily by GlcNAc6ST2 and GlcNAc6ST3, under physiological conditions in mice.

Major differences in glycosylation and fucosyltransferase expression in low-grade versus high-grade bladder cancer cell lines.

Bladder cancer is the ninth most frequently diagnosed cancer worldwide, and there is a need to develop new biomarkers for staging and prognosis of this disease. Here we report that cell lines derived from low-grade and high-grade bladder cancers exhibit major differences in expression of glycans in surface glycoproteins. We analyzed protein glycosylation in three low-grade bladder cancer cell lines RT4 (grade-1-2), 5637 (grade-2), and SW780 (grade-1), and three high-grade bladder cancer cell lines J82COT (grade-3), T24 (grade-3) and TCCSUP (grade-4), with primary bladder epithelial cells, A/T/N, serving as a normal bladder cell control. Using a variety of approaches including flow cytometry, immunofluorescence, glycomics and gene expression analysis, we observed that the low-grade bladder cancer cell lines RT4, 5637 and SW780 express high levels of the fucosylated Lewis-X antigen (Lex, CD15) (Galß1-4(Fucα1-3)GlcNAcß1-R), while normal bladder epithelial A/T/N cells lack Lex expression. T24 and TCCSUP cells also lack Lex, whereas J82COT cells express low levels of Lex. Glycomics analyses revealed other major differences in fucosylation and sialylation of N-glycans between these cell types. O-glycans are highly differentiated, as RT4 cells synthesize core 2-based O-glycans that are lacking in the T24 cells. These differences in glycan expression correlated with differences in RNA expression levels of their cognate glycosyltransferases, including α1-3/4-fucosyltransferase genes. These major differences in glycan structures and gene expression profiles between low- and high-grade bladder cancer cells suggest that glycans and glycosyltransferases are candidate biomarkers for grading bladder cancers.

Lactobacillus rhamnosus JB3 inhibits Helicobacter pylori infection through multiple molecular actions.

BACKGROUND: Eradication of Helicobacter pylori infection is the most direct and effective way for preventing gastric cancer. Lactic acid bacteria are considered as alternative therapeutic agents against H. pylori infection. METHODS: Effects of Lactobacillus rhamnosus JB3 (LR-JB3) on the virulence gene expression of H. pylori and infection-induced cellular responses of AGS cells were investigated by co-cultivating infected AGS cells with different multiplicity of infections (MOIs) of LR-JB3. RESULTS: LR-JB3, specifically at a MOI of 25, suppressed the association ability of H. pylori and its induced IL-8 levels, as well as the mRNA levels of vacA, sabA, and fucT of H. pylori, infection-induced Lewis (Le)x antigen and Toll-like receptor 4 (TLR4) expressions in AGS cells. However, the apoptosis mediated by infection was inhibited by LR-JB3 in a dose-dependent manner. In addition, autoinducer (AI)-2 was observed to have increased the association ability and fucT expression of H. pylori, and Lex antigen and TLR4 expression of AGS cells. Interestingly, an unknown bioactive cue was hypothesized to have been secreted from LR-JB3 at a MOI of 25 to act as an antagonist of AI-2. CONCLUSIONS: LR-JB3 possesses various means to interfere with H. pylori pathogenesis and infection-induced cellular responses of AGS cells to fight against infection.

Therapeutic Effects of an Anti-sialyl Lewis X Antibody in a Murine Model of Allergic Asthma.

Asthma is an allergic disease that causes severe infiltration of leukocytes into the lungs. Leukocyte infiltration is mediated by the binding of sialyl Lewis X (sLex) glycans present on the leukocytes to E-and P-selectins present on the endothelial cells at the sites of inflammation. Here, we found that mouse eosinophils express sLex glycans, and their infiltration into the lungs and proliferation in the bone marrow were significantly suppressed by an anti-sLex monoclonal antibody (mAb) F2 in a murine model of ovalbumin-induced asthma. The percentage of eosinophils in the bronchoalveolar lavage fluid and bone marrow and serum IgE levels decreased significantly in the F2-administered mice. Levels of T helper type 2 (Th2) cytokines and chemokines, involved in IgE class switching and eosinophil proliferation and recruitment, were also decreased in the F2-administered mice. An ex vivo cell rolling assay revealed that sLex glycans mediate the rolling of mouse eosinophils on P-selectin-expressing cells. These results indicate that the mAb F2 exerts therapeutic effects in a murine model of allergen-induced asthma, suggesting that sLex carbohydrate antigen could serve as a novel therapeutic target for allergic asthma.

