bims-supasi 2024-07-07 papers

bims-supasi

Biomed News

on Sulfation pathways and signalling

Issue of 2024–07–07
fourteen papers selected by
Jonathan Wolf Mueller, University of Birmingham

Sulfated motifs in heparan sulfate inhibit Streptococcus pneumoniae adhesion onto fibronectin and attenuate corneal infection.
Loss of 3-O-sulfotransferase enzymes, Hs3st3a1 and Hs3st3b1, reduces kidney and glomerular size and disrupts glomerular architecture.
Chondroitin sulfate glycan sulfation patterns influence histochemical labeling of perineuronal nets: a comparative study of interregional distribution in human and mouse brain.
Identification of heparin-binding amino acid residues in antibody HS4C3 with the potential to design antibodies against heparan sulfate domains.
Interaction of chondroitin sulfate with zwitterionic lipid membranes.
Heparan sulfate-dependent phase separation of CCL5 and its chemotactic activity.
Chondroitin Sulfate Derivative Cross-Linking of Decellularized Heart Valve for the Improvement of Mechanical Properties, Hemocompatibility, and Endothelialization.
Chemical approaches to the sulfation of small molecules: current progress and future directions.
The effect of combined hydrolyzed type 2 collagen, methylsulfonylmethane, glucosamine sulfate and chondroitin sulfate supplementation on knee osteoarthritis symptoms.
A method for rapid and reliable quantification of VEGF-cell binding activity.
Insight into binding of endogenous neurosteroid ligands to the sigma-1 receptor.
Keratan sulfate proteoglycan: putative template for neuroblast migratory and axonal fascicular pathways and fetal expression in globus pallidus, thalamus, and olfactory bulb.
RNA Adduction Resulting from the Metabolic Activation of Myristicin by P450 Enzymes and Sulfotransferases.
Novel human recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in a glyco-engineered Escherichia coli strain.

Proteoglycan Res. 2023 Jul 01. pii: e9. [Epub ahead of print]1(3):

Sulfated motifs in heparan sulfate inhibit Streptococcus pneumoniae adhesion onto fibronectin and attenuate corneal infection.

Atsuko Hayashida, Hajirah N Saeed, Fuming Zhang, Yuefan Song, Jian Liu, William C Parks, Paulo J M Bispo, Pyong Woo Park.

  A large number of bacterial pathogens bind to host extracellular matrix (ECM) components. For example, many Gram-negative and Gram-positive pathogens express binding proteins for fibronectin (FN) on their cell surface. Mutagenesis studies of bacterial FN-binding proteins have demonstrated their importance in pathogenesis in preclinical animal models. However, means to draw on these findings to design therapeutic approaches that specifically target FN-bacteria interactions have not been successful because bacterial pathogens can elaborate several FN-binding proteins and also because FN is an essential protein and likely a nondruggable target. Here we report that select heparan compounds potently inhibit Streptococcus pneumoniae infection of injured corneas in mice. Using intact heparan sulfate (HS) and heparin (HP), heparinase-digested fragments of HS, HP oligosaccharides, and chemically or chemoenzymatically modified heparan compounds, we found that inhibition of S. pneumoniae corneal infection by heparan compounds is not mediated by simple charge effects but by a selective sulfate group. Removal of 2-O-sulfates significantly inhibited the ability of HP to inhibit S. pneumoniae corneal infection, whereas the addition of 2-O-sulfates to heparosan (H) significantly increased H's ability to inhibit bacterial corneal infection. Proximity ligation assays indicated that S. pneumoniae attaches directly to FN fibrils in the corneal epithelial ECM and that HS and HP specifically inhibit this binding interaction in a 2-O-sulfate-dependent manner. These data suggest that heparan compounds containing 2-O-sulfate groups protect against S. pneumoniae corneal infection by inhibiting bacterial attachment to FN fibrils in the subepithelial ECM of injured corneas. Moreover, 2-O-sulfated heparan compounds significantly inhibited corneal infection in immunocompromised hosts, by a clinical keratitis isolate of S. pneumoniae, and also when topically administered in a therapeutic manner. These findings suggest that the administration of nonanticoagulant 2-O-sulfated heparan compounds may represent a plausible approach to the treatment of S. pneumoniae keratitis.

Keywords:  Streptococcus pneumoniae; bacterial adhesion; corneal epithelial cell; extracellular matrix; fibronectin; heparan sulfate; heparin

DOI:  https://doi.org/10.1002/pgr2.9
Matrix Biol. 2024 Jun 27. pii: S0945-053X(24)00090-8. [Epub ahead of print]

Loss of 3-O-sulfotransferase enzymes, Hs3st3a1 and Hs3st3b1, reduces kidney and glomerular size and disrupts glomerular architecture.

NIDCD/NIDCR Genomics and Computational Biology Core

  Heparan sulfate (HS) is an important component of the kidney anionic filtration barrier, the glomerular basement membrane (GBM). HS chains attached to proteoglycan protein cores are modified by sulfotransferases in a highly ordered series of biosynthetic steps resulting in immense structural diversity due to negatively charged sulfate modifications. 3-O-sulfation is the least abundant modification generated by a family of seven isoforms but creates the most highly sulfated HS domains. We analyzed the kidney phenotypes in the Hs3st3a1, Hs3st3b1 and Hs3st6 -knockout (KO) mice, the isoforms enriched in kidney podocytes. Individual KO mice show no overt kidney phenotype, although Hs3st3b1 kidneys were smaller than wildtype (WT). Furthermore, Hs3st3a1-/-; Hs3st3b1-/- double knockout (DKO) kidneys were smaller but also had a reduction in glomerular size relative to wildtype (WT). Mass spectrometry analysis of kidney HS showed reduced 3-O-sulfation in Hs3st3a1-/- and Hs3st3b1-/-, but not in Hs3st6-/- kidneys. Glomerular HS showed reduced HS staining and reduced ligand-and-carbohydrate engagement (LACE) assay, a tool that detects changes in binding of growth factor receptor-ligand complexes to HS. Interestingly, DKO mice have increased levels of blood urea nitrogen, although no differences were detected in urinary levels of albumin, creatinine and nephrin. Finally, transmission electron microscopy showed irregular and thickened GBM and podocyte foot process effacement in the DKO compared to WT. Together, our data suggest that loss of 3-O-HS domains disrupts the kidney glomerular architecture without affecting the glomerular filtration barrier and overall kidney function.

