Fucoidans belong to a structurally heterogeneous class of sulfated polysaccharides isolated from brown algae. They have a wide spectrum of biological activities. The complex structures of these polysaccharides hinder structure-activity relationships determination. Fucoidan sulfatases can make useful tools for the determination of the fine chemical structure of fucoidans. In this study, identification and preparation of two recombinant sulfatases able to catalyze the cleavage of sulfate groups from fragments of fucoidan molecules is described for the first time. Two genes of sulfatases swf1 and swf4 of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T were cloned and the proteins were produced in Escherichia coli cells. Sulfatases SWF1 and SWF4 are assigned to S1_17 and S1_25 subfamilies of formylglycine-dependent enzymes of S1 family (SulfAtlas). Some molecular and biochemical characteristics of recombinant fucoidan sulfatases have been studied. Detailed specificity and catalytic features of sulfatases were determined using various sulfated fucooligosaccharides. Structures of products produced by SWF1 and SWF4 were established by nuclear magnetic resonance (NMR) spectroscopy. Based on the obtained data, the enzymes are classified as fucoidan exo-2O-sulfatase (SWF1) and fucoidan exo-3O-sulfatase (SWF4). In addition, we demonstrated the sequential action of sulfatases on 2,3-di-O-sulfated fucooligosacchrides, which indicates an exolitic degradation pathway of fucoidan by a marine bacterium W. fucanilytica CZ1127T.

Sulfated glycans are ubiquitous nutrient sources for microbial communities that have coevolved with eukaryotic hosts. Bacteria metabolize sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognize their glycan substrate remain poorly understood. Here, we use structural biology to determine how sulfatases from the human gut microbiota recognize sulfated glycans. We reveal seven new carbohydrate sulfatase structures spanning four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent novel structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity toward related but distinct glycan targets. Collectively, these data reveal that carbohydrate sulfatases are highly selective for the glycan component of their substrate. These data provide new approaches for probing sulfated glycan metabolism while revealing the roles carbohydrate sulfatases play in host glycan catabolism.

Glycosaminoglycan (GAG) sulfatases, which catalyze the hydrolysis of sulfate esters from GAGs, belong to a large and conserved sulfatase family. Bacterial GAG sulfatases are essential in the process of sulfur cycling and are useful for the structural analysis of GAGs. Only a few GAG-specific sulfatases have been studied in detail and reported to date. Herein, the GAG-degrading Photobacterium sp. FC615 was isolated from marine sediment, and a novel Δ4,5hexuronate-2-O-sulfatase (PB2SF) was identified from this bacterium. PB2SF specifically removed 2-O-sulfate from the unsaturated hexuronate residue located at the non-reducing end of GAG oligosaccharides produced by GAG lyases. A structural model of PB2SF was constructed through a homology-modeling method. Six conserved amino acids around the active site were chosen for further analysis using site-directed mutagenesis. N113A, K141A, K141H, H143A, H143K, H205A, and H205K mutants exhibited only feeble activity, while the H310A, H310K, and D52A mutants were totally inactive, indicating that these conserved residues, particularly Asp52 and His310, were essential in the catalytic mechanism. Furthermore, bioinformatic analysis revealed that GAG sulfatases with specific degradative properties clustered together in the neighbor-joining phylogenetic tree. Based on this finding, 60 Δ4,5hexuronate-2-O-sulfatases were predicted in the NCBI protein database, and one with relatively low identity to PB2SF was characterized to confirm our prediction. Moreover, the signature sequences of bacterial Δ4,5hexuronate-2-O-sulfatases were identified. With the reported signature motifs, the sulfatase sequence of the Δ4,5hexuronate-2-O-sulfatase family could be simply identified before cloning. Taken together, the results of this study should aid in the identification and further application of novel GAG sulfatases.

