bims-supasi Biomed News
on Sulfation pathways and signalling
Issue of 2022‒04‒17
sixteen papers selected by
Jonathan Wolf Mueller
University of Birmingham

  1. Am J Physiol Cell Physiol. 2022 Apr 13.
      Heparan sulfate is a widely expressed polysaccharide in the extracellular matrix and on the cell surface. 3-O-sulfated heparan sulfate represents only a small percentage of heparan sulfate from biological sources. However, this subpopulation is closely associated with biological functions of heparan sulfate. The 3-O-sulfated heparan sulfate is biosynthesized by heparan sulfate 3-O-sulfotransferase, which exists in seven different isoforms. This review article summarizes the recent progress on the substrate specificity studies of different 3-O-sulfotransferase isoforms involving the use of homogeneous oligosaccharide substrates and crystal structural analysis. The article also reviews a newly developed LC-MS/MS based method to analyze the level of 3-O-sulfated heparan sulfate with high sensitivity and quantitative capability. This newly emerged technology will provide new tools to study the structure and function relationship of heparan sulfate.
    Keywords:  heparan sulfate; heparin; proteoglycans; sulfotransferases
  2. ACS Chem Biol. 2022 Apr 14.
      Heparan sulfate (HS) and chondroitin sulfate (CS) are two structurally distinct natural polysaccharides. Here, we report the synthesis of a library of seven structurally homogeneous HS and CS chimeric dodecasaccharides (12-mers). The synthesis was accomplished using six HS biosynthetic enzymes and four CS biosynthetic enzymes. The chimeras contain a CS domain on the reducing end and a HS domain on the nonreducing end. The synthesized chimeras display anticoagulant activity as measured by both in vitro and ex vivo experiments. Furthermore, the anticoagulant activity of H/C 12-mer 5 is reversible by protamine, a U.S. Food and Drug Administration-approved polypeptide to neutralize anticoagulant drug heparin. Our findings demonstrate the synthesis of unnatural HS-CS chimeric oligosaccharides using natural biosynthetic enzymes, offering a new class of glycan molecules for biological research.
  3. Biosci Biotechnol Biochem. 2022 Apr 15. pii: zbac057. [Epub ahead of print]
      Glycosaminoglycans (GAGs) are found in the various tissues and are involved in many physiological functions. Since the rhesus monkey (Macaca mulatta) is the most widely used nonhuman primate in biomedical research, an understanding of the compositions of GAGs in their tissues is important. The aim of the study was to determine the content and sulfation pattern of disaccharides contained in several tissues of the rhesus monkey. The chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chain was extracted from several tissues of female and male rhesus monkeys. Compositional analysis was performed after digestion with chondroitinases ABC and ACI to reveal the sulfation pattern of the CS/DS hybrid chain. This study revealed that the major CS/DS disaccharide units present in the tissues were A- and C-types. The E- and iE-types were specifically distributed not only in the tracheal tissue but also in gastrointestinal tissues.
    Keywords:  chondroitin sulfate; dermatan sulfate; glycosaminoglycan; rhesus monkey; sulfation pattern
  4. Chem Asian J. 2022 Apr 15.
      Heparan sulfate (HS) is a highly sulfated natural carbohydrate that plays crucial roles in cancer, inflammation, and angiogenesis. Heparanase (HPSE) is the sole HS degrading endoglycosidase that cleaves HS at structure-dependent sites along the polysaccharide chain. Overexpression of HPSE by cancer cells correlates with increased tumor size and enhanced metastasis. Previously we have shown that a tetramer HS mimetic is a potent HPSE inhibitor displaying remarkable anticancer activity in vivo . Building on that work, we report the synthesis and testing of a novel library of single entity trimer glycolipid mimetics that effectively inhibit HPSE at low nanomolar concentrations. A lipophilic arm was introduced to assess whether an improvement of pharmacokinetics and plasma residence time would offset the reduction in charge and multivalency. Preclinical tests in a mouse syngeneic model showed effective tumor growth inhibition by the tetramer but not the trimer glycomimetic.
