bims-supasi Biomed News
on Sulfation pathways and signalling
Issue of 2023‒03‒05
ten papers selected by
Jonathan Wolf Mueller
University of Birmingham

  1. Front Mol Biosci. 2023 ;10 1146685
      Heparan sulfates (HSs) are the main components in the glycocalyx which covers endothelial cells and modulates vascular homeostasis through interactions with multiple Heparan sulfate binding proteins (HSBPs). During sepsis, heparanase increases and induces HS shedding. The process causes glycocalyx degradation, exacerbating inflammation and coagulation in sepsis. The circulating heparan sulfate fragments may serve as a host defense system by neutralizing dysregulated Heparan sulfate binding proteins or pro-inflammatory molecules in certain circumstances. Understanding heparan sulfates and heparan sulfate binding proteins in health and sepsis is critical to decipher the dysregulated host response in sepsis and advance drug development. In this review, we will overview the current understanding of HS in glycocalyx under septic condition and the dysfunctional heparan sulfate binding proteins as potential drug targets, particularly, high mobility group box 1 (HMGB1) and histones. Moreover, several drug candidates based on heparan sulfates or related to heparan sulfates, such as heparanase inhibitors or heparin-binding protein (HBP), will be discussed regarding their recent advances. By applying chemical or chemoenzymatic approaches, the structure-function relationship between heparan sulfates and heparan sulfate binding proteins is recently revealed with structurally defined heparan sulfates. Such homogenous heparan sulfates may further facilitate the investigation of the role of heparan sulfates in sepsis and the development of carbohydrate-based therapy.
    Keywords:  HMGB1 (high mobility group box 1); heparan sulfate; heparan sulfate binding proteins; heparin; histones
  2. Biochem Biophys Res Commun. 2023 Feb 20. pii: S0006-291X(23)00223-1. [Epub ahead of print]652 103-111
      Hepatocellular carcinoma (HCC) is an aggressive tumor triggered by various factors such as virus infection and alcohol abuse. Glucuronomannan polysaccharide (Gx) is a subtype of fucoidans that possesses many bioactivities, but its anti-tumor activities in HCC have not been reported. In this paper, the anti-tumor effects of glucuronomannan oligosaccharides (Gx) and its sulfated derivatives (GxSy) on hepatocarcinoma Huh7.5 cells were investigated. The anti-proliferation, anti-metastasis activities, and underlying mechanism of Gx and GxSy on Huh7.5 cells were analyzed and compared by MTT, wound healing, transwell, and western blotting assays, respectively. Results showed that the best anti-proliferation effects were G4S1 and G4S2 among 13 drugs, which were 38.67% and 30.14%, respectively. The cell migration rates were significantly inhibited by G2S1, G4S2, G6S2, and unsulfated Gn. In addition, cell invasion effects treated with G4S1, G4S2, and G6S1 decreased to 48.62%, 36.26%, and 42.86%, respectively. Furthermore, sulfated G4 regulated the expression of (p-) FAK and MAPK pathway, and sulfated G6 down-regulated the MAPK signaling pathway while activating the PI3K/AKT pathway. On the contrary, sulfated G2 and unsulfated Gx had no inhibited effects on the FAK-mTOR pathway. These results indicated that sulfated Gx derivatives have better anti-tumor activities than unsulfated Gx in cell proliferation and metastasis process in vitro, and those properties depend on the sulfation group levels. Moreover, degrees of polymerization of Gx also played a vital role in mechanisms and bioactivities. This finding shows the structure-activity relationship for developing and applying the marine oligosaccharide candidates.
