GlcNAc6ST2/CHST4 Is Essential for the Synthesis of R-10G-Reactive Keratan Sulfate/Sulfated N-Acetyllactosamine Oligosaccharides in Mouse Pleural Mesothelium.

We recently showed that 6-sulfo sialyl N-acetyllactosamine (LacNAc) in O-linked glycans recognized by the CL40 antibody is abundant in the pleural mesothelium under physiological conditions and that these glycans undergo complementary synthesis by GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5) in mice. GlcNAc6ST3 is essential for the synthesis of R-10G-positive keratan sulfate (KS) in the brain. The predicted minimum epitope of the R-10G antibody is a dimeric asialo 6-sulfo LacNAc. Whether R-10G-reactive KS/sulfated LacNAc oligosaccharides are also present in the pleural mesothelium was unknown. The question of which GlcNAc6STs are responsible for R-10G-reactive glycans was an additional issue to be clarified. Here, we show that R-10G-reactive glycans are as abundant in the pulmonary pleura as CL40-reactive glycans and that GlcNAc6ST3 is only partially involved in the synthesis of these pleural R-10G glycans, unlike in the adult brain. Unexpectedly, GlcNAc6ST2 is essential for the synthesis of R-10G-positive KS/sulfated LacNAc oligosaccharides in the lung pleura. The type of GlcNAc6ST and the magnitude of its contribution to KS glycan synthesis varied among tissues in vivo. We show that GlcNAc6ST2 is required and sufficient for R-10G-reactive KS synthesis in the lung pleura. Interestingly, R-10G immunoreactivity in KSGal6ST (encoded by Chst1) and C6ST1 (encoded by Chst3) double-deficient mouse lungs was markedly increased. MUC16, a mucin molecule, was shown to be a candidate carrier protein for pleural R-10G-reactive glycans. These results suggest that R-10G-reactive KS/sulfated LacNAc oligosaccharides may play a role in mesothelial cell proliferation and differentiation. Further elucidation of the functions of sulfated glycans synthesized by GlcNAc6ST2 and GlcNAc6ST3, such as R-10G and CL40 glycans, in pathological conditions may lead to a better understanding of the underlying mechanisms of the physiopathology of the lung mesothelium.

Hemolysis is associated with altered heparan sulfate of the endothelial glycocalyx and with local complement activation in thrombotic microangiopathies.

The complement system plays a key role in the pathophysiology of kidney thrombotic microangiopathies (TMA), as illustrated by atypical hemolytic uremic syndrome. But complement abnormalities are not the only drivers of TMA lesions. Among other potential pathophysiological actors, we hypothesized that alteration of heparan sulfate (HS) in the endothelial glycocalyx could be important. To evaluate this, we analyzed clinical and histological features of kidney biopsies from a monocentric, retrospective cohort of 72 patients with TMA, particularly for HS integrity and markers of local complement activation. The role of heme (a major product of hemolysis) as an HS-degrading agent in vitro, and the impact of altering endothelial cell (ECs) HS on their ability to locally activate complement were studied. Compared with a positive control, glomerular HS staining was lower in 57 (79%) patients with TMA, moderately reduced in 20 (28%), and strongly reduced in 37 (51%) of these 57 cases. Strongly reduced HS density was significantly associated with both hemolysis at the time of biopsy and local complement activation (C3 and/or C5b-9 deposits). Using primary endothelial cells (HUVECs, Glomerular ECs), we observed decreased HS expression after short-term exposure to heme, and that artificial HS degradation by exposure to heparinase was associated with local complement activation. Further, prolonged exposure to heme modulated expression of several key genes of glycocalyx metabolism involved in coagulation regulation (C5-EPI, HS6ST1, HS3ST1). Thus, our study highlights the impact of hemolysis on the integrity of endothelial HS, both in patients and in endothelial cell models. Hence, acute alteration of HS may be a mechanism of heme-induced complement activation.

A role for endothelial alpha-mannosidase MAN1C1 in radiation-induced immune cell recruitment.

