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Heparan sulfate proteoglycans (HSPGs) act as signaling co‐receptors by interaction of their sulfated glycosaminoglycan chains with numerous signaling molecules. In breast cancer, the function of heparan sulfate 2‐O‐sulfotransferase (HS2ST1), the enzyme mediating 2‐O‐sulfation of HS, is largely unknown. Hence, a comparative study on the functional consequences of HS2ST1 overexpression and siRNA knockdown was performed in the breast cancer cell lines MCF‐7 and MDA‐MB‐231. HS2ST1 overexpression inhibited Matrigel invasion, while its knockdown reversed the phenotype. Likewise, cell motility and adhesion to fibronectin and laminin were affected by altered HS2ST1 expression. Phosphokinase array screening revealed a general decrease in signaling via multiple pathways. Fluorescent ligand binding studies revealed altered binding of fibroblast growth factor 2 (FGF‐2) to HS2ST1‐expressing cells compared with control cells. HS2ST1‐overexpressing cells showed reduced MAPK signaling responses to FGF‐2, and altered expression of epidermal growth factor receptor (EGFR), E‐cadherin, Wnt‐7a, and Tcf4. The increased viability of HS2ST1‐depleted cells was reduced to control levels by pharmacological MAPK pathway inhibition. Moreover, MAPK inhibitors generated a phenocopy of the HS2ST1‐dependent delay in scratch wound repair. In conclusion, HS2ST1 modulation of breast cancer cell invasiveness is a compound effect of altered E‐cadherin and EGFR expression, leading to altered signaling via MAPK and additional pathways. Our results suggest that, in breast cancer, high levels of HS2ST1 result in structural changes in heparan sulfate and altered growth factor binding, which leads to attenuated signaling through the MAPK and additional pathways. Reduced signaling and expression of E‐cadherin and epidermal growth factor receptor (EGFR) is associated with reduced viability, adhesion, migration, and invasion of breast cancer cells.

ObjectivesHeparosan is used as the starting polysaccharide sulfated using sulfotransferase to generate fully elaborate heparin, a widely used clinical drug. However, the preparation of heparosan and enzymes was considered tedious since such material must be prepared in separate fermentation batches. In this study, a commonly admitted probiotic, Escherichia coli strain Nissle 1917 (EcN), was engineered to intracellularly express sulfotransferases and, simultaneously, secreting heparosan into the culture medium.ResultsThe engineered strain EcN::T7M, carrying the λDE3 region of BL21(DE3) encoding T7 RNA polymerase, expressed the sulfotransferase domain (NST) of human N-deacetylase/N-sulfotransferase-1 (NDST-1) and the catalytic domain of mouse 3-O-sulfotransferase-1 (3-OST-1) in a flask. The fed-batch fermentation of EcN::T7M carrying the plasmid expressing NST was carried out, which brought the yield of NST to 0.21 g/L and the yield of heparosan to 0.85 g/L, respectively. Furthermore, the heparosan was purified, characterized by 1H nuclear magnetic resonance (NMR), and sulfated by NST using 3′-phosphoadenosine-5′-phosphosulfate (PAPS) as the sulfo donor. The analysis of element composition showed that over 80% of disaccharide repeats of heparosan were N-sulfated.ConclusionsThese results indicate that EcN::T7M is capable of preparing sulfotransferase and heparosan at the same time. The EcN::T7M strain is also a suitable host for expressing exogenous proteins driven by tac promoter and T7 promoter.

