Phenotype-related differential alpha-2,6- or alpha-2,3-sialylation of glycoprotein N-glycans in human chondrocytes.
ABSTRACT: Sialic acids frequently occur at the terminal positions of glycoprotein N-glycans present at chondrocyte surfaces or in the cartilage matrix. Sialic acids are transferred to glycoproteins in either alpha-2,3 or alpha-2,6 linkage by specific sialyltransferases (SiaTs) and can potentially affect cell functions and cell-matrix interactions. The present study aimed to assess the relationship between the expression of the human chondrocyte phenotype and the sialylation of chondrocyte glycoprotein N-glycans.The transcription of 5 SiaT was quantified using real-time Reverse transcription polymerase chain reaction (RT-PCR) assays. N-glycan analysis was performed using LC-ESI-MS. Primary human chondrocytes were cultured in monolayer or alginate beads and compared to the chondrocyte cell lines C-28/I2 and SW1353. In addition, effects of interleukin-1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) on primary cells were assessed.Primary human chondrocytes predominantly express alpha-2,6-specific SiaTs and accordingly, alpha-2,6-linked sialic acid residues in glycoprotein N-glycans. In contrast, the preponderance of alpha-2,3-linked sialyl residues and, correspondingly, reduced levels of alpha-2,6-specific SiaTs are associated with the altered chondrocyte phenotype of C-28/I2 and SW1353 cells. Importantly, a considerable shift towards alpha-2,3-linked sialic acids and alpha-2,3-specific SiaT mRNA levels occurred in primary chondrocytes treated with IL-1beta or tumour necrosis factor-alpha (TNF-alpha).The expression of the differentiated chondrocyte phenotype is linked to the ratio of alpha-2,6- to alpha-2,3-linked sialic acids in chondrocyte glycoprotein N-glycans. A shift towards altered sialylation might contribute to impaired cell-matrix interactions in disease conditions.
Project description:Human parainfluenza virus type 3 (hPIV3) recognizes both ?2,3- and ?2,6-linked sialic acids, whereas human parainfluenza virus type 1 (hPIV1) recognizes only ?2,3-linked sialic acids. To identify amino acid residues that confer ?2,6-linked sialic acid recognition of hPIV3, amino acid residues in or neighboring the sialic acid binding pocket of the hPIV3 hemagglutinin-neuraminidase (HN) glycoprotein were substituted for the corresponding residues of hPIV1 HN. Hemadsorption assay with sialyl linkage-modified red blood cells indicated that amino acid residues at positions 275, 277, 372, and 426 contribute to ?2,6-linked sialic acid recognition of the HN3 glycoprotein.
Project description:Human IgG antibodies containing terminal alpha 2,6-linked sialic acid on their Fc N-glycans have been shown to reduce antibody-dependent cell-mediated cytotoxicity and possess anti-inflammatory properties. Although terminal sialylation on complex N-glycans can happen via either an alpha 2,3-linkage or an alpha 2,6-linkage, sialic acids on human serum IgG Fc are almost exclusively alpha 2,6-linked. Recombinant IgGs expressed in Chinese hamster ovary (CHO) cells, however, have sialic acids through alpha 2,3-linkages because of the lack of the alpha 2,6-sialyltransferase gene. The impact of different sialylation linkages to the structure of IgG has not been determined. In this work, we investigated the impact of different types of sialylation to the conformational stability of IgG through hydrogen/deuterium exchange (HDX) and limited proteolysis experiments. When human-derived and CHO-expressed IgG1 were analyzed by HDX, sialic acid-containing glycans were found to destabilize the CH2 domain in CHO-expressed IgG, but not human-derived IgG. When structural isomers of sialylated glycans were chromatographically resolved and identified in the limited proteolysis experiment, we found that only alpha 2,3-linked sialic acid on the 6-arm (the major sialylated glycans in CHO-expressed IgG1) destabilizes the CH2 domain, presumably because of the steric effect that decreases the glycan-CH2 domain interaction. The alpha 2,6-linked sialic acid on the 3-arm (the major sialylated glycan in human-derived IgG), and the alpha 2,3-linked sialic acid on the 3-arm, do not have this destabilizing effect.