    Keywords:  Carbohydrates; Colorectal cancer; Glycolipids; Heparan Sulfate; Heparin
  5. J Virol. 2022 Apr 14. e0204221
      As an important neurotropic enterovirus, enterovirus 71 (EV71) is occasionally associated with severe neurological diseases and high mortality rates in infants and young children. Understanding the interaction between host factors and EV71 will play a vital role in developing antivirals and optimizing vaccines. Here, we performed a genome-wide CRISPR-Cas9 knockout screen and revealed that scavenger receptor class B member 2 (SCARB2), solute carrier family 35 member B2 (SLC35B2), and beta-1,3-glucuronyltransferase 3 (B3GAT3) are essential in facilitating EV71 replication. Subsequently, the exploration of molecular mechanisms suggested that the knockout of SLC35B2 or B3GAT3, not SCARB2, led to a remarkable decrease in the binding of EV71 to cells and internalization into cells. Furthermore, we found that the infection efficiency for EV71 was positively correlated with the level of host cell sulfation, not simply with the amount of heparan sulfate, suggesting that an unidentified sulfated protein(s) must contribute to EV71 infection. In support of this idea, we screened possible sulfated proteins among the proteinous receptors for EV71 and confirmed that SCARB2 could uniquely interact with both tyrosyl protein sulfotransferases in humans. We then performed mass spectrometric analysis of SCARB2, identifying five sites with tyrosine sulfation. The function verification test indicated that there were more than five tyrosine-sulfated sites on SCARB2. Finally, we constructed a model for EV71 entry in which both heparan sulfate and SCARB2 are regulated by SLC35B2 and act cooperatively to support viral binding, internalization, and uncoating. Taken together, this is the first time that we performed the pooled CRISPR-Cas9 genetic screening to investigate the interplay of host cells and EV71. Furthermore, we found that a novel host factor, SLC35B2, played a dual role in regulating the overall sulfation comprising heparan sulfate sulfation and protein tyrosine sulfation, which are critical for EV71 entry. IMPORTANCE As the most important nonpolio neurotropic enterovirus lacking specific treatments, EV71 can transmit to the central nervous system, leading to severe and fatal neurological complications in infants and young children. The identification of new factors that facilitate or inhibit EV71 replication is crucial to uncover the mechanisms of viral infection and pathogenesis. To date, only a few host factors involved in EV71 infection have been characterized. Herein, we conducted a genome-wide CRISPR-Cas9 functional knockout (GeCKO) screen for the first time to study EV71 in HeLa cells. The screening results are presented as a ranked list of candidates, including 518 hits in the positive selection that facilitate EV71 replication and 1,044 hits in the negative selection that may be essential for cell growth and survival or for suppressing EV71 infection. We subsequently concentrated on the top three hits in the positive selection: SCARB2, SLC35B2, and B3GAT3. The knockout of any of these three genes confers strong resistance against EV71 infection. We confirmed that EV71 infection is codependent on two receptors, heparan sulfate and SCARB2. We also identified a host entry factor, SLC35B2, indirectly facilitating EV71 infection through regulation of the host cell sulfation, and determined a novel posttranslational modification, protein tyrosine sulfation existing in SCARB2. This study revealed that EV71 infectivity exhibits a significant positive correlation with the level of cellular sulfation regulated by SLC35B2. Due to the sulfation pathway being required for many distinct viruses, including but not limited to EV71 and respiratory syncytial virus (RSV), which were tested in this study, SLC35B2 represents a target of broad-spectrum antiviral therapy.
