bims-supasi 2023-09-10 papers

bims-supasi

Biomed News

on Sulfation pathways and signalling

Issue of 2023–09–10
eightteen papers selected by
Jonathan Wolf Mueller, University of Birmingham

Advances and challenges in biotechnological production of chondroitin sulfate and its oligosaccharides.
Endothelial Glycocalyx Degradation Patterns in Sepsis-Associated Pediatric Acute Respiratory Distress Syndrome: A Single Center Retrospective Observational Study.
Sequence variety in the CC' loop of Siglec-8/9/3 determines the recognitions to sulfated oligosaccharides.
Advanced culture strategy shows varying bioactivities of sulfated polysaccharides of Poria cocos.
Low-molecular weight sulfated marine polysaccharides: Promising molecules to prevent neurodegeneration in mucopolysaccharidosis IIIA?
Characterization of a New Family of 6-Sulfo-N-Acetylglucosaminidases.
Emodin Inhibits the Indoxyl Sulfate-Induced trans-Differentiation of Vascular Smooth Muscle Cells through Upregulating Thrombospondin-1.
Achieving Molecular Recognition of Structural Analogues in Surface-Enhanced Raman Scattering Spectroscopy: Inducing Charge and Geometry Complementarity to Mimic Molecular Docking.
Chemical Synthesis and Biochemical Properties of Cholestane-5α,6β-diol-3-sulfonate, A Non-hydrolysable Analogue of cholestane-5α,6β-diol-3β-sulfate.
Anorexia Nervosa and Adrenal Hormones: A Systematic Review and Meta-analysis.
CD44 targeting nanodrug based on chondroitin sulfate for melanoma therapy by inducing mitochondrial apoptosis pathways.
Considerations for defining +80 Da mass shifts in mass spectrometry-based proteomics: phosphorylation and beyond.
Regulation of Glycoprotein VI-Dependent Platelet Activation and Thrombus Formation by Heparan Sulfate Proteoglycan Perlecan.
TPST2-mediated receptor tyrosine sulfation enhances leukocidin cytotoxicity and S. aureus infection.
Occurrence of Hydroxytyrosol, Tyrosol and Their Metabolites in Italian Cheese.
Engineering of SH2 Domains for the Recognition of Protein Tyrosine O-Sulfation Sites.
Docking and Molecular Dynamics Simulations Clarify Binding Sites for Interactions of Novel Marine Sulfated Glycans with SARS-CoV-2 Spike Glycoprotein.
Cortisol, cortisone and DHEAS in epidermis and scales of fish Aphanius fasciatus: HPLC-MS/MS measurement of stress indicators as proxies for natural and human-induced factors.

Int J Biol Macromol. 2023 Sep 01. pii: S0141-8130(23)03447-5. [Epub ahead of print]253(Pt 1): 126551

Advances and challenges in biotechnological production of chondroitin sulfate and its oligosaccharides.

Weijiao Zhang, Ruirui Xu, Jiamin Chen, Haibo Xiong, Yang Wang, Bo Pang, Guocheng Du, Zhen Kang.

  Chondroitin sulfate (CS) is a member of glycosaminoglycans (GAGs) and has critical physiological functions. CS is widely applied in medical and clinical fields. Currently, the supply of CS relies on traditional animal tissue extraction methods. From the perspective of medical applications, the biggest drawback of animal-derived CS is its uncontrollable molecular weight and sulfonated patterns, which are key factors affecting CS activities. The advances of cell-free enzyme catalyzed systems and de novo biosynthesis strategies have paved the way to rationally regulate CS sulfonated pattern and molecular weight. In this review, we first present a general overview of biosynthesized CS and its oligosaccharides. Then, the advances in chondroitin biosynthesis, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) synthesis and regeneration, and CS biosynthesis catalyzed by sulfotransferases are discussed. Moreover, the progress of mining and expression of chondroitin depolymerizing enzymes for preparation of CS oligosaccharides is also summarized. Finally, we analyze and discuss the challenges faced in synthesizing CS and its oligosaccharides using microbial and enzymatic methods. In summary, the biotechnological production of CS and its oligosaccharides is a promising method in addressing the drawbacks associated with animal-derived CS and enabling the production of CS oligosaccharides with defined structures.

Keywords:  3′-Phosphoadenosine-5′-phosphosulfate; Chondroitin sulfate; Glycosaminoglycan; Oligosaccharides; Sulfotransferases

DOI:  https://doi.org/10.1016/j.ijbiomac.2023.126551
J Intensive Care Med. 2023 Sep 06. 8850666231200162

Endothelial Glycocalyx Degradation Patterns in Sepsis-Associated Pediatric Acute Respiratory Distress Syndrome: A Single Center Retrospective Observational Study.

Colin J Sallee, Joseph A Hippensteel, Kristen R Miller, Kaori Oshima, Andrew T Pham, Robert P Richter, John Belperio, Yamila L Sierra, Andreas Schwingshackl, Peter M Mourani, Eric P Schmidt, Anil Sapru, Aline B Maddux.