Project description:Microarrays containing 45 different lectins were analyzed to identify global changes in the glycosylation of serum glycoproteins from mice exposed to whole-body γ-radiation. The results showed that radiation exposure increased and decreased the relative amounts of α-2,3- and α-2,6-sialic acids, respectively. The expression of α-2,3- and α-2,6-sialyltransferase genes in the liver was analyzed to determine whether changes in their expression were responsible for the sialic acid changes. The increase in α-2,3-sialic acid correlated with St3gal5 upregulation after radiation exposure; however, a decrease in St6gal1 expression was not observed. Analysis of a PCR array of genes expressed in irradiated mouse livers revealed that irradiation did not alter the expression of most of the included genes. These results suggest that glycomic screening of serum glycoproteins using lectin microarrays can be a powerful tool for identifying radiation-induced changes in the post-translational addition of sugar moieties to proteins. In addition, the results indicate that altered sialylation of glycoproteins may be an initial response to acute radiation exposure.
Project description:Osteoarthritis (OA) is the most common degenerative joint disease characterized by articular cartilage degradation and joint degeneration. The articular cartilage is mainly formed by chondrocytes and a collagen-proteoglycan extracellular matrix that contains high levels of glycosylated proteins. It was reported that the shift from glycoproteins containing ?-2,6-linked sialic acids to those that contain ?-2,3 was associated with the onset of common types of arthritis. However, the pathophysiology of ?-2,3-sialylation in cartilage has not been yet elucidated. We show that cartilage from osteoarthritic patients expresses high levels of the ?-2,3-sialylated transmembrane mucin receptor, known as podoplanin (PDPN). Additionally, the Maackia amurensis seed lectin (MASL), that can be utilized to target PDPN, attenuates the inflammatory response mediated by NF-kB activation in primary chondrocytes and protects human cartilage breakdown ex vivo and in an animal model of arthritis. These findings reveal that specific lectins targeting ?-2,3-sialylated receptors on chondrocytes might effectively inhibit cartilage breakdown. We also present a computational 3D molecular model for this interaction. These findings provide mechanistic information on how a specific lectin could be used as a novel therapy to treat degenerative joint diseases such as osteoarthritis.
Project description:Influenza A viruses pose a major public health threat by causing seasonal epidemics and sporadic pandemics. Their epidemiological success relies on airborne transmission from person to person; however, the viral properties governing airborne transmission of influenza A viruses are complex. Influenza A virus infection is mediated via binding of the viral haemagglutinin (HA) to terminally attached ?2,3 or ?2,6 sialic acids on cell surface glycoproteins. Human influenza A viruses preferentially bind ?2,6-linked sialic acids whereas avian influenza A viruses bind ?2,3-linked sialic acids on complex glycans on airway epithelial cells. Historically, influenza A viruses with preferential association with ?2,3-linked sialic acids have not been transmitted efficiently by the airborne route in ferrets. Here we observe efficient airborne transmission of a 2009 pandemic H1N1 (H1N1pdm) virus (A/California/07/2009) engineered to preferentially bind ?2,3-linked sialic acids. Airborne transmission was associated with rapid selection of virus with a change at a single HA site that conferred binding to long-chain ?2,6-linked sialic acids, without loss of ?2,3-linked sialic acid binding. The transmissible virus emerged in experimentally infected ferrets within 24 hours after infection and was remarkably enriched in the soft palate, where long-chain ?2,6-linked sialic acids predominate on the nasopharyngeal surface. Notably, presence of long-chain ?2,6-linked sialic acids is conserved in ferret, pig and human soft palate. Using a loss-of-function approach with this one virus, we demonstrate that the ferret soft palate, a tissue not normally sampled in animal models of influenza, rapidly selects for transmissible influenza A viruses with human receptor (?2,6-linked sialic acids) preference.
