Proteinuria converts hepatic heparan sulfate to an effective proprotein convertase subtilisin kexin type 9 enzyme binding partner.

Hepatic uptake of triglyceride-rich remnant lipoproteins is mediated by the low-density lipoprotein receptor, a low-density lipoprotein receptor related protein and the heparan sulfate proteoglycan, syndecan-1. Heparan sulfate proteoglycan also mediates low-density lipoprotein receptor degradation by a regulator of cholesterol homeostasis, proprotein convertase subtilisin kexin type 9 (PCSK9), thereby hampering triglyceride-rich remnant lipoproteins uptake. In this study, we investigated the effects of proteinuria on PCSK9, hepatic heparan sulfate proteoglycan and plasma triglyceride-rich remnant lipoproteins. Adriamycin-injected rats developed proteinuria, elevated triglycerides and total cholesterol (all significantly increased). Proteinuria associated with triglycerides and total cholesterol and serum PCSK9 (all significant associations) without loss of the low-density lipoprotein receptor as evidenced by immunofluorescence staining and western blotting. In proteinuric rats, PCSK9 accumulated in sinusoids, whereas in control rats PCSK9 was localized in the cytoplasm of hepatocytes. Molecular profiling revealed that the heparan sulfate side chains of heparan sulfate proteoglycan to be hypersulfated in proteinuric rats. Competition assays revealed sulfation to be a major determinant for PCSK9 binding. PCSK9 partly colocalized with hypersulfated heparan sulfate in proteinuric rats, but not in control rats. Hence, proteinuria induces hypersulfated hepatic heparan sulfate proteoglycans, increasing their affinity to PCSK9. This might impair hepatic triglyceride-rich remnant lipoproteins uptake, causing proteinuria-associated dyslipidemia. Thus, our study reveals PCSK9/heparan sulfate may be a novel target to control dyslipidemia.

Expression and purification of recombinant extracellular sulfatase HSulf-2 allows deciphering of enzyme sub-domain coordinated role for the binding and 6-O-desulfation of heparan sulfate.

Through their ability to edit 6-O-sulfation pattern of Heparan sulfate (HS) polysaccharides, Sulf extracellular endosulfatases have emerged as critical regulators of many biological processes, including tumor progression. However, study of Sulfs remains extremely intricate and progress in characterizing their functional and structural features has been hampered by limited access to recombinant enzyme. In this study, we unlock this critical bottleneck, by reporting an efficient expression and purification system of recombinant HSulf-2 in mammalian HEK293 cells. This novel source of enzyme enabled us to investigate the way the enzyme domain organization dictates its functional properties. By generating mutants, we confirmed previous studies that HSulf-2 catalytic (CAT) domain was sufficient to elicit arylsulfatase activity and that its hydrophilic (HD) domain was necessary for the enzyme 6-O-endosulfatase activity. However, we demonstrated for the first time that high-affinity binding of HS substrates occurred through the coordinated action of both domains, and we identified and characterized 2 novel HS binding sites within the CAT domain. Altogether, our findings contribute to better understand the molecular mechanism governing HSulf-2 substrate recognition and processing. Furthermore, access to purified recombinant protein opens new perspectives for the resolution of HSulf structure and molecular features, as well as for the development of Sulf-specific inhibitors.

Loss of endothelial sulfatase-1 after experimental sepsis attenuates subsequent pulmonary inflammatory responses.

Sepsis patients are at increased risk for hospital-acquired pulmonary infections, potentially due to postseptic immunosuppression known as the compensatory anti-inflammatory response syndrome (CARS). CARS has been attributed to leukocyte dysfunction, with an unclear role for endothelial cells. The pulmonary circulation is lined by an endothelial glycocalyx, a heparan sulfate-rich layer essential to pulmonary homeostasis. Heparan sulfate degradation occurs early in sepsis, leading to lung injury. Endothelial synthesis of new heparan sulfates subsequently allows for glycocalyx reconstitution and endothelial recovery. We hypothesized that remodeling of the reconstituted endothelial glycocalyx, mediated by alterations in the endothelial machinery responsible for heparan sulfate synthesis, contributes to CARS. Seventy-two hours after experimental sepsis, coincident with glycocalyx reconstitution, mice demonstrated impaired neutrophil and protein influx in response to intratracheal lipopolysaccharide (LPS). The postseptic reconstituted glycocalyx was structurally remodeled, with enrichment of heparan sulfate disaccharides sulfated at the 6-O position of glucosamine. Increased 6-O-sulfation coincided with loss of endothelial sulfatase-1 (Sulf-1), an enzyme that specifically removes 6-O-sulfates from heparan sulfate. Intravenous administration of Sulf-1 to postseptic mice restored the pulmonary response to LPS, suggesting that loss of Sulf-1 was necessary for postseptic suppression of pulmonary inflammation. Endothelial-specific knockout mice demonstrated that loss of Sulf-1 was not sufficient to induce immunosuppression in non-septic mice. Knockdown of Sulf-1 in human pulmonary microvascular endothelial cells resulted in downregulation of the adhesion molecule ICAM-1. Taken together, our study indicates that loss of endothelial Sulf-1 is necessary for postseptic suppression of pulmonary inflammation, representing a novel endothelial contributor to CARS.

