Beta3Gn-T7 Is a Keratan Sulfate β1,3 N-Acetylglucosaminyltransferase in the Adult Brain

International audienceKeratan 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 β1,3 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

GlcNAc6ST2/CHST4 Is Essential for the Synthesis of R-10G-Reactive Keratan Sulfate/Sulfated <i>N</i>-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

Edition of heparan sulfate by endosulfatases monitored by HILIC chromatography coupled to mass spectrometry

International audienceGlycosaminoglycans (GAGs) are anionic polysaccharides of remarkable molecular complexity involved invarious biological and physio-pathological processes. The determination of structure-functionrelationships among these molecules is of great interest; however, the complex structure of GAGs, ofwhich heparan sulfate (HS) is the most challenging representative, and the lack of tools for decipheringcomplex GAG sequences has restricted advances in the GAGs field. In fact, at the molecular level, HSconstitutive disaccharide units can be modified by acetylation, epimerization, and sulfation at multiplepositions by highly regulated biosynthetic machinery. These modifications are completed by a post-synthetic editing process involving endosulfatases that finely tune the sulfate code along the HS chain.In humans, HSulf-1 and HSulf-2 are extracellular sulfatases that regioselectively remove the 6-O-sulfategroups from HS. HSulfs action alters HS ligand binding properties and modulates multiple signalingpathways. To gain new insights into the functional properties of HSulf enzymes, we set up a robust andresolving analytical method based on hydrophilic interaction liquid chromatography (HILIC) coupled withmass spectrometry (MS). This method allowed the structural determination of the enzyme productsfrom various sulfated oligosaccharide substrates and the monitoring of the 6-O-sulfate hydrolysis ofnatural sulfated substrates by HSulf enzymes. HILIC-MS methods are developed in our laboratory toallow the separation of GAG-sulfated oligosaccharides by size and sulfate patern. A specificmethodology was developed to monitor the progress of the enzyme reaction catalyzed by theendosulfatase HSulfs on various heparin (Hp)-derived oligosaccharides and characterize both thestructure and the kinetics of the formation of the enzyme products. We followed the desulfation reactionon various heparin-oligosaccharide substrates over time. The reaction conditions of the heparinoligosaccharide substrates with the enzymes were optimized by adjusting buffer parameters to allowmonitoring of the reaction by HILIC-MS. We observed the sequential hydrolysis of 6-O-sulfate groupswithin the sulfated oligosaccharides, confirming that HSulfs act processively while desulfating theirsubstrates. In addition, we compared a variety of HP oligosaccharides to determine the influence of thesulfated motifs on the HSulf enzyme activity, taking us a step further in deciphering the catalyticmechanism of these specific editing enzymes

Edition of heparan sulfate by endosulfatases monitored by HILIC chromatography coupled to mass spectrometry

International audienceGlycosaminoglycans (GAGs) are anionic polysaccharides of remarkable molecular complexity involved invarious biological and physio-pathological processes. The determination of structure-functionrelationships among these molecules is of great interest; however, the complex structure of GAGs, ofwhich heparan sulfate (HS) is the most challenging representative, and the lack of tools for decipheringcomplex GAG sequences has restricted advances in the GAGs field. In fact, at the molecular level, HSconstitutive disaccharide units can be modified by acetylation, epimerization, and sulfation at multiplepositions by highly regulated biosynthetic machinery. These modifications are completed by a post-synthetic editing process involving endosulfatases that finely tune the sulfate code along the HS chain.In humans, HSulf-1 and HSulf-2 are extracellular sulfatases that regioselectively remove the 6-O-sulfategroups from HS. HSulfs action alters HS ligand binding properties and modulates multiple signalingpathways. To gain new insights into the functional properties of HSulf enzymes, we set up a robust andresolving analytical method based on hydrophilic interaction liquid chromatography (HILIC) coupled withmass spectrometry (MS). This method allowed the structural determination of the enzyme productsfrom various sulfated oligosaccharide substrates and the monitoring of the 6-O-sulfate hydrolysis ofnatural sulfated substrates by HSulf enzymes. HILIC-MS methods are developed in our laboratory toallow the separation of GAG-sulfated oligosaccharides by size and sulfate patern. A specificmethodology was developed to monitor the progress of the enzyme reaction catalyzed by theendosulfatase HSulfs on various heparin (Hp)-derived oligosaccharides and characterize both thestructure and the kinetics of the formation of the enzyme products. We followed the desulfation reactionon various heparin-oligosaccharide substrates over time. The reaction conditions of the heparinoligosaccharide substrates with the enzymes were optimized by adjusting buffer parameters to allowmonitoring of the reaction by HILIC-MS. We observed the sequential hydrolysis of 6-O-sulfate groupswithin the sulfated oligosaccharides, confirming that HSulfs act processively while desulfating theirsubstrates. In addition, we compared a variety of HP oligosaccharides to determine the influence of thesulfated motifs on the HSulf enzyme activity, taking us a step further in deciphering the catalyticmechanism of these specific editing enzymes

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

International audienceSialyl 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

Edition of Heparan sulfate by human endosulfatases monitored by several analytical techniques

