Loss of NDST1 N-sulfotransferase activity is associated with autosomal recessive intellectual disability.

Intellectual Disability (ID) is the major cause of handicap, affecting nearly 3% of the general population, and is highly genetically heterogenous with more than a thousand genes involved. Exome sequencing performed in two independent families identified the same missense variant, p.(Gly611Ser), in the NDST1 (N-deacetylase/N-sulfotransferase member 1) gene. This variant had been previously found in ID patients of two other families but has never been functionally characterized. The NDST1 gene encodes a bifunctional enzyme that catalyzes both N-deacetylation and N-sulfation of N-acetyl-glucosamine residues during heparan sulfate (HS) biosynthesis. This step is essential because it influences the downstream enzymatic modifications and thereby determines the overall structure and sulfation degree of the HS polysaccharide chain. To discriminate between a rare polymorphism and a pathogenic variant, we compared the enzymatic properties of wild-type and mutant NDST1 proteins. We found that the p.(Gly611Ser) variant results in a complete loss of N-sulfotransferase activity while the N-deacetylase activity is retained. NDST1 shows the highest and the most homogeneous expression in the human cerebral structures compared to the other members of the NDST gene family. These results indicate that a loss of NDST1 N-sulfation activity is associated with impaired cognitive functions.

Histone H3Y99sulf regulates hepatocellular carcinoma responding to hypoxia.

Histone H3 tyrosine-99 sulfation (H3Y99sulf) is a recently identified histone mark that can cross-talk with H4R3me2a to regulate gene transcription, but its role in cancer biology is less studied. Here, we report that H3Y99sulf is a cancer-associated histone mark that can mediate hepatocellular carcinoma (HCC) cells responding to hypoxia. Hypoxia-stimulated SNAIL pathway elevates the expression of PAPSS2, which serves as a source of adenosine 3'-phosphate 5'-phos-phosulfate for histone sulfation and results in upregulation of H3Y99sulf. The transcription factor TDRD3 is the downstream effector of H3Y99sulf-H4R3me2a axis in HCC. It reads and co-localizes with the H3Y99sulf-H4R3me2a dual mark in the promoter regions of HIF1A and PDK1 to regulate gene transcription. Depletion of SULT1B1 can effectively reduce the occurrence of H3Y99sulf-H4R3me2a-TDRD3 axis in gene promoter regions and lead to downregulation of targeted gene transcription. Hypoxia-inducible factor 1-alpha and PDK1 are master regulators for hypoxic responses and cancer metabolism. Disruption of the H3Y99sulf-H4R3me2a-TDRD3 axis can inhibit the expression and functions of hypoxia-inducible factor 1-alpha and PDK1, resulting in suppressed proliferation, tumor growth, and survival of HCC cells suffering hypoxia stress. The present study extends the regulatory and functional mechanisms of H3Y99sulf and improves our understanding of its role in cancer biology.

Crystal structure of tick tyrosylprotein sulfotransferase reveals the activation mechanism of the tick anticoagulant protein madanin.

Ticks pose a substantial public health risk as they transmit various pathogens. This concern is related to the adept blood-sucking strategy of ticks, underscored by the action of the anticoagulant, madanin, which is known to exhibit an approximately 1000-fold increase in anticoagulant activity following sulfation of its two tyrosine residues, Tyr51 and Tyr54. Despite this knowledge, the molecular mechanism underlying sulfation by tick tyrosylprotein sulfotransferase (TPST) remains unclear. In this study, we successfully prepared tick TPST as a soluble recombinant enzyme. We clarified the method by which this enzyme proficiently sulfates tyrosine residues in madanin. Biochemical analysis using a substrate peptide based on madanin and tick TPST, along with the analysis of the crystal structure of the complex and docking simulations, revealed a sequential sulfation process. Initial sulfation at the Tyr51 site augments binding, thereby facilitating efficient sulfation at Tyr54. Beyond direct biochemical implications, these findings considerably improve our understanding of tick blood-sucking strategies. Furthermore, combined with the utility of modified tick TPST, our findings may lead to the development of novel anticoagulants, promising avenues for thrombotic disease intervention and advancements in the field of public health.

Processing of a plant peptide hormone precursor facilitated by posttranslational tyrosine sulfation.