   BACKGROUND: Sepsis-associated destruction of the pulmonary microvascular endothelial glycocalyx (EGCX) creates a vulnerable endothelial surface, contributing to the development of acute respiratory distress syndrome (ARDS). Constituents of the EGCX shed into circulation, glycosaminoglycans and proteoglycans, may serve as biomarkers of endothelial dysfunction. We sought to define the patterns of plasma EGCX degradation products in children with sepsis-associated pediatric ARDS (PARDS), and test their association with clinical outcomes.
METHODS: We retrospectively analyzed a prospective cohort (2018-2020) of children (≥1 month to <18 years of age) receiving invasive mechanical ventilation for acute respiratory failure for ≥72 h. Children with and without sepsis-associated PARDS were selected from the parent cohort and compared. Blood was collected at time of enrollment. Plasma glycosaminoglycan disaccharide class (heparan sulfate, chondroitin sulfate, and hyaluronan) and sulfation subtypes (heparan sulfate and chondroitin sulfate) were quantified using liquid chromatography tandem mass spectrometry. Plasma proteoglycans (syndecan-1) were measured through an immunoassay.
RESULTS: Among the 39 mechanically ventilated children (29 with and 10 without sepsis-associated PARDS), sepsis-associated PARDS patients demonstrated higher levels of heparan sulfate (median 639 ng/mL [interquartile range, IQR 421-902] vs 311 [IQR 228-461]) and syndecan-1 (median 146 ng/mL [IQR 32-315] vs 8 [IQR 8-50]), both p = 0.01. Heparan sulfate subtype analysis demonstrated greater proportions of N-sulfated disaccharide levels among children with sepsis-associated PARDS (p = 0.01). Increasing N-sulfated disaccharide levels by quartile were associated with severe PARDS (n = 9/29) with the highest quartile including >60% of the severe PARDS patients (test for trend, p = 0.04). Higher total heparan sulfate and N-sulfated disaccharide levels were independently associated with fewer 28-day ventilator-free days in children with sepsis-associated PARDS (all p < 0.05).
CONCLUSIONS: Children with sepsis-associated PARDS exhibited higher plasma levels of heparan sulfate disaccharides and syndecan-1, suggesting that EGCX degradation biomarkers may provide insights into endothelial dysfunction and PARDS pathobiology.

Keywords:  acute respiratory distress syndrome; glycocalyx; glycosaminoglycan; mechanical ventilation; pediatric intensive care; pediatrics; proteoglycan; sepsis

DOI:  https://doi.org/10.1177/08850666231200162
Comput Struct Biotechnol J. 2023 ;21 4159-4171

Sequence variety in the CC' loop of Siglec-8/9/3 determines the recognitions to sulfated oligosaccharides.

Yucheng Wang, Yujie Peng, Rui Long, Peiting Shi, Yinghao Zhang, De-Xin Kong, Jinshui Zheng, Xiaocong Wang.

  Siglecs are important lectins found in different types of immune cells and function as regulatory molecules by recognizing self-associated glycans and converting extracellular interactions into signals for inhibiting immune cell functions. Although many Siglecs have been found to show broad specificities and recognize different types of sulfated oligosaccharides, Siglec-8 and Siglec-9 displayed a high degree of specificity for sialyl N-acetyllactosamine (sLacNAc) with sulfations at O6-positions of the galactose (6'-sulfation) and N-acetylglucosamine (6-sulfation), respectively. Siglec-3 was recently discovered to bind sLacNAc both sulfations. In addition to a conserved arginine residue for binding to sialic acid residue, the sequence variety in the CC' loop may provide binding specificities to sulfated oligosaccharides in Siglecs. Thus, the present study employed molecular models to study the impact of different residues in the CC' loops of Siglec-8/9/3 to the recognitions of 6-sulfations in Gal and/or GlcNAc of sLacNAc. The negatively charged residues in the CC' loop of Siglec-9 formed unfavorable electrostatic repulsions with the 6-sulfate in Gal and resulted no recognitions, in contrast to the favorable interactions formed between the positively charged residues in the CC' loop of Siglec-8 and the 6-sulfate in Gal resulting strong specificity. A two-state binding model was proposed for Siglec-3 recognizing 6-sulfations in Gal and GlcNAc of sLacNAc, as the neutral residues in the CC' loop of Siglec-3 could not form strong favorable interactions to lock the 6-sulfate in Gal within a single binding pose or strong unfavorable interactions to repel the 6-sulfate in Gal. The oligosaccharide adopted two distinctive binding poses and oriented the sulfate groups to form interactions with residues in the CC' loop and G-strand. The present study provided a structural mechanism for the sequence variety in the CC' loop of Siglec-8/9/3 determining the recognitions to the sulfated oligosaccharides and offered insights into the binding specificities for Siglecs.

