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.

Introduction: Heparan sulfate (HS) in the vascular endothelial glycocalyx (eGC) is a critical regulator of blood vessel homeostasis. Trauma results in HS shedding from the eGC, but the impact of trauma on HS structural modifications that could influence mechanisms of vascular injury and repair has not been evaluated. Moreover, the effect of eGC HS shedding on endothelial cell (EC) homeostasis has not been fully elucidated. The objectives of this work were to characterize the impact of trauma on HS sulfation and determine the effect of eGC HS shedding on the transcriptional landscape of vascular ECs. Methods: Plasma was collected from 25 controls and 49 adults admitted to a level 1 trauma center at arrival and 24 h after hospitalization. Total levels of HS and angiopoietin-2, a marker of pathologic EC activation, were measured at each time point. Enzymatic activity of heparanase, the enzyme responsible for HS shedding, was determined in plasma from hospital arrival. Liquid chromatography-tandem mass spectrometry was used to characterize HS di-/tetrasaccharides in plasma. In vitro work was performed using flow conditioned primary human lung microvascular ECs treated with vehicle or heparinase III to simulate human heparanase activity. Bulk RNA sequencing was performed to determine differentially expressed gene-enriched pathways following heparinase III treatment. Results: We found that heparanase activity was increased in trauma plasma relative to controls, and HS levels at arrival were elevated in a manner proportional to injury severity. Di-/tetrasaccharide analysis revealed lower levels of 3-O-sulfated tetramers with a concomitant increase in ΔIIIS and ΔIIS disaccharides following trauma. Admission levels of total HS and specific HS sulfation motifs correlated with 24-h angiopoietin-2 levels, suggesting an association between HS shedding and persistent, pathological EC activation. In vitro pathway analysis demonstrated downregulation of genes that support cell junction integrity, EC polarity, and EC senescence while upregulating genes that promote cell differentiation and proliferation following HS shedding. Discussion: Taken together, our findings suggest that HS cleavage associated with eGC injury may disrupt homeostatic EC signaling and influence biosynthetic mechanisms governing eGC repair. These results require validation in larger, multicenter trauma populations coupled with in vivo EC-targeted transcriptomic and proteomic analyses.

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here, we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii, transfer sulfate in vitro to the luciferin substrate, vargulin. We find luciferin sulfotransferases (LSTs) of ostracods are not orthologous to known LSTs of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Reuse of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.

The gradual deterioration of articular cartilage was thought to be the central event in osteoarthritis (OA), but recent studies demonstrated the importance of low-grade synovitis in the progression of OA. The Syndecan (SDC) family of membrane proteoglycans is known to be involved in the regulation of inflammation, but there is limited evidence considering the role of syndecans in OA synovitis. Our study aimed to investigate the hip OA synovial membrane expression patterns of SDC1, SDC2 and SDC4, as well as exostosins and sulfotransferases (enzymes involved in the polymerisation and modification of syndecans\' heparan sulphate chains). Synovial membrane samples of patients with OA (24) were divided into two groups according to their Krenn synovitis score severity. The immunohistochemical expressions of SDC1, SDC2, SDC4, EXT1, EXT2, NDST1 and NDST2 in synovial intima and subintima were then analysed and compared with the control group (patients with femoral neck fracture). According to our study, the immunoexpression of SDC1, NDST1 and EXT2 is significantly increased in the intimal cells of OA synovial membrane in patients with lower histological synovitis scores and SDC4 in patients with higher synovitis scores, in comparison with non-OA controls. The difference in the expression of SDC2 among the OA and non-OA groups was insignificant. SDC1, SDC4, NDST1 and EXT2 seem to be involved as inflammation moderators in low-grade OA synovitis and, therefore, should be further investigated as potential markers of disease progression and therapeutic goals.

We previously found that feeding rats with broccoli or cauliflower leads to the formation of characteristic DNA adducts in the liver, intestine and various other tissues. We identified the critical substances in the plants as 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate and its degradation product 1-MIM-OH. DNA adduct formation and the mutagenicity of 1-MIM-OH in cell models were drastically enhanced when human sulfotransferase (SULT) 1A1 was expressed. The aim of this study was to clarify the role of SULT1A1 in DNA adduct formation by 1-MIM-OH in mouse tissues in vivo. Furthermore, we compared the endogenous mouse Sult1a1 and transgenic human SULT1A1 in the activation of 1-MIM-OH using genetically modified mouse strains. We orally treated male wild-type (wt) and Sult1a1-knockout (ko) mice, as well as corresponding lines carrying the human SULT1A1-SULT1A2 gene cluster (tg and ko-tg), with 1-MIM-OH. N2-(1-MIM)-dG and N6-(1-MIM)-dA adducts in DNA were analysed using isotope-dilution UPLC-MS/MS. In the liver, caecum and colon adducts were abundant in mice expressing mouse and/or human SULT1A1, but were drastically reduced in ko mice (1.2-10.6% of wt). In the kidney and small intestine, adduct levels were high in mice carrying human SULT1A1-SULT1A2 genes, but low in wt and ko mice (1.8-6.3% of tg-ko). In bone marrow, adduct levels were very low, independently of the SULT1A1 status. In the stomach, they were high in all four lines. Thus, adduct formation was primarily controlled by SULT1A1 in five out of seven tissues studied, with a strong impact of differences in the tissue distribution of mouse and human SULT1A1. The behaviour of 1-MIM-OH in these models (levels and tissue distribution of DNA adducts; impact of SULTs) was similar to that of methyleugenol, classified as \"probably carcinogenic to humans\". Thus, there is a need to test 1-MIM-OH for carcinogenicity in animal models and to study its adduct formation in humans consuming brassicaceous foodstuff.