A Atractylodes lancea polysaccharide inhibits metastasis of human osteosarcoma U-2 OS cells by blocking sialyl Lewis X (sLex )/E-selectin binding.

In this study, a new water and alkaline-soluble polysaccharide (ALP), with an average molecular weight of 6.63 × 104  Da, was successfully purified from the rhizomes of Atractylodes lancea. GC analysis demonstrated that ALP was a kind of glucan. The effect of the ALP on the interaction between E-selectin and sialyl Lewis X (sLex ) was examined in human osteosarcoma U-2 OS cells. It was obvious that the expression of sLex antigen on the surface of U-2 OS cells was visible under fluorescence microscopy. The addition of ALP (0.5, 1 and 2 mg/mL) resulted in a marked inhibition on the adhesion, migration and invasion of U-2 OS cells to human umbilical vein endothelial cells (HUVECs), which was achieved by the decreased sLex expression on U-2 OS cells. Additionally, the induction of apoptosis can be observed in U-2 OS cells following ALP treatment using TUNEL staining and Annexin V-FITC/PI double-staining analysis on flow cytometry. In conclusion, these results indicated that ALP exerted anti-metastatic activity towards osteosarcoma cells via inhibition of sLex /E-selectin binding, which suggested that ALP could be a potent agent for human osteosarcoma intervention.

Glycoengineering of chimeric antigen receptor (CAR) T-cells to enforce E-selectin binding.

Tissue colonization (homing) by blood-borne cells critically hinges on the ability of the cells to adhere to vascular endothelium with sufficient strength to overcome prevailing hemodynamic shear stress. These adhesive interactions are most effectively engendered via binding of the endothelial lectin E-selectin (CD62E) to its cognate ligand, sialyl Lewis-X (sLe X ), displayed on circulating cells. Although chimeric antigen receptor (CAR) T-cell immunotherapy holds promise for treatment of various hematologic and non-hematologic malignancies, there is essentially no information regarding the efficiency of CAR T-cell homing. Accordingly, we performed integrated biochemical studies and adhesion assays to examine the capacity of human CAR T-cells to engage E-selectin. Our data indicate that CAR T-cells do not express sLe X and do not bind E-selectin. However, enforced sLe X display can be achieved on human CAR T-cells by surface fucosylation, with resultant robust E-selectin binding under hemodynamic shear. Importantly, following intravascular administration into mice, fucosylated human CAR-T cells infiltrate marrow with 10-fold higher efficiency than do unfucosylated cells. Collectively, these findings indicate that custom installation of sLe X programs tissue colonization of vascularly administered human CAR T-cells, offering a readily translatable strategy to augment tissue delivery, thereby lowering the pertinent cell dosing and attendant cell production burden, for CAR T-cell immunotherapy applications.

Detecting substrate glycans of fucosyltransferases with fluorophore-conjugated fucose and methods for glycan electrophoresis.

Like sialylation, fucose usually locates at the nonreducing ends of various glycans on glycoproteins and constitutes important glycan epitopes. Detecting the substrate glycans of fucosyltransferases is important for understanding how these glycan epitopes are regulated in response to different growth conditions and external stimuli. Here we report the detection of these glycans on glycoproteins as well as in their free forms via enzymatic incorporation of fluorophore-conjugated fucose using FUT2, FUT6, FUT7, FUT8 and FUT9. Specifically, we describe the detection of the substrate glycans of these enzymes on fetal bovine fetuin, recombinant H1N1 viral neuraminidase and therapeutic antibodies. The detected glycans include complex and high-mannose N-glycans. By establishing a series of precursors for the synthesis of Lewis X and sialyl Lewis X structures, we not only provide convenient electrophoresis methods for studying glycosylation but also demonstrate the substrate specificities and some kinetic features of these enzymes. Our results support the notion that fucosyltransferases are key targets for regulating the synthesis of Lewis X and sialyl Lewis X structures.

The physiological and pathological roles and applications of sialyl Lewis x, a common carbohydrate ligand of the three selectins.