Keywords:  Binding pose; Energy prediction; GLYCAM; Molecular model; Sialic acid; Siglecs; Two-state binding

DOI:  https://doi.org/10.1016/j.csbj.2023.08.014
Int J Biol Macromol. 2023 Sep 01. pii: S0141-8130(23)03566-3. [Epub ahead of print]253(Pt 1): 126669

Advanced culture strategy shows varying bioactivities of sulfated polysaccharides of Poria cocos.

Mei-Kuang Lu, Chi-Hsein Chao, Yu-Chi Hsu.

  This study compares the bioactivity of six sulfated polysaccharides derived from glucose- and sucrose-feeding extracted from P. cocos. Anti-inflammatory potentials of these polysaccharides were evaluated by pretreating lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells. Of the tested polysaccharides, the sulfated polysaccharide derived from sucrose-feeding at the concentration of 40 g/l (referred to as "suc 40") exhibited the highest anti-inflammatory activity, of 83 %, and 33 % inhibition of IL-6 and TNF-α secretion, respetively. It achieved this by inhibiting the p-38 and c-Jun N-terminal kinase (JNK) MAPK signaling pathways. On the other hand, the sulfated polysaccharide derived from glucose-feeding at a concentration of 20 g/l (referred to as "glc 20") demonstrated the greatest anti-lung cancer activity. This was achieved by inducing apoptotic-related molecules, such as poly (ADP-ribose) polymerase (PARP) and CHOP. Furthermore, glc 20 had the highest contents of sulfate, fucose, and mannose compared to the other tested polysaccharides. This suggests that the composition of monosaccharide residues are critical factors influencing the anti-inflammatory and anti-cancer activities of these sulfated polysaccharides. Overall, this study highlights the potential of sulfated polysaccharides derived from P. cocos to function as bioactive compounds with anti-inflammatory and anti-cancer properties.

Keywords:  Anti-cancer; Anti-inflammation; P. cocos; Sulfated polysaccharides

DOI:  https://doi.org/10.1016/j.ijbiomac.2023.126669
Carbohydr Polym. 2023 Nov 15. pii: S0144-8617(23)00679-3. [Epub ahead of print]320 121214

Low-molecular weight sulfated marine polysaccharides: Promising molecules to prevent neurodegeneration in mucopolysaccharidosis IIIA?

Noemi Veraldi, Isabelle Dentand Quadri, Yohan van de Looij, Laura Malaguti Modernell, Corinne Sinquin, Agata Zykwinska, Benjamin B Tournier, Fabien Dalonneau, Honglian Li, Jin-Ping Li, Philippe Millet, Romain Vives, Sylvia Colliec-Jouault, Ariane de Agostini, Eduardo Farias Sanches, Stéphane V Sizonenko.

  Mucopolysaccharidosis IIIA is a hereditary disease caused by mutations in the sulfamidase enzyme that participates in catabolism of heparan sulfate (HS), leading to HS fragment accumulation and multisystemic failure. No cure exists and death occurs around the second decade of life. Two low molecular weight highly sulfated compounds derived from marine diabolican and infernan exopolysaccharides (A5_3 and A5_4, respectively) with heparanase inhibiting properties were tested in a MPSIIIA cell line model, resulting in limited degradation of intracellular HS. Next, we observed the effects of intraperitoneal injections of the diabolican derivative A5_3 from 4 to 12 weeks of age on MPSIIIA mice. Brain metabolism and microstructure, levels of proteins and genes involved in MPSIIIA brain pathophysiology were also investigated. 1H-Magnetic Resonance Spectroscopy (MRS) indicated deficits in energetic metabolism, tissue integrity and neurotransmission at both 4 and 12 weeks in MPSIIIA mice, with partial protective effects of A5_3. Ex-vivo Diffusion Tensor Imaging (DTI) showed white matter microstructural damage in MPSIIIA, with noticeable protective effects of A5_3. Protein and gene expression assessments displayed both pro-inflammatory and pro-apoptotic profiles in MPSIIIA mice, with benefits of A5_3 counteracting neuroinflammation. Overall, derivative A5_3 was well tolerated and was shown to be efficient in preventing brain metabolism failure and inflammation, resulting in preserved brain microstructure in the context of MPSIIIA.

Keywords:  Heparan sulfate; Magnetic spectroscopy resonance; Marine polysaccharides; Mucopolysaccharidosis

DOI:  https://doi.org/10.1016/j.carbpol.2023.121214
J Biol Chem. 2023 Sep 01. pii: S0021-9258(23)02242-1. [Epub ahead of print] 105214

Characterization of a New Family of 6-Sulfo-N-Acetylglucosaminidases.

Rajneesh K Bains, Seyed A Nasseri, Feng Liu, Jacob F Wardman, Peter Rahfeld, Stephen G Withers.

