Sulfatase-Induced In Situ Formulation of Antineoplastic Supra-PROTACs.

Proteolysis targeting chimera (PROTAC) technology is an innovative strategy for cancer therapy, which, however, suffers from poor targeting delivery and limited capability for protein of interest (POI) degradation. Here, we report a strategy for the in situ formulation of antineoplastic Supra-PROTACs via intracellular sulfatase-responsive assembly of peptides. Coassembling a sulfated peptide with two ligands binding to ubiquitin VHL and Bcl-xL leads to the formation of a pro-Supra-PROTAC, in which the ratio of the two ligands is rationally optimized based on their protein binding affinity. The resulting pro-Supra-PROTAC precisely undergoes enzyme-responsive assembly into nanofibrous Supra-PROTACs in cancer cells overexpressing sulfatase. Mechanistic studies reveal that the pro-Supra-PROTACs selectively cause apparent cytotoxicity to cancer cells through the degradation of Bcl-xL and the activation of caspase-dependent apoptosis, during which the rationally optimized ligand ratio improves the bioactivity for POI degradation and cell death. In vivo studies show that in situ formulation enhanced the tumor accumulation and retention of the pro-Supra-PROTACs, as well as the capability for inhibiting tumor growth with excellent biosafety when coadministrating with chemodrugs. Our findings provide a new approach for enzyme-regulated assembly of peptides in living cells and the development of PROTACs with high targeting delivering and POI degradation efficiency.

Biocatalytic Conversion of Carrageenans for the Production of 3,6-Anhydro-D-galactose.

Marine biomass stands out as a sustainable resource for generating value-added chemicals. In particular, anhydrosugars derived from carrageenans exhibit a variety of biological functions, rendering them highly promising for utilization and cascading in food, cosmetic, and biotechnological applications. However, the limitation of available sulfatases to break down the complex sulfation patterns of carrageenans poses a significant limitation for the sustainable production of valuable bioproducts from red algae. In this study, we screened several carrageenolytic polysaccharide utilization loci for novel sulfatase activities to assist the efficient conversion of a variety of sulfated galactans into the target product 3,6-anhydro-D-galactose. Inspired by the carrageenolytic pathways in marine heterotrophic bacteria, we systematically combined these novel sulfatases with other carrageenolytic enzymes, facilitating the development of the first enzymatic one-pot biotransformation of ι- and κ-carrageenan to 3,6-anhdyro-D-galactose. We further showed the applicability of this enzymatic bioconversion to a broad series of hybrid carrageenans, rendering this process a promising and sustainable approach for the production of value-added biomolecules from red-algal feedstocks.

To gel or not to gel - Tuning the sulfation pattern of carrageenans to expand their field of application.

Carrageenans represent a major cell wall component of red macro algae and, as established gelling and thickening agents, they contribute significantly to a broad variety of commercial applications in the food and cosmetic industry. As a highly sulfated class of linear polysaccharides, their functional properties are strongly related to the sulfation pattern of their carrabiose repeating units. Therefore, the biocatalytic fine-tuning of these polymers by generating tailored sulfation architectures harnessing the hydrolytic activity of sulfatases could be a powerful tool to produce novel polymer structures with tuned properties to expand applications of carrageenans beyond their current use. To contribute to this vision, we sought to identify novel carrageenan sulfatases by studying several putative carrageenolytic clusters in marine heterotrophic bacteria. This approach revealed two novel formylglycine-dependent sulfatases from Cellulophaga algicola DSM 14237 and Cellulophaga baltica DSM 24729 with promiscuous hydrolytic activity towards the sulfated galactose in the industrially established ι- and κ-carrageenan, converting them into α- and ß-carrageenan, respectively, and enabling the production of a variety of novel pure and hybrid carrageenans. The rheological analysis of these enzymatically generated structures revealed significantly altered physicochemical properties that may open the gate to a variety of novel carrageenan-based applications.

Cloning and Characterization of a Novel N-Acetyl-D-galactosamine-4-O-sulfate Sulfatase, SulA1, from a Marine Arthrobacter Strain.

