bims-supasi Biomed News
on Sulfation pathways and signalling
Issue of 2022‒02‒27
23 papers selected by
Jonathan Wolf Mueller
University of Birmingham

  1. Int J Mol Sci. 2022 Feb 10. pii: 1963. [Epub ahead of print]23(4):
      Heparan sulfate (HS) is a type of glycosaminoglycan that plays a key role in a variety of biological functions in neurology, skeletal development, immunology, and tumor metastasis. Biosynthesis of HS is initiated by a link of xylose to Ser residue of HS proteoglycans, followed by the formation of a linker tetrasaccharide. Then, an extension reaction of HS disaccharide occurs through polymerization of many repetitive units consisting of iduronic acid and N-acetylglucosamine. Subsequently, several modification reactions take place to complete the maturation of HS. The sulfation positions of N-, 2-O-, 6-O-, and 3-O- are all mediated by specific enzymes that may have multiple isozymes. C5-epimerization is facilitated by the epimerase enzyme that converts glucuronic acid to iduronic acid. Once these enzymatic reactions have been completed, the desulfation reaction further modifies HS. Apart from HS biosynthesis, the degradation of HS is largely mediated by the lysosome, an intracellular organelle with acidic pH. Mucopolysaccharidosis is a genetic disorder characterized by an accumulation of glycosaminoglycans in the body associated with neuronal, skeletal, and visceral disorders. Genetically modified animal models have significantly contributed to the understanding of the in vivo role of these enzymes. Their role and potential link to diseases are also discussed.
    Keywords:  biosynthesis; heparan sulfate; knockout mice; lysosome
  2. Viruses. 2022 Feb 17. pii: 413. [Epub ahead of print]14(2):
      Coronaviruses (CoVs) are common among humans and many animals, causing respiratory or gastrointestinal diseases. Currently, only a few antiviral drugs against CoVs are available. Especially for SARS-CoV-2, new compounds for treatment of COVID-19 are urgently needed. In this study, we characterize the antiviral effects of two high-sulfated glycosaminoglycan (GAG) derivatives against SARS-CoV-2 and bovine coronaviruses (BCoV), which are both members of the Betacoronavirus genus. The investigated compounds are based on hyaluronan (HA) and chondroitin sulfate (CS) and exhibit a strong inhibitory effect against both CoVs. Yield assays were performed using BCoV-infected PT cells in the presence and absence of the compounds. While the high-sulfated HA (sHA3) led to an inhibition of viral growth early after infection, high-sulfated CS (sCS3) had a slightly smaller effect. Time of addition assays, where sHA3 and sCS3 were added to PT cells before, during or after infection, demonstrated an inhibitory effect during all phases of infection, whereas sHA3 showed a stronger effect even after virus absorbance. Furthermore, attachment analyses with prechilled PT cells revealed that virus attachment is not blocked. In addition, sHA3 and sCS3 inactivated BCoV by stable binding. Analysis by quantitative real-time RT PCR underlines the high potency of the inhibitors against BCoV, as well as B.1-lineage, Alpha and Beta SARS-CoV-2 viruses. Taken together, these results demonstrated that the two high-sulfated GAG derivatives exhibit low cytotoxicity and represent promising candidates for an anti-CoV therapy.
    Keywords:  SARS-CoV-2; antiviral activity; bovine coronavirus; high-sulfated glycosaminoglycans
  3. Sci Rep. 2022 02 23. 12(1): 3075
      Proteoglycans are proteins that are modified with glycosaminoglycan chains. Chondroitin sulfate proteoglycans (CSPGs) are currently being exploited as targets for drug-delivery in various cancer indications, however basic knowledge on how CSPGs are internalized in tumor cells is lacking. In this study we took advantage of a recombinant CSPG-binding lectin VAR2CSA (rVAR2) to track internalization and cell fate of CSPGs in tumor cells. We found that rVAR2 is internalized into cancer cells via multiple internalization mechanisms after initial docking on cell surface CSPGs. Regardless of the internalization pathway used, CSPG-bound rVAR2 was trafficked to the early endosomes in an energy-dependent manner but not further transported to the lysosomal compartment. Instead, internalized CSPG-bound rVAR2 proteins were secreted with exosomes to the extracellular environment in a strictly chondroitin sulfate-dependent manner. In summary, our work describes the cell fate of rVAR2 proteins in tumor cells after initial binding to CSPGs, which can be further used to inform development of rVAR2-drug conjugates and other therapeutics targeting CSPGs.