  Sulfation is widespread in Nature, and plays an important role in modulating biological function. Among the strategies developed by microbes to access sulfated oligosaccharides as a nutrient source is the production of 6-sulfoGlcNAcases to selectively release 6-sulfoGlcNAc from target oligosaccharides. Thus far, all 6-sulfoGlcNAcases identified have belonged to the large GH20 family of β-hexosaminidases. Ηere, we identify and characterize a new, highly specific non-GH20 6-sulfoGlcNAcase from Streptococcus pneumoniae TIGR4, Sp_0475 with a greater than 110,000-fold preference towards N-acetyl-β-D-glucosamine-6-sulfate substrates over the non-sulfated version. Sp_0475 shares distant sequence homology with enzymes of the GH20 and with the newly formed GH163 family. However, the sequence similarity between them is sufficiently low that Sp_0475 has been assigned as the founding member of a new glycoside hydrolase family, GH185. By combining results from site-directed mutagenesis with mechanistic studies and bioinformatics we provide insight into the substrate specificity, mechanism, and key active site residues of Sp_0475. Enzymes of the GH185 family follow a substrate-assisted mechanism, consistent with their distant homology to the GH20 family, but the catalytic residues involved are quite different. Taken together, our results highlight in more detail how microbes can degrade sulfated oligosaccharides for nutrients.

Keywords:  CAZy; DUF4838; bioinformatics; carbohydrate; enzyme; glycobiology; glycoside hydrolases; kinetics; sulfated oligosaccharides; transglycosylation

DOI:  https://doi.org/10.1016/j.jbc.2023.105214
J Vasc Res. 2023 Sep 05. 1-11

Emodin Inhibits the Indoxyl Sulfate-Induced trans-Differentiation of Vascular Smooth Muscle Cells through Upregulating Thrombospondin-1.

Weidong Zhang, Jing Chen, Xiao Tan, Pan Zhang, Xialian Xu, Xiaoqiang Ding, Shuan Zhao, Shi Jin.

   BACKGROUND: Indoxyl sulfate (IS) is a protein-bound uremic toxin with vascular toxicity. The primary cause of death in uremic patients on maintenance hemodialysis is vascular disease, and it had been reported that vascular smooth muscle cells (VSMCs) trans-differentiation (VT) plays a vital role in the context of vascular diseases, but the underlying mechanisms remain obscure. Thrombospondin-1 (TSP-1) participates in vascular calcification by keeping the balance of extracellular matrix, but its role in IS-induced VT is unclear.
METHODS: In this study, clinical specimens, animal models, and in vitro VSMCs were used to investigate the role of TSP-1 in IS induced VT and the potential therapeutic methods.
RESULTS: We found that TSP-1 was significantly decreased in arterial samples from uremic patients, animal models, and in VSMCs after IS treatment. Downregulation of TSP-1 sufficiently induced the trans-differentiation genotypes of VSMCs.
CONCLUSION: Emodin, the main monomer extracted from rhubarb, could alleviate IS-induced VT in vitro by upregulating TSP-1. Taken together, IS induces VT by downregulating TSP-1. Emodin might be a candidate drug to alleviate VT under IS treatment.

Keywords:  Emodin; Indoxyl sulfate; Thrombospondin-1; Vascular smooth muscle cells; trans-differentiation

DOI:  https://doi.org/10.1159/000532028
Angew Chem Int Ed Engl. 2023 Sep 07. e202309610

Achieving Molecular Recognition of Structural Analogues in Surface-Enhanced Raman Scattering Spectroscopy: Inducing Charge and Geometry Complementarity to Mimic Molecular Docking.

Shi Xuan Leong, Ya-Chuan Kao, Xuemei Han, Zhong Wei Poh, Jaslyn Ru Ting Chen, Emily Xi Tan, Yong Xiang Leong, Yih Hong Lee, Wei Xuan Teo, George W Yip, Yulin Lam, Xing Yi Ling.

  Molecular recognition of complex isomeric biomolecules remains challenging in surface-enhanced Raman scattering (SERS) spectroscopy due to their small Raman cross-sections and/or poor surface affinities. To date, the use of molecular probes has achieved excellent molecular sensitivities but still suffers from poor spectral specificity. Here, we induce 'charge and geometry complementarity' between probe and analyte as a key strategy to achieve high spectral specificity for effective SERS molecular recognition of structural analogues. We employ 4-mercaptopyridine (MPY) as the probe, and chondroitin sulfate (CS) disaccharides with isomeric sulfation patterns as our proof-of-concept study. Our experimental and in silico studies reveal that 'charge and geometry complementarity' between MPY's binding pocket and the CS sulfation patterns drives the formation of site-specific, multidentate interactions at the respective CS isomerism sites, which 'locks' each CS in its analogue-specific complex geometry, akin to molecular docking events. Leveraging the resultant spectral fingerprints, we achieve > 97% classification accuracy for 4 CSs and 5 potential structural interferences, as well as attain multiplex CS quantification with < 3 % prediction error. These insights could enable practical SERS differentiation of biologically important isomers to meet the burgeoning demand for fast-responding applications across various fields such as biodiagnostics, food and environmental surveillance.

