bims-supasi 2022-05-15 papers

bims-supasi

Biomed News

on Sulfation pathways and signalling

Issue of 2022–05–15
twenty papers selected by
Jonathan Wolf Mueller, University of Birmingham

6-O-sulfation of endothelial glycocalyx heparan sulfates regulates systemic coagulation.
Effect of high glucose on glycosaminoglycans in cultured retinal endothelial cells and rat retina.
Functions of chondroitin/dermatan sulfate containing GalNAc4,6-disulfate.
The glycosaminoglycan interactome 2.0.
A Highly Active Chondroitin Sulfate Lyase ABC for Enzymatic Depolymerization of Chondroitin Sulfate.
The Loss of Endothelial Glycocalyx in Diabetic Retinopathy.
Sulfation modification of dopamine in brain regulates aggregative behavior of animals.
Evaluating the Effects of Chondroitin Sulfate and Hyaluronic Acid in Modifying the Properties of Collagen-Based Matrices.
Low-Molecular Weight Protamine Overcomes Chondroitin Sulfate Inhibition of Neural Regeneration.
Analysis of the aggregation mechanism of chondroitin sulfate/chitosan particles and fabrication of hydrogel cell scaffolds.
Sulfated Hyaluronan Binds to Heparanase and Blocks Its Enzymatic and Cellular Actions in Carcinoma Cells.
CHST11-mediated microenvironment events contribute to pulmonary fibrosis and cancer progression.
Myeloid Sulf Deletion and Immune Dysfunction in Multiple Sulfatase Deficiency.
Maternal Zika Virus Infection Alters Offspring Hippocampal Glycan Sulfation Patterns in Nonhuman Primates.
Sulfated Undaria pinnatifida polysaccharide inhibits the formation of kidney stones by inhibiting HK-2 cell damage and reducing the adhesion of nano‑calcium oxalate crystals.
Sulfated phenolic acids in plants.
In vitroevaluation of anti-aging and regenerative properties of dermatan sulfate for skin care.
Differential Neutralization of Unfractionated Heparin and Enoxaparin by Andexanet Alfa.
Comparative Studies on the Interaction of Heparins from Various Origins with Heparin Cofactor II and Antithrombin.
Inhibition of Human Sulfotransferases by Phthalate Monoesters.

FASEB J. 2022 May;36 Suppl 1

6-O-sulfation of endothelial glycocalyx heparan sulfates regulates systemic coagulation.

Kaori Oshima, Pavel Davizon-Castillo, Giovanny Hernandez, Christine Baird, Marilyn Manco-Johnson, Eric Schmidt.

OBJECTIVE: The endothelial glycocalyx is a glycosaminoglycan-rich layer that lines the surface of all vessels and is critical for vascular homeostasis, including the maintenance of an anticoagulant vascular surface. Heparan sulfate (HS), a linear polysaccharide composed of repeating disaccharide units of glucosamine and iduronic acid, is the major glycosaminoglycan comprising the endothelial glycocalyx. The mechanism by which glycocalyx HS regulates coagulation is unknown. As the biological function of HS derives from its unique pattern of sulfation (2-O-, 6-O-, and/or N-sulfation) within constituent disaccharides, we hypothesized that the degree of heparan sulfate sulfation critically regulates systemic hemostasis.
METHODS: We performed thromboelastography on whole blood collected from tamoxifen-treated VE-Cadherin-Cre-ERT2-Sulf1/2 knockout mice, which have loss of endothelial sulf-1 (a 6-O specific HS sulfatase) and thus increased heparan sulfate 6-O-sulfation at the endothelial surface. Tamoxifen-treated floxed mice (no Cre recombinase) served as controls. To determine the effects of HS in clot initiation and propagation, separate from platelet contributions, we treated platelet-poor plasma from healthy donors (pooled and deidentified) with highly-sulfated HS (HS09) and lower-sulfated HS (HS01) and measured endogenous thrombin potential with calibrated automated thrombography. To explore the potential mechanism by which HS functions as anticoagulant, we repeated calibrated automated thrombography with antithrombin-III-, heparin cofactor-II depleted plasma.
RESULTS: Mice lacking endothelial sulf-1 demonstrated increased R-time, decreased angle, and increased K-time compared to tamoxifen-treated controls, suggesting that increased endothelial-surface 6-O HS sulfation decreases coagulation. In platelet poor plasma, highly-sulfated HS09 attenuated thrombin generation in dose-dependent manner while such effect was not observed with the same dose of less-sulfated HS01. Repeated analysis using platelet poor plasma depleted of antithrombin-III and of heparin cofactor-II showed dose-dependent decrease of thrombin generation with HS09, suggesting that HS exerts this anticoagulant effect independently of heparin-like activation of antithrombin-III.
CONCLUSION: The anticoagulant effect of endothelial-surface HS is dependent on 6-O sulfation of its constituent disaccharides, and this effect is mediated by the mechanisms distinct from well-known heparin anticoagulation mechanisms.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R3891
Glycobiology. 2022 May 12. pii: cwac029. [Epub ahead of print]

Effect of high glucose on glycosaminoglycans in cultured retinal endothelial cells and rat retina.

Gaganpreet Kaur, Yuefan Song, Ke Xia, Kevin McCarthy, Fuming Zhang, Robert J Linhardt, Norman R Harris.