In the past decades, the roles of carbohydrates in living organisms and their potential use in many fields have been extensively investigated. Sialyl Lewis x (sLex), a member of body carbohydrate, is an inherent blood-type tetrasaccharide on the surface of different cells, the lymphocyte, neutrophil, some T cells, multiple tumor cells and so on. SLex is a common ligand of the three selectins, L-selectin, E-selectin and P-selectin, and plays important roles in multiple physiological phenomenas by interacting with selectins. Under normal physiological conditions, sLex can affect the immune process and fertilization process. Lower expression of sLex could cause leukocyte adhesion defects (LAD) II. Overexpression of sLex on the other hand has been linked to several cancers including melanoma, breast, pancreatic, liver, lung, head and neck, ovarian, bladder carcinomas and some blood disease including Hodgkin disease, some B cell chronic lymphocytic leukemias, acute lymphoblastic leukemias, and most acute nonlymphocytic leukemias. This paper mainly reviews the physiological functions and pathological effects of sLex and its applications in disease diagnosis, drug delivery, gene transfer and medical molecular imaging. We aim to help researchers and other readers quickly grasp the physiological and pathological roles and its medical applications of sLex, and give some suggestions for research directions.

Upregulation of Fucosyltransferase 3, 8 and protein O-Fucosyltransferase 1, 2 genes in esophageal cancer stem-like cells (CSLCs).

Recently, studies have shown that Fucosylation plays an important role in the invasion and metastatic process of CSLCs. Understanding the expression pattern of fucosyltransferase (FUT) genes may help to suggest better-targeted therapy strategies for esophageal squamous cell carcinoma (ESCC). The study aimed to address the expression pattern of FUT gene variants in esophageal CSLCs and parental adherent cells. Sphere formation method was used to enrich CSLCs. Expression of FUT genes was examined in tumor sphere and parental adherent cells using the RT-PCR method and then relative expression of detected variants was performed by the Real-Time PCR method in both groups. The detected FUTs, also, were assessed in fresh ESCC tumors and the matched healthy controls. Analysis of The cell surface carbohydrate Lewis x (LeX, CD15) was performed by flow cytometry. Molecular analysis showed that the expression of FUT 3, 8 and POFUT1, 2 genes in tumorsphere were significantly higher than parental adherent cells. Analysis of fresh ESCC tumor tissues and the matched healthy controls showed that FUT8 and POFUT1, 2 genes in contrast to FUT 3 have higher expression in tumor tissues than controls. Flow cytometric analyses revealed that tumorsphere and their parent cells do not differ significantly in Lewis x surface marker. The present study showed that FUT 3, 8 and POFUT1, 2 genes upregulated in esophageal CSLCs in comparison to adherent cells. Understanding the expression pattern of FUT gene variants may help to suggest better-targeted therapy strategies for ESCC.

L1CAM as an E-selectin Ligand in Colon Cancer.

Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLeX) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 (FUT6) coding for the α1,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLeX in CRC. The SW620FUT6 cell line expressed high levels of sLeX antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation.

Selectins-The Two Dr. Jekyll and Mr. Hyde Faces of Adhesion Molecules-A Review.

Selectins belong to a group of adhesion molecules that fulfill an essential role in immune and inflammatory responses and tissue healing. Selectins are glycoproteins that decode the information carried by glycan structures, and non-covalent interactions of selectins with these glycan structures mediate biological processes. The sialylated and fucosylated tetrasaccharide sLex is an essential glycan recognized by selectins. Several glycosyltransferases are responsible for the biosynthesis of the sLex tetrasaccharide. Selectins are involved in a sequence of interactions of circulated leukocytes with endothelial cells in the blood called the adhesion cascade. Recently, it has become evident that cancer cells utilize a similar adhesion cascade to promote metastases. However, like Dr. Jekyll and Mr. Hyde's two faces, selectins also contribute to tissue destruction during some infections and inflammatory diseases. The most prominent function of selectins is associated with the initial stage of the leukocyte adhesion cascade, in which selectin binding enables tethering and rolling. The first adhesive event occurs through specific non-covalent interactions between selectins and their ligands, with glycans functioning as an interface between leukocytes or cancer cells and the endothelium. Targeting these interactions remains a principal strategy aimed at developing new therapies for the treatment of immune and inflammatory disorders and cancer. In this review, we will survey the significant contributions to and the current status of the understanding of the structure of selectins and the role of selectins in various biological processes. The potential of selectins and their ligands as therapeutic targets in chronic and acute inflammatory diseases and cancer will also be discussed. We will emphasize the structural characteristic of selectins and the catalytic mechanisms of glycosyltransferases involved in the biosynthesis of glycan recognition determinants. Furthermore, recent achievements in the synthesis of selectin inhibitors will be reviewed with a focus on the various strategies used for the development of glycosyltransferase inhibitors, including substrate analog inhibitors and transition state analog inhibitors, which are based on knowledge of the catalytic mechanism.