Project description:Receptor recognition and binding is the first step in the viral cycle. It has been established that Newcastle Disease Virus (NDV) interacts with sialylated molecules such as gangliosides and glycoproteins at the cell surface. Nevertheless, the specific receptor(s) that mediate virus entry are not well known. We have analysed the role of the sialic acid linkage in the early steps of the viral infection cycle. Pretreatment of ELL-0 cells with both ?2,3 and ?2,6 specific sialidases led to the inhibition of NDV binding, fusion and infectivity, which were restored after ?2,3(N)- and ?2,6(N)-sialyltransferase incubation. Moreover, ?2,6(N)-sialyltransferases also restored NDV activities in ?2-6-linked sialic acid deficient cells. Competition with ?2-6 sialic acid-binding lectins led to a reduction in the three NDV activities (binding, fusion and infectivity) suggesting a role for ?2-6- linked sialic acid in NDV entry. We conclude that both ?2-3- and ?2-6- linked sialic acid containing glycoconjugates may be used for NDV infection. NDV was able to efficiently bind, fuse and infect the ganglioside-deficient cell line GM95 to a similar extent to that of its parental MEB4, suggesting that gangliosides are not essential for NDV binding, fusion and infectivity. Nevertheless, the fact that the interaction of NDV with cells deficient in N-glycoprotein expression such as Lec1 was less efficient prompted us to conclude that NDV requires N-linked glycoproteins for efficient attachment and entry into the host cell.
Project description:We studied whether selective inhibitors of cyclic nucleotide hydrolysing phosphodiesterase (PDE) isoenzymes influence IL-1beta-induced nitric oxide (NO) release from human articular chondrocytes. In addition, the pattern of PDE isoenzymes contributing to cyclic nucleotide hydrolysis in human chondrocytes was characterized. Chondrocytes were isolated from human osteoarthritic cartilage and cultured in alginate beads. IL-1beta-induced chondrocyte products (nitric oxide and prostaglandin E(2)) were measured in culture supernatants after 48 h incubation time. PDE activities were assessed in chondrocyte lysates. Inducible nitric oxide synthase (iNOS) and PDE4A-D proteins were detected by immunoblotting. The selective PDE4 inhibitors Piclamilast and Roflumilast partially attenuated IL-1beta-induced NO production whereas selective inhibitors of PDE2 (EHNA), PDE3 (Motapizone) or PDE5 (Sildenafil) were inactive. Indomethacin reversed the reduction of IL-1beta-induced NO by PDE4 inhibitors. It was shown that autocrine prostaglandin E(2) (PGE(2)) enabled PDE4 inhibitors to reduce IL-1beta-induced NO in this experimental setting. Major PDE4 and PDE1 activities were identified in chondrocyte lysates whereas only minor activities of PDE2, 3 and 5 were found. IL-1beta and cyclic AMP-mimetics upregulated PDE4 activity and this was associated with an augmentation of PDE4B2 protein. Based on the view that nitric oxide contributes to cartilage degradation in osteoarthritis our study suggests that PDE4 inhibitors may have chondroprotective effects.
Project description:beta 1,4-N-Acetylgalactosaminyltransferase (beta 1,4GalNAc-transferase) and alpha 2,3-sialyltransferase are both involved in the biosynthesis of the Sda blood group antigen, which is also present in cells of large intestine. The expression of these enzymes and of alpha 2,6-sialyltransferase activity towards N-acetyl-lactosamine was investigated in rat intestinal cells and correlated with both cell differentiation and extent of postnatal maturation. The beta 1,4GalNAc-transferase activity was exclusively found in epithelial cells of the large intestine, preferentially in the proximal segments suggesting a proximal-distal gradient of expression. The beta 1,4GalNAc-transferase and alpha 2,3-sialyltransferase activity towards N-acetyl-lactosamine were expressed in all cell fractions of the colonic crypt, with a maximum activity in the deeply located cells; therefore Sda antigen biosynthesis appears to occur preferentially at a specific stage of cell differentiation. By using N-acetyl-lactosamine as an acceptor, the predominant sialyltransferase in the colon cells was that capable of adding sialic acid in the alpha 2,3- linkage, whereas in the ileum cells the major enzyme was that forming the alpha 2,6-isomer. There were dramatic changes in the expression of colonic beta 1,4GalNac-transferase and of alpha 2,6-sialyltransferase activity towards N-acetyl-lactosamine during postnatal maturation. The former enzyme, practically absent at birth, increased slowly in the first days of life and then rapidly after weaning; by contrast, the latter enzyme was largely expressed only in newborn animals. As the colonic alpha 2,3-sialyltransferase activity towards N-acetyl-lactosamine did not change during the postnatal period, the ratio between the alpha 2,6- and alpha 2,3-sialyltransferase activities was reversed after weaning.