  The endothelial glycocalyx regulates vascular permeability, inflammation, and coagulation, and acts as a mechanosensor. The loss of glycocalyx can cause endothelial injury and contribute to several microvascular complications, and therefore may promote diabetic retinopathy. Studies have shown a partial loss of retinal glycocalyx in diabetes, but with few molecular details of the changes in glycosaminoglycan (GAG) composition. Therefore, the purpose of our study was to investigate the effect of hyperglycemia on GAGs of the retinal endothelial glycocalyx. GAGs were isolated from rat retinal microvascular endothelial cells (RRMECs), media, and retinas, followed by LC-MS assays. qRT-PCR was used to study mRNA transcripts of the enzymes involved in GAG biosynthesis. Hyperglycemia significantly increased the shedding of heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). There were no changes to the levels of HS in RRMEC monolayers grown in high-glucose media, but the levels of CS and HA decreased dramatically. Similarly, while HA decreased in the retinas of diabetic rats, the total GAG and CS levels increased. Hyperglycemia in RRMECs caused a significant increase in the mRNA levels of the enzymes involved in GAG biosynthesis (including EXTL-1,2,3, EXT-1,2, ChSY-1,3, and HAS-2,3), with these increases potentially being compensatory responses to overall glycocalyx loss. Both RRMECs and retinas of diabetic rats exhibited glucose-induced alterations in the disaccharide compositions and sulfation of HS and CS, with the changes in sulfation including N,6-O-sulfation on HS and 4-O-sulfation on CS.

Keywords:  Diabetic Retinopathy; Endothelial Glycocalyx; Glycosaminoglycans; Hyperglycemia

DOI:  https://doi.org/10.1093/glycob/cwac029
Glycobiology. 2022 May 12. pii: cwac030. [Epub ahead of print]

Functions of chondroitin/dermatan sulfate containing GalNAc4,6-disulfate.

Osami Habuchi.

  Chondroitin sulfate (CS) and dermatan sulfate (DS) containing GalNAc4,6-disulfate (GalNAc4S6S) were initially discovered in marine animals. Following the discovery, these glycosaminoglycans have been found in various animals including human. In the biosynthesis of CS/DS containing GalNAc4S6S, three groups of sulfotransferases are involved; chondroitin 4-sulfotransferases (C4STs), dermatan 4-sulfotransferase-1 (D4ST-1) and GalNAc 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST). GalNAc4S-6ST and its products have been shown to play important roles in the abnormal pathological conditions such as central nervous system injury, cancer development, abnormal tissue fibrosis, development of osteoporosis, and infection with viruses or nematodes. CS/DS containing GalNAc4S6S has been shown to increase with the functional differentiation of mast cells, macrophages and neutrophils. Genetic approaches using knockout or knockdown of GalNAc4S-6ST, blocking of the epitopes containing GalNAc4S6S by specific antibodies and chemical technology that enabled the synthesis of oligosaccharides with defined sulfation patterns have been applied successfully to these investigations. These studies contributed significantly to the basic understanding of the functional roles of CS/DS containing GalNAc4S6S in various abnormal conditions, and appear to provide promising clues to the development of possible measures to treat them.

Keywords:  cancer metastasis/chondroitin sulfate E/CHST15/GalNAc4S-6ST/neurite outgrowth

DOI:  https://doi.org/10.1093/glycob/cwac030
Am J Physiol Cell Physiol. 2022 May 11.

The glycosaminoglycan interactome 2.0.

Sylvain D Vallet, Coline Berthollier, Sylvie Ricard-Blum.

  Glycosaminoglycans (GAGs) are complex linear polysaccharides, which are covalently attached to core proteins (except for hyaluronan) to form proteoglycans. They play key roles in the organization of the extracellular matrix, and at the cell surface where they contribute to the regulation of cell signaling and of cell adhesion. To explore the mechanisms and pathways underlying their functions, we have generated an expanded dataset of 4290 interactions corresponding to 3464 unique GAG-binding proteins, four times more than the first version of the GAG interactome (Vallet and Ricard-Blum, 2021 J Histochem Cytochem 69:93-104). The increased size of the GAG network is mostly due to the addition of GAG-binding proteins captured from cell lysates and biological fluids by affinity chromatography and identified by mass spectrometry. We review here the interaction repertoire of natural GAGs and of synthetic sulfated hyaluronan, the specificity and molecular functions of GAG-binding proteins, and the biological processes and pathways they are involved in. This dataset is also used to investigate the differences between proteins binding to iduronic acid-containing GAGs (dermatan sulfate and heparin/heparan sulfate) and those interacting with GAGs lacking iduronic acid (chondroitin sulfate, hyaluronan, and keratan sulfate).

Keywords:  Extracellular matrix; Glycosaminoglycans; Interaction networks

DOI:  https://doi.org/10.1152/ajpcell.00095.2022
Polymers (Basel). 2022 Apr 27. pii: 1770. [Epub ahead of print]14(9):

A Highly Active Chondroitin Sulfate Lyase ABC for Enzymatic Depolymerization of Chondroitin Sulfate.

Xiao-Man Fan, Jia-Ying Huang, Xiao-Min Ling, Wei Wei, Wen-Bin Su, Ye-Wang Zhang.

  Enzymatic preparation of low-molecular-weight chondroitin sulfate (LMWCS) has received increasing attention. In this work, a chondroitin sulfate lyase ABC (Chon-ABC) was successfully cloned, expressed, and characterized. The Km and Vmax of the Chon-ABC were 0.54 mM and 541.3 U mg-1, respectively. The maximal activity was assayed as 500.4 U mg-1 at 37 °C in pH 8.0 phosphate buffer saline. The half-lives of the Chon-ABC were 133 d and 127 min at 4 °C and 37 °C, respectively. Enzymatic preparation of LMWCS was performed at room temperature for 30 min. The changes between the substrate and product were analyzed with mass spectrometry (MS), high-performance liquid chromatography (HPLC), gel permeation chromatography (GPC), and nuclear magnetic resonance (NMR). Overall, the Chon-ABC from Bacteroides thetaiotaomicron is competitive in large-scale enzymatic preparation of LMWCS for its high activity, stability, and substrate specificity.

Keywords:  Bacteroides thetaiotaomicron; characterization; depolymerization; high activity; low-molecular-weight chondroitin sulfate

DOI:  https://doi.org/10.3390/polym14091770
FASEB J. 2022 May;36 Suppl 1

The Loss of Endothelial Glycocalyx in Diabetic Retinopathy.