    Keywords:  CRISPR screening; enterovirus 71; host entry factor; tyrosine sulfation
  6. Int J Mol Sci. 2022 Mar 29. pii: 3769. [Epub ahead of print]23(7):
      Signalling activities are tightly regulated to control cellular responses. Heparan sulfate proteoglycans (HSPGs) at the cell membrane and extracellular matrix regulate ligand availability and interaction with a range of key receptors. SULF1 and SULF2 enzymes modify HSPG sulfation by removing 6-O sulfates to regulate cell signalling but are considered functionally identical. Our in vitro mRNA and protein analyses of two diverse human endothelial cell lines, however, highlight their markedly distinct regulatory roles of maintaining specific HSPG sulfation patterns through feedback regulation of HS 6-O transferase (HS6ST) activities and highly divergent roles in vascular endothelial growth factor (VEGF) and Transforming growth factor β (TGFβ) cell signalling activities. Unlike Sulf2, Sulf1 over-expression in dermal microvascular HMec1 cells promotes TGFβ and VEGF cell signalling by simultaneously upregulating HS6ST1 activity. In contrast, Sulf1 over-expression in venous ea926 cells has the opposite effect as it attenuates both TGFβ and VEGF signalling while Sulf2 over-expression maintains the control phenotype. Exposure of these cells to VEGF-A, TGFβ1, and their inhibitors further highlights their endothelial cell type-specific responses and integral growth factor interactions to regulate cell signalling and selective feedback regulation of HSPG sulfation that additionally exploits alternative Sulf2 RNA-splicing to regulate net VEGF-A and TGFβ cell signalling activities.
    Keywords:  Sulf1; Sulf2; TGFβ cell signalling; VEGF cell signalling; angiogenesis
  7. AACE Clin Case Rep. 2022 Mar-Apr;8(2):8(2): 89-92
      Background/Objective: Variants in PAPSS2 (3'-phosphoadenosine 5'-phosphosulfate synthetase 2) present with varying degrees of brachyolmia (short trunk, platyspondyly, mild long-bone abnormalities). Our objective is to present the phenotype of male and female siblings with the same novel inactivating variant in PAPSS2.Case Report: A Jordanian female (case 1), born to consanguineous parents, was referred at 10 years of age for short stature (SS). She had a normal laboratory workup, including normal growth hormone stimulation testing. Spinal x-rays done for clinical scoliosis revealed platyspondyly. She attained an adult height of 143.5 cm (-3 SD). Years later, her brother (case 2) was referred at 21 months of age for SS. His laboratory workup and bone age were normal. His growth velocity declined at 6 years of age, but normal growth factors did not suggest growth hormone deficiency. When he returned during puberty, disproportionate body measurements were noted. A skeletal survey revealed platyspondyly, increasing suspicion of growth plate pathology. Exome sequencing in the family revealed a homozygous variant, p.His496Pro (H496P) in PAPSS2 (NM_004670.3:c.1487A>C). Both parents carried the same variant.
    Discussion: PAPSS2 assists with the sulfonation of dehydroepiandrosterone (DHEA) to DHEA sulfate and the sulfonation of proteoglycans in the cartilage, necessary for endochondral bone formation. PAPSS2-inactivating variants present with skeletal dysplasia and elevated DHEA levels.
    Conclusion: This novel variant in PAPSS2 manifested with mild brachyolmia but disproportionate SS in male and female siblings. Biochemical phenotype with low circulating DHEA sulfate and high DHEA levels reflect a sulfonation defect.
    Keywords:  DHEA; DHEA, dehydroepiandrosterone; DHEA-S; DHEA-S, dehydroepiandrosterone sulfate; PAPSS2; PAPSS2, 3′-phosphoadenosine 5′-phosphosulfate synthetase 2 gene; PAPSS2, 3′-phosphoadenosine 5′-phosphosulfate synthetase 2 protein; SS, short stature; disproportionate short stature; idiopathic short stature
  8. Am J Physiol Cell Physiol. 2022 Apr 13.
      Perlecan, a basement membrane-type heparan sulfate proteoglycan, is an important molecule in the functional diversity of organisms because of the diversity of its glycan chains and the multifunctionality of its core proteins. Human diseases associated with perlecan have been identified using gene-deficient mice. Two human diseases related to perlecan have been reported. One is Silverman-Handmaker type Dyssegmental Dysplasia, resulting from complete loss of function of the HSPG2 gene which encods perlecan core protein which maps to chromosome 1p36. The other is Schwartz-Jampel syndrome from partial loss of function of the HSPG2 gene. Subsequent in vivo and in vitrostudies have revealed the organ-specific functions of perlecan, suggesting its involvement in the pathogenesis of various human diseases. In this review, we discuss the role of perlecan in human diseases and summarize our knowledge about perlecan as a future therapeutic target to treat the related diseases and for healthy longevity.