  4. Am J Physiol Cell Physiol. 2022 Feb 23.
      Heparan sulfate (HS) is a linear polysaccharide attached to a core protein, forming heparan sulfate proteoglycans (HSPGs) that are ubiquitously expressed on the surface of almost all mammalian cells and the extracellular matrix. HS orchestrates the binding of various signal molecules to their receptors, thus, regulating many biological processes, including homeostasis, metabolism, and various pathological processes. Due to its wide distribution and negatively charged properties, HS is exploited by many viruses as a co-factor to attach to host cells. Therefore, inhibition of the interaction between virus and HS is proposed as a promising approach to mitigate viral infection, including SARS-CoV-2. In this review, we summarize the interaction manners of HS with viruses with focus on significant pathogenic RNA viruses, including alphaviruses, flaviviruses, and coronaviruses. We also provide an overview of the challenges we may face when using HS-mimetics as antivirals for clinical treatment. More studies are needed to provide a further understanding of the interplay between HS and viruses both in vitro and in vivo, which will favor the development of specific antiviral inhibitors.
    Keywords:  Flavivirus; Heparan sulfate; SARS-CoV-2; alphavirus; coronavirus
  5. Pharmaceuticals (Basel). 2022 Feb 21. pii: 258. [Epub ahead of print]15(2):
      With the increased prevalence of new SARS-CoV-2 variants of concern, such as Delta and Omicron, the COVID-19 pandemic has become an ongoing human health disaster, killing millions worldwide. SARS-CoV-2 invades its host through the interaction of its spike (S) protein with a host cell receptor, angiotensin-converting enzyme 2 (ACE2). In addition, heparan sulfate (HS) on the surface of host cells plays an important role as a co-receptor for this viral pathogen-host cell interaction. Our previous studies demonstrated that many sulfated glycans, such as heparin, fucoidans, and rhamnan sulfate have anti-SARS-CoV-2 activities. In the current study, a small library of sulfated glycans and highly negatively charged compounds, including pentosan polysulfate (PPS), mucopolysaccharide polysulfate (MPS), sulfated lactobionic acid, sulodexide, and defibrotide, was assembled and evaluated for binding to the S-proteins and inhibition of viral infectivity in vitro. These compounds inhibited the interaction of the S-protein receptor-binding domain (RBD) (wild type and different variants) with immobilized heparin, a highly sulfated HS, as determined using surface plasmon resonance (SPR). PPS and MPS showed the strongest inhibition of interaction of heparin and S-protein RBD. The competitive binding studies showed that the IC50 of PPS and MPS against the S-protein RBD binding to immobilized heparin was ~35 nM and ~9 nM, respectively, much lower than the IC50 for soluble heparin (IC50 = 56 nM). Both PPS and MPS showed stronger inhibition than heparin on the S-protein RBD or spike pseudotyped lentiviral particles binding to immobilized heparin. Finally, in an in vitro cell-based assay, PPS and MPS exhibited strong antiviral activities against pseudotyped viral particles of SARS-CoV-2 containing wild-type or Delta S-proteins.
    Keywords:  SARS-CoV-2; heparan sulfate; heparin; mucopolysaccharide polysulfate; pentosan polysulfate; surface plasmon resonance
  6. Drug Metab Dispos. 2022 Feb 22. pii: DMD-MR-2021-000478. [Epub ahead of print]
      Sulfotransferases are ubiquitous enzymes that transfer a sulfo group (SO3) from the universal cofactor donor PAPS to a broad range of acceptor substrates. In humans, the cytosolic sulfotransferases (SULTs) are involved in the sulfation of endogenous compounds such as steroids, neurotransmitters, hormones and bile acids as well as xenobiotics including drugs, toxins and environmental chemicals. The Golgi associated membrane-bound sulfotransferases are involved in post-translational modification of macromolecules from glycosaminoglycans to proteins. The sulfation of small molecules can have profound biological effects on the functionality of the acceptor, including activation, deactivation or enhanced metabolism and elimination. Sulfation of macromolecules has been shown to regulate a number of physiological and pathophysiological pathways by enhancing binding affinity to regulatory proteins or binding partners. Over the last 25 years, crystal structures of these enzymes have provided a wealth of information on the mechanisms of this process and the specificity of these enzymes. This review will focus on the general commonalities of the sulfotransferases, from enzyme structure to catalytic mechanism as well as providing examples into how structural information is being utilized to either design drugs that inhibit sulfotransferases or to modify the enzymes to improve drug synthesis. Significance Statement This manuscript honors Dr. Masahiko Negishi's contribution to our understanding of sulfotransferases mechanism, specificity and their roles in biology by analyzing the crystal structures that have been solved over the last 25 years.