Gaganpreet Kaur, Yuefan Song, Ke Xia, Diana Cruz-Topete, Kevin McCarthy, Fuming Zhang, Robert Linhardt, Norman Harris.

INTRODUCTION: The vascular endothelium is lined with the endothelial glycocalyx, composed of core proteins and glycosaminoglycans (GAGs). The loss of glycocalyx can cause endothelial injury and could contribute to the progression of retinopathy. The glycocalyx thickness has been shown to decrease in the diabetic retina; however, changes in individual components of the retinal glycocalyx have yet to be determined. Therefore, the present study examined hyperglycemia-induced changes in retinal glycocalyx components.
METHODS: Type-1 diabetic rats and primary rat retinal microvascular endothelial cells (RRMECs) exposed to high glucose were used to mimic diabetes. Western blots and qRT-PCR were used to analyze the expression of core proteins. Further, GAG composition was studied using LC-MS.
RESULTS: Both retinal endothelial cells and rat retina exhibited a significant decrease in mRNA transcripts and protein levels of syndecan-3 under hyperglycemic condition, whereas syndecan-1 expression was significantly increased. Further, we observed a substantial loss of glypican-1 in RRMECs with high glucose, but a significant increase in the retina of diabetic rats. RRMECs in high glucose exhibited a significant decrease in chondroitin sulfate (CS) and hyaluronic acid (HA), but no change in heparan sulfate (HS) levels. In addition, 4-O-sulfated CS residues and 2-O-N-sulfated HS residues were reduced, whereas N-sulfated and 6-O-N-sulfated HS residues were increased in retinal endothelial cells treated with high glucose. Lastly, we observed a significant increase in media levels of syndecan-1, HA, HS, and CS, indicating increased shedding of glycocalyx components under hyperglycemia.
CONCLUSIONS: Our findings suggest that the loss of endothelial glycocalyx in the diabetic retina could be a result of a loss of the proteoglycan syndecan-3 (and possibly glypican-1) as well as the GAGs CS and HA.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R3558
Natl Sci Rev. 2022 Apr;9(4): nwab163

Sulfation modification of dopamine in brain regulates aggregative behavior of animals.

Bing Chen, Xiwen Tong, Xia Zhang, Wanying Gui, Guoming Ai, Lihua Huang, Ding Ding, Jiangxu Zhang, Le Kang.

  Behavioral plasticity and the underlying neuronal plasticity represent a fundamental capacity of animals to cope with environmental stimuli. Behavioral plasticity is controlled by complex molecular networks that act under different layers of regulation. While various molecules have been found to be involved in the regulation of plastic behaviors across species, less is known about how organisms orchestrate the activity of these molecules as part of a coherent behavioral response to varying environments. Here we discover a mechanism for the regulation of animal behavioral plasticity involving molecular sulfation in the brain, a modification of substrate molecules by sulfotransferase (ST)-catalyzed addition of a sulfonate group (SO3) from an obligate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the substrates. We investigated aggregation behaviors of migratory locusts, which are well-known for extreme phase change plasticity triggered by population density. The processes of PAPS biosynthesis acted efficiently on induction of locust behavioral transition: Inhibition of PAPS synthesis solicited a behavioral shift from gregarious to solitarious states; external PAPS dosage, by contrast, promoted aggregation in solitarious locusts. Genetic or pharmacological intervention in the sulfation catalyzation resulted into pronounced solitarizing effects. Analysis of substrate-specific STs suggests a widespread involvement of sulfated neurotransmitters in the behavioral response. Dopamine in the brain was finally identified to be actively sulfate conjugated, and the sulfate conjugation enhanced the free DA-mediated behavioral aggregation. Similar results in Caenorhabditis elegans and mice indicate that sulfation may be involved more broadly in the modulation of animal aggregation. These findings reveal a general mechanism that effectively regulates animal social-like behavioral plasticity, possibly through sulfation-mediated modification of neural networks.

Keywords:  aggregation; behavioral plasticity; dopamine; migratory locust; sulfation

DOI:  https://doi.org/10.1093/nsr/nwab163
FASEB J. 2022 May;36 Suppl 1

Evaluating the Effects of Chondroitin Sulfate and Hyaluronic Acid in Modifying the Properties of Collagen-Based Matrices.

Marcos G Cortes-Medina, Andrew Bushman, Peter Beshay, Jonathan J Adorno, Miles M Menyhert, Riley Hildebrand, Alex Avendano, Jonathan W Song.

The extracellular matrix (ECM) enveloping cells in living tissue is comprised of signaling and structural support molecules. The ECM is vital to maintenance of normal tissue as it controls cell signaling and is a semiporous barrier to interstitial fluid flow. Two important types of ECM molecules are fibrillar collagen (e.g. type I) and glycosaminoglycans (GAGs). In certain solid tumors, hyaluronic acid (HA) and chondroitin sulfate (CS) are two types of GAG molecules that are known to be unusually abundant. These GAGs are negatively charged with the propensity to imbibe aqueous fluid and promote tissue swelling. While many studies have described the effect each molecule has on shaping ECM properties, studies describing how the CS and HA interact with each other are much less common. This study uses an integrated approach to characterize the effects of HA and CS on fluid transport (hydraulic permeability), stiffness (indentation modulus), collagen polymerization kinetics (turbidity), and matrix microarchitecture (pore size and fiber radius). Utilizing a microfluidic approach to study hydraulic permeability observed that the addition of GAGs decreased convective fluid transport. Indenting the hydrogels with a macroscale indenter demonstrated no changes in stiffness when adding CS, while HA, and CS/HA increased hydrogel stiffness. Analysis of confocal reflectance microscopy images showed increases in both pore size and fiber radius with the addition of both GAG molecules. Finally, turbidity measurements concluded that adding GAGs to a collagen gel accelerated its polymerization, with the change in fiber radius being the primary determinant in enhanced fibril formation. This study provides additional evidence that GAGs play an important role in defining physical properties of the ECM and demonstrates further ways into controllably modifying collagen based hydrogels via the addition of native ECM molecules.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R4151
Front Cell Dev Biol. 2022 ;10 865275

Low-Molecular Weight Protamine Overcomes Chondroitin Sulfate Inhibition of Neural Regeneration.