Radiation therapy damages tumors and normal tissues, probably in part through the recruitment of immune cells. Endothelial high-mannose N-glycans are, in particular, involved in monocyte-endothelium interactions. Trimmed by the class I α-mannosidases, these structures are quite rare in normal conditions. Here, we show that the expression of the endothelial α-mannosidase MAN1C1 protein decreases after irradiation. We modeled two crucial steps in monocyte recruitment by developing in vitro real-time imaging models. Inhibition of MAN1C1 expression by siRNA gene silencing increases the abundance of high-mannose N-glycans, improves the adhesion of monocytes on endothelial cells in flow conditions and, in contrast, decreases radiation-induced transendothelial migration of monocytes. Consistently, overexpression of MAN1C1 in endothelial cells using lentiviral vectors decreases the abundance of high-mannose N-glycans and monocyte adhesion and enhances transendothelial migration of monocytes. Hence, we propose a role for endothelial MAN1C1 in the recruitment of monocytes, particularly in the adhesion step to the endothelium.

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.

Design and Synthesis of 6-O-Phosphorylated Heparan Sulfate Oligosaccharides to Inhibit Amyloid ß Aggregation.

Dysregulation of amyloidogenic proteins and their abnormal processing and deposition in tissues cause systemic and localized amyloidosis. Formation of amyloid ß (Aß) fibrils that deposit as amyloid plaques in Alzheimer's disease (AD) brains is an earliest pathological hallmark. The polysulfated heparan sulfate (HS)/heparin (HP) is one of the non-protein components of Aß deposits that not only modulates Aß aggregation, but also acts as a receptor for Aß fibrils to mediate their cytotoxicity. Interfering with the interaction between HS/HP and Aß could be a therapeutic strategy to arrest amyloidosis. Here we have synthesized the 6-O-phosphorylated HS/HP oligosaccharides and reported their competitive effects on the inhibition of HP-mediated Aß fibril formation in vitro using a thioflavin T fluorescence assay and a tapping mode atomic force microscopy.

Beta3Gn-T7 Is a Keratan Sulfate ß1,3 N-Acetylglucosaminyltransferase in the Adult Brain.

Keratan sulfate (KS) glycan is covalently attached to a core protein of proteoglycans. KS is abundant in neuropils and presents densely in close proximity to the perineuronal region of the perineuronal net-positive neurons in the adult brain under physiological conditions. We previously showed that the synthesis of KS positive for the R-10G antibody in the adult brain is mediated by GlcNAc-6-sulfotransferase 3 (GlcNAc6ST3; encoded by Chst5). Deficiency in both GlcNAc6ST3 and GlcNAc6ST1, encoded by Chst2, completely abolished KS. Protein-tyrosine phosphatase receptor type z1 (Ptprz1)/phosphacan was identified as a KS scaffold. KS requires the extension of GlcNAc by ß1,3 N-acetylglucosaminyltransferase (Beta3Gn-T). Members of the Beta3Gn-T family involved in the synthesis of adult brain KS have not been identified. In this study, we show by a method of gene targeting that Beta3Gn-T7, encoded by B3gnt7, is a major Beta3Gn-T for the synthesis of KS in neuropils and the perineuronal region in the adult brain. Intriguingly, the B3gnt7 gene is selectively expressed in oligodendrocyte precursor cells (OPCs) and oligodendrocytes similar to that of GlcNAc6ST3. These results indicate that Beta3Gn-T7 in oligodendrocyte lineage cells may play a role in the formation of neuropils and perineuronal nets in the adult brain through the synthesis of R-10G-positive KS-modified proteoglycan.

Synthesis of new sulfated disaccharides for the modulation of TLR4-dependent inflammation.

Natural sulfated glycans are key players in inflammation through TLR4 activation; therefore synthetic exogenous sulfated saccharides can be used to downregulate inflammation processes. We have designed and synthesized new sulfated compounds based on small and biocompatible carbohydrates that are able to cross the BBB. A suitable protected donor and acceptor, obtained from a unique precursor, have been stereoselectively glycosylated to give an orthogonally protected cellobiose disaccharide. Selective deprotection and sulfation allowed the syntheses of four differentially sulfated disaccharides, which have been characterized by NMR, HRMS and MS/MS. Together with their partially protected precursors, the new compounds were tested on HEK-TLR4 cells. Our results show the potential of small oligosaccharides to modulate TLR4 activity, confirming the need for sulfation and the key role of the 6-sulfate groups to trigger TLR4 signalization.

Geniac: Automatic Configuration GENerator and Installer for nextflow pipelines.