Keywords:  Isomers; Molecular probes; Surface-enhanced Raman scattering; molecular recognition; noncovalent interactions

DOI:  https://doi.org/10.1002/anie.202309610
J Steroid Biochem Mol Biol. 2023 Sep 06. pii: S0960-0760(23)00151-6. [Epub ahead of print] 106396

Chemical Synthesis and Biochemical Properties of Cholestane-5α,6β-diol-3-sulfonate, A Non-hydrolysable Analogue of cholestane-5α,6β-diol-3β-sulfate.

Philippe de Médina, Silia Ayadi, Régis Soulès, Bruno Payre, Sandrine Rup-Jacques, Sandrine Silvente-Poirot, Mohammad Samadi, Marc Poirot.

  Cholestane-3β,5α,6β-triol (CT) is a primary metabolite of 5,6-epoxycholesterols (5,6-EC) that is catalyzed by the cholesterol-5,6-epoxide hydrolase (ChEH). CT is a well-known biomarker for Niemann-Pick disease type C (NP-C), a progressive inherited neurodegenerative disease. On the other hand, CT is known to be metabolized by the 11β-hydroxysteroid-dehydrogenase of type 2 (11β-HSD2) into a tumor promoter named oncosterone that stimulates the growth of breast cancer tumors. Sulfation is a major metabolic transformation leading to the production of sulfated oxysterols. The production of cholestane-5α,6β-diol-3β-O-sulfate (CDS) has been reported in breast cancer cells. However, no data related to CDS biological properties have been reported so far. These studies have been hampered because sulfate esters of sterols and steroids are rapidly hydrolyzed by steroid sulfatase to give free steroids and sterols. In order to get insight into the biological properties of CDS, we report herein the synthesis and the characterization of cholestane-5α,6β-diol-3β-sulfonate (CDSN), a non-hydrolysable analogue of CDS. We show that CDSN is a potent inhibitor of 11β-HSD2 that blocks oncosterone production on cell lysate. The inhibition of oncosterone biosynthesis of a whole cell assay was observed but resulst from the blocage by CDSN of the uptake of CT in MCF-7 cells. While CDSN inhibits MCF-7 cell proliferation, we found that it potentiates the cytotoxic activity of post-lanosterol cholesterol biosynthesis inhibitors such as tamoxifen and PBPE. This effect was associated with an increase of free sterol accumulation and the appearance of giant multilamellar bodies, a structural feature reminiscent of Type C Niemann-Pick disease cells and consistant with a possible inhibition by CDSN of NPC1. Altogether, our data showed that CDSN is biologically active and that it is a valuable tool to study the biological properties of CDS and more specifically its impact on immunity and viral infection.

Keywords:  NPC1; autophagy; cancer; cell death; cell death MLB; cholesterol; metabolism; oxysterol; oxysterol sulfate; proliferation; sterols; uptake

DOI:  https://doi.org/10.1016/j.jsbmb.2023.106396
Eur J Endocrinol. 2023 Sep 05. pii: lvad123. [Epub ahead of print]

Anorexia Nervosa and Adrenal Hormones: A Systematic Review and Meta-analysis.

Subhanudh Thavaraputta, Patompong Ungprasert, Selma F Witchel, Pouneh K Fazeli.

   OBJECTIVE: Anorexia nervosa is a primary psychiatric disorder characterized by self-induced negative energy balance. A number of hormonal responses and adaptations occur in response to starvation and low body weight including changes in adrenocortical hormones. Our objective was to systematically review adrenocortical hormone levels in anorexia nervosa.
DESIGN/METHODS: We searched MEDLINE and EMBASE for studies that reported at least one adrenocortical hormone, including dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEA-S), progesterone, 17-hydroxyprogesterone, pregnenolone, cortisol (serum, urine, cerebrospinal fluid, and hair sample), aldosterone, androstenedione and testosterone in patients with anorexia nervosa and normal-weight healthy controls from inception until October 2021. Means and standard deviations for each hormone were extracted from the studies to calculate a mean difference (MD). A Pooled MD was then calculated by combining MDs of each study using the random-effects model.
RESULTS: We included a total of 101 studies with over 2500 females with anorexia nervosa. Mean cortisol levels were significantly higher in anorexia nervosa as compared to normal-weight controls for multiple forms of measurement, including morning cortisol, 12-hour and 24-hour pooled serum cortisol, 24-hour urine cortisol, and after an overnight dexamethasone suppression test. In contrast, mean serum total testosterone and DHEA-S levels were significantly lower among patients with anorexia nervosa.
CONCLUSIONS: Women with anorexia nervosa have higher cortisol levels and lower DHEA-S and testosterone levels compared to women without anorexia nervosa. This finding is important to consider when evaluating low-weight women for disorders involving the adrenal axis, especially Cushing's syndrome.