Most peptide hormones and growth factors are matured from larger inactive precursor proteins by proteolytic processing and further posttranslational modification. Whether or how posttranslational modifications contribute to peptide bioactivity is still largely unknown. We address this question here for TWS1 (Twisted Seed 1), a peptide regulator of embryonic cuticle formation in Arabidopsis thaliana. Using synthetic peptides encompassing the N- and C-terminal processing sites and the recombinant TWS1 precursor as substrates, we show that the precursor is cleaved by the subtilase SBT1.8 at both the N and the C termini of TWS1. Recognition and correct processing at the N-terminal site depended on sulfation of an adjacent tyrosine residue. Arginine 302 of SBT1.8 was found to be required for sulfotyrosine binding and for accurate processing of the TWS1 precursor. The data reveal a critical role for posttranslational modification, here tyrosine sulfation of a plant peptide hormone precursor, in mediating processing specificity and peptide maturation.

Electron Capture vs Transfer Dissociation for Site Determination of Tryptic Peptide Tyrosine Sulfation: Direct Detection of Fibrinogen Sulfation Sites and Identification of Novel Isobaric Interferences.

Tyrosine sulfation, an understudied but crucial post-translational modification, cannot be directly detected in conventional nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) due to the extreme sulfate lability. Here, we report the detection of sulfate-retaining fragments from LC-electron capture dissociation (ECD) and nanoLC-electron transfer higher energy collision dissociation (EThcD). Sulfopeptide candidates were identified by Proteome Discoverer and MSFragger analysis of nanoLC-HCD MS/MS data and added to inclusion lists for LC-ECD or nanoLC-EThcD MS/MS. When this approach failed, targeted LC-ECD with fixed m/z isolation windows was performed. For the plasma protein fibrinogen, the known pyroglutamylated sulfopeptide QFPTDYDEGQDDRPK from the beta chain N-terminus was identified despite a complete lack of sulfate-containing fragment ions. The peptide QVGVEHHVEIEYD from the gamma-B chain C-terminus was also identified as sulfated or phosphorylated. This sulfopeptide is not annotated in Uniprot but was previously reported. MSFragger further identified a cysteine-containing peptide from the middle of the gamma chain as sulfated and deamidated. NanoLC-EThcD and LC-ECD MS/MS confirmed the two former sulfopeptides via sulfate-retaining fragment ions, whereas an unexpected fragmentation pattern was observed for the third sulfopeptide candidate. Manual interpretation of the LC-ECD spectrum revealed two additional isobaric identifications: a trisulfide-linked cysteinyl-glycine or a carbamidomethyl-dithiothreiotol covalent adduct. Synthesis of such adducts confirmed the latter identity.

Sulfoproteomics Workflow with Precursor Ion Accurate Mass Shift Analysis Reveals Novel Tyrosine Sulfoproteins in the Golgi.

Tyrosine sulfation in the Golgi of secreted and membrane proteins is an important post-translational modification (PTM). However, its labile nature has limited analysis by mass spectrometry (MS), a major reason why no sulfoproteome studies have been previously reported. Here, we show that a phosphoproteomics experimental workflow, which includes serial enrichment followed by high resolution, high mass accuracy MS, and tandem MS (MS/MS) analysis, enables sulfopeptide coenrichment and identification via accurate precursor ion mass shift open MSFragger database search. This approach, supported by manual validation, allows the confident identification of sulfotyrosine-containing peptides in the presence of high levels of phosphorylated peptides, thus enabling these two sterically and ionically similar isobaric PTMs to be distinguished and annotated in a single proteomic analysis. We applied this approach to isolated interphase and mitotic rat liver Golgi membranes and identified 67 tyrosine sulfopeptides, corresponding to 26 different proteins. This work discovered 23 new sulfoproteins with functions related to, for example, Ca2+-binding, glycan biosynthesis, and exocytosis. In addition, we report the first preliminary evidence for crosstalk between sulfation and phosphorylation in the Golgi, with implications for functional control.

Knockout of the intellectual disability-linked gene Hs6st2 in mice decreases heparan sulfate 6-O-sulfation, impairs dendritic spines of hippocampal neurons, and affects memory.