A previously uncharacterized sulfotransferase was identified, capable of catalyzing the formation of adenosine phosphosulfate from adenosine monophosphate. Structure-guided engineering increased its catalytic activity by 93%, demonstrating the potential of rational enzyme design for cofactor biosynthesis.A synthetic pathway for 3′-phosphoadenosine-5′-phosphosulfate (PAPS) biosynthesis was constructed using adenine and d-ribose precursors, enabling high-titer production of PAPS. This pathway provides a robust platform for the sustainable supply of this essential cofactor.Integrating the PAPS biosynthesis and regeneration pathway into an engineered Escherichia coli strain facilitated the de novo biosynthesis of chondroitin sulfate A (CSA). This achievement highlights the potential of the pathway to support industrial-scale production of valuable sulfated compounds. Sulfonated compounds are widely utilized in feed additives, daily commodities, industrial manufacturing, and healthcare applications. Their production relies on the sulfonate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS). This study identified a novel adenine-sulfotransferase with sulfotransferase activity toward adenosine monophosphate for adenosine phosphosulfate formation. The identified enzyme was rationally engineered, yielding the mutant BtaAPSSTH8M/L117D, which exhibited a 0.93-fold increase in sulfotransfer efficiency. The mutant BtaAPSSTH8M/L117D was subsequently combined with additional enzymes to reconstruct what we term the RPA pathway, enabling the synthesis of PAPS at titers of 7.6 g/l and 5.03 g/l in Escherichia coli and Bacillus subtilis, respectively, using adenine and ribose as substrates. The RPA pathway was further integrated into the chondroitin producing strain E. coli GZ17, to construct E. coli CSA-02, which produced 1.89 g/l chondroitin sulfate A (CSA) with a sulfation rate of 76%. These results offer a promising way to enhance the biosynthesis of sulfonated compounds in microbial cell factories. [Display omitted] In this study, rational engineering of BtaAPSST via H8M/L117D mutations enhanced sulfotransferase efficiency by 0.93-fold, enabling efficient adenosine 5′-phosphosulfate (APS) biosynthesis. Reconstitution of the RPA pathway supported high-level-3′-phosphoadenosine-5′-phosphosulfate (PAPS) production (7.6 g/l in Escherichia coli, 5.03 g/l in Bacillus subtilis), demonstrating the scalability across divergent microbial hosts. Further integration of the RPA pathway into a chondroitin-producing strain yielded 1.89 g/l chondroitin sulfate A with a 76% sulfation degree, confirming effective metabolic coupling. The system robustness demonstrated under laboratory conditions, combined with reproducible PAPS production, supports classification at Technology Readiness Level 4 (TRL4). To address the critical bottleneck in the industrial production of sulfonated products, an enhanced synthesis pathway for the common sulfo donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) was developed. Through key enzyme mining and engineering, an efficient microbial platform was established for endogenous PAPS generation and de novo synthesis of sulfonated products.

Chondroitin sulfate E (CS-E) is a vital sulfated glycosaminoglycan with diverse biological functions and therapeutic potential. This study marks a significant milestone by achieving the first successful microbial production of chondroitin 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) in Escherichia coli , enabling recombinant CS-E biosynthesis. Initially, we identified sulfotransferases capable of converting chondroitin sulfate A (CS-A) to CS-E, but these enzymes were non-functional when expressed in E. coli . Moreover, there is no experimentally derived three-dimensional structure available for this specific sulfotransferase in the protein databases. To overcome this challenge, we developed a 3D model of GalNAc4S-6ST using AlphaFold2 and employed PROSS stability design to identify mutations that enhance enzyme solubility and stability with different N-terminal truncations. Experimental validation of these mutations led to the identification of several functional enzymes. Among various E. coli strains tested for enzyme expression, Origami B (DE3) emerged as the most effective host. This facilitated the enzymatic conversion of CS-A to CS-E, achieving a conversion rate of over 50%, and marking the first successful biosynthesis of animal-free CS-E. These findings represent a significant advancement towards the large-scale synthesis of CS-E using cost-effective carbon sources, offering a sustainable alternative to traditional sourcing from endangered animals like sharks. Key points • Functional expression of GalNAc4S-6ST in a simple prokaryote was accomplished. • First-time biosynthesis of animal-free chondroitin sulfate E was accomplished.