    Keywords:  SULT; Structure/function/mechanism; heparin; phase II drug metabolism; protein structure; sulfation; sulfotransferases
  7. Front Pediatr. 2021 ;9 803732
      Arylsulfatase B is an enzyme present in the lysosomes that involves in the breakdown of large sugar molecules known as glycosaminoglycans (GAGs). Arylsulfatase B chemically modifies two GAGs, namely, dermatan sulfate and chondroitin sulfate, by removing the sulfate group. Mutations in the gene encoding the arylsulfataseB enzyme causes lysosomal storage disorder, mucopolysaccharidosis type VI (MPS VI), or Maroteaux-Lamy syndrome. In this study, we report a case of congenital hearing loss with mild pigmentary changes in the retina, indicative of Usher syndrome, and a missense variant reported as likely pathogenic for MPS VI. Sequencing results identified a pathogenic missense variant p.Arg1746Gln in the CDH23 gene. However, another missense variant ARSB:p.Arg159Cys was reported as likely pathogenic to the treating physician. Mutations in ARSB gene have been associated with MPS VI. Subsequently, ARSB enzyme activity was found low twice in dried blood spot (DBS), suggestive of MPS VI. The patient did not have the clinical features of MPS VI, but considering the wide clinical spectrum, progressive nature of MPS VI, and the fact that a treatment for MPS VI is available to prevent disease progression, further biochemical, enzymatic, and in silico studies were performed to confirm the pathogenicity of this variant. In silico tools predicted this variant to be pathogenic. However, the results of urine and serum GAGs and ARSB enzyme levels measured from patient's fibroblast were found normal. Based on clinical and biochemical findings, ARSB:p.Arg159Cys is likely benign and did not support the diagnosis of MPS VI. However, CDH23:p.Arg1746Gln, a pathogenic variant, supports the underlying cause of hearing loss. This study highlights the importance of a robust correlation between genetic results and clinical presentation, and biochemical and enzymatic studies, to achieve a differential diagnosis.
    Keywords:  Cdh23; arylsulfatase B; glycosaminoglycans; non-syndromic hearing loss; whole exome sequencing
  8. Yi Chuan. 2022 Feb 20. 44(2): 178-186
      Protein tyrosine sulfation (PTS) is an important post-translational modification that regulates a variety of physiological and pathological processes. However, PTS is unstable and lacks effective enrichment methods, which make it difficult to be detected in biological samples. In this study, we detected the tyrosine sulfation modification level of total proteins in developing zebrafish embryos by using high-resolution mass spectrometer, Orbitrap Exploris 480. A total of 26 proteins with tyrosine sulfation were detected, including membrane proteins, secreted proteins, cytoplasmic proteins and nucleoproteins. This study established a methodology in detecting protein tyrosine sulfation modification in embryonic zebrafish, which paved the way for evaluating the biological function of PTS.
    Keywords:  high resolution mass spectrometry; protein tyrosine sulfation; zebrafish
  9. Rev Bras Ginecol Obstet. 2022 Feb;44(2): 142-153
      OBJECTIVE:  To examine the possible effects of adrenal prohormones in the prediction of clinical and metabolic abnormalities in women with polycystic ovary syndrome (PCOS).METHODS:  The present study enrolled 299 normal cycling non-PCOS, 156 normoandrogenemic, and 474 hyperandrogenemic women with PCOS. Baseline characteristics were compared using a chi-squared test or analysis of variance (ANOVA) as appropriate. The roles of adrenal prohormones and their ratios with total testosterone in predicting co-occurring morbidities in women PCOS were evaluated using univariate and multivariate logistic regression analyses.