Distinct human α(1,3)-fucosyltransferases drive Lewis-X/sialyl Lewis-X assembly in human cells.

In humans, six α(1,3)-fucosyltransferases (α(1,3)-FTs: FT3/FT4/FT5/FT6/FT7/FT9) reportedly fucosylate terminal lactosaminyl glycans yielding Lewis-X (LeX; CD15) and/or sialyl Lewis-X (sLeX; CD15s), structures that play key functions in cell migration, development, and immunity. Prior studies analyzing α(1,3)-FT specificities utilized either purified and/or recombinant enzymes to modify synthetic substrates under nonphysiological reaction conditions or molecular biology approaches wherein α(1,3)-FTs were expressed in mammalian cell lines, notably excluding investigations using primary human cells. Accordingly, although significant insights into α(1,3)-FT catalytic properties have been obtained, uncertainty persists regarding their human LeX/sLeX biosynthetic range across various glycoconjugates. Here, we undertook a comprehensive evaluation of the lactosaminyl product specificities of intracellularly expressed α(1,3)-FTs using a clinically relevant primary human cell type, mesenchymal stem cells. Cells were transfected with modified mRNA encoding each human α(1,3)-FT, and the resultant α(1,3)-fucosylated lactosaminyl glycoconjugates were analyzed using a combination of flow cytometry and MS. The data show that biosynthesis of sLeX is driven by FTs-3, -5, -6, and -7, with FT6 and FT7 having highest potency. FT4 and FT9 dominantly biosynthesize LeX, and, among all FTs, FT6 holds a unique capacity in creating sLeX and LeX determinants across protein and lipid glycoconjugates. Surprisingly, FT4 does not generate sLeX on glycolipids, and neither FT4, FT6, nor FT9 synthesizes the internally fucosylated sialyllactosamine VIM-2 (CD65s). These results unveil the relevant human lactosaminyl glycans created by human α(1,3)-FTs, providing novel insights on how these isoenzymes stereoselectively shape biosynthesis of vital glycoconjugates, thereby biochemically programming human cell migration and tuning human immunologic and developmental processes.

Mutation W222L at the Receptor Binding Site of Hemagglutinin Could Facilitate Viral Adaption from Equine Influenza A(H3N8) Virus to Dogs.

An outbreak of respiratory disease caused by the equine-origin influenza A(H3N8) virus was first detected in dogs in 2004 and since then has been enzootic among dogs. Currently, the molecular mechanisms underlying host adaption of this virus from horses to dogs is unknown. Here, we have applied quantitative binding, growth kinetics, and immunofluorescence analyses to elucidate these mechanisms. Our findings suggest that a substitution of W222L in the hemagglutinin of the equine-origin A(H3N8) virus facilitated its host adaption to dogs. This mutation increased binding avidity of the virus specifically to receptor glycans with N-glycolylneuraminic acid (Neu5Gc) and sialyl Lewis X (SLeX) motifs. We have demonstrated these motifs are abundantly located in the submucosal glands of dog trachea. Our findings also suggest that in addition to the type of glycosidic linkage (e.g., α2,3-linkage or α2,6-linkage), the type of sialic acid (Neu5Gc or 5-N-acetyl neuraminic acid) and the glycan substructure (e.g., SLeX) also play an important role in host tropism of influenza A viruses.IMPORTANCE Influenza A viruses (IAVs) cause a significant burden on human and animal health, and mechanisms for interspecies transmission of IAVs are far from being understood. Findings from this study suggest that an equine-origin A(H3N8) IAV with mutation W222L at its hemagglutinin increased binding to canine-specific receptors with sialyl Lewis X and Neu5Gc motifs and, thereby, may have facilitated viral adaption from horses to dogs. These findings suggest that in addition to the glycosidic linkage (e.g., α2,3-linked and α2,6-linked), the substructure in the receptor saccharides (e.g., sialyl Lewis X and Neu5Gc) could present an interspecies transmission barrier for IAVs and drive viral mutations to overcome such barriers.