Sulfation is gaining increased interest due to the role of sulfate in the bioactivity of many polysaccharides of marine origin. Hence, sulfatases, enzymes that control the degree of sulfation, are being more extensively researched. In this work, a novel sulfatase (SulA1) encoded by the gene sulA1 was characterized. The sulA1-gene is located upstream of a chondroitin lyase encoding gene in the genome of the marine Arthrobacter strain (MAT3885). The sulfatase was produced in Escherichia coli. Based on the primary sequence, the enzyme is classified under sulfatase family 1 and the two catalytic residues typical of the sulfatase 1 family-Cys57 (post-translationally modified to formyl glycine for function) and His190-were conserved. The enzyme showed increased activity, but not improved stability, in the presence of Ca2+, and conserved residues for Ca2+ binding were identified (Asp17, Asp18, Asp277, and Asn278) in a structural model of the enzyme. The temperature and pH activity profiles (screened using p-nitrocatechol sulfate) were narrow, with an activity optimum at 40-50 °C and a pH optimum at pH 5.5. The Tm was significantly higher (67 °C) than the activity optimum. Desulfation activity was not detected on polymeric substrates, but was found on GalNAc4S, which is a sulfated monomer in the repeated disaccharide unit (GlcA-GalNAc4S) of, e.g., chondroitin sulfate A. The position of the sulA1 gene upstream of a chondroitin lyase gene and combined with the activity on GalNAc4S suggests that there is an involvement of the enzyme in the chondroitin-degrading cascade reaction, which specifically removes sulfate from monomeric GalNAc4S from chondroitin sulfate degradation products.

Tuning of CHO secretional machinery improve activity of secreted therapeutic sulfatase 150-fold.

Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. The recombinant production of such sulfatases suffers greatly from both low product activity and yield, further limiting accessibility for patient groups. To mitigate the low product activity, we have investigated cellular properties through computational evaluation of cultures with varying media conditions and comparison of two CHO clones with different levels of one active sulfatase variant. Transcriptome analysis identified 18 genes in secretory pathways correlating with increased sulfatase production. Experimental validation by upregulation of a set of three key genes improved the specific enzymatic activity at varying degree up to 150-fold in another sulfatase variant, broadcasting general production benefits. We also identified a correlation between product mRNA levels and sulfatase activity that generated an increase in sulfatase activity when expressed with a weaker promoter. Furthermore, we suggest that our proposed workflow for resolving bottlenecks in cellular machineries, to be useful for improvements of cell factories for other biologics as well.

A novel iPSC model reveals selective vulnerability of neurons in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is an ultra-rare, inherited lysosomal storage disease caused by mutations in the gene sulfatase modifying factor 1 (SUMF1). MSD is characterized by the functional deficiency of all sulfatase enzymes, leading to the storage of sulfated substrates including glycosaminoglycans (GAGs), sulfolipids, and steroid sulfates. Patients with MSD experience severe neurological impairment, hearing loss, organomegaly, corneal clouding, cardiac valve disease, dysostosis multiplex, contractures, and ichthyosis. Here, we generated a novel human model of MSD by reprogramming patient peripheral blood mononuclear cells to establish an MSD induced pluripotent stem cell (iPSC) line (SUMF1 p.A279V). We also generated an isogenic control iPSC line by correcting the pathogenic variant with CRISPR/Cas9 gene editing. We successfully differentiated these iPSC lines into neural progenitor cells (NPCs) and NGN2-induced neurons (NGN2-iN) to model the neuropathology of MSD. Mature neuronal cells exhibited decreased SUMF1 gene expression, increased lysosomal stress, impaired neurite outgrowth and maturation, reduced sulfatase activities, and GAG accumulation. Interestingly, MSD iPSCs and NPCs did not exhibit as severe of phenotypes, suggesting that as neurons differentiate and mature, they become more vulnerable to loss of SUMF1. In summary, we demonstrate that this human iPSC-derived neuronal model recapitulates the cellular and biochemical features of MSD. These cell models can be used as tools to further elucidate the mechanisms of MSD pathology and for the development of therapeutics.

Tandem mass spectrometric assay of N-acetylglucosamine-6-sulfatase for multiplex analysis of mucopolysaccharidosis-IIID in dried blood spots.

Previously we developed a multiplex liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay using dried blood spots for all subtypes of mucopolysaccharidoses (MPS) except MPS-IIID. Here we show that the MPS-IIID enzyme N-acetylglucosamine-6-sulfatase (GNS) is inhibited in dried blood spot (DBS) extracts, but activity can be recovered if the extract is diluted to reduce the concentrations of endogenous inhibitors. The new GNS assay displays acceptable characteristics including linearity in product formation with incubation time and amount of enzyme, low variability, and ability to distinguish MPS-IIID-affected from healthy patients using DBS. The assay can be added to the LC-MS/MS multiplex panel for all MPS subtypes requiring ∼2 min per newborn for the LC-MS/MS run.

Structure and functional impact of glycosaminoglycan modification of HSulf-2 endosulfatase revealed by atomic force microscopy and mass spectrometry.

