Chemical Synthesis of Δ-4,5 Unsaturated Heparan Sulfate Oligosaccharides for Biomarker Discovery.

A methodology is described that can provide heparan sulfate oligosaccharides having a Δ4,5-double bond, which are needed as analytical standards and biomarkers for mucopolysaccharidoses. It is based on chemical oligosaccharide synthesis followed by modification of the C-4 hydroxyl of the terminal uronic acid moiety as methanesulfonate. This leaving group is stable under conditions used to remove temporary protecting groups, O-sulfation, and hydrogenolysis. Treatment with NaOH results in elimination of the methanesulfonate and formation of a Δ4,5-double bond.

Treating pain in patients with Ehlers-Danlos syndrome : Multidisciplinary management of a multisystemic disease.

BACKGROUND: The clinical picture of people with Ehlers-Danlos syndromes (EDS) is complex and involves a variety of potential causes of pain. This poses major challenges to patients and healthcare professionals alike in terms of diagnosis and management of the condition. OBJECTIVES: The aim of the article was to provide an overview of the specific pain management needs of patients with EDS and address their background. MATERIAL AND METHODS: A selective literature search was performed to highlight the current state of research on pain management in EDS patients. RESULTS: Affected patients require multimodal pain management considering their individual needs, disease-specific features, and comorbidities. CONCLUSION: Medical awareness and evidence need to be further improved to enhance the medical care situation of these patients with complex needs.

Synthetic Heparanase Inhibitors Can Prevent Herpes Simplex Viral Spread.

Herpes simplex virus (HSV-1) employs heparan sulfate (HS) as receptor for cell attachment and entry. During late-stage infection, the virus induces the upregulation of human heparanase (Hpse) to remove cell surface HS allowing viral spread. We hypothesized that inhibition of Hpse will prevent viral release thereby representing a new therapeutic strategy for HSV-1. A range of HS-oligosaccharides was prepared to examine the importance of chain length and 2-O-sulfation of iduronic moieties for Hpse inhibition. It was found that hexa- and octasaccharides potently inhibited the enzyme and that 2-O-sulfation of iduronic acid is tolerated. Computational studies provided a rationale for the observed structure-activity relationship. Treatment of human corneal epithelial cells (HCEs) infected with HSV-1 with the hexa- and octasaccharide blocked viral induced shedding of HS which significantly reduced spread of virions. The compounds also inhibited migration and proliferation of immortalized HCEs thereby providing additional therapeutic properties.

Well-Defined Heparin Mimetics Can Inhibit Binding of the Trimeric Spike of SARS-CoV-2 in a Length-Dependent Manner.

The emergence of new SARS-CoV-2 variants and the dangers of long-covid necessitate the development of broad-acting therapeutics that can reduce viral burden. SARS-CoV-2 employs heparan sulfate (HS) as an initial cellular attachment factor, and therefore, there is interest in developing heparin as a therapeutic for SARS-CoV-2. Its use is, however, complicated by structural heterogeneity and the risk of causing bleeding and thrombocytopenia. Here, we describe the preparation of well-defined heparin mimetics by a controlled head-to-tail assembly of HS oligosaccharides having an alkyne or azide moiety by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Alkyne- and azide-containing sulfated oligosaccharides were prepared from a common precursor by modifying an anomeric linker with 4-pentynoic acid and by enzymatic extension with an N-acetyl-glucosamine having an azide moiety at C-6 (GlcNAc6N3), respectively, followed by CuAAC. The process of enzymatic extension with GlcNAc6N3 followed by CuAAC with the desired alkyne-containing oligosaccharides could be repeated to give compounds composed of 20 and 27 monosaccharides, respectively. The heparin mimetics could inhibit the binding of the SARS-CoV-2 spike or RBD to immobilized heparin or to Vero E6 cells. The inhibitory potency increased with increasing chain length, and a compound composed of four sulfated hexasaccharides linked by triazoles had a similar potency as unfractionated heparin. Sequence analysis and HS microarray binding studies with a wide range of RBDs of variants of concern indicate that they have maintained HS-binding capabilities and selectivities. The heparin mimetics exhibit no or reduced binding to antithrombin-III and platelet factor 4, respectively, which are associated with side effects.

Chemoenzymatic Synthesis of Heparan Sulfate Oligosaccharides having a Domain Structure.