Glucosinolates, a characteristic group of specialized metabolites found in Brassicales plants, are converted to toxic isothiocyanates upon herbivory. Several insect herbivores, including the cabbage stem flea beetle (Psylliodes chrysocephala), prevent glucosinolate activation by forming desulfo-glucosinolates. Here we investigated the molecular basis of glucosinolate desulfation in P. chrysocephala, an important pest of oilseed rape. Enzyme activity assays with crude beetle protein extracts revealed that glucosinolate sulfatase (GSS) activity is associated with the gut membrane and has narrow substrate specificity towards the benzenic glucosinolate sinalbin. In agreement with GSS activity localization in vivo, we identified six genes encoding arylsulfatase-like enzymes with a predicted C-terminal transmembrane domain, of which five showed GSS activity upon heterologous expression in insect cells. PcGSS1 and PcGSS2 used sinalbin and indol-3-ylmethyl glucosinolate as substrates, respectively, whereas PcGSS3, PcGSS4, and PcGSS5 showed weak activity in enzyme assays. RNAi-mediated knock-down of PcGSS1 and PcGSS2 expression in adult beetles confirmed their function in vivo. In a phylogenetic analysis of coleopteran and lepidopteran arylsulfatases, the P. chrysocephala GSSs formed a cluster within a coleopteran-specific sulfatase clade distant from the previously identified GSSs of the diamondback moth, Plutella xylostella, suggesting an independent evolution of GSS activity in ermine moths and flea beetles.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death world-wide. Recently, we showed that COPD is associated with gene polymorphisms in SUMF1, a master regulator of sulfatases. Sulfatases are involved in extracellular matrix remodeling and activated by SUMF1, but their role in the lung is poorly described. We aimed to examine how sulfatases are affected in the airways of patients with COPD compared to ever smokers and never smokers. We observed that mRNA expression of the sulfatases GALNS, GNS and IDS was increased, while protein expression of many sulfatases was decreased in COPD fibroblasts. Several sulfatases, including GALNS, IDS, and SGSH, showed increased activity in COPD fibroblasts. Examination of different sulfatases by immunofluorescence showed that IDS, ARSB, GNS and SGSH in fibroblasts were localized to sites other than their reported destination. Using a master panel from different organs, RNA expression of all sulfatases could be observed in lung tissue. Additionally, immunohistochemistry on lung biopsies indicated differing expression of sulfatases in COPD patients. In conclusion, mRNA, protein expression, sulfatase activity levels, and localization of sulfatases are altered in lung fibroblasts and lung tissue from COPD patients and may be mechanistically important in COPD pathogenesis. This could contribute to the understanding of the disease mechanism in COPD and in the long run, to lead to more individualized therapies.

Heparan sulfate proteoglycans (HSPGs) use highly sulfated polysaccharide side-chains to interact with several key growth factors and morphogens, thereby regulating their accessibility and biological activity. Various sulfotransferases and sulfatases with differing specificities control the pattern of HSPG sulfation, which is functionally critical. Among these enzymes in the mouse are two secreted 6-O-endosulfatases, Sulf1 and Sulf2, which modify HSPGs in the extracellular matrix and on the cell surface. The roles of Sulf1 and Sulf2 during normal development are not well understood.

Changes in sulfation of cartilage glycosaminoglycans as mediated by sulfatases can regulate growth factor signaling. The aim of this study was to analyze expression patterns of recently identified extracellular sulfatases Sulf-1 and Sulf-2 in articular cartilage and chondrocytes.

The 6-O-endosulfatases (sulfs) are important enzymatic components involved in the regulation of heparan sulfate by altering the sulfatation pattern. Specifically in the kidney, sulfs have been implicated in the glomerular podocyte-endothelial cell crosstalk and in the preservation of the glomerular filtration barrier (GFB) in different mouse models. Since it has been shown that in zebrafish larvae, Sulf1, Sulf2a, and Sulf2b are expressed in the pronephric kidney we set out to establish if a reduction in sulf expression leads to GFB dysfunction. Here, we show that a reduced sulf expression following morpholino (MO) induced knockdown in zebrafish larvae promotes damage to the GFB leading to renal plasma protein loss from the circulation. Moreover, a combined knockdown of Sulf1, Sulf2a, and Sulf2b is associated with severe morphologic changes including narrowing of the fenestration between glomerular endothelial cells as well as thickening of the glomerular basement membrane and podocyte foot process effacement, suggesting that glomerular damage is an underlying cause of the circulatory protein loss observed after MO injection. Additionally, we show that a decrease in sulf expression reduces the bioavailability of VegfA in the glomerulus of the pronephros, which may contribute to the structural changes observed in the glomeruli of morphant fish. Furthermore, consistent with previous results, knockdown of the sulfs is associated with arteriovenous malformations in particular in the tail region of the larvae. Overall, taken together our results suggest that 6-O-endosulfatases are important in the preservation of GFB integrity and a reduction in their expression levels induces phenotypic changes that are indicative of renal protein loss.