    RESULTS:  Adrenal hyperandrogenism per dehydroepiandrosterone sulfate (DHEAS) levels were found in 32% of women with PCOS. In non-PCOS women, dehydroepiandrosterone (DHEA) and its sulfate had no predictive role concerning clinical, anthropometric, and metabolic parameters. In PCOS women, mainly in the hyperandrogenemic group, DHEA showed to be a significant predictor against most anthropometric-metabolic index abnormalities (odds ratio [OR] = 0.36-0.97; p < 0.05), and an increase in triglycerides (TG) levels (OR = 0.76; p = 0.006). Dehydroepiandrosterone sulfate presented a few predictive effects regarding PCOS-associated disorders. In controls, DHEAS predicted against the increase in estimated average glucose (OR= 0.38; p = 0.036). In the normoandrogenic group, it predicted against elevation in the waist/hip ratio (WHR) (OR= 0.59; p = 0.042), and in hyperandrogenemic PCOS women, it predicted against abnormality in the conicity index (CI) (OR = 0.31; p = 0.028).
    CONCLUSION:  Dehydroepiandrosterone was shown to be a better predictor of abnormal anthropometric and biochemical parameters in women with PCOS than DHEAS. Thus, regarding adrenal prohormones, DHEA measurement, instead of DHEAS, should be preferred in PCOS management. The effects of androgen prohormones on the prediction of PCOS abnormalities are weak.
  10. J Leukoc Biol. 2022 Feb 21.
      Primary bile acids (BAs), products of cholesterol metabolism and clearance, are synthesized in the liver and released into the intestine to facilitate the digestion and absorption of lipids. BAs are further converted by gut commensal bacteria into secondary colonic BAs and the metabolism disorder is closely linked to cholestatic liver diseases via regulating immune response. However, the effect and underlying mechanism of these host-microorganism biliary metabolites on T lymphocyte remain unclear. In the current study, we synthesized a sulfated product of lithocholic acid (LCA), lithocholic acid 3-sulfate (LCA-3-S), and investigated the binding affinity of the BAs metabolites on RORγt, the transcription factor of IL-17A. Our results demonstrated that the sulfate of LCA, LCA-3-S, exhibited better effect than its oxidated metabolite, 3-oxo-LCA, binding to RORγt. The results further demonstrated that LCA-3-S selectively suppressed Th17 cell differentiation without influence on Th1, Th2, and Treg cells. Collectively, we synthesized the sulfated biliary metabolite LCA-3-S and demonstrated that LCA-3-S selectively inhibited Th17 cell differentiation by targeting RORγt, indicating that metabolite disorder of BAs resulting in the decrease of LCA-3-S probably contributes to the pathogenesis of cholestatic liver diseases.
    Keywords:  RORγt; Th17 cells; lithocholic 3-sulfate
  11. Physiol Res. 2021 Dec 30. 70(Suppl4): S617-S634
      As gestational diabetes mellitus (GDM) is both a frequent and serious complication, steroid levels in pregnancy are extremely elevated and their role in pregnancy is crucial, this review focuses on the role of steroids and related substances in the GDM pathophysiology. Low SHBG levels are associated with insulin resistance and hyperinsulinemia, while also predicting a predisposition to GDM. Other relevant agents are placental hormones such as kisspeptin and CRH, playing also an important role beyond pregnancy, but which are synthesized here in smaller amounts in the hypothalamus. These hormones affect both the course of pregnancy as well as the synthesis of pregnancy steroids and may also be involved in the GDM pathophysiology. Steroids, whose biosynthesis is mainly provided by the fetal adrenal glands, placenta, maternal adrenal glands, and both maternal and fetal livers, are also synthesized in limited amounts directly in the pancreas and may influence the development of GDM. These substances involve the sulfated ?5 steroids primarily acting via modulating different ion channels and influencing the development of GDM in different directions, mostly diabetogenic progesterone and predominantly anti-diabetic estradiol acting both in genomic and non-genomic way, androgens associated with IR and hyperinsulinemia, neuroactive steroids affecting the pituitary functioning, and cortisol whose production is stimulated by CRH but which suppresses its pro-inflammatory effects. Due to the complex actions of steroids, studies assessing their predominant effect and studies assessing their predictive values for estimating predisposition to GDM are needed.
  12. ACS Chem Biol. 2022 Feb 23.
      Sulfotransferases (STs) are ubiquitous enzymes that participate in a vast number of biological processes involving sulfuryl group (SO3) transfer. 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is the universal ST cofactor, serving as the "active sulfate" source in cells. Herein, we report the synthesis of three fluorinated PAPS analogues that bear fluorine or trifluoromethyl substituents at the C2 or C8 positions of adenine and their evaluation as substitute cofactors that enable ST activity to be quantified and real-time-monitored by fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy. Using plant AtSOT18 and human SULT1A3 as two model enzymes, we reveal that the fluorinated PAPS analogues show complementary properties with regard to recognition by enzymes and the working 19F NMR pH range and are attractive versatile tools for studying STs. Finally, we developed an 19F NMR assay for screening potential inhibitors against SULT1A3, thereby highlighting the possible use of fluorinated PAPS analogues for the discovery of drugs for ST-related diseases.