Heparan sulfate (HS) has a domain structure in which regions that are modified by epimerization and sulfonation (NS domains) are interspersed by unmodified fragments (NA domains). There is data to support that domain organization of HS can regulate binding of proteins, however, such model has been difficult to probe. Here, we report a chemoenzymatic methodology that can provide HS oligosaccharides composed of two or more NS domains separated by NA domains of different length. It is based on the chemical synthesis of a HS oligosaccharide that enzymatically was extended by various GlcA-GlcNAc units and terminated in GlcNAc having an azido moiety at C-6 position. HS oligosaccharides having an azide and alkyne moiety could be assembled by copper catalyzed alkyne-azide cycloaddition to give compounds having various NS domains separated by unsulfonated regions. Competition binding studies showed that the length of an NA domain modulates the binding of the chemokines CCL5 and CXCL8.

Molecular dynamics-based descriptors of 3-O-Sulfated Heparan sulfate as contributors of protein binding specificity.

Glycosaminoglycans are linear periodic and anionic polysaccharides found in the extracellular matrix, involved in a range of key biochemical processes as a result of their interactions with a variety of protein partners. Due to the template-less synthesis, high flexibility and charge of GAGs, as well as the multipose binding of GAG ligands to receptors, the specificity of GAG-protein interactions can be difficult to elucidate. In this study we propose a set of MD-based descriptors of unbound Heparan Sulfate hexasaccharides that can be used to characterize GAGs and explain their binding affinity to a set of protein receptors. With the help of experimental data on GAG-protein binding affinity, we were able to further characterize the nature of this interaction in addition to providing a basis for predictor functions of GAG-protein binding specificity.

Synthetic Heparan Sulfate Hydrogels Regulate Neurotrophic Factor Signaling and Neuronal Network Activity.

Three-dimensional (3D) synthetic heparan sulfate (HS) constructs possess promising attributes for neural tissue engineering applications. However, their sulfation-dependent ability to facilitate molecular recognition and cell signaling has not yet been investigated. We hypothesized that fully sulfated synthetic HS constructs (bearing compound 1) that are functionalized with neural adhesion peptides will enhance fibroblast growth factor-2 (FGF2) binding and complexation with FGF receptor-1 (FGFR1) to promote the proliferation and neuronal differentiation of human neural stem cells (hNSCs) when compared to constructs with unsulfated controls (bearing compound 2). We tested this hypothesis in vitro using 2D and 3D substrates consisting of different combinations of HS tetrasaccharides (compounds 3 and 4) and an engineered integrin-binding chimeric peptide (CP), which were assembled using strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry. Results indicated that the adhesion of hNSCs increased significantly when cultured on 2D glass substrates functionalized with chimeric peptide. hNSCs encapsulated in 1-CP hydrogels and cultured in media containing the mitogen FGF2 exhibited significantly higher neuronal differentiation when compared to hNSCs in 2-CP hydrogels. These observations were corroborated by Western blot analysis, which indicated the enhanced binding and retention of both FGF2 and FGFR1 by 1 as well as downstream phosphorylation of extracellular signal-regulated kinases (ERK1/2) and enhanced proliferation of hNSCs. Lastly, calcium activity imaging revealed that both 1 and 2 hydrogels supported the neuronal growth and activity of pre-differentiated human prefrontal cortex neurons. Collectively, these results demonstrate that synthetic HS hydrogels can be tailored to regulate growth factor signaling and neuronal fate and activity.

Sialic acid-containing glycolipids mediate binding and viral entry of SARS-CoV-2.

Emerging evidence suggests that host glycans influence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we reveal that the receptor-binding domain (RBD) of the spike (S) protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (Sia), with preference for monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind to the RBD. The monomeric affinities (Kd = 100-200 µM) of gangliosides for the RBD are similar to another negatively charged glycan ligand of the RBD proposed as a viral co-receptor, heparan sulfate (HS) dp2-dp6 oligosaccharides. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to angiotensin-converting enzyme 2 (ACE2)-expressing cells is decreased following depletion of cell surface Sia levels using three approaches: sialyltransferase (ST) inhibition, genetic knockout of Sia biosynthesis, or neuraminidase treatment. These effects on RBD binding and both pseudotyped and authentic SARS-CoV-2 viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

Influence of saccharide modifications on heparin lyase III substrate specificities.

A library of 23 synthetic heparan sulfate (HS) oligosaccharides, varying in chain length, types, and positions of modifications, was used to analyze the substrate specificities of heparin lyase III enzymes from both Flavobacterium heparinum and Bacteroides eggerthii. The influence of specific modifications, including N-substitution, 2-O sulfation, 6-O sulfation, and 3-O sulfation on lyase III digestion was examined systematically. It was demonstrated that lyase III from both sources can completely digest oligosaccharides lacking O-sulfates. 2-O Sulfation completely blocked cleavage at the corresponding site; 6-O and 3-O sulfation on glucosamine residues inhibited enzyme activity. We also observed that there are differences in substrate specificities between the two lyase III enzymes for highly sulfated oligosaccharides. These findings will facilitate obtaining and analyzing the functional sulfated domains from large HS polymer, to better understand their structure/function relationships in biological processes.