Heparan sulfate (HS) is a linear polysaccharide that plays a key role in cellular signaling networks. HS functions are regulated by its 6-O-sulfation, which is catalyzed by three HS 6-O-sulfotransferases (HS6STs). Notably, HS6ST2 is mainly expressed in the brain and HS6ST2 mutations are linked to brain disorders, but the underlying mechanisms remain poorly understood. To determine the role of Hs6st2 in the brain, we carried out a series of molecular and behavioral assessments on Hs6st2 knockout mice. We first carried out strong anion exchange-high performance liquid chromatography and found that knockout of Hs6st2 moderately decreases HS 6-O-sulfation levels in the brain. We then assessed body weights and found that Hs6st2 knockout mice exhibit increased body weight, which is associated with abnormal metabolic pathways. We also performed behavioral tests and found that Hs6st2 knockout mice showed memory deficits, which recapitulate patient clinical symptoms. To determine the molecular mechanisms underlying the memory deficits, we used RNA sequencing to examine transcriptomes in two memory-related brain regions, the hippocampus and cerebral cortex. We found that knockout of Hs6st2 impairs transcriptome in the hippocampus, but only mildly in the cerebral cortex. Furthermore, the transcriptome changes in the hippocampus are enriched in dendrite and synapse pathways. We also found that knockout of Hs6st2 decreases HS levels and impairs dendritic spines in hippocampal CA1 pyramidal neurons. Taken together, our study provides novel molecular and behavioral insights into the role of Hs6st2 in the brain, which facilitates a better understanding of HS6ST2 and HS-linked brain disorders.

Larsucosterol: endogenous epigenetic regulator for treating chronic and acute liver diseases.

Larsucosterol, a potent endogenous epigenetic regulator, has been reported to play a significant role in lipid metabolism, inflammatory responses, and cell survival. The administration of larsucosterol has demonstrated a reduction in lipid accumulation within hepatocytes and the attenuation of inflammatory responses induced by lipopolysaccharide (LPS) and TNFα in macrophages, alleviating LPS- and acetaminophen (ATMP)-induced multiple organ injury, and decreasing mortalities in animal models. Results from phase 1 and 2 clinical trials have shown that larsucosterol has potential as a biomedicine for the treatment of acute and chronic liver diseases. Recent evidence suggests that larsucosterol is a promising candidate for treating alcohol-associated hepatitis with positive results from a phase 2a clinical trial, and for metabolic dysfunction-associated steatohepatitis (MASH) from a phase 1b clinical trial. In this review, we present a culmination of our recent research efforts spanning two decades. We summarize the discovery, physiological and pharmacological mechanisms, and clinical applications of larsucosterol. Furthermore, we elucidate the pathophysiological pathways of metabolic dysfunction-associated steatotic liver diseases (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), and acute liver injuries. A central focus of the review is the exploration of the therapeutic potential of larsucosterol in treating life-threatening conditions, including acetaminophen overdose, endotoxin shock, MASLD, MASH, hepatectomy, and alcoholic hepatitis.

Crystal structure of activating sulfotransferase SgdX2 involved in biosynthesis of secondary metabolite sungeidine.

Microorganisms synthesize a plethora of complex secondary metabolites, many of which are beneficial to human health, such as anticancer agents and antibiotics. Among these, the Sungeidines are a distinct class of secondary metabolites known for their bulky and intricate structures. They are produced by a specific biosynthetic gene cluster within the genome of the soil-dwelling actinomycete Micromonospora sp. MD118. A notable enzyme in the Sungeidine biosynthetic pathway is the activating sulfotransferase SgdX2. In this pathway, SgdX2 mediates a key sulfation step, after which the product undergoes spontaneous dehydration to yield a Sungeidine compound. To delineate the structural basis for SgdX2's substrate recognition and catalytic action, we have determined the crystal structure of SgdX2 in complex with its sulfate donor product, 3'-phosphoadenosine 5'-phosphate (PAP), at a resolution of 1.6 Å. Although SgdX2 presents a compact overall structure, its core elements are conserved among other activating sulfotransferases. Our structural analysis reveals a unique substrate-binding pocket that accommodates bulky, complex substrates, suggesting a specialized adaptation for Sungeidine synthesis. Moreover, we have constructed a substrate docking model that provides insights into the molecular interactions between SgdX2 and Sungeidine F, enhancing our understanding of the enzyme's specificity and catalytic mechanism. The model supports a general acid-base catalysis mechanism, akin to other sulfotransferases, and underscores the minor role of disordered regions in substrate recognition. This integrative study of crystallography and computational modeling advances our knowledge of microbial secondary metabolite biosynthesis and may facilitate the development of novel biotechnological applications.