  13. Metabolites. 2022 Jan 19. pii: 91. [Epub ahead of print]12(2):
      Athletes are often exposed to extreme physical stress during training or competitions. The consequent activation of the hypothalamus-hypophysis-adrenal (HPA) axis results in intensified steroid hormone production in the adrenal cortex. We determined the impact of an acute extreme physical stress on adrenal and gonadal steroidogenesis in healthy male professional athletes (n = 40). The subjects underwent an extreme physical load test until total voluntary fatigue between 14:00 and 18:00 when the hormone levels are relatively stable. Blood was taken before the start (baseline), at the peak load (peak), and 30 min following completion of the exercise (recovery). The vital parameters, lactate levels, and blood levels of the 14 steroid hormones were recorded. The multivariate statistical analysis of the results revealed that all monitored hormone levels increased upon stress. Significant changes in steroid concentrations were detected at peak versus baseline, peak versus recovery, and at baseline versus recovery. The mineralocorticoid (including aldosterone and corticosterone), glucocorticoid (11-deoxycortisol and cortisol), and androgen (androstenedione, dehydroepiandrosterone, and dehydroepiandrosterone sulfate) pathways, as well as gonadal testosterone synthesis are activated simultaneously under extreme physical load. The profiling of adrenal and gonadal steroid biosynthesis in athletes may help the characterization of their loading capacity.
    Keywords:  load capacity; physical exercise; steroid
  14. IUBMB Life. 2022 Feb 26.
      Hyaluronan (HA) is a component of the extracellular matrix (ECM) it is the main non-sulfated glycosaminoglycan able to modulate cell behavior in the healthy and tumor context. Sulfated hyaluronan (sHA) is a biomaterial derived from chemical modifications of HA, since this molecule is not naturally sulfated. The HA sulfation modifies several properties of the native molecule, acquiring antitumor properties in different cancers. In this study, we evaluated the action of sHA of ~30-60 kDa with different degrees of sulfation (0.7 sHA1 and 2.5 sHA3) on tumor cells of a breast, lung, and colorectal cancer model and its action on other cells of the tumor microenvironment, such as endothelial and monocytes/macrophage cells. Our data showed that in breast and lung tumor cells, sHA3 is able to modulate cell viability, cytotoxicity, and proliferation, but no effects were observed on colorectal cancer cells. In 3D cultures of breast and lung cancer cells, sHA3 diminished the size of the tumorsphere and modulated total HA levels. In these tumor models, treatment of monocytes/macrophages with sHA3 showed a downregulation of the expression of angiogenic factors. We also observed a decrease in endothelial cell migration and modulation of the hyaluronan-binding protein TSG-6. In the breast in vivo xenograft model, monocytes/macrophages preincubated with sHA1 or sHA3 decreased tumor vasculature, TSG-6 and HA levels. Besides, in silico analysis showed an association of TSG-6, HAS2, and IL-8 with biological processes implicated in the progression of the tumor. Taken together, our data indicate that sHA in a breast and lung tumor context is able to induce an antiangiogenic action on tumor cells as well as in monocytes/macrophages (Mo/MØ) by modulation of endothelial migration, angiogenic factors, and vessel formation.
    Keywords:  angiogenesis; monocytes/macrophages; sulfated Hyaluronan; tumor microenvironment
  15. Int J Mol Sci. 2022 Feb 09. pii: 1950. [Epub ahead of print]23(4):
      Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children and infants. To date, there is no effective vaccine available against RSV. Heparan sulfate is a type of glycosaminoglycan that aids in the attachment of the RSV to the host cell membrane via the G protein. In the present study, the effect of amino acid substitution on the structure and stability of the ectodomain G protein was studied. Further, it was investigated whether mutation (K117A) in the CX3C motif of G protein alters the binding with heparan sulfate. The point mutation significantly affects the conformational stability of the G protein. The mutant protein showed a low binding affinity with heparan sulfate as compared to the wild-type G protein, as determined by fluorescence quenching, isothermal titration calorimetry (ITC), and molecular docking studies. The low binding affinity and decreased stability suggested that this mutation may play an important role in prevention of attachment of virion to the host cell receptors. Collectively, this investigation suggests that mutation in the CX3C motif of G protein may likely improve the efficacy and safety of the RSV vaccine.