The human sulfatase HSulf-2 is one of only two known endosulfatases that play a decisive role in modulating the binding properties of heparan sulfate proteoglycans on the cell surface and in the extracellular matrix. Recently, HSulf-2 was shown to exhibit an unusual post-translational modification consisting of a sulfated glycosaminoglycan chain. This study describes the structural characterization of this glycosaminoglycan (GAG) and provides new data on its impact on the catalytic properties of HSulf-2. The unrevealed nature of this GAG chain is identified as a chondroitin/dermatan sulfate (CS/DS) mixed chain, as shown by mass spectrometry combined with NMR analysis. It consists primarily of 6-O and 4-O monosulfated disaccharide units, with a slight predominance of the 4-O-sulfation. Using atomic force microscopy, we show that this unique post-translational modification dramatically impacts the enzyme hydrodynamic volume. We identified human hyaluronidase-4 as a secreted hydrolase that can digest HSulf-2 GAG chain. We also showed that HSulf-2 is able to efficiently 6-O-desulfate antithrombin III binding pentasaccharide motif, and that this activity was enhanced upon removal of the GAG chain. Finally, we identified five N-glycosylation sites on the protein and showed that, although required, reduced N-glycosylation profiles were sufficient to sustain HSulf-2 integrity.

Synthesis and Biological Activity of Sulfamate-Adamantane Derivatives as Glucosinolate Sulfatase Inhibitors.

The glucosinolate-myrosinase system, exclusively found in the Brassicaceae family, is a main defense strategy against insect resistance. The efficient detoxification activity of glucosinolate sulfatases (GSSs) has successfully supported the feeding of Plutella xylostella on cruciferous plants. With the activity of GSSs hampered in P. xylostella, the toxic isothiocyanates produced from glucosinolates severely impair larval growth and adult reproduction. Therefore, inhibitors of GSSs have been suggested as an alternative approach to controlling P. xylostella. Herein, we synthesized eight adamantyl-possessing sulfamate derivatives as novel inhibitors of GSSs. Adam-20-S exhibited the most potent GSS inhibitory activity, with an IC50 value of 9.04 mg/L. The suppression of GSSs by Adam-20-S impaired glucosinolate metabolism to produce more toxic isothiocyanates in P. xylostella. Consequently, the growth and development of P. xylostella were significantly hindered when feeding on the host plant. Our study may help facilitate the development of a comprehensive pest management strategy that combines insect detoxification enzyme inhibitors with plant chemical defenses.

Assessment of changes in Streptococcus pyogenes levels using N-acetylgalactosamine-6-sulfatase marker and pharyngeal airway space with appliance therapy in mouth breathers - An ELISA-based study.

Background: The frequency of adenotonsillar hypertrophy in mouth-breathing children when compared to the average found in the general population is considered to be higher. Mouth breathing is considered as one of the causative factors for tonsillitis in children. Through continuous irritation on tonsillar wall, tonsils swell up and inflammation develops. Purpose: The purpose of the study is to evaluate Streptococcus pyogenes count using colony-forming units (CFUs) and N-acetylgalactosamine-6-sulfatase side chain marker on ELISA (enzyme linked immunosorbent assay) in mouth breathers and to establish its correlation with pharyngeal airway space pre- and post-oral screen appliance therapy. Materials and Methods: A total number of 24 (n) mouth breathers aged between 5 and 12 years were included in the study and given oral screen appliance therapy. The subjects were evaluated for the various parameters before the delivery of a habit-breaking appliance and then reevaluated for the same parameters (presence of S. pyogenes and its counts, size of tonsils, and pharyngeal airway space dimensions) after 6 months of appliance usage. Results: A statistically significant difference was seen in levels of S. pyogenes using ELISA and CFUs. Furthermore, statistically significant difference was observed in Friedman tonsil scoring and pharyngeal airway space and pre- and post-oral screen appliance therapy. Conclusion: Oral screen appliance therapy reduced the frequency of occurrence of tonsillitis in mouth breathers by decreasing the counts of S. pyogenes bacteria. Upper and lower pharyngeal airway space dimensions were increased after 6 months of appliance therapy in mouth breathers.

Immobilized Enzymes on Magnetic Beads for Separate Mass Spectrometric Investigation of Human Phase II Metabolite Classes.

The human body has evolved to remove xenobiotics through a multistep clearance process. Non-endogenous metabolites are converted through a series of phase I and different phase II enzymes into compounds with higher hydrophilicity. These compounds are important for diverse research fields such as toxicology, nutrition, biomarker discovery, doping control, and microbiome metabolism. One of the challenges in these research fields has been the investigation of the two major phase II modifications, sulfation and glucuronidation, and the corresponding unconjugated aglycon independently. We have now developed a new methodology utilizing an immobilized arylsulfatase and an immobilized ß-glucuronidase to magnetic beads for treatment of human urine samples. The enzyme activities remained the same compared to the enzyme in solution. The separate mass spectrometric investigation of each metabolite class in a single sample was successfully applied to obtain the dietary glucuronidation and sulfation profile of 116 compounds. Our new chemical biology strategy provides a new tool for the investigation of metabolites in biological samples with the potential for broad-scale application in metabolomics, nutrition, and microbiome studies.