Dermatan sulfate (DS) is a type of glycosaminoglycan present in the extracellular matrix, and which is related to tissue strength, structure, and healing. Dermatan 4- O -sulfotransferase 1 (D4ST1) is an enzyme that catalyzes the transfer of a sulfate group to the N -acetylgalactosamine residue of dermatan, resulting in mature DS. Biallelic loss-of-function variants in the carbohydrate sulfotransferase 14 ( CHST14 ) gene encoding D4ST1, induce defective DS biosynthesis. DS deficiency causes severe connective tissue fragility and deformities in humans (musculocontractural Ehlers–Danlos Syndrome [mcEDS]) and mice ( Chst14 gene knockout [ Chst14 -/- ] mice). Many patients with mcEDS experience gastrointestinal symptoms such as constipation, diverticula, diverticulitis, and perforation. However, pathogenesis of these symptoms has not been systematically investigated. Therefore, we sought to determine the effects of DS deficiency on the colon using Chst14 -/- mice. We found that collagen fibrils were abnormally arranged in the submucosa of the colon. The mice also exhibited accelerated colonic contraction. Unexpectedly, no significant aggravation of dextran sulfate sodium-induced colitis was observed in Chst14 -/- mice compared with wild-type mice. These findings suggest a physiological role of DS in the colon and may shed light on the potential mechanisms underlying the gastrointestinal symptoms of mcEDS.

Tyrosine sulfation is a posttranslational modification common in peptides and proteins synthesized by the secretory pathway in most eukaryotes. In plants, this modification is critical for the biological activities of a subset of peptide hormones such as PSK and PSY1. In animals, tyrosine sulfation is catalyzed by Golgi-localized type II transmembrane proteins called tyrosylprotein sulfotransferases (TPSTs). However, no orthologs of animal TPST genes have been found in plants, suggesting that plants have evolved plant-specific TPSTs structurally distinct from their animal counterparts. To investigate the mechanisms of tyrosine sulfation in plants, we purified TPST activity from microsomal fractions of Arabidopsis MM2d cells, and identified a 62-kDa protein that specifically interacts with the sulfation motif of PSY1 precursor peptide. This protein is a 500-aa type I transmembrane protein that shows no sequence similarity to animal TPSTs. A recombinant version of this protein expressed in yeast catalyzed tyrosine sulfation of both PSY1 and PSK precursor polypeptide in vitro, indicating that the newly identified protein is indeed an Arabidopsis (At)TPST. AtTPST is expressed throughout the plant body, and the highest levels of expression are in the root apical meristem. A loss-of-function mutant of AtTPST displayed a marked dwarf phenotype accompanied by stunted roots, pale green leaves, reduction in higher order veins, early senescence, and a reduced number of flowers and siliques. Our results indicate that plants and animals independently acquired tyrosine sulfation enzymes through convergent evolution.

Neuronal development is the result of a multitude of neural migrations, which require extensive cell-cell communication. These processes are modulated by extracellular matrix components, such as heparan sulfate (HS) polysaccharides. HS is molecularly complex as a result of nonrandom modifications of the sugar moieties, including sulfations in specific positions. We report here mutations in HS 6-O-sulfotransferase 1 (HS6ST1) in families with idiopathic hypogonadotropic hypogonadism (IHH). IHH manifests as incomplete or absent puberty and infertility as a result of defects in gonadotropin-releasing hormone neuron development or function. IHH-associated HS6ST1 mutations display reduced activity in vitro and in vivo, suggesting that HS6ST1 and the complex modifications of extracellular sugars are critical for normal development in humans. Genetic experiments in Caenorhabditis elegans reveal that HS cell-specifically regulates neural branching in vivo in concert with other IHH-associated genes, including kal-1, the FGF receptor, and FGF. These findings are consistent with a model in which KAL1 can act as a modulatory coligand with FGF to activate the FGF receptor in an HS-dependent manner.

Cytosolic sulfotransferases (SULTs), one of the predominant phase II drug metabolizing enzymes (DME), play important roles in metabolism of xeno- and endobiotics to generate their sulfo-conjugates. These sulfo-conjugates often have biological activities but are difficult to study, because even though only small amounts are required to evaluate their efficacy and safety, chemical or biological synthesis of sulfo-conjugatesis is often challenging. Previously, we constructed a DME expression system for cytochrome P450 and UGT, using yeast cells, and successfully produced xenobiotic metabolites in a whole-cell-dependent manner. In this study, we developed a yeast expression system for human SULTs, including SULT1A1, 1A3, 1B1, 1C4, 1E1, and 2A1, in Saccharomyces cerevisiae and examined its sulfo-conjugate productivity. The recombinant yeast cells expressing each of the SULTs successfully produced several hundred milligram per liter of xeno- or endobioticsulfo-conjugates within 6 h. This whole-cell-dependent biosynthesis enabled us to produce sulfo-conjugates without the use of 3’-phosphoadenosine-5’-phosphosulfate, an expensive cofactor. Additionally, the production of regiospecific sulfo-conjugates of several polyphenols was possible with this method, making this novel yeast expression system a powerful tool for uncovering the metabolic pathways and biological actions of sulfo-conjugates.