    Keywords:  CX3C motif; G protein; RSV; fluorescence binding; glycosaminoglycan; heparan sulfate; isothermal titration calorimetry; molecular docking
  16. Antioxidants (Basel). 2022 Feb 11. pii: 361. [Epub ahead of print]11(2):
      Hydrogen sulfide (H2S) was the third gasotransmitter to be recognized as a cytoprotectant. A recent study demonstrated that exogenous supplementation of H2S ameliorates functional insufficiency in chronic kidney disease (CKD). However, how the H2S system is impaired by CKD has not been elucidated. The uremic toxin indoxyl sulfate (IS) is known to accumulate in CKD patients and harm the renal tubular cells. This study therefore treated the proximal tubular cells, LLC-PK1, with IS to see how IS affects H2S formation. Our results showed that H2S release from LLC-PK1 cells was markedly attenuated by IS when compared with control cells. The H2S donors NaHS and GYY-4137 significantly attenuated IS-induced tubular damage, indicating that IS impairs H2S formation. Interestingly, IS downregulated the H2S-producing enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST), and these effects could be reversed by inhibition of the IS receptor, aryl hydrocarbon receptor (AhR). As transcription factor specificity protein 1 (Sp1) regulates the gene expression of H2S-producing enzymes, we further showed that IS significantly decreased the DNA binding activity of Sp1 but not its protein expression. Blockade of AhR reversed low Sp1 activity caused by IS. Moreover, exogenous H2S supplementation attenuated IS-mediated superoxide formation and depletion of the cellular glutathione content. These results clearly indicate that IS activates AhR, which then attenuates Sp1 function through the regulation of H2S-producing enzyme expression. The attenuation of H2S formation contributes to the low antioxidant defense of glutathione in uremic toxin-mediated oxidative stress, causing tubular cell damage.
    Keywords:  chronic kidney disease; hydrogen sulfide; indoxyl sulfate; oxidative stress; specificity protein 1; tubulotoxicity
  17. Antioxidants (Basel). 2022 Feb 16. pii: 400. [Epub ahead of print]11(2):
      Kidney disease patients may have concurrent chronic kidney disease-associated mineral bone disorder and hypertension. Cardiovascular disease (CVD) and neuropathy occur due to kidney failure-induced accumulation of uremic toxins in the body. Indoxyl sulfate (IS), a product of indole metabolism in the liver, is produced from tryptophan by the intestinal flora and is ultimately excreted through the kidneys. Hemodialysis helps renal failure patients eliminate many nephrotoxins, except for IS, which leads to a poor prognosis. Although the impacts of IS on cardiac and renal development have been well documented using mouse and rat models, other model organisms, such as zebrafish, have rarely been studied. The zebrafish genome shares at least 70% similarity with the human genome; therefore, zebrafish are ideal model organisms for studying vertebrate development, including renal development. In this study, we aimed to investigate the impact of IS on the development of zebrafish embryos, especially cardiac and renal development. At 24 h postfertilization (hpf), zebrafish were exposed to IS at concentrations ranging from 2.5 to 10 mM. IS reduced survival and the hatching rate, caused cardiac edema, increased mortality, and shortened the body length of zebrafish embryos. In addition, IS decreased heart rates and renal function. IS affected zebrafish development via the ROS and MAPK pathways, which subsequently led to inflammation in the embryos. The results suggest that IS interferes with cardiac and renal development in zebrafish embryos, providing new evidence about the toxicity of IS to aquatic organisms and new insights for the assessment of human health risks. Accordingly, we suggest that zebrafish studies can ideally complement mouse model studies to allow the simultaneous and comprehensive investigation of the physiological impacts of uremic endotheliotoxins, such as IS, on cardiac and renal development.