With the advent of high-throughput biotechnological platforms and their ever-growing capacity, life science has turned into a digitized, computational and data-intensive discipline. As a consequence, standard analysis with a bioinformatics pipeline in the context of routine production has become a challenge such that the data can be processed in real-time and delivered to the end-users as fast as possible. The usage of workflow management systems along with packaging systems and containerization technologies offer an opportunity to tackle this challenge. While very powerful, they can be used and combined in many multiple ways which may differ from one developer to another. Therefore, promoting the homogeneity of the workflow implementation requires guidelines and protocols which detail how the source code of the bioinformatics pipeline should be written and organized to ensure its usability, maintainability, interoperability, sustainability, portability, reproducibility, scalability and efficiency. Capitalizing on Nextflow, Conda, Docker, Singularity and the nf-core initiative, we propose a set of best practices along the development life cycle of the bioinformatics pipeline and deployment for production operations which target different expert communities including i) the bioinformaticians and statisticians ii) the software engineers and iii) the data managers and core facility engineers. We implemented Geniac (Automatic Configuration GENerator and Installer for nextflow pipelines) which consists of a toolbox with three components: i) a technical documentation available at https://geniac.readthedocs.io to detail coding guidelines for the bioinformatics pipeline with Nextflow, ii) a command line interface with a linter to check that the code respects the guidelines, and iii) an add-on to generate configuration files, build the containers and deploy the pipeline. The Geniac toolbox aims at the harmonization of development practices across developers and automation of the generation of configuration files and containers by parsing the source code of the Nextflow pipeline.

ARIAweb: a server for automated NMR structure calculation.

Nuclear magnetic resonance (NMR) spectroscopy is a method of choice to study the dynamics and determine the atomic structure of macromolecules in solution. The standalone program ARIA (Ambiguous Restraints for Iterative Assignment) for automated assignment of nuclear Overhauser enhancement (NOE) data and structure calculation is well established in the NMR community. To ultimately provide a perfectly transparent and easy to use service, we designed an online user interface to ARIA with additional functionalities. Data conversion, structure calculation setup and execution, followed by interactive visualization of the generated 3D structures are all integrated in ARIAweb and freely accessible at https://ariaweb.pasteur.fr.

Glycosylation changes in inflammatory diseases.

Glycosylation is one of the most important modifications of proteins and lipids, and cell surface glycoconjugates are thought to play important roles in a variety of biological functions including cell-cell and cell-substrate interactions, bacterial adhesion, cell immunogenicity and cell signaling. Alterations of glycosylation are observed in a number of inflammatory diseases. Pro-inflammatory cytokines have been shown to modulate cell surface glycosylation by regulating the expression of glycosyltransferases and sulfotransferases involved in the biosynthesis of glycan chains, inducing the expression of specific carbohydrate antigens at the cell surface that can be recognized by different types of lectins or by bacterial adhesins, contributing to the development of diseases. Glycosylation can also regulate biological functions of immune cells by recruiting leukocytes to inflammation sites with pro- or anti-inflammatory effects. Cell surface proteoglycans provide a large panel of binding sites for many mediators of inflammation, and regulate their bio-availability and functions. In this review, we summarize the current knowledge of the glycosylation changes occurring in mucin type O-linked glycans, glycosaminoglycans, as well as in glycosphingolipids, with a particular focus on cystic fibrosis and neurodegenerative diseases, and their consequences on cell interactions and disease progression.

The Emerging Roles of Heparan Sulfate 3-O-Sulfotransferases in Cancer.

Alteration in the expression of heparan sulfate (HS)-modifying enzymes has been frequently observed in cancer. Consequently, dysregulation of the HS biosynthetic machinery results in dramatic changes in the HS structure, thereby impacting a range of pivotal cellular processes involved in tumorigenesis and cancer progression including proliferation, migration, apoptosis, and immune escape. HS 3-O-sulfotransferases (HS3STs) catalyse the maturation step of glucosaminyl 3-O-sulfation within HS chains. Although seven HS3ST isozymes have been described in human, 3-O-sulfation is a rare modification and only a few biological processes have been described to be influenced by 3-O-sulfated HS. An aberrant expression of HS3STs has been reported in a variety of cancers. Thus, it was suggested that changes in the expression of these enzymes as a result of tumorigenesis or tumor growth may critically influence cancer cell behavior. In accordance with this assumption, a number of studies have documented the epigenetic repression of HS3ST2 and HS3ST3A in many cancers. However, the situation is not so clear, and there is accumulating evidence that HS3ST2, HS3ST3A, HS3ST3B, and HS3ST4 may also act as tumor-promoting enzymes in a number of cancer cells depending on their phenotypes and molecular signatures. In this mini-review, we focus on the recent insights regarding the abnormal expression of HS3STs in cancer and discuss the functional consequences on tumor cell behavior. In term of clinical outcome, further investigations are needed to explore the potential value of HS3STs and/or their 3-O-sulfated products as targets for therapeutic strategies in cancer treatment.