Natalia Kulesskaya, Ekaterina Mugantseva, Rimante Minkeviciene, Natalia Acosta, Ari Rouhiainen, Juha Kuja-Panula, Mikhail Kislin, Sami Piirainen, Mikhail Paveliev, Heikki Rauvala.

  Protamine is an arginine-rich peptide that replaces histones in the DNA-protein complex during spermatogenesis. Protamine is clinically used in cardiopulmonary bypass surgery to neutralize the effects of heparin that is required during the treatment. Here we demonstrate that protamine and its 14-22 amino acid long fragments overcome the neurite outgrowth inhibition by chondroitin sulfate proteoglycans (CSPGs) that are generally regarded as major inhibitors of regenerative neurite growth after injuries of the adult central nervous system (CNS). Since the full-length protamine was found to have toxic effects on neuronal cells we used the in vitro neurite outgrowth assay to select a protamine fragment that retains the activity to overcome the neurite outgrowth inhibition on CSPG substrate and ended up in the 14 amino acid fragment, low-molecular weight protamine (LMWP). In contrast to the full-length protamine, LMWP displays very low or no toxicity in our assays in vitro and in vivo. We therefore started studies on LMWP as a possible drug lead in treatment of CNS injuries, such as the spinal cord injury (SCI). LMWP mimicks HB-GAM (heparin-binding growth-associated molecule; pleiotrophin) in that it overcomes the CSPG inhibition on neurite outgrowth in primary CNS neurons in vitro and inhibits binding of protein tyrosine phosphatase (PTP) sigma, an inhibitory receptor in neurite outgrowth, to its CSPG ligand. Furthermore, the chondroitin sulfate (CS) chains of the cell matrix even enhance the LMWP-induced neurite outgrowth on CSPG substrate. In vivo studies using the hemisection and hemicontusion SCI models in mice at the cervical level C5 revealed that LMWP enhances recovery when administered through intracerebroventricular or systemic route. We suggest that LMWP is a promising drug lead to develop therapies for CNS injuries.

Keywords:  animal models; chondroitin sulfates; low-molecular weight protamine; protamine; spinal cord injury

DOI:  https://doi.org/10.3389/fcell.2022.865275
Int J Biol Macromol. 2022 May 07. pii: S0141-8130(22)00983-7. [Epub ahead of print]

Analysis of the aggregation mechanism of chondroitin sulfate/chitosan particles and fabrication of hydrogel cell scaffolds.

Makoto Yamazaki, Kazutoshi Iijima.

  In this study, the aggregation mechanism of polyion complex (PIC) particles from chitosan (CHI) and chondroitin sulfate C (CS) in phosphate-buffered saline (PBS) was analyzed, and a novel method for the fabrication of hydrogels via aggregation was developed. The PBS induced a decrease in the ζ-potential of the CS/CHI PIC particles, increase in their diameter, and aggregation in a concentration-dependent manner. The hydrogels prepared by mixing CS/CHI PIC particle dispersion and PBS showed the PIC components, with porous structure, high swelling ratio (161.4 ± 13.3%), and high storage moduli (26.2 ± 1.4 kPa). By mixing PBS with suspended adhesive cells and CS/CHI PIC particle dispersion, hydrogels with high cell-loading efficiency were successfully synthesized. The loaded cells within the hydrogels exhibited high viability, uniform distribution, and formation of cell aggregates. These results indicate that CS/CHI-based hydrogels have a potential application as three-dimensional scaffolds for cell culture in tissue engineering.

Keywords:  Hydrogels; Polyion complexes; Polysaccharides

DOI:  https://doi.org/10.1016/j.ijbiomac.2022.05.027
Int J Mol Sci. 2022 May 02. pii: 5055. [Epub ahead of print]23(9):

Sulfated Hyaluronan Binds to Heparanase and Blocks Its Enzymatic and Cellular Actions in Carcinoma Cells.

Jia Shi, Riku Kanoya, Yurina Tani, Sodai Ishikawa, Rino Maeda, Sana Suzuki, Fumiya Kawanami, Naoko Miyagawa, Katsuhiko Takahashi, Teruaki Oku, Ami Yamamoto, Kaori Fukuzawa, Motowo Nakajima, Tatsuro Irimura, Nobuaki Higashi.

  We examined whether sulfated hyaluronan exerts inhibitory effects on enzymatic and biological actions of heparanase, a sole endo-beta-glucuronidase implicated in cancer malignancy and inflammation. Degradation of heparan sulfate by human and mouse heparanase was inhibited by sulfated hyaluronan. In particular, high-sulfated hyaluronan modified with approximately 2.5 sulfate groups per disaccharide unit effectively inhibited the enzymatic activity at a lower concentration than heparin. Human and mouse heparanase bound to immobilized sulfated hyaluronan. Invasion of heparanase-positive colon-26 cells and 4T1 cells under 3D culture conditions was significantly suppressed in the presence of high-sulfated hyaluronan. Heparanase-induced release of CCL2 from colon-26 cells was suppressed in the presence of sulfated hyaluronan via blocking of cell surface binding and subsequent intracellular NF-κB-dependent signaling. The inhibitory effect of sulfated hyaluronan is likely due to competitive binding to the heparanase molecule, which antagonizes the heparanase-substrate interaction. Fragment molecular orbital calculation revealed a strong binding of sulfated hyaluronan tetrasaccharide to the heparanase molecule based on electrostatic interactions, particularly characterized by interactions of (-1)- and (-2)-positioned sulfated sugar residues with basic amino acid residues composing the heparin-binding domain-1 of heparanase. These results propose a relevance for sulfated hyaluronan in the blocking of heparanase-mediated enzymatic and cellular actions.