Schistosomiasis, otherwise known as bilharzia or snail fever, is a disease that usually affects poor people and people exposed to poor sanitation. The disease affects over 200 million people worldwide annually. Schistosomiasis has been treated using a single drug, praziquantel, since the 1970s and this is resulting in schistosomes becoming resistant. Therefore, there is an urgent need to develop new antischistosoma drugs and vaccines. This study focuses on identifying potential antischistosomal compounds from the plant Salvia fruticosa. We virtually screened a library of 163 S fruticosa compounds by docking against Schistosoma mansoni sulfotransferase (SmSULT) using the PyRx software. Docking scores ranged from -4.7 to -9.3 kcal/mol. Compounds with binding affinity of -7.6 or stronger were subjected to drug-likeness assessments using the DataWarrior software. We also employed the PAINS removal tool to filter off false-positive results. Twelve compounds passed the drug-likeness screen, and these were subjected to in silico toxicity predictions to determine their mutagenic, tumorigenic and reproductive potential. Seven compounds were predicted to be nontoxic. After considering the toxicity analysis results and drug scores of the compounds, we identified rosmarinic acid and hispidulin as qualifying for further evaluation as potential drugs against schistosomiasis. Free energy calculations using the fastDRH webserver and molecular dynamics simulations using CABS-flex showed that the receptor-ligand complexes for the 2 lead compounds are stable under physiological conditions. We recommend that rosmarinic acid and hispidulin be used as hit compounds for the development of potential antischistosomal drugs.

Cytosolic sulfotransferases (SULTs) are cytosolic enzymes that catalyze the transfer of sulfonate group to key endogenous compounds, altering the physiological functions of their substrates. SULT enzymes catalyze the O-sulfonation of hydroxy groups or N-sulfonation of amino groups of substrate compounds. In this study, we report the discovery of C-sulfonation of α,β-unsaturated carbonyl groups mediated by a new SULT enzyme, SULT7A1, and human SULT1C4. Enzymatic assays revealed that SULT7A1 is capable of transferring the sulfonate group from 3\'-phosphoadenosine 5\'-phosphosulfate to the α-carbon of α,β-unsaturated carbonyl-containing compounds, including cyclopentenone prostaglandins as representative endogenous substrates. Structural analyses of SULT7A1 suggest that the C-sulfonation reaction is catalyzed by a novel mechanism mediated by His and Cys residues in the active site. Ligand-activity assays demonstrated that sulfonated 15-deoxy prostaglandin J2 exhibits antagonist activity against the prostaglandin receptor EP2 and the prostacyclin receptor IP. Modification of α,β-unsaturated carbonyl groups via the new prostaglandin-sulfonating enzyme, SULT7A1, may regulate the physiological function of prostaglandins in the gut. Discovery of C-sulfonation of α,β-unsaturated carbonyl groups will broaden the spectrum of potential substrates and physiological functions of SULTs.

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.

Polychlorinated biphenyls (PCBs) are persistent organic toxicants derived from legacy pollution sources and their formation as inadvertent byproducts of some current manufacturing processes. Metabolism of PCBs is often a critical component in their toxicity, and relevant metabolic pathways usually include their initial oxidation to form hydroxylated polychlorinated biphenyls (OH-PCBs). Subsequent sulfation of OH-PCBs was originally thought to be primarily a means of detoxication; however, there is strong evidence that it may also contribute to toxicities associated with PCBs and OH-PCBs. These contributions include either the direct interaction of PCB sulfates with receptors or their serving as a localized precursor for OH-PCBs. The formation of PCB sulfates is catalyzed by cytosolic sulfotransferases, and, when transported into the serum, these metabolites may be retained, taken up by other tissues, and subjected to hydrolysis catalyzed by intracellular sulfatase(s) to regenerate OH-PCBs. Dynamic cycling between PCB sulfates and OH-PCBs may lead to further metabolic activation of the resulting OH-PCBs. Ultimate toxic endpoints of such processes may include endocrine disruption, neurotoxicities, and many others that are associated with exposures to PCBs and OH-PCBs. This review highlights the current understanding of the complex roles that PCB sulfates can have in the toxicities of PCBs and OH-PCBs and research on the varied mechanisms that control these roles.

Sulfotransferases (SULTs) are phase II metabolizing enzymes catalyzing the sulfoconjugation from the co-factor 3\'-Phosphoadenosine 5\'-Phosphosulfate (PAPS) to a wide variety of endogenous compounds, drugs and natural products. Although SULT1A1 and SULT1A3 share 93% identity, SULT1A1, the most abundant SULT isoform in humans, exhibits a broad substrate range with specificity for small phenolic compounds, while SULT1A3 displays a high affinity toward monoamine neurotransmitters like dopamine. To elucidate the factors determining the substrate specificity of the SULT1 isoenzymes, we studied the dynamic behavior and structural specificities of SULT1A1 and SULT1A3 by using molecular dynamics (MD) simulations and ensemble docking of common and specific substrates of the two isoforms. Our results demonstrated that while SULT1A1 exhibits a relatively rigid structure by showing lower conformational flexibility except for the lip (loop L1), the loop L2 and the cap (L3) of SULT1A3 are extremely flexible. We identified protein residues strongly involved in the recognition of different substrates for the two isoforms. Our analyses indicated that being more specific and highly flexible, the structure of SULT1A3 has particularities in the binding site, which are crucial for its substrate selectivity.