Keywords:  DHEA; DHEA-S; aldosterone; anorexia nervosa; cortisol; progesterone; testosterone

DOI:  https://doi.org/10.1093/ejendo/lvad123
Carbohydr Polym. 2023 Nov 15. pii: S0144-8617(23)00720-8. [Epub ahead of print]320 121255

CD44 targeting nanodrug based on chondroitin sulfate for melanoma therapy by inducing mitochondrial apoptosis pathways.

Yan Li, Huiwen Hou, Zengmei Liu, Wen Tang, Jie Wang, Lu Lu, Jiaai Fu, Didi Gao, Feiyan Zhao, XinQing Gao, Peixue Ling, Fengshan Wang, Feng Sun, Haining Tan.

  Neovascularization is crucial to the occurrence and progression of tumors, and the development of antiangiogenic drugs has essential theoretical value and clinical significance. However, antiangiogenesis therapy alone cannot meet the needs of tumor therapy. Meanwhile, polysaccharides are ideal drug carriers with promising applications in drug modification and delivery. In this research, we developed a novel redox and acid sensitive nanodrug (CDDP-CS-Cys-EA, CCEA) composed of chondroitin sulfate (CS), antiangiogenic peptide (endostatin2-alft1, EA) and chemotherapeutic drug (cisplatin, CDDP). CCEA exhibited redox and acid responsiveness, better blood hemocompatibility (hemolysis rate < 5 %), the ability to target tumors (CD44-mediated endocytosis), and strong antiangiogenesis and antitumor characteristics in vitro. Moreover, CCEA showed excellent antitumor activity and low toxicity in B16 xenograft mice. It also has been confirmed that CCEA induced tumor cell apoptosis through promoting the expression of Bax, suppressing the expression of Bcl-2, decreasing mitochondrial membrane potential, releasing cytochrome C (Cyto C), and enhancing the activities of Caspase 9 and Caspase 3. The results of this paper provided a theoretical basis and insight for the development of antitumor drugs.

Keywords:  Antiangiogenic peptide; Antitumor; Chondroitin sulfate; Cisplatin; Tumor microenvironment

DOI:  https://doi.org/10.1016/j.carbpol.2023.121255
Chem Commun (Camb). 2023 Sep 08.

Considerations for defining +80 Da mass shifts in mass spectrometry-based proteomics: phosphorylation and beyond.

Leonard A Daly, Christopher J Clarke, Allen Po, Sally O Oswald, Claire E Eyers.

Post-translational modifications (PTMs) are ubiquitous and key to regulating protein function. Understanding the dynamics of individual PTMs and their biological roles requires robust characterisation. Mass spectrometry (MS) is the method of choice for the identification and quantification of protein modifications. This article focusses on the MS-based analysis of those covalent modifications that induce a mass shift of +80 Da, notably phosphorylation and sulfation, given the challenges associated with their discrimination and pinpointing the sites of modification on a polypeptide chain. Phosphorylation in particular is highly abundant, dynamic and can occur on numerous residues to invoke specific functions, hence robust characterisation is crucial to understanding biological relevance. Showcasing our work in the context of other developments in the field, we highlight approaches for enrichment and site localisation of phosphorylated (canonical and non-canonical) and sulfated peptides, as well as modification analysis in the context of intact proteins (top down proteomics) to explore combinatorial roles. Finally, we discuss the application of native ion-mobility MS to explore the effect of these PTMs on protein structure and ligand binding.

DOI: https://doi.org/10.1039/d3cc02909c
Int J Mol Sci. 2023 Aug 28. pii: 13352. [Epub ahead of print]24(17):

Regulation of Glycoprotein VI-Dependent Platelet Activation and Thrombus Formation by Heparan Sulfate Proteoglycan Perlecan.

Isabella Provenzale, Ilaria De Simone, Jonathan M Gibbins, Johan W M Heemskerk, Paola E J van der Meijden, Chris I Jones.

  Proteoglycans form a heterogeneous family of proteins with covalently bound sulfated glycosaminoglycans. The extracellular matrix proteoglycan perlecan has been proposed to bind to the platelet- and megakaryocyte-specific receptor G6bB, co-regulating platelet glycoprotein VI (GPVI) signaling. The derived non-sulfate proteoglycan endorepellin was previously shown to enhance platelet adhesion via the collagen receptor, integrin α2β1. Here, we compared the roles of perlecan and other matrix proteoglycans in platelet responses and thrombus formation. We used multi-color flow cytometry to measure the degranulation and integrin αIIbβ3 activation of washed platelets in response to various proteoglycans and collagen-related peptide (CRP), the GPVI agonist. Perlecan, but not endorepellin, enhanced the CRP-induced activation of platelets in a time- and concentration-dependent manner. Similar to collagen, immobilized perlecan, but not other proteoglycans, supported static platelet adhesion and spreading. In-flowed whole-blood perlecan diminished shear-dependent platelet adhesion, while it enforced GPVI-dependent thrombus formation-to a larger extent than endorepellin-to induce more contracted aggregates of activated platelets. We concluded that the sulfated proteoglycan perlecan enhances GPVI-dependent platelet responses extending to thrombus formation, but it does so at the expense of reduced adhesion of platelets under flow.