Inflammation plays a vital role in the development of diabetic nephropathy, but the underlying regulatory mechanisms are only partially understood. Our previous studies demonstrated that, during acute inflammation, endothelial heparan sulfate (HS) contributes to the adhesion and transendothelial migration of leukocytes into perivascular tissues by direct interaction with l-selectin and the presentation of bound chemokines. In the current study, we aimed to assess the role of endothelial HS on chronic renal inflammation and fibrosis in a diabetic nephropathy mouse model. To reduce sulfation of HS specifically in the endothelium, we generated Ndst1f/fTie2Cre+ mice in which N-deacetylase/N-sulfotransferase-1 (Ndst1), the gene that initiates HS sulfation modifications in HS biosynthesis, was expressly ablated in endothelium. To induce diabetes, age-matched male Ndst1f/fTie2Cre- (wild type) and Ndst1f/fTie2Cre+ mice on a C57Bl/6J background were injected intraperitoneally with streptozotocin (STZ) (50 mg/kg) on five consecutive days (N = 10–11/group). Urine and plasma were collected. Four weeks after diabetes induction the animals were sacrificed and kidneys were analyzed by immunohistochemistry and qRT-PCR. Compared to healthy controls, diabetic Ndst1f/fTie2Cre- mice showed increased glomerular macrophage infiltration, mannose binding lectin complement deposition and glomerulosclerosis, whereas these pathological reactions were prevented significantly in the diabetic Ndst1f/fTie2Cre+ animals (all three p < 0.01). In addition, the expression of the podocyte damage marker desmin was significantly higher in the Ndst1f/fTie2Cre− group compared to the Ndst1f/fTie2Cre+ animals (p < 0.001), although both groups had comparable numbers of podocytes. In the cortical tubulo-interstitium, similar analyses show decreased interstitial macrophage accumulation in the diabetic Ndst1f/fTie2Cre+ animals compared to the diabetic Ndst1f/fTie2Cre− mice (p < 0.05). Diabetic Ndst1f/fTie2Cre+ animals also showed reduced interstitial fibrosis as evidenced by reduced density of αSMA-positive myofibroblasts (p < 0.01), diminished collagen III deposition (p < 0.001) and reduced mRNA expression of collagen I (p < 0.001) and fibronectin (p < 0.001). Our studies indicate a pivotal role of endothelial HS in the development of renal inflammation and fibrosis in diabetic nephropathy in mice. These results suggest that HS is a possible target for therapy in diabetic nephropathy.

Sulfonation, primarily facilitated by sulfotransferases, plays a crucial role in the detoxification pathways of endogenous substances and xenobiotics, promoting metabolism and elimination. Traditionally, this bioconversion has been attributed to a family of human cytosolic sulfotransferases (hSULTs) known for their high sequence similarity and dependence on 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as a sulfo donor. However, recent studies have revealed the presence of PAPS-dependent sulfotransferases within gut commensals, indicating that the gut microbiome may harbor a diverse array of sulfotransferase enzymes and contribute to detoxification processes via sulfation. In this study, we investigated the prevalence of sulfotransferases in members of the human gut microbiome. Interestingly, we stumbled upon PAPS-independent sulfotransferases, known as aryl-sulfate sulfotransferases (ASSTs). Our bioinformatics analyses revealed that members of the gut microbial genus Sutterella harbor multiple asst genes, possibly encoding multiple ASST enzymes within its members. Fluctuations in the microbes of the genus Sutterella have been associated with various health conditions. For this reason, we characterized 17 different ASSTs from Sutterella wadsworthensis 3_1_45B. Our findings reveal that SwASSTs share similarities with E. coli ASST but also exhibit significant structural variations and sequence diversity. These differences might drive potential functional diversification and likely reflect an evolutionary divergence from their PAPS-dependent counterparts.