    Keywords:  Anti-hepatocarcinoma; Glucuronomannan oligosaccharides; Mechanism; Structure-activity relationship; Sulfation
  3. Int J Biol Macromol. 2023 Feb 24. pii: S0141-8130(23)00418-X. [Epub ahead of print]235 123525
      Gellan gum (GG) is a biodegradable polysaccharide and forms thermosensitive hydrogels by a helix-mediated mechanism. Unfortunately, the wide use of GG in tissue engineering has been restricted due to its dramatically higher gelation temperature than normal body temperature. Here, we show that partial sulfation of GG affords a cytocompatible body temperature-responsive hydrogel with an interesting thermoreversibility at 42 °C. The partial sulfation of GG was confirmed by FTIR, EDX and elemental analyses. The sulfated GGs (SGGs) had a higher swelling ratio and degradation in PBS compared to the neat GG. Based on the results of rheometry analysis, the SGG with a degree of sulfation of 0.27 (H3 sample) showed a gelation temperature close to the physiological temperature. In addition, the drop in mechanical properties of SGGs was compensated by a further calcium-mediated ionic crosslinking, where Young's modulus of H3 increased from 10.6 ± 1.9 kPa up to 38.4 ± 5.5 kPa. Finally, we showed that the partial sulfation reaction of GG is a simple and mild strategy to modify chemical structure of GG, and to produce a cytocompatible, body temperature-responsive hydrogel compared to other modifying reactions such as oxidation reaction.
    Keywords:  Gelation temperature; Gellan gum; Hydrogel; Hydrogen bonding; Sulfation
  4. Nat Commun. 2023 Mar 03. 14(1): 1226
      Netrin-1 is a bifunctional chemotropic guidance cue that plays key roles in diverse cellular processes including axon pathfinding, cell migration, adhesion, differentiation, and survival. Here, we present a molecular understanding of netrin-1 mediated interactions with glycosaminoglycan chains of diverse heparan sulfate proteoglycans (HSPGs) and short heparin oligosaccharides. Whereas interactions with HSPGs act as platform to co-localise netrin-1 close to the cell surface, heparin oligosaccharides have a significant impact on the highly dynamic behaviour of netrin-1. Remarkably, the monomer-dimer equilibrium of netrin-1 in solution is abolished in the presence of heparin oligosaccharides and replaced with highly hierarchical and distinct super assemblies leading to unique, yet unknown netrin-1 filament formation. In our integrated approach we provide a molecular mechanism for the filament assembly which opens fresh paths towards a molecular understanding of netrin-1 functions.
  5. Drug Test Anal. 2023 Feb 26.
      Sulfated metabolites have shown to have potential as long-term markers (LTMs) of anabolic-androgenic steroid (AAS) abuse. The compatibility of gas chromatography-mass spectrometry (GC-MS) with trimethylsilyl (TMS)-derivatives of non-hydrolysed sulfated steroids has been demonstrated, where, after derivatisation, generally, two closely eluting isomers are formed that both have the same molecular ion [M-H2 SO4 ]•+ . Sulfated reference standards are in limited commercial availability and therefore the current knowledge of the GC-MS behaviour of these compounds is mainly based on sulfating and analysing the available standard reference material. This procedure can unfortunately not cover all of the current known LTMs as these are often not available as pure substance. Therefore, in theory, some metabolites could be missed as they exhibit alternative behaviour. To investigate the matter, in-house sulfated reference materials that bear resemblance to known sulfated LTMs were analysed on GC-MS in their TMS-derivatised non-hydrolysed state. The (alternative) gas chromatographic and mass spectrometric behaviour was mapped, evaluated and linked to the corresponding steroid structures. Afterwards, using fraction collection, known sulfated LTMs were isolated from excretion urine to confirm the observed findings. The categories that were selected were mono-hydroxy-diones, 17-methyl-3,17-diols and 17-keto-3,16-diols as these are commonly encountered AAS conformations. The ability to predict the GC-MS behaviour of non-hydrolysed sulfated AAS metabolites is the corner stone of finding new metabolites. This knowledge is also essential for e.g., understanding AAS detection analyses, for the mass spectrometric characterization of metabolites of new designer steroids or when one needs to characterize an unknown steroid structure.