Keywords:  NF-κB; cell invasion in 3D culture; chemokine; fragment molecular orbital method; heparan sulfate degradation; heparanase; heparin; sulfated hyaluronan

DOI:  https://doi.org/10.3390/ijms23095055
FASEB J. 2022 May;36 Suppl 1

CHST11-mediated microenvironment events contribute to pulmonary fibrosis and cancer progression.

Chien Hsiu Li, Ming-Hsien Chan, Micheal Hsiao.

The Carbohydrate sulfotransferase (CHST) family is essential for Chondroitin Sulfate synthesis and is associated with cancer progression. Comparing all CHST families, we found that CHST11 has prognostic value than other families. CHST11 is related to the overall survival rate in the pan-cancer profile, among which lung adenocarcinoma is the most significant. From pulmonary fibrosis to lung cancer, the expression of CHST11 showed a linear increase. We analyzed the CHST11 interactome, and the results indicated that CHST11 contributes to diverse canonical pathways, including immune microenvironment, fibrosis, and cancer progression. Based on the prediction results of Ingenuity Pathway Analysis (IPA), we speculate that CHST11 can secrete IL1B and IL6 signals to activate the upstream regulator IRF8. This is also the key to the fact that IRF8 has contributed to cystic fibrosis and lung cancer progression. In addition, IRF8 can also transactivate CD80 and CD86 to reform the tumor microenvironments and immune response. By reversing the CHST11-IL6/IL1B-IRF8-CD80/CD86 mediated axis, we confirmed that VE821 and LY2603618 have the value of drug repurposing. Our study proves novel insight into how CHST11 contributes to cystic fibrosis and lung cancer by reprogramming the immune microenvironment.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R3279
FASEB J. 2022 May;36 Suppl 1

Myeloid Sulf Deletion and Immune Dysfunction in Multiple Sulfatase Deficiency.

Anna K Whitehead, Xinping Yue.

INTRODUCTION: Multiple sulfatase deficiency (MSD) is a rare autosomal recessive disease due to pathogenic mutations in the sulfatase-modifying factor 1 (SUMF1) gene. SUMF1 encodes the formylglycine-generating enzyme responsible for an activating posttranslational modification of all sulfatases (17 in humans), and deficiency in these sulfatases results in accumulation of sulfated sugars, lipids, and steroids. The heparan sulfate (HS) 6-O-endosulfatases, commonly referred to as the Sulfs (Sulf1 and Sulf2), are among the sulfatases affected by SUMF1mutation. Unlike other sulfatases which degrade sulfated molecules in lysosomes, the Sulfs remodel the 6-O-sulfation status of HS outside of cells and thus modulate the functions of HS binding growth factors and cytokines. Although the systemic symptoms of MSD can be variable due to varying degrees of deficiency across all sulfatases, common features of MSD include hepatosplenomegaly and recurrent pneumonia. As HS has been shown to play important regulatory roles in host immunity, the objective of the current study was to investigate the role of the Sulfs in myeloid lineage development and function and to examine how myeloid Sulf deficiency contributes to the symptomatology of MSD.
METHODS: To generate a mouse model with myeloid specific deletion of the Sulfs, we crossed Sulf1 and Sulf2 double floxed mice with mice carrying the Cre recombinase under control of lysozyme 2 (Lyz2-cre). The role of the Sulfs in macrophage function was examined both in vitro in bone marrow derived macrophages and in vivo using the lipopolysaccharide (LPS) lung injury and inflammation model. Finally, Western blotting was performed to evaluate how myeloid Sulf deficiency alters transforming growth factor (TGF)-β signaling.
RESULTS: The myeloid specific Sulf deficient mice had enlarged spleens (231.0 ± 51.1 mg) compared to the control mice (Lyz2-cre+/- alone with wildtype Sulfs, 95.4 ± 7.0 mg, P = 0.039), and some (5-10%) died prematurely. Bone marrow derived macrophages from myeloid Sulf deficient mice exhibited increased proliferation but reduced ability to phagocytose Escherichia Coli (E. Coli) in vitrocompared to the control macrophages. Following intratracheal LPS (0.1 mg/kg) challenge (4 hours), myeloid Sulf deficient mice had significantly less neutrophils (1.8x105 ± 0.6x105 cells) in the bronchoalveolar lavage fluid (BALF) compared to the control mice (6.3x105 ± 1.0x105 cells, P = 0.0076). In contrast, coadministration of recombinant Sulf2 with LPS in wildtype mice lead to greater neutrophil recruitment in the airspace. Finally, enhanced phosphorylation of Smad2/3 (TGF-β signal transducers) was found in spleens and lungs from myeloid specific Sulf deficient mice compared to the controls.
CONCLUSION: Our study reveals several important findings: the Sulfs play important roles in myeloid lineage development and function, myeloid Sulf deficiency contributes to the immune dysregulation observed in MSD, and enhanced TGF-β signaling in the absence of the Sulfs might be responsible for the immune abnormality observed in myeloid Sulf deficient mice and in MSD. Our study builds on the foundation for targeting HS or the Sulfs in the treatment of MSD and potentially other myeloproliferative pathologies.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R3030
FASEB J. 2022 May;36 Suppl 1

Maternal Zika Virus Infection Alters Offspring Hippocampal Glycan Sulfation Patterns in Nonhuman Primates.

Kimberly M Alonge, Orlando Cervantes, Kendra Francis, Jarrad Scarlett, Michael Gale, Lakshmi Rajagopal, Charles Frevert, Kristina Adams Waldorf, Michael Schwartz.