Chondroitin sulfate/dermatan sulfate (CS/DS) sulfatases are potential tools for structural and functional studies of CD/DS chains. In our previous study, a CS/DS 4-O-endosulfatase (endoVB4SF) was identified from a marine bacterium (Wang et al., 2015). Herein, another CS/DS 4-O-sulfatase (exoPB4SF) was identified from a Photobacterium sp. ExoPB4SF shares an 83% identity with endoVB4SF but showed strict exolytic activity. Comparative studies were performed for both enzymes on the basis of biochemical features, substrate-degrading patterns and three-dimensional structures. exoPB4SF exhibited a wider temperature and pH adaptability and better thermostability than endoVB4SF. Furthermore, exoPB4SF is a strict exolytic sulfatase that only releases the sulfate group from the GalNAc residue located at the reducing end, whereas endoVB4SF preferentially removed sulfate esters from the reducing end toward the non-reducing end though its directional degradation property was not strict. In addition, the structure of endoVB4SF was determined by X-ray crystallography at 1.95 Å. It adopts a globular conformation with two monomers per asymmetric unit. The exoPB4SF structure was constructed by homology modeling. Molecular docking results showed that although the residues around the catalytic center are conserved, the residues at the active site of endoVB4SF adopted a more favorable conformation for the binding of long CS/DS chains than those of exoPB4SF, which may explain why the two highly homogenous sulfatases possessed different action patterns. The results of this study provide insight into the structure-function relationship of CS/DS endo- and exosulfatases for the first time.

Many algae are rich sources of sulfated polysaccharides with biological activities. The physicochemical/rheological properties and biological activities of sulfated polysaccharides are affected by the pattern and number of sulfate moieties. Sulfation of carbohydrates is catalyzed by carbohydrate sulfotransferases (CHSTs) while modification of sulfate moieties on sulfated polysaccharides was presumably catalyzed by sulfatases including formylglycine-dependent sulfatases (FGly-SULFs). Post-translationally modification of Cys to FGly in FGly-SULFs by sulfatase modifiying factors (SUMFs) is necessary for the activity of this enzyme. The aims of this study are to mine for sequences encoding algal CHSTs, FGly-SULFs and putative SUMFs from the fully sequenced algal genomes and to infer their phylogenetic relationships to their well characterized counterparts from other organisms. Algal sequences encoding CHSTs, FGly-SULFs, SUMFs, and SUMF-like proteins were successfully identified from green and brown algae. However, red algal FGly-SULFs and SUMFs were not identified. In addition, a group of SUMF-like sequences with different gene structure and possibly different functions were identified for green, brown and red algae. The phylogeny of these putative genes contributes to the corpus of knowledge of an unexplored area. The analyses of these putative genes contribute toward future production of existing and new sulfated carbohydrate polymers through enzymatic synthesis and metabolic engineering.

Heparan sulfate proteoglycans (HSPGs) are control elements in Wnt signaling, which bind extracellularly to Wnt ligands and regulate their ability to interact with signal transduction receptors on the cell surface. Sulf-1 and Sulf-2 are novel extracellular sulfatases that act on internal glucosamine-6-sulfate (6S) modifications within HSPGs and thereby modulate HSPG interactions with various signaling molecules, including Wnt ligands. Emerging evidence indicates the importance of reactivated Wnt signaling in a number of cancers, including pancreatic adenocarcinoma.

Sulfatases cleave sulfate groups from various molecules and constitute a biologically and industrially important group of enzymes. However, the number of sulfatases whose substrate has been characterized is limited in comparison to the huge diversity of sulfated compounds, yielding functional annotations of sulfatases particularly prone to flaws and misinterpretations. In the context of the explosion of genomic data, a classification system allowing a better prediction of substrate specificity and for setting the limit of functional annotations is urgently needed for sulfatases. Here, after an overview on the diversity of sulfated compounds and on the known sulfatases, we propose a classification database, SulfAtlas (http://abims.sb-roscoff.fr/sulfatlas/), based on sequence homology and composed of four families of sulfatases. The formylglycine-dependent sulfatases, which constitute the largest family, are also divided by phylogenetic approach into 73 subfamilies, each subfamily corresponding to either a known specificity or to an uncharacterized substrate. SulfAtlas summarizes information about the different families of sulfatases. Within a family a web page displays the list of its subfamilies (when they exist) and the list of EC numbers. The family or subfamily page shows some descriptors and a table with all the UniProt accession numbers linked to the databases UniProt, ExplorEnz, and PDB.