    Keywords:  Perlecan; Schwartz-Jampel syndrome; chondrodysplasia; heparan sulfate proteoglycan; neuromuscular disorders
  9. Mol Cell Biochem. 2022 Apr 13.
      In utero hyperglycemia has consequences on future outcomes in the offsprings. We had earlier shown that in utero hyperglycemia impacts proteoglycans/glycosaminoglycans, one of the key molecules involved in brain development. Hypothalamic HSPGs such as syndecan-1 and syndecan-3 are well known for their involvement in feeding behavior. Therefore, studies were carried out to determine the effect of maternal hyperglycemia on the expression of HSPGs in the hypothalamus of offspring brain. Results revealed increased protein abundance of Syndecan-1 and -3 as well as glypican-1 in postnatal adults from hyperglycemic mothers. This was associated with increased hyperphagia and increased expression of Neuropeptide Y. These results indicate the likely consequences on offsprings exposed to in utero hyperglycemia on its growth.
    Keywords:  Feeding behavior; Heparan sulfate proteoglycans; Hypothalamus; In utero hyperglycemia; Sulfated glycosaminoglycans
  10. Matrix Biol. 2022 Apr 08. pii: S0945-053X(22)00053-1. [Epub ahead of print]
      Hyaluronan (HA) is an extracellular matrix component that regulates a variety of physiological and pathological processes. The function of HA depends both on its overall amount and on its size, properties that are controlled by HA synthesizing and degrading enzymes. The lack of inhibitors that can specifically block individual HA degrading enzymes has hampered attempts to understand the contribution of individual hyaluronidases to different physiological and pathological processes. CEMIP is a recently discovered hyaluronidase that cleaves HA through mechanisms and under conditions that are distinct from those of other hyaluronidases such as HYAL1 or HYAL2. The role of its hyaluronidase activity in physiology and disease is poorly understood. Here, we characterized a series of sulfated HA derivatives (sHA) with different sizes and degrees of sulfation for their ability to inhibit specific hyaluronidases. We found that highly sulfated sHA derivatives potently inhibited CEMIP hyaluronidase activity. One of these compounds, designated here as sHA3.7, was characterized further and shown to inhibit CEMIP with considerable selectivity over other hyaluronidases. Inhibition of CEMIP with sHA3.7 in fibroblasts, which are the main producers of HA in the interstitial matrix, increased the cellular levels of total and high molecular weight HA, while decreasing the fraction of low molecular weight HA fragments. Genetic deletion of CEMIP in mouse embryonic fibroblasts (MEFs) produced analogous results and confirmed that the effects of sHA3.7 on HA levels were mediated by CEMIP inhibition. Importantly, both CEMIP deletion and its inhibition by sHA3.7 suppressed fibroblast proliferation, while promoting differentiation into myofibroblasts, as reflected in a lack of CEMIP in myofibroblasts within skin wounds in experimental mice. By contrast, adipogenic and osteogenic differentiation were attenuated upon CEMIP loss or inhibition. Our results demonstrate the importance of CEMIP for the HA metabolism, proliferation and differentiation of fibroblasts, and suggest that inhibition of CEMIP with sulfated HA derivatives such as sHA3.7 has potential utility in pathological conditions that are dependent on CEMIP function.