    Keywords:  Doping; gas chromatography; mass spectrometry; steroids; sulfates
  6. Drug Metab Dispos. 2023 Mar 02. pii: DMD-AR-2022-001218. [Epub ahead of print]
      The human pharmacokinetics, metabolism, and excretion of [14C]-ganaxolone (GNX) were characterized in healthy male subjects (n = 8) following a single 300 mg (150 mCi) oral dose. GNX exhibited a short half-life of 4 h in plasma, while total radioactivity had a half-life of 413 h indicating extensive metabolism to long-lived metabolites. Identification of the major GNX circulating metabolites required extensive isolation and purification for LC-MS/MS analysis, together with in vitro studies, NMR spectroscopy and synthetic chemistry support. This revealed that the major routes of GNX metabolism involved hydroxylation at the 16a-hydroxy position, stereoselective reduction of the 20-ketone to afford the corresponding 20a-hydroxysterol, and sulfation of the 3a-hydroxy group. This latter reaction yielded an unstable tertiary sulfate, which eliminated the elements of H2SO4 to introduce a double bond in the A ring. A combination of these pathways, together with oxidation of the 3b-methyl substituent to a carboxylic acid and sulfation at the 20a position, led to the major circulating metabolites in plasma, termed M2 and M17. These studies, which led to the complete or partial identification of no less than 59 metabolites of GNX, demonstrated the high complexity of the metabolic fate of this drug in humans, and demonstrated that the major circulating products in plasma can result from multiple sequential processes that may not be easily replicated in animals or with animal or human in vitro systems. Significance Statement Studies on the metabolism of GNX in humans revealed a complex array of products that circulated in plasma, the two major components of which were formed via an unexpected multi-step pathway. Complete structural characterization of these (disproportionate) human metabolites required extensive in vitro studies, along with contemporary mass spectrometry, NMR spectroscopy, and synthetic chemistry efforts, which served to underscore the limitations of traditional animal studies in predicting major circulating metabolites in man.
    Keywords:  drug metabolism; metabolite identification
  7. Int J Biol Macromol. 2023 Feb 28. pii: S0141-8130(23)00740-7. [Epub ahead of print] 123846
      Appearance of senescent beta cells in the pancreas leads to the onset of type 2 diabetes (T2D). The structural analysis of a sulfated fuco-manno-glucuronogalactan (SFGG) indicated SFGG had the backbones of interspersing 1, 3-linked β-D-GlcpA residues, 1, 4-linked α-D-Galp residues, and alternating 1, 2-linked α-D-Manp residues and 1, 4-linked β-D-GlcpA residues, sulfated at C6 of Man residues, C2/C3/C4 of Fuc residues and C3/C6 of Gal residues, and branched at C3 of Man residues. SFGG effectively alleviated senescence-related phenotypes in vitro and in vivo, including cell cycle, senescence-associated β-galactosidase, DNA damage and senescence-associated secretory phenotype (SASP) -associated cytokines and hall markers of senescence. SFGG also alleviated beta cell dysfunction in insulin synthesis and glucose-stimulated insulin secretion. Mechanistically, SFGG attenuated senescence and improved beta cell function via PI3K/Akt/FoxO1 signaling pathway. Therefore, SFGG could be used for beta cell senescence treatment and alleviation of the progression of T2D.
    Keywords:  PI3K/Akt/FoxO1; Pancreatic beta cell senescence; Sulfated fuco-manno-glucuronogalactan