Corneal epithelial wound repair involves the migration of epithelial cells to cover the defect followed by the proliferation of the cells to restore thickness. Heparan sulfate proteoglycans (HSPGs) are ubiquitous extracellular molecules that bind to a plethora of growth factors, cytokines, and morphogens and thereby regulate their signaling functions. Ligand binding by HS chains depends on the pattern of four sulfation modifications, one of which is 6-O-sulfation of glucosamine (6OS). SULF1 and SULF2 are highly homologous, extracellular endosulfatases, which post-synthetically edit the sulfation status of HS by removing 6OS from intact chains. The SULFs thereby modulate multiple signaling pathways including the augmentation of Wnt/ß-catenin signaling. We found that wounding of mouse corneal epithelium stimulated SULF1 expression in superficial epithelial cells proximal to the wound edge. Sulf1⁻/⁻, but not Sulf2⁻/⁻, mice, exhibited a marked delay in healing. Furthermore, corneal epithelial cells derived from Sulf1⁻/⁻ mice exhibited a reduced rate of migration in repair of a scratched monolayer compared to wild-type cells. In contrast, human primary corneal epithelial cells expressed SULF2, as did a human corneal epithelial cell line (THCE). Knockdown of SULF2 in THCE cells also slowed migration, which was restored by overexpression of either mouse SULF2 or human SULF1. The interchangeability of the two SULFs establishes their capacity for functional redundancy. Knockdown of SULF2 decreased Wnt/ß-catenin signaling in THCE cells. Extracellular antagonists of Wnt signaling reduced migration of THCE cells. However in SULF2- knockdown cells, these antagonists exerted no further effects on migration, consistent with the SULF functioning as an upstream regulator of Wnt signaling. Further understanding of the mechanistic action of the SULFs in promoting corneal repair may lead to new therapeutic approaches for the treatment of corneal injuries.

Biochemical, physiological and genomic comparisons of two Pseudomonas strains, assigned previously to the Pseudomonas jessenii subgroup, which are efficient SDS-degraders were carried out. A GO enrichment analysis showed that the genomes of SDS-degraders encode more genes connected with bacterial cell wall biosynthesis and alkanesulfonate monooxygenase activity than their closest relatives from the P. jessenii subgroup. A transcriptomic analysis of the most promising strain exposed to detergent suggests that although SDS can be later utilized as a carbon source, in early stages it influences cell envelope integrity, causing a global stress response followed by cell wall modification and induction of repair mechanisms. Genomes of the analyzed strains from P. jessenii group encode multiple putative sulfatases and their enzymatic activity was experimentally verified, which led to the identification of three novel enzymes exhibiting activity toward SDS. Two of the novel alkylsulfatases showed their highest activity at pH 8.0 and the temperature of 60°C or 70°C. One of the enzymes retained its activity even after 1 h of incubation at 60°C. Ions like K+ and Mg2+ enhanced enzymatic activity of both proteins, whereas Cu2+ or EDTA had inhibitory effects.

Brassica plants have glucosinolate (GLs)-myrosinase defense mechanisms to deter herbivores. However, Plutella xylostella specifically feeds on Brassica vegetables. The larvae possess three glucosinolate sulfatases (PxGSS1-3) that compete with plant myrosinase for shared GLs substrates and produce nontoxic desulfo-GLs (deGLs). Although PxGSSs are considered potential targets for pest control, the lack of a comprehensive review has hindered the development of PxGSSs-targeted pest control methods. Recent advances in integrative multi-omics analysis, substrate-enzyme kinetics, and molecular biological techniques have elucidated the evolutionary origin and functional diversity of these three PxGSSs. This review summarizes research progress on PxGSSs over the past 20 years, covering sequence properties, evolution, protein modification, enzyme activity, structural variation, substrate specificity, and interaction scenarios based on functional diversity. Finally, we discussed the potential applications of PxGSSs-targeted pest control technologies driven by artificial intelligence, including CRISPR/Cas9-mediated gene drive, transgenic plant-mediated RNAi, small-molecule inhibitors, and peptide inhibitors. These technologies have the potential to overcome current management challenges and promote the development and field application of PxGSSs-targeted pest control.