Cancer cells induce immune escape via glycocalyx changes controlled by the telomeric protein TRF2.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells with strong immunosuppressive activity that promote tumor growth. In this study, we describe a mechanism by which cancer cells control MDSCs in human cancers by upregulating TRF2, a protein required for telomere stability. Specifically, we showed that the TRF2 upregulation in cancer cells has extratelomeric roles in activating the expression of a network of genes involved in the biosynthesis of heparan sulfate proteoglycan, leading to profound changes in glycocalyx length and stiffness, as revealed by atomic force microscopy. This TRF2-dependent regulation facilitated the recruitment of MDSCs, their activation via the TLR2/MyD88/IL-6/STAT3 pathway leading to the inhibition of natural killer recruitment and cytotoxicity, and ultimately tumor progression and metastasis. The clinical relevance of these findings is supported by our analysis of cancer cohorts, which showed a correlation between high TRF2 expression and MDSC infiltration, which was inversely correlated with overall patient survival.

Biogenesis and structure of a type VI secretion baseplate.

To support their growth in a competitive environment and cause pathogenesis, bacteria have evolved a broad repertoire of macromolecular machineries to deliver specific effectors and toxins. Among these multiprotein complexes, the type VI secretion system (T6SS) is a contractile nanomachine that targets both prokaryotic and eukaryotic cells. The T6SS comprises two functional subcomplexes: a bacteriophage-related tail structure anchored to the cell envelope by a membrane complex. As in other contractile injection systems, the tail is composed of an inner tube wrapped by a sheath and built on the baseplate. In the T6SS, the baseplate is not only the tail assembly platform, but also docks the tail to the membrane complex and hence serves as an evolutionary adaptor. Here we define the biogenesis pathway and report the cryo-electron microscopy (cryo-EM) structure of the wedge protein complex of the T6SS from enteroaggregative Escherichia coli (EAEC). Using an integrative approach, we unveil the molecular architecture of the whole T6SS baseplate and its interaction with the tail sheath, offering detailed insights into its biogenesis and function. We discuss architectural and mechanistic similarities but also reveal key differences with the T4 phage and Mu phage baseplates.

The Pro-Tumoral Activity of Heparan Sulfate 3-O-Sulfotransferase 3B (HS3ST3B) in Breast Cancer MDA-MB-231 Cells Is Dependent on the Expression of Neuropilin-1.

Heparan sulfate 3-O-sulfotransferases (HS3STs) catalyze the maturation step of heparan sulfate (HS) 3-O-sulfation. This modification is relatively rare. Moreover, only a few biological processes have been described to be influenced by 3-O-sulfated HS, and few ligands have been identified so far. Among them, neuropilin-1 (Nrp1) was reported to exhibit tumor-promoting properties by enhancing the action of various growth factors. We recently demonstrated that transient overexpression of HS3ST2, 3B or 4 enhanced the proliferation of breast cancer MDA-MB-231 cells and promote efficient protection against pro-apoptotic stimuli. Hence, we hypothesized that the pro-tumoral activity of these HS3STs could depend on the expression of Nrp1. To test this, MDA-MB-231 cells were stably transfected with a construct encoding HS3ST3B and the expression of Nrp1 was down-regulated by RNA interference. First, we confirmed that stable expression of HS3ST3B effectively increased cell proliferation and viability. Silencing the expression of Nrp1 markedly attenuated the promoting effects of HS3ST3B, while the same treatment had only a moderate effect on the behavior of the parental cells. Altogether, our findings support the idea that the tumor-promoting effects of HS3ST3B could be dependent on the expression of Nrp1 in cancer cells.

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2) displays an unexpected subcellular localization in the plasma membrane.