    Keywords:  ROS; indoxyl sulfate (IS); inflammation; mitogen-activated protein kinase (MAPK) pathway; zebrafish model
  18. PLoS Genet. 2022 Feb 22. 18(2): e1010067
      Chondroitin/dermatan sulfate (CS/DS) proteoglycans are indispensable for animal development and homeostasis but the large number of enzymes involved in their biosynthesis have made CS/DS function a challenging problem to study genetically. In our study, we generated loss-of-function alleles in zebrafish genes encoding CS/DS biosynthetic enzymes and characterized the effect on development in single and double mutants. Homozygous mutants in chsy1, csgalnact1a, csgalnat2, chpfa, ust and chst7, respectively, develop to adults. However, csgalnact1a-/- fish develop distinct craniofacial defects while the chsy1-/- skeletal phenotype is milder and the remaining mutants display no gross morphological abnormalities. These results suggest a high redundancy for the CS/DS biosynthetic enzymes and to further reduce CS/DS biosynthesis we combined mutant alleles. The craniofacial phenotype is further enhanced in csgalnact1a-/-;chsy1-/- adults and csgalnact1a-/-;csgalnact2-/- larvae. While csgalnact1a-/-;csgalnact2-/- was the most affected allele combination in our study, CS/DS is still not completely abolished. Transcriptome analysis of chsy1-/-, csgalnact1a-/- and csgalnact1a-/-;csgalnact2-/- larvae revealed that the expression had changed in a similar way in the three mutant lines but no differential expression was found in any of fifty GAG biosynthesis enzymes identified. Thus, zebrafish larvae do not increase transcription of GAG biosynthesis genes as a consequence of decreased CS/DS biosynthesis. The new zebrafish lines develop phenotypes similar to clinical characteristics of several human congenital disorders making the mutants potentially useful to study disease mechanisms and treatment.
  19. Front Plant Sci. 2022 ;13 823668
      Fucoidanases are endo-fucoidanases (also known as endo-fucanases) that catalyze hydrolysis of α-glycosidic linkages in fucoidans, a family of sulfated fucose-rich polysaccharides primarily found in the cell walls of brown seaweeds. Fucoidanases are promising tools for producing bioactive fucoidan oligosaccharides for a range of biomedical applications. High sulfation degree has been linked to high bioactivity of fucoidans. In this study, a novel fucoidanase, Fhf2, was identified in the genome of the aerobic, Gram-negative marine bacterium Formosa haliotis. Fhf2 was found to share sequence similarity to known endo-α(1,4)-fucoidanases (EC from glycoside hydrolase family 107. A C-terminal deletion mutant Fhf2∆484, devoid of 484 amino acids at the C-terminus, with a molecular weight of approximately 46 kDa, was constructed and found to be more stable than the full-length Fhf2 protein. Fhf2∆484 showed endo-fucoidanase activity on fucoidans from different seaweed species including Fucus evanescens, Fucus vesiculosus, Sargassum mcclurei, and Sargassum polycystum. The highest activity was observed on fucoidan from F. evanescens. The Fhf2∆484 enzyme was active at 20-45°C and at pH 6-9 and had optimal activity at 37°C and pH 8. Additionally, Fhf2∆484 was found to be calcium-dependent. NMR analysis showed that Fhf2∆484 catalyzed hydrolysis of α(1,4) linkages between L-fucosyl moieties sulfated on C2 (similar to Fhf1 from Formosa haliotis), but Fhf2∆484 in addition released oligosaccharides containing a substantial amount of 2,4-disulfated fucose residues. The data thus suggest that the Fhf2∆484 enzyme could be a valuable candidate for producing highly sulfated oligosaccharides applicable for fucoidan bioactivity investigations.
    Keywords:  FTIR; Fucus evanescens; Sargassum mcclurei; T9SS; calcium dependency; sulfation
  20. ACS Chem Biol. 2022 Feb 24.
      Heparin products are widely used clinical anticoagulants essential in the practice of modern medicine. Low-molecular-weight heparins (LMWHs) are currently prepared by the controlled chemical or enzymatic depolymerization of unfractionated heparins (UFHs) that are extracted from animal tissues. In many clinical applications, LMWHs have displaced UFHs and currently comprise over 60% of the heparin market. In the past, our laboratory has made extensive efforts to prepare bioengineered UFHs relying on a chemoenzymatic process to address concerns about animal-sourced UFHs. The current study describes the use of a novel chemoenzymatic process to prepare a chemobiosynthetic LMWH from a low-molecular-weight heparosan. The resulting chemobiocatalytic LMWH matches most of the United States pharmacopeial specifications for enoxaparin, a LMWH prepared through the base-catalyzed depolymerization of animal-derived UFH.