Keywords:  collagen-related peptide; endorepellin; glycoprotein VI; perlecan; platelet spreading; proteoglycan

DOI:  https://doi.org/10.3390/ijms241713352
Front Immunol. 2023 ;14 1242330

TPST2-mediated receptor tyrosine sulfation enhances leukocidin cytotoxicity and S. aureus infection.

Jie He, Xianggui Yang, Kai Yang, Honglin Xu, Cheng Chen, Junxiong Wang, Jun Zeng.

   Background: An essential fact underlying the severity of Staphylococcus aureus (S. aureus) infection is the bicomponent leukocidins released by the pathogen to target and lyse host phagocytes through specific binding cell membrane receptors. However, little is known about the impact of post-transcriptional modification of receptors on the leukocidin binding.
Method: In this study, we used small interfering RNA library (Horizon/Dharmacon) to screen potential genes that affect leukocidin binding on receptors. The cell permeability was investigated through flow cytometry measuring the internalization of 4',6-diamidino-2-phenylindole. Expression of C5a anaphylatoxin chemotactic receptor 1 (C5aR1), sulfated C5aR1 in, and binding of 6x-His-tagged Hemolysin C (HlgC) and Panton-Valentine leukocidin (PVL) slow-component to THP-1 cell lines was detected and analyzed via flow cytometry. Bacterial burden and Survival analysis experiment was conducted in WT and myeloid TPST-cko C57BL/6N mice.
Results: After short hairpin RNA (shRNA) knockdown of TPST2 gene in THP-1, HL-60, and RAW264.7, the cytotoxicity of HlgAB, HlgCB, and Panton-Valentine leukocidin on THP-1 or HL-60 cells was decreased significantly, and the cytotoxicity of HlgAB on RAW264.7 cells was also decreased significantly. Knockdown of TPST2 did not affect the C5aR1 expression but downregulated cell surface C5aR1 tyrosine sulfation on THP-1. In addition, we found that the binding of HlgC and LukS-PV on cell surface receptor C5aR1 was impaired in C5aR1+TPST2- and C5aR1-TPST2- cells. Phagocyte knockout of TPST2 protects mice from S. aureus infection and improves the survival of mice infected with S. aureus.
Conclusion: These results indicate that phagocyte TPST2 mediates the bicomponent leukocidin cytotoxicity by promoting cell membrane receptor sulfation modification that facilitates its binding to leukocidin S component.

Keywords:  TPST2; leukocidin; macrophage; sepsis; tyrosine sulfation

DOI:  https://doi.org/10.3389/fimmu.2023.1242330
Molecules. 2023 Aug 23. pii: 6204. [Epub ahead of print]28(17):

Occurrence of Hydroxytyrosol, Tyrosol and Their Metabolites in Italian Cheese.

Danilo Giusepponi, Carolina Barola, Elisabetta Bucaletti, Simone Moretti, Fabiola Paoletti, Andrea Valiani, Raffaella Branciari, Roberta Galarini.

  Tyrosol (T) and hydroxytyrosol (HT) are phenyl alcohol polyphenols with well-recognized health-promoting properties. They are widely diffused in several vegetables, especially in olive products (leaves, fruits and oil). Therefore, they could be present in food produced from herbivorous animals such as in milk and cheese. In this study, an analytical method to determine T, HT and some of their phase II metabolites (sulphates and glucuronides) in cheese was developed and validated. Samples were extracted with an acidic mixture of MeOH/water 80/20 (v/v) and, after a low temperature clean-up, the extracts were evaporated and injected in a liquid-chromatography coupled with high resolution mass spectrometry (LC-Q-Orbitrap). A validation study demonstrated satisfactory method performance characteristics (selectivity, linearity, precision, recovery factors, detection and quantification limits). The developed protocol was then applied to analyze 36 Italian cheeses made from ewe, goat and cow milk. The sum of detected compounds (T, tyrosol sulfate, hydroxytyrosol-3-O-sulfate and hydroxytyrosol-4-O-sulfate) reached as high as 2300 µg kg-1 on a dry weight basis, although in about 45% of cow cheeses it did not exceed 50 µg kg-1. Ewe cheeses were significantly richer of polyphenols (sum) as well as HT sulfate metabolites than cow cheeses. In conclusion, results shows that cheese cannot be considered an important dietary source of these valuable compounds.