BACKGROUND: Heparan sulfate (HS) 3-O-sulfation can be catalysed by seven 3-O-sulfotransferases (HS3STs) in humans, still it is the rarest modification in HS and its biological function is yet misunderstood. HS3ST2 and HS3ST3B exhibit the same activity in vitro. They are however differently expressed in macrophages depending on cell environment, which suggests that they may be involved in distinct cellular processes. Here, we hypothesized that both isozymes might also display distinct subcellular localizations. METHODS: The subcellular distribution of HS3ST2 and HS3ST3B was analysed by using overexpression systems in HeLa cells. The localization of endogenous HS3ST2 was confirmed by immunostaining in primary macrophages. RESULTS: We found that HS3ST3B was only localized in the Golgi apparatus and no difference between full-length enzyme and truncated construct depleted of its catalytic domain was observed. In contrast, HS3ST2 was clearly visualized at the plasma membrane. Its truncated form remained in the Golgi apparatus, meaning that the catalytic domain might support correct addressing of HS3ST2 to cell surface. Moreover, we found a partial co-localization of HS3ST2 with syndecan-2 in HeLa cells and primary macrophages. Silencing the expression of this proteoglycan altered the localization of HS3ST2, which suggests that syndecan-2 is required to address the isozyme outside of the Golgi apparatus. CONCLUSIONS: We demonstrated that HS3ST3B is a Golgi-resident isozyme, while HS3ST2 is addressed to the plasma membrane with syndecan-2. GENERAL SIGNIFICANCE: The membrane localization of HS3ST2 suggests that this enzyme may participate in discrete processes that occur at the cell surface.

The heparan sulfate 3-O-sulfotransferases (HS3ST) 2, 3B and 4 enhance proliferation and survival in breast cancer MDA-MB-231 cells.

Heparan sulfate 3-O-sulfotransferases (HS3STs) catalyze the final maturation step of heparan sulfates. Although seven HS3ST isozymes have been described in human, 3-O-sulfation is a relatively rare modification, and only a few biological processes have been described to be influenced by 3-O-sulfated motifs. A conflicting literature has recently reported that HS3ST2, 3A, 3B and 4 may exhibit either tumor-promoting or anti-oncogenic properties, depending on the model used and cancer cell phenotype. Hence, we decided to compare the consequences of the overexpression of each of these HS3STs in the same cellular model. We demonstrated that, unlike HS3ST3A, the other three isozymes enhanced the proliferation of breast cancer MDA-MB-231 and BT-20 cells. Moreover, the colony forming capacity of MDA-MB-231 cells was markedly increased by the expression of HS3ST2, 3B and 4. No notable difference was observed between the three isozymes, meaning that the modifications catalyzed by each HS3ST had the same functional impact on cell behavior. We then demonstrated that overexpression of HS3ST2, 3B and 4 was accompanied by increased activation of c-Src, Akt and NF-κB and up-regulation of the anti-apoptotic proteins survivin and XIAP. In line with these findings, we showed that HS3ST-transfected cells are more resistant to cell death induction by pro-apoptotic stimuli or NK cells. Altogether, our findings demonstrate that HS3ST2, 3B and 4 share the same pro-tumoral activity and support the idea that these HS3STs could compensate each other for loss of their expression depending on the molecular signature of cancer cells and/or changes in the tumor environment.

Use of Toll-like receptor assays for the detection of bacterial contaminations in icodextrin batches released for peritoneal dialysis.

Icodextrin is a starch derivative used for preparing solutions of peritoneal dialysis. Unfortunately, peptidoglycans (PGN) and lipopolysaccharides (LPS) have been reported to contaminate certain icodextrin batches and to contribute to the development of sterile peritonitis. The decision of selecting or rejecting icodextrin batches is however difficult, because of limitations in the detection of these bacterial contaminants. Besides monocyte activation tests of cytokine release, a number of bio-assays using stably TLR-transfected cell lines have been developed. Here, we compared the efficacy of TLR2- and TLR4-transfected cells to detect bacterial contamination with the responses of monocytes exposed to the same icodextrin samples. In contrast to monocyte models of cytokine release, we found that TLR2- and TLR4-transfected cell lines are highly sensitive to detect little PGN and LPS contaminations in the presence of icodextrin. With the intent to increase PGN reactivity, mutanolysin was used to generate soluble fragments in icodextrin samples. We found that such an enzymatic treatment led to an enhanced response of TLR2-transfected cells, even though parental icodextrin samples were poorly reactive. Altogether, these findings indicate that the use of TLR2- and TLR4-transfected cell lines is a valuable approach for helping to the decision of selecting icodextrin batches for peritoneal dialysis.

Radiation-induced changes in the glycome of endothelial cells with functional consequences.