Project description:The differentiated phenotype of chondrocyte is lost in pathological situations and after interleukin (IL)-1beta challenge. Wnt proteins and the inorganic pyrophosphate (PP(i)) transporter Ank regulate the differentiation process in many cell types. We investigated the possible contribution of Ank and/or PP(i) to the maintenance of the differentiated chondrocyte phenotype with special care to Wnt signaling. Primary articular chondrocytes lost their phenotype upon IL-1beta challenge, with cessation of type II collagen and Sox-9 expression. Ank expression and PP(i) transport were strongly reduced by IL-1beta, whereas Wnt-5a was the only Wnt protein increased. Transient overexpression of Ank counteracted most of IL-1beta effects on Type II collagen, Sox-9, and Wnt-5a expression. When resting chondrocytes were transfected with a siRNA against Ank, this reproduced the phenotype induced by IL-1beta. In both cases, no markers for hypertrophic chondrocytes were detected. The conditioned supernatant from chondrocytes knocked-down for Ank contained Wnt-5a, which activated Tcf/Lef reporter plasmids and promoted translocation of beta-catenin into the nucleus without activating the c-Jun N-terminal kinase (JNK) pathway. Supplementation with PP(i) compensated for most effects of Ank deficiency on Type II collagen, Sox-9, and Wnt-5 expression, both in IL-1beta and Ank knock-down conditions. Phenotype changes induced by IL-1beta were also supported by activation of the JNK pathway, but this latter was not sensitive to PP(i) supplementation. Altogether our data demonstrate that the transport of PP(i) by ANK contributed to the maintenance of the differentiated phenotype of chondrocyte by controlling the canonical Wnt pathway in a Wnt-5a-dependent manner.
Project description:Intra-articular injection of local anesthetics (LAs) is a common procedure for therapeutic purposes. However, LAs have been found toxic to articular cartilage, and hyaluronan may attenuate this toxicity. In this study we investigated whether hyaluronan attenuated lidocaine-induced chondrotoxicity, and if so, to elucidate the underlying mechanisms.Human chondrocyte cell line SW1353 and newly isolated murine chondrocytes were incubated in culture medium containing hyaluronan and/or lidocaine for 72 h. Cell viability was evaluated using MTT assay. Cell apoptosis was detected with DAPI staining, caspase 3/7 activity assay and flow cytometry. Cell cycle distributions, ROS levels and mitochondrial membrane potential (ΔΨm) were determined using flow cytometry. The expression of p53 and p53-regulated gene products was measured with Western blotting.Lidocaine (0.005%-0.03%) dose-dependently decreased the viability of SW1353 cells. This local anesthetic (0.015%, 0.025%) induced apoptosis, G2/M phase arrest and loss of ΔΨm, and markedly increased ROS production in SW1353 cells. Hyaluronan (50-800 μg/mL) alone did not affect the cell viability, but co-treatment with hyaluronan (200 μg/mL) significantly attenuated lidocaine-induced apoptosis and other abnormalities in SW1353 cells. Furthermore, co-treatment with lidocaine and hyaluronan significantly decreased the levels of p53 and its transcription targets Bax and p21 in SW1353 cells, although treatment with lidocaine alone did not significantly change these proteins. Similar results were obtained in ex vivo cultured murine chondrocytes.Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro through inhibiting the p53-dependent mitochondrial apoptotic pathway.