Keywords:  LC-Q-Orbitrap; cheese; glucuronate metabolites; hydroxytyrosol; sulphated metabolites; tyrosol

DOI:  https://doi.org/10.3390/molecules28176204
Methods Mol Biol. 2023 ;2705 293-305

Engineering of SH2 Domains for the Recognition of Protein Tyrosine O-Sulfation Sites.

Sean Paul Waldrop, Wei Niu, Jiantao Guo.

  Protein engineering has brought advances to industrial processes, biomaterials, nanotechnology, biosensors, and biomedical applications. This chapter will focus on the engineering of Src Homology 2 domains (SH2) to act as an antibody mimetic for the recognition of sulfotyrosine-containing peptides or proteins. In comparison to anti-sulfotyrosine antibodies, SH2 mutants have much smaller size and can be heterologously expressed and purified in large quantity at low cost. This chapter will describe the use of phage display to identify a sulfotyrosine-binding SH2 mutant and the subsequent enrichment of sulfotyrosine-containing peptides in complex biological samples.

Keywords:  Phage display; Protein engineering; Protein tyrosine O-sulfation; Src Homology 2 domain; Sulfotyrosine

DOI:  https://doi.org/10.1007/978-1-0716-3393-9_16
Molecules. 2023 Sep 03. pii: 6413. [Epub ahead of print]28(17):

Docking and Molecular Dynamics Simulations Clarify Binding Sites for Interactions of Novel Marine Sulfated Glycans with SARS-CoV-2 Spike Glycoprotein.

Priyanka Samanta, Sushil K Mishra, Vitor H Pomin, Robert J Doerksen.

  The entry of SARS-CoV-2 into the host cell is mediated by its S-glycoprotein (SGP). Sulfated glycans bind to the SGP receptor-binding domain (RBD), which forms a ternary complex with its receptor angiotensin converting enzyme 2. Here, we have conducted a thorough and systematic computational study of the binding of four oligosaccharide building blocks from novel marine sulfated glycans (isolated from Pentacta pygmaea and Isostichopus badionotus) to the non-glycosylated and glycosylated RBD. Blind docking studies using three docking programs identified five potential cryptic binding sites. Extensive site-targeted docking and molecular dynamics simulations using two force fields confirmed only two binding sites (Sites 1 and 5) for these novel, highly charged sulfated glycans, which were also confirmed by previously published reports. This work showed the structural features and key interactions driving ligand binding. A previous study predicted Site 2 to be a potential binding site, which was not observed here. The use of several molecular modeling approaches gave a comprehensive assessment. The detailed comparative study utilizing multiple modeling approaches is the first of its kind for novel glycan-SGP interaction characterization. This study provided insights into the key structural features of these novel glycans as they are considered for development as potential therapeutics.

Keywords:  SARS-CoV-2 spike glycoprotein; binding free energy; cryptic binding sites; glycosaminoglycans; molecular docking; molecular dynamics

DOI:  https://doi.org/10.3390/molecules28176413
Sci Total Environ. 2023 Sep 06. pii: S0048-9697(23)05525-0. [Epub ahead of print] 166900

Cortisol, cortisone and DHEAS in epidermis and scales of fish Aphanius fasciatus: HPLC-MS/MS measurement of stress indicators as proxies for natural and human-induced factors.

Giovanna Mazzi, Matteo Feltracco, Luca Altavilla, Agata Alterio, Elena Barbaro, Mara Bortolini, Stefano Malavasi, Andrea Gambaro.

  Fish health can be affected by a multitude of stressors. Acute and chronic stress assessment via specific hormones monitoring has become a trending research topic. Common investigated matrices are blood and plasma, but recently less invasive substrates have been identified. As chemical composition of skin mucus/epidermis has been demonstrated to link with acute stress, and of scales with chronic stress in fish, the aim of the study was firstly to improve the determination of three stress hormones, namely cortisol (COL), cortisone (CON), and dehydroepiandrosterone-3-sulfate (DHEAS), in skin mucus/epidermis and scales of Aphanius fasciatus. Secondly, an evaluation of the impact of different environments on hormones concentrations was carried out. A liquid chromatography coupled to tandem mass spectrometry method (HPLC-MS/MS) and a preanalytical procedure were validated to determine COL, CON and DHEAS. This methodology was applied to compare a pull of field-collected fish with a pull of fish housed in the laboratory for one year. Our results highlighted a significant presence of cortisol and cortisone in epidermis of the latter pull (averagely 0.10 and 0.14 ng mg-1, respectively), while in the first pull both hormones were much less concentrated (averagely 0.006 and 0.008 ng mg-1, respectively). Scales of both pulls showed presence of hormones, with a higher concentration for fish housed in the laboratory, although a relevant difference in concentration was found only for cortisone. DHEAS was always below the limit of detection.

Keywords:  Acute/chronic stress; Aphanius fasciatus; Biomonitoring; Liquid chromatography coupled to tandem mass spectrometry; Scales/epidermis of fish; Stress hormones

DOI:  https://doi.org/10.1016/j.scitotenv.2023.166900