Local vaginal bioelectrical impedance can predict preterm delivery in mice.

Preterm birth is a serious pregnancy complication that affects neonatal mortality, morbidity, and long-term neurological prognosis. Predicting spontaneous preterm delivery (PTD) is important for its management. While excluding the risk of PTD is important, identifying women at high risk of PTD is imperative for medical intervention. Currently used PTD prediction parameters in clinical practice have shown high negative predictive values, but low positive predictive values. We focused on sulfated and sialylated glycocalyx changes in the uterus and vagina prior to the onset of parturition and explored the potential of electrophysiological detection of these changes as a PTD prediction parameter with a high positive predictive value. In vivo local vaginal bioelectrical impedance (VZ) was measured using two different mouse PTD models. PTD was induced in ICR mice through the subcutaneous injection of mifepristone or local intrauterine injection of lipopolysaccharide (LPS). The PTD rates were 100% and 60% post-administration of mifepristone (16-20 h, n = 4) and LPS (12-24 h, n = 20), respectively. The local VZ values (15 and 10 h after mifepristone or LPS treatment, respectively) were significantly lower in the PTD group than in the non-PTD group. Receiver operator characteristic (ROC) curve analysis of VZ at 125 kHz as a predictor of PTD showed an area under the ROC curve of 1.00 and 0.77 and positive predictive values of 1.00 and 0.86, for the mifepristone and LPS models, respectively, suggesting that local VZ value can predict PTD. Histological examination of the LPS-treated model 6 h post-treatment revealed increased expression of sulfomucins and/or sulfated proteoglycans and sialomucins in the cervical epithelium, cervical stroma and vaginal stroma. In conclusion, local VZ values can determine sulfated and sialylated glycocalyx alterations within the uterus and vagina and might be a useful PTD prediction parameter.

Mass spectrometry analysis of phosphotyrosine-containing proteins.

Tyrosine phosphorylation is a crucial posttranslational modification that is involved in various aspects of cell biology and often has functions in cancers. It is necessary not only to identify the specific phosphorylation sites but also to quantify their phosphorylation levels under specific pathophysiological conditions. Because of its high sensitivity and accuracy, mass spectrometry (MS) has been widely used to identify endogenous and synthetic phosphotyrosine proteins/peptides across a range of biological systems. However, phosphotyrosine-containing proteins occur in extremely low abundance and they degrade easily, severely challenging the application of MS. This review highlights the advances in both quantitative analysis procedures and enrichment approaches to tyrosine phosphorylation before MS analysis and reviews the differences among phosphorylation, sulfation, and nitration of tyrosine residues in proteins. In-depth insights into tyrosine phosphorylation in a wide variety of biological systems will offer a deep understanding of how signal transduction regulates cellular physiology and the development of tyrosine phosphorylation-related drugs as cancer therapeutics.

Complex roles for sulfation in the toxicities of polychlorinated biphenyls.

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.

"Glyco-sulfo barcodes" regulate chemokine receptor function.

Chemokine ligands and receptors regulate the directional migration of leukocytes. Post-translational modifications of chemokine receptors including O-glycosylation and tyrosine sulfation have been reported to regulate ligand binding and resulting signaling. Through in silico analyses, we determined potential conserved O-glycosylation and sulfation sites on human and murine CC chemokine receptors. Glyco-engineered CHO cell lines were used to measure the impact of O-glycosylation on CC chemokine receptor CCR5, while mutation of tyrosine residues and treatment with sodium chlorate were performed to determine the effect of tyrosine sulfation. Changing the glycosylation or tyrosine sulfation on CCR5 reduced the receptor signaling by the more positively charged CCL5 and CCL8 more profoundly compared to the less charged CCL3. The loss of negatively charged sialic acids resulted only in a minor effect on CCL3-induced signal transduction. The enzymes GalNAc-T1 and GalNAc-T11 were shown to be involved in the process of chemokine receptor O-glycosylation. These results indicate that O-glycosylation and tyrosine sulfation are involved in the fine-tuning and recognition of chemokine interactions with CCR5 and the resulting signaling.