The "in and out" of glucosamine 6-O-sulfation: the 6th sense of heparan sulfate.

The biological properties of Heparan sulfate (HS) polysaccharides essentially rely on their ability to bind and modulate a multitude of protein ligands. These interactions involve internal oligosaccharide sequences defined by their sulfation patterns. Amongst these, the 6-O-sulfation of HS contributes significantly to the polysaccharide structural diversity and is critically involved in the binding of many proteins. HS 6-O-sulfation is catalyzed by 6-O-sulfotransferases (6OSTs) during biosynthesis, and it is further modified by the post-synthetic action of 6-O-endosulfatases (Sulfs), two enzyme families that remain poorly characterized. The aim of the present review is to summarize the contribution of 6-O-sulfates in HS structure/function relationships and to discuss the present knowledge on the complex mechanisms regulating HS 6-O-sulfation.

Extracellular endosulfatase Sulf-2 harbors a chondroitin/dermatan sulfate chain that modulates its enzyme activity.

Sulfs represent a class of unconventional sulfatases which provide an original post-synthetic regulatory mechanism for heparan sulfate polysaccharides and are involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human Sulf-2 (HSulf-2) harbors a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate-binding domain. We demonstrate that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo. In addition, we show that mammalian hyaluronidase acts as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a proteoglycan-related enzyme and its GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute to clarifying the conflicting data on the activities of the Sulfs.

RHO GTPases: from new partners to complex immune syndromes.

Ras homology (RHO) GTPases are signalling proteins that have crucial roles in triggering multiple immune functions. Through their interactions with a broad range of effectors and kinases, they regulate cytoskeletal dynamics, cell polarity and the trafficking and proliferation of immune cells. The activity and localization of RHO GTPases are highly controlled by classical families of regulators that share consensus motifs. In this Review, we describe the recent discovery of atypical modulators and partners of RHO GTPases, which bring an additional layer of regulation and plasticity to the control of RHO GTPase activities in the immune system. Furthermore, the development of large-scale genetic screening has now enabled researchers to identify dysregulation of RHO GTPase signalling pathways as a cause of many immune system-related diseases. We discuss the mutations that have been identified in RHO GTPases and their signalling circuits in patients with rare diseases. The discoveries of new RHO GTPase partners and genetic mutations in RHO GTPase signalling hubs have uncovered unsuspected layers of crosstalk with other signalling pathways and may provide novel therapeutic opportunities for patients affected by complex immune or broader syndromes.

HS and Inflammation: A Potential Playground for the Sulfs?

Heparan sulfate (HS) is a complex polysaccharide abundantly found in extracellular matrices and cell surfaces. HS participates in major cellular processes, through its ability to bind and modulate a wide array of signaling proteins. HS/ligand interactions involve saccharide domains of specific sulfation pattern. Assembly of such domains is orchestrated by a complex biosynthesis machinery and their structure is further regulated at the cell surface by post-synthetic modifying enzymes. Amongst them, extracellular sulfatases of the Sulf family catalyze the selective removal of 6-O-sulfate groups, which participate in the binding of many proteins. As such, increasing interest arose on the regulation of HS biological properties by the Sulfs. However, studies of the Sulfs have so far been essentially restricted to the fields of development and tumor progression. The aim of this review is to survey recent data of the literature on the still poorly documented role of the Sulfs during inflammation, and to widen the perspectives for the study of this intriguing regulatory mechanism toward new physiopathological processes.

Mass spectrometry analysis of the human endosulfatase Hsulf-2.