Zika virus (ZIKV) infection during pregnancy can impair offspring hippocampal neurocircuit formation and cognitive function, but underlying mechanisms driving this neuronal miswiring remain unknown. During fetal development, axonal migration, pathfinding, and synaptogenesis are strongly influenced by extracellular chondroitin and dermatan sulfate-glycosaminoglycans (CS/DS-GAGs), which consist of six differentially sulfated isomers (CS-A, B (DS), C, D, E, O). The relative abundance of these CS/DS isomers can influence functions ranging from circuit plasticity (CS-C) and maturation (CS-A, B) to axonal growth attraction (CS-D) and repulsion (CS-E). Using LC-MS/MS to analyze the relative abundance of each CS/DS isomer in cortical and hippocampal tissue obtained from normal 145d fetal Macaca nemestrina nonhuman primates (NHP), a key difference between cortex and hippocampus included the presence of a less 'mature' glycan matrix environment in the latter region. This finding is supported by a 1.6-fold increase in the neuroplastic CS-C (p=0.002) and 1.7-fold increase in the neuronal attractant CS-D (p=0.0001) isomers, along with a 39%-decrease in axonal repulsive CS-E (p<0.0001), a 22%-decrease in synapse stabilizing CS-B/DS (p=0.06), and a 6%-decrease in the matrix maturing CS-A (p=0.02) isomers. The hippocampus also exhibited a 51%-decrease in the nonsulfated CS-O (p=0.0001) isomer compared to the adjacent cortex, with the resultant hypersulfation of hippocampal CS/DS-GAGs (p=0.0001) predicted to increase binding of positively charged extracellular growth factors. These differences in CS/DS-GAGs between brain regions suggest that whereas the cortex provides a matured matrix environment capable of supporting newly formed circuitry, hippocampal CS/DS-GAGs retain a developmental sulfation patterning that allows for additional neuronal migration, axon growth, and synapse formation. With this background, we then tested whether 3rd trimester congenital ZIKV infection (fetal d120-d124) in M. nemestrina influences offspring CS/DS-GAG sulfation patterns 21d later. We found that whereas no change was observed in the cortex, offspring exposed to congenital ZIKV exhibited a 33%-decrease in CS-D (p=0.01) and a 1.5x-increase in CS-O (p=0.03) in the hippocampus. This ZIKV-driven reduction of the axonal attractant CS-D can be predicted to limit hippocampal neurocircuit formation, potentially exacerbated by reduced retention of growth factors resulting from the increase in nonsulfated CS-O, which is consistent with previous evidence of reduced hippocampal neurocircuit formation after maternal ZIKV infection. Overall, these results imply that maternal ZIKV infection during late-stage fetal neurodevelopment 1) targets the immature hippocampal CS/DS-GAGs over the matured cortex, 2) identifies recoding of CS/DS-GAGs as a novel target for ZIKV infection, and 3) offers a novel and plausible mechanism to explain the deleterious impact of maternal ZIKV on offspring development.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.R4337
Mater Sci Eng C Mater Biol Appl. 2021 Nov 25. pii: S0928-4931(21)00704-9. [Epub ahead of print] 112564

Sulfated Undaria pinnatifida polysaccharide inhibits the formation of kidney stones by inhibiting HK-2 cell damage and reducing the adhesion of nano‑calcium oxalate crystals.

Xue-Wu Chen, Xin-Yuan Sun, Gu-Hua Tang, Jian-Ming Ouyang.

   OBJECTIVE: The formation of kidney stone is closely related to cell injury and crystal adhesion.
METHOD: The sulfur trioxide-pyridine method was used to sulfate raw Undaria pinnatifida polysaccharide (UPP) with a molecular weight (Mw) of 8.33 kDa. Four polysaccharides with the sulfate group (-OSO3-) contents of 1.59% (UPP0), 6.03% (UPP1), 20.83% (UPP2), and 36.39% (UPP3) were obtained. The antioxidant activity of the four UPPs, the difference in oxidative damage inflicted by nano-CaOx monohydrate (nano-COM) on human proximal tubular epithelial (HK-2) cells before and after protection by UPPs, and the inhibitory effect on nano-COM adhesion were explored.
RESULTS: Structural characterization showed that sulfation was successful. As the -OSO3- content in the UPPs was increased, the antioxidant activity and capability of the UPPs to regulate the growth of calcium oxalate (CaOx) crystals gradually increased. The damage caused by nano-COM crystals to HK-2 cells under protection by UPPs was weakened. This effect enhanced cell viability, enabled the maintenance of good cell morphology, reduced reactive oxygen species (ROS) levels, and inhibited the decrease in mitochondrial membrane potential, as well as decreased the eversion of phosphatidylserine (PS) and the expression of the adhesion proteins osteopontin (OPN), heat shock protein (HSP 90), and Annexin A1 (ANXA1). The adhesion of nano-COM to HK-2 cells was inhibited under the protection by UPPs.
CONCLUSION: UPP3 with the highest content of -OSO3- presented the best antioxidant activity and crystal regulation ability, while UPP2 with the second highest -OSO3- content showed optimal cell protection ability and crystal adhesion inhibition ability. The biological activity of UPPs was regulated by Mw and -OSO3- content. UPP2 with moderate -OSO3- content may become a potential drug for preventing CaOx stones.

Keywords:  Adhesion protein; Calcium oxalate kidney stones; Cell protection; Sulfation modification; Undaria pinnatifida polysaccharide

DOI:  https://doi.org/10.1016/j.msec.2021.112564
Planta. 2022 May 13. 255(6): 124

Sulfated phenolic acids in plants.

Klara Supikova, Andrea Kosinova, Martin Vavrusa, Lucie Koplikova, Anja François, Jiri Pospisil, Marek Zatloukal, Ron Wever, Aloysius Hartog, Jiri Gruz.