The 3-O-sulfation of heparan sulfate modulates protein binding and lyase degradation.

Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.

In vitro affinity maturation of broader and more-potent variants of the HIV-1-neutralizing antibody CAP256-VRC26.25.

Three variable 2 (V2) loops of HIV-1 envelope glycoprotein (Env) trimer converge at the Env apex to form the epitope of an important classes of HIV-1 broadly neutralizing antibodies (bNAbs). These V2-glycan/apex antibodies are exceptionally potent but less broad (∼60 to 75%) than many other bNAbs. Their CDRH3 regions are typically long, acidic, and tyrosine sulfated. Tyrosine sulfation complicates efforts to improve these antibodies through techniques such as phage or yeast display. To improve the breadth of CAP256-VRC26.25 (VRC26.25), a very potent apex antibody, we adapted and extended a B cell display approach. Specifically, we used CRISPR/Cas12a to introduce VRC26.25 heavy- and light-chain genes into their respective loci in a B cell line, ensuring that each cell expresses a single VRC26.25 variant. We then diversified these loci through activation-induced cytidine deaminase-mediated hypermutation and homology-directed repair using randomized CDRH3 sequences as templates. Iterative sorting with soluble Env trimers and further randomization selected VRC26.25 variants with successively improving affinities. Three mutations in the CDRH3 region largely accounted for this improved affinity, and VRC26.25 modified with these mutations exhibited greater breadth and potency than the original antibody. Our data describe a broader and more-potent form of VRC26.25 as well as an approach useful for improving the breadth and potency of antibodies with functionally important posttranslational modifications.

Nuclear magnetic resonance-based structural elucidation of novel marine glycans and derived oligosaccharides.

Marine glycans of defined structures are unique representatives among all kinds of structurally complex glycans endowed with important biological actions. Besides their unique biological properties, these marine sugars also enable advanced structure-activity relationship (SAR) studies given their distinct and defined structures. However, the natural high molecular weights (MWs) of these marine polysaccharides, sometimes even bigger than 100 kDa, pose a problem in many biophysical and analytical studies. Hence, the preparation of low MW oligosaccharides becomes a strategy to overcome the problem. Regardless of the polymeric or oligomeric lengths of these molecules, structural elucidation is mandatory for SAR studies. For this, nuclear magnetic resonance (NMR) spectroscopy plays a pivotal role. Here, we revisit the NMR-based structural elucidation of a series of marine sulfated poly/oligosaccharides discovered in our laboratory within the last 2 years. This set of structures includes the α-glucan extracted from the bivalve Marcia hiantina; the two sulfated galactans extracted from the red alga Botryocladia occidentalis; the fucosylated chondroitin sulfate isolated from the sea cucumber Pentacta pygmaea; the oligosaccharides produced from the fucosylated chondroitin sulfates from this sea cucumber species and from another species, Holothuria floridana; and the sulfated fucan from this later species. Specific 1H and 13C chemical shifts, generated by various 1D and 2D homonuclear and heteronuclear NMR spectra, are exploited as the primary source of information in the structural elucidation of these marine glycans.

Enhanced tyrosine sulfation is associated with chronic kidney disease-related atherosclerosis.

BACKGROUND: Chronic kidney disease (CKD) accelerates atherosclerosis, but the mechanisms remain unclear. Tyrosine sulfation has been recognized as a key post-translational modification (PTM) in regulation of various cellular processes, and the sulfated adhesion molecules and chemokine receptors have been shown to participate in the pathogenesis of atherosclerosis via enhancement of monocyte/macrophage function. The levels of inorganic sulfate, the essential substrate for the sulfation reaction, are dramatically increased in patients with CKD, which indicates a change of sulfation status in CKD patients. Thus, in the present study, we detected the sulfation status in CKD patients and probed into the impact of sulfation on CKD-related atherosclerosis by targeting tyrosine sulfation function. RESULTS: PBMCs from individuals with CKD showed higher amounts of total sulfotyrosine and tyrosylprotein sulfotransferase (TPST) type 1 and 2 protein levels. The plasma level of O-sulfotyrosine, the metabolic end product of tyrosine sulfation, increased significantly in CKD patients. Statistically, O-sulfotyrosine and the coronary atherosclerosis severity SYNTAX score positively correlated. Mechanically, more sulfate-positive nucleated cells in peripheral blood and more abundant infiltration of sulfated macrophages in deteriorated vascular plaques in CKD ApoE null mice were noted. Knockout of TPST1 and TPST2 decreased atherosclerosis and peritoneal macrophage adherence and migration in CKD condition. The sulfation of the chemokine receptors, CCR2 and CCR5, was increased in PBMCs from CKD patients. CONCLUSIONS: CKD is associated with increased sulfation status. Increased sulfation contributes to monocyte/macrophage activation and might be involved in CKD-related atherosclerosis. Inhibition of sulfation may suppress CKD-related atherosclerosis and is worthy of further study.