The distribution of the proteoglycan FORSE-1 in the developing mouse central nervous system.

Glycosylation is a major post-translational modification in which a carbohydrate known as a glycan is enzymatically attached to target proteins which regulate protein folding and stability. Glycans are strongly expressed in the developing nervous system where they play multiple roles during development. The importance of these glycan epitopes in neural development is highlighted by a group of conditions known as congenital disorders of glycosylation which lead to psychomotor difficulties, mental retardation, lissencephaly, microencephaly and epilepsy. One of these glycan epitopes, known as Lewis X, is recognised by the FORSE-1 antibody and is regionally expressed in the developing nervous system. In this study, we report the regional and temporal expression patterns of FORSE-1 immunolabelling during the periods of neurogenesis, gliogenesis and axonogenesis in developing mouse nervous system. We demonstrate the localisation of FORSE-1 on subsets of neuroepithelial cells and radial glial cells, and in compartments corresponding to axon tract formation. These spatial, temporal and regional expression patterns are suggestive of roles in the determination of different cell lineages and in the patterning of white matter during development, and help provide insights into the neuroanatomical regions affected by congenital disorders of glycosylation.

Hsa-miR-370 inhibited P-selectin-induced cell adhesion in human colon adenocarcinoma cells.

Sialyl Lewis x (sLex) is a minimal recognition motif for ligands of P-selectin and plays an important role in tumor cell adhesion and migration. Thus, targeting sLex could be an effective method to prevent tumor metastasis. In this study, we aimed to identify a microRNA (miRNA) which is capable to suppress the expression of sLex. MicroRNAs which may target ST3GAL4 were predicted by the online tools. Colo 320 HSR human colon adenocarcinoma cells were employed. The transcriptional and translational levels of ST3GAL4 were evaluated by western blotting and Real-time quantitative polymerase chain reaction. Cell adhesion and spread were assessed with or without hsa-miR-370 treatment. It was shown that hsa-miR-370 inhibited the expression of sLex in colo-320 cells, which repressed the binding of P-selectin, and led to reduced cell attachment and spread. Our results found that P-selectin-induced elevations of p-p38 and p-PI3K levels were significantly inhibited by hsa-miR-370, indicating that repressed sLex level is able to reduce the P-selectin binding and therefore eliminating the P-selectin-induced activation of p38 and PI3K signaling. In conclusion, we found that hsa-miR-370 specifically inhibits the expression of sLex, represses cell adhesion and spreading in colo-320 cells. Our study provides a possible effective treatment against tumor invasion.

Bone Morphogenetic Protein Promotes Lewis X Stage-Specific Embryonic Antigen 1 Expression Thereby Interfering with Neural Precursor and Stem Cell Proliferation.

The glycoprotein Prominin-1 and the carbohydrate Lewis X stage-specific embryonic antigen 1 (LeX-SSEA1) both have been extensively used as cell surface markers to purify neural stem cells (NSCs). While Prominin-1 labels a specialized membrane region in NSCs and ependymal cells, the specificity of LeX-SSEA1 expression and its biological significance are still unknown. To address these issues, we have here monitored the expression of the carbohydrate in neonatal and adult NSCs and in their progeny. Our results show that the percentage of immunopositive cells and the levels of LeX-SSEA1 immunoreactivity both increase with postnatal age across all stages of the neural lineage. This is associated with decreased proliferation in precursors including NSCs, which accumulate the carbohydrate at the cell surface while remaining quiescent. Exposure of precursors to bone morphogenetic protein (BMP) increases LEX-SSEA1 expression, which promotes cell cycle withdrawal by a mechanism involving LeX-SSEA1-mediated interaction at the cell surface. Conversely, interference with either BMP signaling or with LeX-SSEA1 promotes proliferation to a similar degree. Thus, in the postnatal germinal niche, the expression of LeX-SSEA1 increases with age and exposure to BMP signaling, thereby downregulating the proliferation of subependymal zone precursors including NSCs. Stem Cells 2017;35:2417-2429.