Dissecting structure-function of 3-O-sulfated heparin and engineered heparan sulfates.

Heparan sulfate (HS) polysaccharides are master regulators of diverse biological processes via sulfated motifs that can recruit specific proteins. 3-O-sulfation of HS/heparin is crucial for anticoagulant activity, but despite emerging evidence for roles in many other functions, a lack of tools for deciphering structure-function relationships has hampered advances. Here, we describe an approach integrating synthesis of 3-O-sulfated standards, comprehensive HS disaccharide profiling, and cell engineering to address this deficiency. Its application revealed previously unseen differences in 3-O-sulfated profiles of clinical heparins and 3-O-sulfotransferase (HS3ST)­specific variations in cell surface HS profiles. The latter correlated with functional differences in anticoagulant activity and binding to platelet factor 4 (PF4), which underlies heparin-induced thrombocytopenia, a known side effect of heparin. Unexpectedly, cells expressing the HS3ST4 isoenzyme generated HS with potent anticoagulant activity but weak PF4 binding. The data provide new insights into 3-O-sulfate structure-function and demonstrate proof of concept for tailored cell-based synthesis of next-generation heparins.

Heparan Sulfate Proteoglycans as Attachment Factor for SARS-CoV-2.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing an unprecedented global pandemic demanding the urgent development of therapeutic strategies. Microarray binding experiments, using an extensive heparan sulfate (HS) oligosaccharide library, showed that the receptor binding domain (RBD) of the spike of SARS-CoV-2 can bind HS in a length- and sequence-dependent manner. A hexasaccharide composed of IdoA2S-GlcNS6S repeating units was identified as the minimal binding epitope. Surface plasmon resonance showed the SARS-CoV-2 spike protein binds with a much higher affinity to heparin (K D = 55 nM) compared to the RBD (K D = 1 µM) alone. It was also found that heparin does not interfere in angiotensin-converting enzyme 2 (ACE2) binding or proteolytic processing of the spike. However, exogenous administered heparin or a highly sulfated HS oligosaccharide inhibited RBD binding to cells. Furthermore, an enzymatic removal of HS proteoglycan from physiological relevant tissue resulted in a loss of RBD binding. The data support a model in which HS functions as the point of initial attachment allowing the virus to travel through the glycocalyx by low-affinity high-avidity interactions to reach the cell membrane, where it can engage with ACE2 for cell entry. Microarray binding experiments showed that ACE2 and HS can simultaneously engage with the RBD, and it is likely no dissociation between HS and RBD is required for binding to ACE2. The results highlight the potential of using HS oligosaccharides as a starting material for therapeutic agent development.

Structure, Immunogenicity, and Conformation-Dependent Receptor Binding of the Postfusion Human Metapneumovirus F Protein.

Human metapneumovirus (hMPV) is an important cause of acute viral respiratory infection. As the only target of neutralizing antibodies, the hMPV fusion (F) protein has been a major focus for vaccine development and targeting by drugs and monoclonal antibodies (MAbs). While X-ray structures of trimeric prefusion and postfusion hMPV F proteins from genotype A, and monomeric prefusion hMPV F protein from genotype B have been determined, structural data for the postfusion conformation for genotype B is lacking. We determined the crystal structure of this protein and compared the structural differences of postfusion hMPV F between hMPV A and B genotypes. We also assessed the receptor binding properties of the hMPV F protein to heparin and heparan sulfate (HS). A library of HS oligomers was used to verify the HS binding activity of hMPV F, and several compounds showed binding to predominantly prefusion hMPV F, but had limited binding to postfusion hMPV F. Furthermore, MAbs to antigenic sites III and the 66-87 intratrimeric epitope block heparin binding. In addition, we evaluated the efficacy of postfusion hMPV B2 F protein as a vaccine candidate in BALB/c mice. Mice immunized with hMPV B2 postfusion F protein showed a balanced Th1/Th2 immune response and generated neutralizing antibodies against both subgroup A2 and B2 hMPV strains, which protected the mice from hMPV challenge. Antibody competition analysis revealed the antibodies generated by immunization target two known antigenic sites (III and IV) on the hMPV F protein. Overall, this study provides new characteristics of the hMPV F protein, which may be informative for vaccine and therapy development. IMPORTANCE Human metapneumovirus (hMPV) is an important cause of viral respiratory disease. In this paper, we report the X-ray crystal structure of the hMPV fusion (F) protein in the postfusion conformation from genotype B. We also assessed binding of the hMPV F protein to heparin and heparan sulfate, a previously reported receptor for the hMPV F protein. Furthermore, we determined the immunogenicity and protective efficacy of postfusion hMPV B2 F protein, which is the first study using a homogenous conformation of the protein. Antibodies generated in response to vaccination give a balanced Th1/Th2 response and target two previously discovered neutralizing epitopes.