   MAIN CONCLUSION: Sulfated phenolic acids are widely occurring metabolites in plants, including fruits, vegetables and crops. The untargeted UHPLC-QTOF-MS metabolomics of more than 50 samples from plant, fungi and algae lead to the discovery of a small group of sulfated metabolites derived from phenolic acids. These compounds were detected in land plants for the first time. In this study, zosteric acid, 4-(sulfooxy)benzoic acid, 4-(sulfoooxy)phenylacetic acid, ferulic acid 4-sulfate and/or vanillic acid 4-sulfate were detected in a number of edible species/products, including oat (Avena sativa L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), tomato (Solanum lycopersicum L.), carrot (Daucus carota subsp. Sativus Hoffm.), broccoli (Brassica oleracea var. Italica Plenck), celery (Apium graveolens L.), cabbage (Brassica oleracea convar. sabauda L.), banana tree (Musa tropicana L.), pineapple fruit (Ananas comosus L.), radish bulb (Raphanus sativus L.) and olive oil (Olea europaea L.). The structural identification of sulfated compounds was performed by comparing retention times and mass spectral data to those of synthesized standards. In addition to above-mentioned compounds, isoferulic acid 3-sulfate and caffeic acid 4-sulfate were putatively identified in celery bulb (Apium graveolens L.) and broccoli floret (Brassica oleracea var. Italica Plenck), respectively. While sulfated phenolic acids were quantified in concentrations ranging from 0.34 to 22.18 µg·g-1 DW, the corresponding non-sulfated acids were mostly undetected or present at lower concentrations. The subsequent analysis of oat symplast and apoplast showed that they are predominantly accumulated in the symplast (> 70%) where they are supposed to be biosynthesized by sulfotransferases.

Keywords:  Edible species; LC–MS; Mass spectrometry; Metabolomics; Phenolic acids; Sulfated compounds

DOI:  https://doi.org/10.1007/s00425-022-03902-6
FASEB J. 2022 May;36 Suppl 1

In vitroevaluation of anti-aging and regenerative properties of dermatan sulfate for skin care.

Patricia Galvez-Martin, Daniel Martinez-Puig, Cristina Soto-Fernández, Jessica Romero-Rueda.

INTRODUCTION: Dermatan sulfate (DS) is a linear polysaccharide classified as a sulfated glycosaminoglycan (GAGs). GAGs form an integral part of the skin and make up 0.1-0.3% of total skin weight. DS is covalently attached to the core proteins to form proteoglycans (PGs). PGs predominates in dermis presenting higher level of DS than other sulfated GAGs. The intrinsic aging processes of the skin entails the reduction of total sulfated GAGs levels causing a thinning of the dermis and modifying the quality of the skin. DS possess strong biological activity that can be modulated to improve skin quality. This study was performed to determine the effects of DS on Human Dermal Fibroblasts (HDF) to evaluate its anti-aging and regenerative properties.
METHODS: Human Dermal Fibroblasts (HDF) were isolated from a sample of the foreskin of a neonatal caucasian donor (Lonza). The effect of the DS on cell proliferation was assessed at 24 h and 48 h in HDF, using the method bromodeoxyuridine (BrdU) incorporation, detected by ELISA. The cell migration was evaluated in HDF at 48 h using the Oris™ Cell Migration Assay (Platypus Technologies). To monitor the migration progress, the cells were labelled with calcein. The wound healing capacity of DS was evaluated in HDF using the scratch test. The inductive capacity on Extracellular matrix proteins (ECMp) synthesis was evaluated by immunolocalization-ELISA quantifying type I and type III collagen and elastin.
RESULTS: The results showed that DS induced HDF proliferation after 24 h and 48 h of treatment at all tested concentrations. A dose-dependent increase in proliferation was observed after 48 h of treatment of these cells, reaching a maximum increase of 5.10 (± 0.82) fold at the highest concentration tested (5 mg/mL) compared to uHDF. Regarding HDF cell migration, no significant differences were observed at 24 h after treatment with DS. The results showed a significant improvement in the in vitro wound healing capacity of HDF treated with 0.25 and 1 mg/mL of DS, with an increase in the healed area of 30.28% (± 4.27%) and 23.76% (± 8.87%) respectively, compared to uHDF. Regarding the ECMp synthesis-stimulating capacity of DS, the results showed a significant increase of both type III Collagen (50.98 ± 34.48% increase) and Elastin (32.25 ± 2.61%) levels in HDF treated for 72 h with 1 mg/mL of DS compared to uHDF. HDF treated with 0.25 mg/mL also showed higher type I Collagen production than uHDF, although the increase (30.96 ±7.04%) was not statistically significant.
CONCLUSIONS: The results showed that DS improves the proliferation and regeneration of HDF present in dermis. In addition, DS increases the synthesis of ECMp as collagen type III and elastin, promoting the maintenance and functionality of the dermis. All these properties support the regenerative and anti-aging activity of DS acting against intrinsic aging in dermis.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.L7681
Clin Appl Thromb Hemost. 2022 Jan-Dec;28:28 10760296221099934

Differential Neutralization of Unfractionated Heparin and Enoxaparin by Andexanet Alfa.

Joseph Lewis, Omer Iqbal, Walter Jeske, Debra Hoppensteadt, Fakiha Siddiqui, Jawed Fareed.

   INTRODUCTION: Andexanet alfa (andexanet) is an approved antidote used to reverse the bleeding effects of Direct Oral Anticoagulant (Direct-Xa agents) agents because it reverses anti-Xa activity. Unfractionated heparin (UFH) and low molecular weight heparins (LMWHs) exhibit anti-Xa activity. The purpose is to investigate the neutralization of UFH and LMWH by andexanet in activated clotting time (ACT), thrombelastography (TEG), and anti-Xa due to the protamine sulfate shortage.
METHODS: UFH and LMWH were studied with andexanet, PS, or saline as potential reversal agents/controls at varying concentrations in ACT, TEG, and anti-Xa and compared to each other.
RESULTS: Andexanet partially neutralized both drugs several TEG parameters at high andexanet concentrations, but it was not as effective as protamine sulfate in any of the assays used. Most TEG parameters were correlated with andexanet concentration. In ACT, significant neutralization was demonstrated at many andexanet concentrations for UFH, but not LMWH. UFH was completely neutralized by PS in ACT, while LMWH was partially neutralized by PS in ACT. Andexanet alfa was a less effective neutralization agent than the protamine sulfate as it only partially neutralized UFH in ACT and was ineffective at neutralizing LMWH when tested at the same concentration as PS (10 ug/mL).
CONCLUSION: Andexanet partially neutralized UFH and LMWH with variability between assays, necessitating investigation into assay-dependent differences.