The human 6-O-endosulfatases HSulf-1 and -2 catalyze the region-selective hydrolysis of the 6-O-sulfate group of the glucosamine residues within sulfated domains of heparan sulfate, thereby ensuring a unique and original post-biosynthetic modification of the cell surface proteoglycans. While numerous studies point out the role of HSulf-2 in crucial physiological processes as well as in pathological conditions particularly in cancer, its structural organization in two chains and its functional properties remain poorly understood. In this study, we report the first characterization by mass spectrometry (MS) of HSulf-2. An average molecular weight of 133,115 Da was determined for the whole enzyme by MALDI-TOF MS, i.e. higher than the naked amino acid backbone (98,170 Da), highlighting a significant contribution of post-translational modifications. The HSulf-2 protein sequence was determined by Nano-LC-MS/MS, leading to 63% coverage and indicating at least four N-glycosylation sites at Asn 108, 147, 174 and 217. These results provide a platform for further structural investigations of the HSulf enzymes, aiming at deciphering the role of each chain in the substrate binding and specificities and in the catalytic activities.

Heparan sulfate in chronic kidney diseases: Exploring the role of 3-O-sulfation.

One of the main feature of chronic kidney disease is the development of renal fibrosis. Heparan Sulfate (HS) is involved in disease development by modifying the function of growth factors and cytokines and creating chemokine gradients. In this context, we aimed to understand the function of HS sulfation in renal fibrosis. Using a mouse model of renal fibrosis, we found that total HS 2-O-sulfation was increased in damaged kidneys, whilst, tubular staining of HS 3-O-sulfation was decreased. The expression of HS modifying enzymes significantly correlated with the development of fibrosis with HS3ST1 demonstrating the strongest correlation. The pro-fibrotic factors TGFß1 and TGFß2/IL1ß significantly downregulated HS3ST1 expression in both renal epithelial cells and renal fibroblasts. To determine the implication of HS3ST1 in growth factor binding and signalling, we generated an in vitro model of renal epithelial cells overexpressing HS3ST1 (HKC8-HS3ST1). Heparin Binding EGF like growth factor (HB-EGF) induced rapid, transient STAT3 phosphorylation in control HKC8 cells. In contrast, a prolonged response was demonstrated in HKC8-HS3ST1 cells. Finally, we showed that both HS 3-O-sulfation and HB-EGF tubular staining were decreased with the development of fibrosis. Taken together, these data suggest that HS 3-O-sulfation is modified in fibrosis and highlight HS3ST1 as an attractive biomarker of fibrosis progression with a potential role in HB-EGF signalling.

High sodium diet converts renal proteoglycans into pro-inflammatory mediators in rats.

BACKGROUND: High dietary sodium aggravates renal disease by affecting blood pressure and by its recently shown pro-inflammatory and pro-fibrotic effects. Moreover, pro-inflammatory modification of renal heparan sulfate (HS) can induce tissue remodeling. We aim to investigate if high sodium intake in normotensive rats converts renal HS into a pro-inflammatory phenotype, able to bind more sodium and orchestrate inflammation, fibrosis and lymphangiogenesis. METHODS: Wistar rats received a normal diet for 4 weeks, or 8% NaCl diet for 2 or 4 weeks. Blood pressure was monitored, and plasma, urine and tissue collected. Tissue sodium was measured by flame spectroscopy. Renal HS and tubulo-interstitial remodeling were studied by biochemical, immunohistochemical and qRT-PCR approaches. RESULTS: High sodium rats showed a transient increase in blood pressure (week 1; p<0.01) and increased sodium excretion (p<0.05) at 2 and 4 weeks compared to controls. Tubulo-interstitial T-cells, myofibroblasts and mRNA levels of VCAM1, TGF-ß1 and collagen type III significantly increased after 4 weeks (all p<0.05). There was a trend for increased macrophage infiltration and lymphangiogenesis (both p = 0.07). Despite increased dermal sodium over time (p<0.05), renal concentrations remained stable. Renal HS of high sodium rats showed increased sulfation (p = 0.05), increased L-selectin binding to HS (p<0,05), and a reduction of sulfation-sensitive anti-HS mAbs JM403 (p<0.001) and 10E4 (p<0.01). Hyaluronan expression increased under high salt conditions (p<0.01) without significant changes in the chondroitin sulfate proteoglycan versican. Statistical analyses showed that sodium-induced tissue remodeling responses partly correlated with observed HS changes. CONCLUSION: We show that high salt intake by healthy normotensive rats convert renal HS into high sulfated pro-inflammatory glycans involved in tissue remodeling events, but not in increased sodium storage.