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.

Heparan sulfate proteoglycans as attachment factor for SARS-CoV-2.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing an unprecedented global pandemic demanding the urgent development of therapeutic strategies. Microarray binding experiments using an extensive heparan sulfate (HS) oligosaccharide library showed that the receptor binding domain (RBD) of the spike of SARS-CoV-2 can bind HS in a length- and sequence-dependent manner. Hexa- and octa-saccharides composed of IdoA2S-GlcNS6S repeating units were identified as optimal ligands. Surface plasma resonance (SPR) showed the SARS-CoV-2 spike protein binds with much higher affinity to heparin (KD = 55 nM) compared to the RBD (KD = 1 uM) alone. We also found that heparin does not interfere in angiotensin-converting enzyme 2 (ACE2) binding or proteolytic processing of the spike. Our data supports a model in which HS functions as the point of initial attachment for SARS-CoV-2 infection. Tissue staining studies using biologically relevant tissues indicate that heparan sulfate proteoglycan (HSPG) is a critical attachment factor for the virus. Collectively, our results highlight the potential of using HS oligosaccharides as a therapeutic agent by inhibiting SARS-CoV-2 binding to target cells.

Neutralizing the pathological effects of extracellular histones with small polyanions.

Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, particularly when liberated by DNases. We report the development of small polyanions (SPAs) (~0.9-1.4 kDa) that interact electrostatically with histones, neutralizing their pathological effects. In vitro, SPAs inhibited the cytotoxic, platelet-activating and erythrocyte-damaging effects of histones, mechanistic studies revealing that SPAs block disruption of lipid-bilayers by histones. In vivo, SPAs significantly inhibited sepsis, deep-vein thrombosis, and cardiac and tissue-flap models of ischemia-reperfusion injury (IRI), but appeared to differ in their capacity to neutralize NET-bound versus free histones. Analysis of sera from sepsis and cardiac IRI patients supported these differential findings. Further investigations revealed this effect was likely due to the ability of certain SPAs to displace histones from NETs, thus destabilising the structure. Finally, based on our work, a non-toxic SPA that inhibits both NET-bound and free histone mediated pathologies was identified for clinical development.

Modular Synthesis of Heparan Sulfate Oligosaccharides Having N-Acetyl and N-Sulfate Moieties.

Heparan sulfates are structurally diverse sulfated polysaccharides that reside at the surface of all animal cells where they can interact with a multitude of proteins, thereby modulating a wide range of physiological and disease processes. We describe here a modular synthetic methodology that can provide libraries of heparan sulfate oligosaccharides that have glucosamine residues modified by different patterns of N-acetyl and N-sulfate moieties. It is based on the use of glycosyl donors that are modified at C2 by an azido- or trifluoromethylphenyl-methanimine moiety, which allowed the selective installation of α-glycosides. The amino protecting groups can be selectively unmasked by a reduction or acid treatment, allowing the installation of N-acetyl and N-sulfate moieties, respectively. In combination with the orthogonal hydroxyl protecting groups levulinic (Lev) ester, thexyldimethylsilyl (TDS) ether, allyloxycarbonate (Alloc), and 9-fluorenylmethyl carbonate (Fmoc), different patterns of O-sulfation can be installed. The methodology was applied to prepare four hexasaccharides that differ in the pattern of N- and O-sulfation. These compounds, together with a number of previously prepared HS oligosaccharides, were printed as a glycan microarray to examine the binding selectivities of several HS-binding proteins.

Pain Symptomatology and Management in Pediatric Ehlers-Danlos Syndrome: A Review.

Ehlers-Danlos syndromes (EDS) are a group of connective tissue disorders that manifest with hyperextensibility of joints and skin, and general tissue fragility. While not a major criterion for clinical diagnosis, pain is a frequently endorsed symptom across subtypes of EDS. As such, the present review aims to summarize research to date on pain characteristics and management, and the relationship between such pain symptomatology and quality of life in pediatric EDS. Characteristics of pain, including theorized etiology, relative intensity and extent of pain are described, as well as descriptions of frequently endorsed pain sites (musculoskeletal, and non-musculoskeletal). Interventions related to the management of musculoskeletal (e.g., pharmaceutical intervention, physical therapy) and non-musculoskeletal pain (e.g., pharmaceutical and psychological interventions) are discussed, highlighting the need for additional research related to pediatric pain management in the context of hypermobility syndromes. In addition, the relationship between pain in pediatric EDS and quality of life is described. Finally, limitations of literature to date are described and recommendations for future lines of research are outlined.