    Keywords:  CEMIP; Hyaluronan (HA); KIAA1199; fibroblast; hyaluronidase; sulfated HA
  11. Bratisl Lek Listy. 2022 ;123(5): 366-371
      AIM: Glucosamine derivatives have been found to have anticancer effects in many cancer cell lines in previous investigations. The effect of glucosamine sulfate on neuroblastoma, however, is uncertain. The potential cytotoxic effects of glucosamine sulfate on the SH-SY5Y cell line were investigated in this study. The underlying mechanisms of this cytotoxicity have also been studied.MATERIAL AND METHODS: In this study, the SH-SY5Y cell lines were used. The cells were treated with various concentrations of glucosamine sulfate (0.3125, 0.625, 1.25 and 2.5 μg/mL) and the viability of the cells was determined using the XTT assay after 24 hours. The quantities of cleaved PARP, BCL-2, 8-Hydroxy-desoxyguanosine (8-oxo-dG), cleaved caspase 3, Bax, total oxidant, and total antioxidant in the cells were determined by ELISA kits.
    RESULTS: At doses of 0.3125, 0.625, 1.25 and 2.5 μg/mL, glucosamine sulfate dramatically reduced cell viability in SH-SY5Y cells (p<0.001). ELISA tests demonstrated that 1.25 μg/mL glucosamine sulfate considerably increased the amounts of 8-oxo-dG, cleaved caspase 3, Bax, cleaved PARP and total oxidant. However, 1.25 μg/mL glucosamine sulfate treatment did not change the quantity of BCL-2 protein.
    CONCLUSIONS: Altogether, glucosamine sulfate produced considerable cytotoxicity in SH-SY5Y cells by triggering oxidative stress, inducing DNA damage, and finally causing apoptosis. In addition, more research is needed to determine the efficacy of glucosamine sulfate as an anticancer drug in the treatment of neuroblastoma (Fig. 5, Ref. 39).
    Keywords:  SH-SY5Y cell; apoptosis ELISA.; cell viability; dna damage; glucosamine sulfate; oxidative stress
  12. Oncogenesis. 2022 Apr 13. 11(1): 16
      Alginate oligosaccharide (AOS) has the function to inhibit tumor progression and the sulfated modification can enhance the antitumor activity. To date, the function and mechanism of sulfated AOS (AOS-SO4) in tumors remain largely elusive. We prepared AOS by the enzymatic degradation of alginate, collected AOS-SO4 by sulfating following the canonical procedure. Using these materials, in vitro assays showed that both AOS and AOS-SO4 elicited antitumor effects in osteosarcoma cells. Sulfated modification significantly enhanced the antitumor activity. In addition, AOS-SO4 had obvious effects on cell cycle arrest, apoptosis, and autophagy induction in vitro and in vivo. Mechanistically, we observed that AOS-SO4 treatment triggered proapoptotic autophagy by inhibiting MEK1/ERK/mTOR signaling. The ERK activator reversed AOS-SO4-induced autophagy. More importantly, we found that KSR1 interacted with MEK1 and functioned as a positive regulator of MEK1 protein in osteosarcoma cells. High KSR1 expression was significantly associated with poor survival in osteosarcoma patients. Together, these results suggest that AOS-SO4 has a better antitumor effect in osteosarcoma by inhibiting MEK1/ERK/mTOR signaling, which is KSR1-dependent; thus, AOS-SO4 can be a new potential therapeutic candidate for the treatment of osteosarcoma.
  13. Biomacromolecules. 2022 Apr 14.
      Polysaccharides are abundant in nature and employed in various biomedical applications ranging from scaffolds for tissue engineering to carriers for drug delivery systems. However, drawbacks such as tedious isolation protocols, contamination, batch-to-batch consistency, and lack of compositional control with regards to stereo- and regioselectivity impede the development and utility of polysaccharides, and thus mimetics are highly sought after. We report a synthetic strategy to regioselectively functionalize poly-amido-saccharides with sulfate or phosphate groups using post-polymerization modification reactions. Orthogonally protected β-lactam monomers, synthesized from D-glucal, undergo anionic ring-opening polymerization to yield polymers with degrees of polymerization of 12, 25, and 50. Regioselective deprotection followed by functionalization and global deprotection affords the sulfated and phosphorylated poly-amido-saccharides. The resulting anionic polymers are water soluble and non-cytotoxic and adopt helical conformations. This new methodology provides access to otherwise inaccessible functional polysaccharide mimetics for biomedical applications.