  21. Mar Drugs. 2022 Feb 03. pii: 121. [Epub ahead of print]20(2):
      We previously reported that rhamnan sulfate (RS) purified from Monostroma nitidum significantly suppressed lipopolysaccharide (LPS)-induced inflammation in cultured human vascular endothelial cells. Here, we analyzed the effect of orally administered RS on LPS-induced damage to mouse organs and vascular endothelium. RS (1 mg) was orally administered daily to BALB/c mice, 50 μg of LPS was intraperitoneally administered on day 8, and Evans blue was injected into the tail vein 6 h later. After 30 min, LPS-treated mice showed pulmonary Evans blue leakage and elevated plasma levels of liver damage markers, whereas this reaction was suppressed in LPS + RS-treated mice. Immunohistochemical and Western blot analysis of mouse organs 24 h after LPS treatment showed significant neutrophil infiltration into the lung, liver, and jejunum tissues of LPS-treated mice and high expression levels of inflammation-related factors in these tissues. Expression levels of these factors were significantly suppressed in LPS + RS-treated mice. Analysis of lung glycocalyx showed a significant reduction in glycocalyx in LPS-treated mice but not in LPS + RS-treated mice. Levels of syndecan-4, one of the glycocalyx components, decreased in LPS-treated mice and increased in LPS + RS-treated mice. The current results suggest that orally administered RS protects organs and vascular endothelium from LPS-induced inflammation and maintains blood circulation.
    Keywords:  Monostroma nitidum; anti-inflammation; endothelium; glycocalyx; rhamnan sulfate
  22. Clin Pharmacol Drug Dev. 2022 Feb 21.
      Isavuconazonium sulfate is the water-soluble prodrug of the novel, broad-spectrum, triazole antifungal agent isavuconazole. This was a first-in-Japanese study assessing the pharmacokinetics, safety, and tolerability of isavuconazonium sulfate. The study was conducted in 2 parts: part 1 (single ascending dose; 100-, 200-, and 400-mg equivalent of isavuconazole oral or intravenous administration); and part 2 (multiple doses for 16 days; 200-mg equivalent of isavuconazole oral or intravenous administration; once-daily administration with a loading regimen every 8 hours for the first 48 hours). A total of 60 and 16 subjects were randomized in part 1 and part 2, respectively. Observed clearance was lower in this study compared to what was previously reported in predominantly White populations and similar to clearance in non-Japanese Asian populations. The range of the plasma isavuconazole concentration in this study was within the range of the pivotal phase 3 study, with no relationship between isavuconazole exposure and either efficacy or safety. There were no serious adverse events, and all reported treatment-emergent adverse events were of mild intensity. This study confirmed that isavuconazonium sulfate was safe and well tolerated in healthy adult Japanese subjects.
    Keywords:  isavuconazole; isavuconazonium sulfate; pharmacokinetics; safety; tolerability
  23. Toxins (Basel). 2022 Jan 26. pii: 100. [Epub ahead of print]14(2):
      The uremic toxin indoxyl sulfate (IS), elevated in chronic kidney disease (CKD), is known to contribute towards progressive cardiovascular disease. IS activates the aryl hydrocarbon receptor (AhR) mediating oxidative stress and endothelial dysfunction via activation of the CYP1A1 pathway. The present study examines AhR inhibition with the antagonist, CH223191, on IS-mediated impairment of vascular endothelial function and disruption of redox balance. The acute effects of IS on endothelium-dependent relaxation were assessed in aortic rings from Sprague Dawley rats exposed to the following conditions: (1) control; (2) IS (300 μM); (3) IS + CH223191 (1 μM); (4) IS + CH223191 (10 μM). Thereafter, tissues were assessed for changes in expression of redox markers. IS reduced the maximum level of endothelium-dependent relaxation (Rmax) by 42% (p < 0.001) compared to control, this was restored in the presence of increasing concentrations of CH223191 (p < 0.05). Rings exposed to IS increased expression of CYP1A1, nitro-tyrosine, NADPH oxidase 4 (NOX4), superoxide, and reduced eNOS expression (p < 0.05). CH223191 (10 μM) restored expression of these markers back to control levels (p < 0.05). These findings demonstrate the adverse impact of IS-mediated AhR activation on the vascular endothelium, where oxidative stress may play a critical role in inducing endothelial dysfunction in the vasculature of the heart and kidneys. AhR inhibition could provide an exciting novel therapy for CVD in the CKD setting.
    Keywords:  aryl hydrocarbon receptor; cardiovascular disease; chronic kidney disease; endothelial dysfunction; indoxyl sulfate; oxidative stress