Tyrosine Sulfation at Antibody Light Chain CDR-1 Increases Binding Affinity and Neutralization Potency to Interleukine-4.

Structure and function of therapeutic antibodies can be modulated by a variety of post-translational modifications (PTM). Tyrosine (Tyr) sulfation is a type of negatively charged PTM that occurs during protein trafficking through the Golgi. In this study, we discovered that an anti-interleukin (IL)-4 human IgG1, produced by transiently transfected HEK293 cells, contained a fraction of unusual negatively charged species. Interestingly, the isolated acidic species exhibited a two-fold higher affinity to IL-4 and a nearly four-fold higher potency compared to the main species. Mass spectrometry (MS) showed the isolated acidic species possessed an +80-Dalton from the expected mass, suggesting an occurrence of Tyr sulfation. Consistent with this hypothesis, we show the ability to control the acidic species during transient expression with the addition of Tyr sulfation inhibitor sodium chlorate or, conversely, enriched the acidic species from 30% to 92% of the total antibody protein when the IL-4 IgG was co-transfected with tyrosylprotein sulfotransferase genes. Further MS and mutagenesis analysis identified a Tyr residue at the light chain complementarity-determining region-1 (CDRL-1), which was sulfated specifically. These results together have demonstrated for the first time that Tyr sulfation at CDRL-1 could modulate antibody binding affinity and potency to a human immune cytokine.

Bioinformatic Analysis of Sulfotransferases from an Unexplored Gut Microbe, Sutterella wadsworthensis 3_1_45B: Possible Roles towards Detoxification via Sulfonation by Members of the Human Gut Microbiome.

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.

Rearrangement of Arylsulfamates and Sulfates to Para-Sulfonyl Anilines and Phenols.

The C(sp2)-aryl sulfonate functional group is found in bioactive molecules, but their synthesis can involve extreme temperatures (>190 °C or flash vacuum pyrolysis) and strongly acidic reaction conditions. Inspired by the 1917 Tyrer industrial process for a sulfa dye that involved an aniline N(sp2)-SO3 intermediate en route to a C(sp2)-SO3 rearranged product, we investigated tributylsulfoammonium betaine (TBSAB) as a milder N-sulfamation to C-sulfonate relay reagent. Initial investigations of a stepwise route involving TBSAB on selected anilines at room temperature enabled the isolation of N(sp2)-sulfamate. Subsequent thermal rearrangement demonstrated the intermediary of a sulfamate en route to the sulfonate; however, it was low-yielding. Investigation of the N-sulfamate to C--sulfonate mechanism through control experiments with variation at the heteroatom positions and kinetic isotope experiments (KIEH/D) confirmed the formation of a key N(sp2)-SO3 intermediate and further confirmed an intermolecular mechanism. Furthermore, compounds without an accessible nitrogen (or oxygen) lone pair did not undergo sulfamation- (or sulfation) -to-sulfonation under these conditions. A one-pot sulfamation and thermal sulfonation reaction was ultimately developed and explored on a range of aniline and heterocyclic scaffolds with high conversions, including N(sp2)-sulfamates (O(sp2)-sulfates) and C(sp2)-sulfonates, in up to 99 and 80% (and 88% for a phenolic example) isolated yield, respectively. Encouragingly, the ability to modulate the ortho-para selectivity of the products obtained was observed under thermal control. A sulfonated analog of the intravenous anesthetic propofol was isolated (88% yield), demonstrating a proof-of-concept modification of a licensed drug alongside a range of nitrogen- and sulfur-containing heterocyclic fragments used in drug discovery.