New Insights into the Plutella xylostella Detoxifying Enzymes: Sequence Evolution, Structural Similarity, Functional Diversity, and Application Prospects of Glucosinolate Sulfatases.

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.

Real-time visualization of sulfatase in living cells and in vivo with a ratiometric AIE fluorescent probe.

Different from the traditional enzymatic hydrolysis strategy, we rationally developed a ratiometric fluorescence probe DQMT-OH with AIE characteristics for sulfatase detection utilizing the "Lock-Key" strategy. It can be successfully used to monitor sulfatase in living cells and in vivo through different fluorescent channels with good cell permeability and low cytotoxicity.

A novel fluorescent probe for rapid detection of sulfatase in vitro and in living cells.

Sulfatase participates in a variety of physiological processes in organisms including hormone regulation, cell signaling, and bacterial pathogenesis. Current sulfatase fluorescent probes can be used to track sulfate esterase overexpression in cancer cells for diagnostic purposes and to understand the pathological activity of sulfate esterase. However, some sulfatase fluorescent probes based on the hydrolysis of the sulfate bond were easily disturbed by the catalytic activity of sulfatase. Herein, we developed the fluorescent probe BQM-NH2 for sulfatase detection, which was based on the quinoline-malononitrile. The probe BQM-NH2 showed a fast response to sulfatase within 1 min and satisfactory sensitivity with a calculated LOD of 1.73 U/L. Importantly, it was successfully used to monitor endogenous sulfate in tumor cells, indicating BQM-NH2 has the potential to monitor sulfatase under physiological and pathological conditions.

Association between SUMF1 polymorphisms and COVID-19 severity.

BACKGROUND: Evidence shows that genetic factors play important roles in the severity of coronavirus disease 2019 (COVID-19). Sulfatase modifying factor 1 (SUMF1) gene is involved in alveolar damage and systemic inflammatory response. Therefore, we speculate that it may play a key role in COVID-19. RESULTS: We found that rs794185 was significantly associated with COVID-19 severity in Chinese population, under the additive model after adjusting for gender and age (for C allele = 0.62, 95% CI = 0.44-0.88, P = 0.0073, logistic regression). And this association was consistent with this in European population Genetics Of Mortality In Critical Care (GenOMICC: OR for C allele = 0.94, 95% CI = 0.90-0.98, P = 0.0037). Additionally, we also revealed a remarkable association between rs794185 and the prothrombin activity (PTA) in subjects (P = 0.015, Generalized Linear Model). CONCLUSIONS: In conclusion, our study for the first time identified that rs794185 in SUMF1 gene was associated with the severity of COVID-19.

Revealing the roles of glycosphingolipid metabolism pathway in the development of keloid: a conjoint analysis of single-cell and machine learning.

Keloid is a pathological scar formed by abnormal wound healing, characterized by the persistence of local inflammation and excessive collagen deposition, where the intensity of inflammation is positively correlated with the size of the scar formation. The pathophysiological mechanisms underlying keloid formation are unclear, and keloid remains a therapeutic challenge in clinical practice. This study is the first to investigate the role of glycosphingolipid (GSL) metabolism pathway in the development of keloid. Single cell sequencing and microarray data were applied to systematically analyze and screen the glycosphingolipid metabolism related genes using differential gene analysis and machine learning algorithms (random forest and support vector machine), and a set of genes, including ARSA,GBA2,SUMF2,GLTP,GALC and HEXB, were finally identified, for which keloid diagnostic model was constructed and immune infiltration profiles were analyzed, demonstrating that this set of genes could serve as a new therapeutic target for keloid. Further unsupervised clustering was performed by using expression profiles of glycosphingolipid metabolism genes to discover keloid subgroups, immune cells, inflammatory factor differences and the main pathways of enrichment between different subgroups were calculated. The single-cell resolution transcriptome landscape concentrated on fibroblasts. By calculating the activity of the GSL metabolism pathway for each fibroblast, we investigated the activity changes of GSL metabolism pathway in fibroblasts using pseudotime trajectory analysis and found that the increased activity of the GSL metabolism pathway was associated with fibroblast differentiation. Subsequent analysis of the cellular communication network revealed the existence of a fibroblast-centered communication regulatory network in keloids and that the activity of the GSL metabolism pathway in fibroblasts has an impact on cellular communication. This contributes to the further understanding of the pathogenesis of keloids. Overall, we provide new insights into the pathophysiological mechanisms of keloids, and our results may provide new ideas for the diagnosis and treatment of keloids.