Distinct Mycoplasma pneumoniae Interactions with Sulfated and Sialylated Receptors.

Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the conducting airways, causing bronchitis and atypical or "walking" pneumonia in humans. M. pneumoniae recognizes sialylated and sulfated oligosaccharide receptors to colonize the respiratory tract, but the contribution of the latter is particularly unclear. We used chamber slides coated with sulfatide (3-O-sulfogalactosylceramide) to provide a baseline for M. pneumoniae binding and gliding motility. As expected, M. pneumoniae bound to surfaces coated with sulfatide in a manner that was dependent on sulfatide concentration and incubation temperature and inhibited by competing dextran sulfate. However, mycoplasmas bound to sulfatide exhibited no gliding motility, regardless of receptor density. M. pneumoniae also bound lactose 3'-sulfate ligated to an inert polymer scaffold, and binding was inhibited by competing dextran sulfate. The major adhesin protein P1 mediates adherence to terminal sialic acids linked α-2,3, but P1-specific antibodies that blocked M. pneumoniae hemadsorption (HA) and binding to the sialylated glycoprotein laminin by 95% failed to inhibit mycoplasma binding to sulfatide, suggesting that P1 does not mediate binding to sulfated galactose. Consistent with this conclusion, the M. pneumoniae HA-negative mutant II-3 failed to bind to sialylated receptors but adhered to sulfatide in a temperature-dependent manner.

Arylsulfatase K inactivation causes mucopolysaccharidosis due to deficient glucuronate desulfation of heparan and chondroitin sulfate.

Mucopolysaccharidoses comprise a group of rare metabolic diseases, in which the lysosomal degradation of glycosaminoglycans (GAGs) is impaired due to genetically inherited defects of lysosomal enzymes involved in GAG catabolism. The resulting intralysosomal accumulation of GAG-derived metabolites consequently manifests in neurological symptoms and also peripheral abnormalities in various tissues like liver, kidney, spleen and bone. As each GAG consists of differently sulfated disaccharide units, it needs a specific, but also partly overlapping set of lysosomal enzymes to accomplish their complete degradation. Recently, we identified and characterized the lysosomal enzyme arylsulfatase K (Arsk) exhibiting glucuronate-2-sulfatase activity as needed for the degradation of heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS). In the present study, we investigated the physiological relevance of Arsk by means of a constitutive Arsk knockout mouse model. A complete lack of glucuronate desulfation was demonstrated by a specific enzyme activity assay. Arsk-deficient mice show, in an organ-specific manner, a moderate accumulation of HS and CS metabolites characterized by 2-O-sulfated glucuronate moieties at their non-reducing ends. Pathophysiological studies reflect a rather mild phenotype including behavioral changes. Interestingly, no prominent lysosomal storage pathology like bone abnormalities were detected. Our results from the Arsk mouse model suggest a new although mild form of mucopolysacharidose (MPS), which we designate MPS type IIB.

A redox-active switch in fructosamine-3-kinases expands the regulatory repertoire of the protein kinase superfamily.

Aberrant regulation of metabolic kinases by altered redox homeostasis substantially contributes to aging and various diseases, such as diabetes. We found that the catalytic activity of a conserved family of fructosamine-3-kinases (FN3Ks), which are evolutionarily related to eukaryotic protein kinases, is regulated by redox-sensitive cysteine residues in the kinase domain. The crystal structure of the FN3K homolog from Arabidopsis thaliana revealed that it forms an unexpected strand-exchange dimer in which the ATP-binding P-loop and adjoining ß strands are swapped between two chains in the dimer. This dimeric configuration is characterized by strained interchain disulfide bonds that stabilize the P-loop in an extended conformation. Mutational analysis and solution studies confirmed that the strained disulfides function as redox "switches" to reversibly regulate the activity and dimerization of FN3K. Human FN3K, which contains an equivalent P-loop Cys, was also redox sensitive, whereas ancestral bacterial FN3K homologs, which lack a P-loop Cys, were not. Furthermore, CRISPR-mediated knockout of FN3K in human liver cancer cells altered the abundance of redox metabolites, including an increase in glutathione. We propose that redox regulation evolved in FN3K homologs in response to changing cellular redox conditions. Our findings provide insights into the origin and evolution of redox regulation in the protein kinase superfamily and may open new avenues for targeting human FN3K in diabetic complications.