  8. Drug Test Anal. 2023 Feb 27.
      In the present study, the application and evaluation of Girard Reagent T (GRT) derivatization for the simultaneous detection and significantly important identification of different phase II methenolone and mesterolone metabolites by LC-MS/(MS) are presented. For the LC-MS analysis of target analytes two complementary isolation methods were developed; a derivatization and shoot method in which native urine is diluted with derivatization reagent and is injected directly to LC-MS and a liquid liquid extraction method, using ethyl acetate at pH 4.5, for the effective isolation of both sulfate and glucuronide metabolites of the named steroids as well as of their free counterparts. For the evaluation of the proposed protocols, urine samples from methenolone and mesterolone excretion studies were analyzed against at least one sample from a different excretion study. Retention times, along with product ion ratios, were evaluated according to the WADA TD2021IDCR requirements, in order to determine maximum detection and identification time windows for each metabolite. Established identification windows obtained after LC-MS/(MS) analysis were further compared with those obtained after GC-MS/(MS) analysis of the same samples from the same excretion studies, for the most common analytes monitored by GC-MS/(MS). Full validation was performed for the developed derivatization and shoot method for the identification of methenolone metabolite, 3α-hydroxy-1-methylen-5α-androstan-17-one-3-glucuronide (mth3). Overall, the GRT derivatization presented herein, offers a tool for the simultaneous sensitive detection of free, intact glucuronide and sulfate metabolites by LC-MS/(MS) that enhance significantly the detection and identification time windows of specific methenolone and mesterolone metabolites for doping control analysis.
    Keywords:  Girard’s T derivatization; LC-MS(/MS); doping control; glucuronide metabolites; mesterolone; methenolone; sulfate metabolites
  9. Front Cell Dev Biol. 2023 ;11 1089028
      Metaplasia, dysplasia, and cancer arise from normal epithelia via a plastic cellular transformation, typically in the setting of chronic inflammation. Such transformations are the focus of numerous studies that strive to identify the changes in RNA/Protein expression that drive such plasticity along with the contributions from the mesenchyme and immune cells. However, despite being widely utilized clinically as biomarkers for such transitions, the role of glycosylation epitopes is understudied in this context. Here, we explore 3'-Sulfo-Lewis A/C, a clinically validated biomarker for high-risk metaplasia and cancer throughout the gastrointestinal foregut: esophagus, stomach, and pancreas. We discuss the clinical correlation of sulfomucin expression with metaplastic and oncogenic transformation, as well as its synthesis, intracellular and extracellular receptors and suggest potential roles for 3'-Sulfo-Lewis A/C in contributing to and maintaining these malignant cellular transformations.
    Keywords:  barrett’s esophagus; gastric metaplasia; glycosylation; intraductal papillary mucinous neoplasm; pancreatic cancer; pancreatic intraepithelial neoplasia; sulfation
  10. Endocrinol Disord. 2022 ;pii: 122. [Epub ahead of print]6(3):
      Sulfoconjugation is the major pathway for thyroid hormone (TH) metabolism, converting T4 to inactive metabolites, T4S, rT3S, and T3S in fetus, via sulfotransferases (SULT) and type 3 deiodinase in gestation. Consistent with high production rate of T4S and rT3S, there are high serum sulfated iodothyronine analogs, including T4S, T3S, rT3S, and 3,3'-T2S (T2S), in ovine and human fetal and preterm infants. Fetal TH metabolic pathways predict T2S as the major TH metabolite in the fetus. Since maternal T2S appears to be quantitatively derived from fetal T3 (the active TH), the amount of T2S in the maternal compartment correlates with fetal thyroid function in sheep. In humans, maternal serum contains high levels of radioimmunoassayable T2S; however, it displays as a peak adjacent to but unidentical to synthetic T2S on HLPC and we named it the W-Compound. Levels of W-Compound increase during pregnancy and peak as high as 20-fold to that of nonpregnant women. Maternal serum levels of W-Compound significantly correlate with fetal T4 and W-compound concentrations but not maternal serum T4 in euthyroid or hyperthyroid women, showing a distinct difference between fetal and maternal in TH metabolism. Fetal T2S is actively transferred to the mother via placenta and the quantity of T2S or its metabolite (W-Compound) in maternal compartment reflects fetal thyroid function. Thus, maternal serum W-Compound may be a biomarker for monitoring fetal thyroid function in utero, although more investigations are needed to determine if it can be used as an alternative strategy for screening/managing congenital hypothyroidism due to dysregulated thyroid hormone metabolism.
    Keywords:  W-Compound; biomarker; congenital hypothyroidism; fetal thyroid function; neonatal screening