International audienceGlycosaminoglycans (GAGs) are anionic polysaccharides of remarkable molecular complexity involved in various biological and physio-pathological processes. The determination of structure-function relationships among these molecules is of great interest; however, the complex structure of GAGs, of which heparan sulfate (HS) is the most challenging representative, and the lack of tools for deciphering complex GAG sequences has restricted advances in the GAGs field. In fact, at the molecular level, HS constitutive disaccharide units can be modified by acetylation, epimerization, and sulfation at multiple positions by highly regulated biosynthetic machinery. These modifications are completed by a post-synthetic editing process involving endosulfatases that finely tune the sulfate code along the HS chain.In humans, HSulf-1 and HSulf-2 are extracellular sulfatases that regioselectively remove the 6-O-sulfate groups from HS. HSulfs action alters HS ligand binding properties and modulates multiple signaling pathways. To gain new insights into the functional properties of HSulf enzymes, we set up a robust and resolving analytical method based on hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (MS). This method allowed the structural determination of the enzyme products from various sulfated oligosaccharide substrates and the monitoring of the 6-O-sulfate hydrolysis of natural sulfated substrates by HSulf enzymes. HILIC-MS methods are developed in our laboratory to allow the separation of GAG-sulfated oligosaccharides by size and sulfate patern. A specific methodology was developed to monitor the progress of the enzyme reaction catalyzed by the endosulfatase HSulfs on various heparin (Hp)-derived oligosaccharides and characterize both the structure and the kinetics of the formation of the enzyme products. We followed the desulfation reaction on various heparin-oligosaccharide substrates over time. The reaction conditions of the heparin oligosaccharide substrates with the enzymes were optimized by adjusting buffer parameters to allow monitoring of the reaction by HILIC-MS. We observed the sequential hydrolysis of 6-O-sulfate groups within the sulfated oligosaccharides, confirming that HSulfs act processively while desulfating their substrates. In addition, we compared a variety of HP oligosaccharides to determine the influence of the sulfated motifs on the HSulf enzyme activity, taking us a step further in deciphering the catalytic mechanism of these specific editing enzyme

Edition of Heparan sulfate by human endosulfatases monitored by several analytical techniques

International audienceGlycosaminoglycans (GAGs) are anionic polysaccharides of remarkable molecular complexity involved in various biological and physio-pathological processes. The determination of structure-function relationships among these molecules is of great interest; however, the complex structure of GAGs, of which heparan sulfate (HS) is the most challenging representative, and the lack of tools for deciphering complex GAG sequences has restricted advances in the GAGs field. In fact, at the molecular level, HS constitutive disaccharide units can be modified by acetylation, epimerization, and sulfation at multiple positions by highly regulated biosynthetic machinery. These modifications are completed by a post-synthetic editing process involving endosulfatases that finely tune the sulfate code along the HS chain.In humans, HSulf-1 and HSulf-2 are extracellular sulfatases that regioselectively remove the 6-O-sulfate groups from HS. HSulfs action alters HS ligand binding properties and modulates multiple signaling pathways. To gain new insights into the functional properties of HSulf enzymes, we set up a robust and resolving analytical method based on hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (MS). This method allowed the structural determination of the enzyme products from various sulfated oligosaccharide substrates and the monitoring of the 6-O-sulfate hydrolysis of natural sulfated substrates by HSulf enzymes. HILIC-MS methods are developed in our laboratory to allow the separation of GAG-sulfated oligosaccharides by size and sulfate patern. A specific methodology was developed to monitor the progress of the enzyme reaction catalyzed by the endosulfatase HSulfs on various heparin (Hp)-derived oligosaccharides and characterize both the structure and the kinetics of the formation of the enzyme products. We followed the desulfation reaction on various heparin-oligosaccharide substrates over time. The reaction conditions of the heparin oligosaccharide substrates with the enzymes were optimized by adjusting buffer parameters to allow monitoring of the reaction by HILIC-MS. We observed the sequential hydrolysis of 6-O-sulfate groups within the sulfated oligosaccharides, confirming that HSulfs act processively while desulfating their substrates. In addition, we compared a variety of HP oligosaccharides to determine the influence of the sulfated motifs on the HSulf enzyme activity, taking us a step further in deciphering the catalytic mechanism of these specific editing enzyme

Deficiency of terminal complement pathway inhibitor promotes neuronal tau pathology and degeneration in mice

Abstract Background The neuronal microtubule-associated protein tau becomes hyperphosphorylated and forms aggregates in tauopathies but the processes leading to this pathological hallmark are not understood. Because tauopathies are accompanied by neuroinflammation and the complement cascade forms a key innate immune pathway, we asked whether the complement system has a role in the development of tau pathology. Findings We tested this hypothesis in two mouse models, which expressed either a central inhibitor of complement or lacked an inhibitor of the terminal complement pathway. Complement receptor-related gene/protein y is the natural inhibitor of the central complement component C3 in rodents. Expressing a soluble variant (sCrry) reduced the number of phospho-tau (AT8 epitope) positive neurons in the brain stem, cerebellum, cortex, and hippocampus of aged P301L mutant tau/sCrry double-transgenic mice compared with tau single-transgenic littermates (JNPL3 line). CD59a is the major inhibitor of formation of the membrane attack complex in mice. Intrahippocampal injection of adeno-associated virus encoding mutant human P301L tau into Cd59a−/− mice resulted in increased numbers of AT8-positive cells compared with wild-type controls. This was accompanied by neuronal and synaptic loss and reduced dendritic integrity. Conclusions Our data in two independent mouse models with genetic changes in key regulators of the complement system support the hypothesis that the terminal pathway has an active role in the development of tau pathology. We propose that inhibition of the terminal pathway may be beneficial in tauopathies

GlcNAc6ST3 is a keratan sulfate sulfotransferase for the protein-tyrosine phosphatase PTPRZ in the adult brain

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Deficiency of terminal complement pathway inhibitor promotes neuronal tau pathology and degeneration in mic