Between the Devil and the Deep Blue Sea-Resveratrol, Sulfotransferases and Sulfatases-A Long and Turbulent Journey from Intestinal Absorption to Target Cells.

For nearly 30 years, resveratrol has attracted the scientific community's interest. This has happened thanks to the so-called French paradox, that is, the paradoxically low mortality from cardiovascular causes in the French population despite a diet rich in saturated fat. This phenomenon has been linked to the consumption of red wine, which contains a relatively high level of resveratrol. Currently, resveratrol is valued for its versatile, beneficial properties. Apart from its anti-atherosclerotic activity, resveratrol's antioxidant and antitumor properties deserve attention. It was shown that resveratrol inhibits tumour growth at all three stages: initiation, promotion, and progression. Moreover, resveratrol delays the ageing process and has anti-inflammatory, antiviral, antibacterial, and phytoestrogenic properties. These favorable biological properties have been demonstrated in vitro and in vivo in animal and human models. Since the beginning of the research on resveratrol, its low bioavailability, mainly due to its rapid metabolism, especially the first-pass effect that leaves almost no free resveratrol in the peripheral circulation, has been indicated as a drawback that has hindered its use. The elucidation of such issues as pharmacokinetics, stability, and the biological activity of resveratrol metabolites is therefore crucial for understanding the biological activity of resveratrol. Second-phase metabolism enzymes are mainly involved in RSV metabolism, e.g., UDP-glucuronyl transferases and sulfotransferases. In the present paper, we took a closer look at the available data on the activity of resveratrol sulfate metabolites and the role of sulfatases in releasing active resveratrol in target cells.

Late infantile form of multiple sulfatase deficiency with a novel missense variant in the SUMF1 gene: case report and review.

BACKGROUND: Multiple sulfatase deficiency (MSD) is a rare lysosomal storage disorder caused due to pathogenic variants in the SUMF1 gene. The SUMF1 gene encodes for formylglycine generating enzyme (FGE) that is involved in the catalytic activation of the family of sulfatases. The affected patients present with a wide spectrum of clinical features including multi-organ involvement. To date, almost 140 cases of MSD have been reported worldwide, with only four cases reported from India. The present study describes two cases of late infantile form of MSD from India and the identification of a novel missense variant in the SUMF1 gene. CASE PRESENTATION: In case 1, a male child presented to us at the age of 6 years. The remarkable presenting features included ichthyosis, presence of irritability, poor social response, thinning of corpus callosum on MRI and, speech regression. Clinical suspicion of MSD was confirmed by enzyme analysis of two sulfatase enzymes followed by gene sequencing. We identified a novel missense variant c.860A > T (p.Asn287Ile) in exon 7 of the SUMF1 gene. In case 2, a two and a half years male child presented with ichthyosis, leukodystrophy and facial dysmorphism. We performed an enzyme assay for two sulfatases, which showed significantly reduced activities thereby confirming MSD diagnosis. CONCLUSION: Overall, present study has added to the existing data on MSD from India. Based on the computational analysis, the novel variant c.860A > T identified in this study is likely to be associated with a milder phenotype and prolonged survival.

Regulation of morphogen pathways by a Drosophila chondroitin sulfate proteoglycan Windpipe.

Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. We found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. Our study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways.

A novel 4-O-endosulfatase with high potential for the structure-function studies of chondroitin sulfate/dermatan sulfate.

The sulfation patterns of chondroitin sulfate (CS)/dermatan sulfate (DS), which encode unique biological information, play critical roles in the various biological functions of CS/DS chains. CS/DS sulfatases, which can specifically hydrolyze sulfate groups, could potentially be essential tools for deciphering and changing the biological information encoded by these sulfation patterns. However, endosulfatase with high activity to efficiently hydrolyze the sulfate groups inside CS/DS polysaccharides have rarely been identified, which hinders the practical applications of CS/DS sulfatases. Herein, a novel CS/DS 4-O-endosulfatase (endoBI4SF) with a strong ability to completely remove 4-O-sulfated groups inside various CS/DS polysaccharides was identified and successfully used to investigate the biological roles of 4-O-sulfated CS/DS in vitro and in vivo. This study provides a much-needed tool to tailor the sulfation patterns and explore the related functions of 4-O-sulfated CS/DS chains in vitro and in vivo.