The chondroitin sulfatases N-acetylgalactosamine-4-sulfatase (ARSB) and galactosamine-N-acetyl-6-sulfatase (GALNS) remove either the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate (C4S) and dermatan sulfate, or the 6-sulfate group of chondroitin 6-sulfate, chondroitin 4,6-disulfate (chondroitin sulfate E), or keratan sulfate. In human prostate cancer tissues, the ARSB activity was reduced and the GALNS activity was increased, compared to normal prostate tissue. In human prostate stem cells, when ARSB was reduced by silencing or GALNS was increased by overexpression, activity of SHP2, the ubiquitous non-receptor tyrosine phosphatase, declined, attributable to increased binding of SHP2 with C4S. This led to increases in phospho-ERK1/2, Myc/Max nuclear DNA binding, DNA methyltransferase (DNMT) activity and expression, and methylation of the Dickkopf Wnt signaling pathway inhibitor (DKK)3 promoter and to reduced DKK3 expression. Since DKK3 negatively regulates Wnt/β-catenin signaling, silencing of ARSB or overexpression of GALNS disinhibited (increased) Wnt/β-catenin signaling. These findings indicate that the chondroitin sulfatases can exert profound effects on Wnt-mediated processes, due to epigenetic effects that modulate Wnt signaling.

Maturation of type I sulfatases requires the conversion of the cysteine (Cys) or serine (Ser) present in the active site to formylglycine (FGly). This activation represents a limiting step during the production of recombinant sulfatases in bacteria and eukaryotic hosts. AslB, YdeM and YidF have been proposed to participate in the activation of sulfatases in Escherichia coli. In this study, we combined in-silico and experimental approaches to study the interaction between Escherichia coli BL21(DE3) AslB and human sulfatases, more specifically iduronate-2-sulfate sulfatase (IDS) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS). In-silico results show that AslB has a higher affinity for the residual motif of GALNS (-9.4kcalmol-1), Cys- and Ser-type, than for the one of IDS (-8.0kcalmol-1). However, the distance between the AslB active residue and the target motif favors the interaction with IDS (4.4Å) more than with GALNS (5.5Å). Experimental observations supported in-silico results where the co-expression of AslB with GALNS Cys- and Ser-type presented an activity increment of 2.0- and 1.5-fold compared to the control cultures, lacking overexpressed AslB. Similarly, IDS activity was increased in 4.6-fold when co-expressed with AslB. The higher sulfatase activity of AslB-IDS suggests that the distance between the AslB active residue and the motif target is a key parameter for the in-silico search of potential sulfatase activators. In conclusion, our results suggest that AslB is involve in the maturation of heterologous human sulfatases in E. coli BL21(DE3), and that it can have important implications in the production of recombinant sulfatases for therapeutic purposes and research.

Multiple sulfatase deficiency (MSD) is a fatal, inherited lysosomal storage disorder characterized by reduced activities of all sulfatases in patients. Sulfatases require a unique post-translational modification of an active-site cysteine to formylglycine that is catalyzed by the formylglycine-generating enzyme (FGE). FGE mutations that affect intracellular protein stability determine residual enzyme activity and disease severity in MSD patients. Here, we show that protein disulfide isomerase (PDI) plays a pivotal role in the recognition and quality control of MSD-causing FGE variants. Overexpression of PDI reduces the residual activity of unstable FGE variants, whereas inhibition of PDI function rescues the residual activity of sulfatases in MSD fibroblasts. Mass spectrometric analysis of a PDI+FGE variant covalent complex allowed determination of the molecular signature for FGE recognition by PDI. Our findings highlight the role of PDI as a disease modifier in MSD, which may also be relevant for other ER-associated protein folding pathologies.

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current 'gold standards' for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated enzymes.

Sulfatases play a biologically important role by cleaving sulfate groups from molecules. They can be identified on the basis of signature sequences within their primary structures, and the largest family, S1, has predictable features that contribute specifically to the recognition and catalytic removal of sulfate groups. However, despite advances in the prediction and understanding of S1 sulfatases, a major question regards the molecular determinants that drive substrate recognition beyond the targeted sulfate group. Here, through analysis of an endo-4S-ι-carrageenan sulfatase (PsS1_19A) from Pseudoalteromonas sp. PS47, particularly X-ray crystal structures in complex with intact substrates, we show that specific recognition of the substrate leaving group components, in this case carbohydrate, provides the enzyme with specificity for its substrate. On the basis of these results we propose a catalytic subsite nomenclature that we anticipate will form a general foundation for understanding and describing the molecular basis of substrate recognition by sulfatases.