  14. Proc Natl Acad Sci U S A. 2022 Apr 19. 119(16): e2201195119
      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.
    Keywords:  peptide hormone; posttranslational modification; proteolytic processing; subtilase; tyrosine sulfation
  15. Horm Metab Res. 2022 Apr;54(4): 232-237
      The purpose of this study was to determine possible cut-off levels of basal DHEA-S percentile rank in the differential diagnosis of patients with Cushing's syndrome (CS) with ACTH levels in the gray zone and normal DHEA-S levels. In this retrospective study including 623 pathologically confirmed CS, the DHEA-S percentile rank was calculated in 389 patients with DHEA-S levels within reference interval. The patients were classified as group 1 (n=265 Cushing's disease; CD), group 2 (n=104 adrenal CS) and group 3 (n=20 ectopic ACTH syndrome).ROC-curve analyses were used to calculate the optimal cut-off level of DHEA-S percentile rank in the reference interval in the differential diagnosis of CS, and the effectiveness of this cut-off level in the identification of the accurate etiology of CS was assessed in patients who were in gray zone according to their ACTH levels. The DHEA-S percentile rank in the reference interval were significantly lower in group 2 compared to the other two groups (p<0.001), while group 1 and group 3 had similar levels. The optimal cut-off level of DHEA-S percentile rank in the reference interval providing differential diagnosis between group 1 and group 2 was calculated as 19.5th percentile (80.8% sensitivity, 81.5% specificity) and the level demonstrated the accurate etiology in 100% of CD and 76% of adrenal CS patients who were in the gray zone. This study showed that the cut-off value of DHEA-S level less than 20% of the reference interval could be used for differential diagnosis of CD and adrenal CS with high sensitivity and specificity, and it should be taken into the initial evaluation.
  16. Steroids. 2022 Apr 08. pii: S0039-128X(22)00073-3. [Epub ahead of print] 109035
      Neurosteroids have been associated with neurodegenerative diseases because they are involved in the modulation of neurotransmitter, neurotropic and neuroprotective actions. Emerging evidence suggests that the enzymes responsible for the synthesis of neurosteroids change during the progression of Alzheimer's disease (AD). The present study aimed to assess the changes in phase I and II enzymes involved in the metabolism of neurosteroids of the progestogen, androgenic and estrogenic steroidogenic pathways and the possibility that the neurosteroids are actively converted into the most abundant metabolites (i.e. glucuronides and sulphates). The gene expression for the phase I and II neurosteroid biosynthetic enzymes were studied in the hippocampus of streptozotocin AD rat model. Male Sprague-Dawley rats were randomly divided into control, sham (saline injected into the hippocampus) and 3 and 12 weeks post-STZ administration (STZ-G3w and STZ-G12w, respectively) groups. Behavioral assessments showed memory impairment in both STZ-injected groups, whereas the formation of amyloid-beta was more pronounced in the STZ-12w group. Gene expression of the hippocampus revealed that glucuronidation and sulphation enzymes transcript of the phase I metabolites were upregulated at the late stage of the disease progression (Hsd17b10, Hsd3b1, Akr1c3 and Cyp19a1) except for Sts. The phase II Sult and Ugt enzymes were mostly upregulated in the STZ-G12w rats (Sult1a1, Sult1e1, Ugt1a1, Ugt1a7c, Ugt1a6, Ugt2b35 and Ugt2b17) and normally expressed in the STZ-G3w group (Sult2a2, Sult2a6, Sult2b1, Ugt2b7, Sult4a1 and Ugt1a7c). In conclusion, changes occur in the phase I and II enzymes transcript of the progestogen, androgenic and estrogenic steroidogenic pathways during the progression of AD.
    Keywords:  Alzheimer’s disease; Sulfotransferase; UDP-Glucuronosyltransferase; neurosteroids; steroidogenic pathways