As it is altered by ionizing radiation, the vascular network is considered as a prime target in limiting normal tissue damage and improving tumor control in radiation therapy. Irradiation activates endothelial cells which then participate in the recruitment of circulating cells, especially by overexpressing cell adhesion molecules, but also by other as yet unknown mechanisms. Since protein glycosylation is an important determinant of cell adhesion, we hypothesized that radiation could alter the glycosylation pattern of endothelial cells and thereby impact adhesion of circulating cells. Herein, we show that ionizing radiation increases high mannose-type N-glycans and decreases glycosaminoglycans. These changes stimulate interactions measured under flow conditions between irradiated endothelial cells and monocytes. Targeted transcriptomic approaches in vitro in endothelial cells and in vivo in a radiation enteropathy mouse model confirm that genes involved in N- and O-glycosylation are modulated by radiation, and in silico analyses give insight into the mechanism by which radiation modifies glycosylation. The endothelium glycome may therefore be considered as a key therapeutic target for modulating the chronic inflammatory response observed in healthy tissues or for participating in tumor control by radiation therapy.

Participation of 3-O-sulfated heparan sulfates in the protection of macrophages by herpes simplex virus-1 glycoprotein D and cyclophilin B against apoptosis.

Heparan sulfates (HS) are involved in numerous biological processes, which rely on their ability to interact with a large panel of proteins. Although the reaction of 3-O-sulfation can be catalysed by the largest family of HS sulfotransferases, very few mechanisms have been associated with this modification and to date, only glycoprotein D (gD) of herpes simplex virus-1 (HSV-1 gD) and cyclophilin B (CyPB) have been well-described as ligands for 3-O-sulfated HS. Here, we hypothesized that both ligands could induce the same responses via a mechanism dependent on 3-O-sulfated HS. First, we checked that HSV-1 gD was as efficient as CyPB to induce the activation of the same signalling events in primary macrophages. We then demonstrated that both ligands efficiently reduced staurosporin-induced apoptosis and modulated the expression of apoptotic genes. In addition to 3-O-sulfated HS, HSV-1 gD was reported to interact with other receptors, including herpes virus entry mediator (HVEM), nectin-1 and -2. Thus, we decided to identify the contribution of each binding site in the responses triggered by HSV-1 gD and CyPB. We found that knock-down of 3-O-sulfotransferase 2, which is the main 3-O-sulfated HS-generating enzyme in macrophages, strongly reduced the responses induced by both ligands. Moreover, silencing the expression of HVEM rendered macrophages unresponsive to either HSV-1 gD and CyPB, thus indicating that both proteins induced the same responses by interacting with a complex formed by 3-O-sulfated HS and HVEM. Collectively, our results suggest that HSV-1 might hijack the binding sites for CyPB in order to protect macrophages against apoptosis for efficient infection.

Tumour-necrosis factor-α induces heparan sulfate 6-O-endosulfatase 1 (Sulf-1) expression in fibroblasts.

Heparan sulfate (HS) 6-O-endosulfatases (Sulfs) have emerged recently as critical regulators of many physiological and pathological processes. By removing 6-O-sulfates from specific HS sequences, they modulate the activities of a variety of growth factors and morphogens, including fibroblast growth factor (FGF)-1. However, little is known about the functions of Sulfs in inflammation. Tumour-necrosis factor (TNF)-α plays an important role in regulating the behaviour of fibroblasts. In this study, we examined the effect of this inflammatory cytokine on the expression of Sulfs in human MRC-5 fibroblasts. Compositional analysis of HS from TNF-α-treated cells showed a strong reduction in the amount of the trisulfated UA2S-GlcNS6S disaccharide, which suggested a selective reaction of 6-O-desulfation. Real-time PCR analysis revealed that TNF-α increased Sulf-1 expression in a dose- and time-dependent manner, via a mechanism involving NF-ĸB, ERK1/2 and p38 MAPK. In addition, we confirmed that cell stimulation with TNF-α was accompanied by the secretion of an active form of Sulf-1. To study the function of Sulf- 1, we examined the responses induced by FGF-1. We showed that ERK1/2 activation and cell proliferation were markedly reduced in TNF-α-treated MRC-5 cells compared with untreated cells. Silencing the expression of Sulf-1 by RNA interference restored the responses induced by FGF-1, which indicated that TNF-α-mediated induction of the sulfatase indeed resulted in alterations of HS biological properties. Taken together, our results indicate that Sulf-1 is responsive to TNF-α stimulation and may function as an autocrine regulator of fibroblast expansion in the course of an inflammatory response.