Keywords:  LMWH; UFH; activated clotting time; andexanet alfa; neutralization; thrombelastography

DOI:  https://doi.org/10.1177/10760296221099934
FASEB J. 2022 May;36 Suppl 1

Comparative Studies on the Interaction of Heparins from Various Origins with Heparin Cofactor II and Antithrombin.

Nausheen Baig, Ahmed Kouta, Mark Jaradeh, Debra Hoppensteadt, Walter Jeske, Jawed Fareed.

INTRODUCTION: Unfractionated heparins (UFH) of mucosal origin are currently obtained from bovine, ovine, and porcine sources. In the USP potency assays, bovine mucosal heparin (BMH) exhibit much lower activities (140 U/mg) in comparison to porcine mucosal heparin (PMH; 195 U/mg) and ovine mucosal heparin (OMH; 200 U/mg). Besides molecular and structural differences, these heparins have differential interactions with serpins such as antithrombin (AT) and heparin cofactor II (HCII). The purpose of this study is to compare the inhibitory profiles of various heparins in systems enriched with AT and HCII.
MATERIALS AND METHODS: The active pharmaceutical ingredients (API) of BMH, OMH, and PMH were obtained from Kin Master (Brazil), Ronssi (China), and Medefil (USA). Bovine (B.LMWH), ovine (O.LMWH), and porcine (P.LMWH) derived low-molecular-weight heparins (LMWH, enoxaparin) were obtained from Ronssi (China). Purified human AT, HCII, and thrombin (IIa) were obtained from Enzyme Research (USA). All heparins (UFH's and LMWH's) were supplemented to AT and HCII enriched buffer systems at various gravimetric and potency equated concentrations. The relative inhibition of IIa was measured in terms of percent inhibition using an amidolytic method employing IIa specific substrates. Percent inhibition was calculated for each concentration in both systems for each of the three heparins. Inhibitory concentrations required to inhibit 50% of thrombin (IC-50) was calculated for each UFH and LMWH in terms of µg/mL and U/mL.
RESULTS: In the AT supplemented system using gravimetric concentrations of various UFH's, PMH and OMH showed comparable values, whereas BMH exhibited much higher IC-50's. However, the LMWH's of various origins showed comparable IC-50 values. In this same system, the USP potency adjusted heparins exhibited comparable results for all UFH's (Figure 1). In the HCII supplemented system, UFH's of various origins exhibited similar IC-50 values in both gravimetric and USP potency adjusted studies. Similarly, the LMWH's when compared in gravimetric amounts demonstrated similar IC-50 values (Figure 2). The relative IC-50 values of the LMWH's in both the AT and HCII enriched systems were higher than the UFH's.
CONCLUSION: These results show that potency adjusted UFH's exhibit comparable inhibition of IIa in AT and HCII enriched systems. The differing BMH results observed in these two systems at gravimetric concentrations may partly be due to differences in charge density and AT-binding consensus sequences. The depolymerized enoxaparin of different origins did not reveal any significant differences in the IC-50 values. This may be due to the chemical modifications that occur during the manufacturing of these agents. Future studies may be warranted to further investigate these cofactors and their impacts on the function of various heparins.

DOI: https://doi.org/10.1096/fasebj.2022.36.S1.L8122
Front Endocrinol (Lausanne). 2022 ;13 868105

Inhibition of Human Sulfotransferases by Phthalate Monoesters.

Hui Huang, Bei-Di Lan, Yu-Jing Zhang, Xiao-Juan Fan, Min-Cui Hu, Guo-Qiang Qin, Fei-Ge Wang, Yue Wu, Tao Zheng, Jun-Hui Liu.

   Objective: This study aimed to investigate the inhibition of human important phase II metabolic enzyme sulfotransferases (SULTs) by phthalate monoesters, which are important metabolites of phthalate esters (PAEs).
Method: Recombinant SULT-catalyzed metabolism of p-nitrophenol (PNP) was employed as the probe reactions of SULTs to investigate the inhibition of 8 kinds of phthalate monoesters towards SULT isoforms. An in vitro incubation system was utilized for preliminary screening, and 100 μM of phthalate monoesters was used. Inhibition kinetics were carried out to determine the inhibition of SULTs by phthalate monoesters.
Result: Multiple phthalate monoesters have been demonstrated to exert strong inhibition potential towards SULT1A1, SULT1B1, and SULT1E1, and no significant inhibition of phthalate monoesters towards SULT1A3 was found. The activity of SULT1A1 was strongly inhibited by mono-hexyl phthalate (MHP), mono-octyl phthalate (MOP), mono-benzyl phthalate (MBZP), and mono-ethylhexyl phthalate (MEHP). Monobutyl phthalate (MBP), MHP, MOP, mono-cyclohexyl phthalate (MCHP), and MEHP significantly inhibited the activity of SULT1B1. MHP, MOP, and MEHP significantly inhibited the activity of SULT1E1. MOP was chosen as the representative phthalate monoester to determine the inhibition kinetic parameters (K i) towards SULT1B1 and SULT1E1. The inhibition kinetic parameters (K i) were calculated to be 2.23 μM for MOP-SULT1B1 and 5.54 μM for MOP-SULT1E1. In silico docking method was utilized to understand the inhibition mechanism of SULT1B1 by phthalate monoesters.
Conclusions: All these information will be beneficial for understanding the risk of phthalate monoester exposure from a new perspective.

Keywords:  enzyme inhibition; in silico docking; in vitro–in vivo extrapolation; phthalate esters (PAEs); sulfotransferases (SULTs)

DOI:  https://doi.org/10.3389/fendo.2022.868105