Keywords:  Heparan sulfate 3-O-sulfotransferase; glomerular basement membrane; heparan sulfate; kidney

DOI:  https://doi.org/10.1016/j.matbio.2024.06.006
bioRxiv. 2024 Jun 21. pii: 2024.02.09.579711. [Epub ahead of print]

Chondroitin sulfate glycan sulfation patterns influence histochemical labeling of perineuronal nets: a comparative study of interregional distribution in human and mouse brain.

Claudia Belliveau, Stéphanie Théberge, Stefanie Netto, Reza Rahimian, Gohar Fakhfouri, Clémentine Hosdey, Maria Antonietta Davoli, Aarun Hendrickson, Kathryn Hao, Bruno Giros, Gustavo Turecki, Kimberly M Alonge, Naguib Mechawar.

Perineuronal nets (PNNs) are a condensed subtype of extracellular matrix that form a net-like coverings around certain neurons in the brain. PNNs are primarily composed of chondroitin sulfate (CS) proteoglycans from the lectican family that consist of CS-glycosaminoglycan (CS-GAG) side chains attached to a core protein. CS disaccharides can exist in various isoforms with different sulfation patterns. Literature suggests that CS disaccharide sulfation patterns can influence the function of PNNs as well as their labeling. This study was conducted to characterize such interregional CS disaccharide sulfation pattern differences in adult human (N = 81) and mouse (N = 19) brains. Liquid chromatography tandem mass spectrometry was used to quantify five different CS disaccharide sulfation patterns, which were then compared to immunolabeling of PNNs using Wisteria Floribunda Lectin (WFL) to identify CS-GAGs and anti-aggrecan to identify CS proteoglycans. In healthy brains, significant regional and species-specific differences in CS disaccharide sulfation and single versus double-labeling pattern were identified. A secondary analysis to investigate how early-life stress (ELS) impacts these PNN features discovered that although ELS increases WFL+ PNN density, the CS-GAG sulfation code and single versus double PNN-labeling distributions remained unaffected in both species. These results underscore PNN complexity in traditional research, emphasizing the need to consider their heterogeneity in future experiments.

DOI: https://doi.org/10.1101/2024.02.09.579711
Glycobiology. 2024 Jul 04. pii: cwae046. [Epub ahead of print]

Identification of heparin-binding amino acid residues in antibody HS4C3 with the potential to design antibodies against heparan sulfate domains.

Lars A A Damen, Thao P Bui, Thierry Wessel, Yong Li, Bart F Straten, Robin Pampiermole, Willeke F Daamen, David G Fernig, Toin H Kuppevelt.

  Heparan sulfate (HS) is a linear polysaccharide with high structural and functional diversity. Detection and localization of HS in tissues can be performed using single chain variable fragment (scFv) antibodies. Although several anti-HS antibodies recognizing different sulfation motifs have been identified, little is known about their interaction with HS. In this study the interaction between the scFv antibody HS4C3 and heparin was investigated. Heparin-binding lysine and arginine residues were identified using a protect and label methodology. Site-directed mutagenesis was applied to further identify critical heparin-binding lysine/arginine residues using immunohistochemical and biochemical assays. In addition, computational docking of a heparin tetrasaccharide towards a 3-D homology model of HS4C3 was applied to identify potential heparin-binding sites. Of the 12 lysine and 15 arginine residues within the HS4C3 antibody, 6 and 9, respectively, were identified as heparin-binding. Most of these residues are located within one of the complementarity determining regions (CDR) or in their proximity. All basic amino acid residues in the CDR3 region of the heavy chain were involved in binding. Computational docking showed a heparin tetrasaccharide close to these regions. Mutagenesis of heparin-binding residues reduced or altered reactivity towards HS and heparin. Identification of heparin-binding arginine and lysine residues in HS4C3 allows for better understanding of the interaction with HS and creates a framework to rationally design antibodies targeting specific HS motifs.

Keywords:  antibodies; heparan sulfate; heparin-binding residues; protect & label; site-specific mutations

DOI:  https://doi.org/10.1093/glycob/cwae046
Chem Phys Lipids. 2024 Jun 29. pii: S0009-3084(24)00042-2. [Epub ahead of print]263 105417

Interaction of chondroitin sulfate with zwitterionic lipid membranes.

Grzegorz Łazarski, Natan Rajtar, Agata Żak, Dorota Jamróz, Mariusz Kepczynski.

  Chondroitin sulfates (CSs) are important components of the extracellular matrix and side chains of membrane proteoglycans. These polysaccharides are, therefore, likely to interact with plasma membranes and play a significant role in modulating cellular functions. So far, the details of the processes occurring at the interface between the extracellular matrix and cellular membranes are not fully understood. In this study, we used experimental methods and atomic-scale molecular dynamics (MD) simulations to reveal the molecular picture of the interactions between CS and phosphocholine (PC) membranes, used as a simplified model of cell membranes. MD simulations reveal that the polysaccharide associates to the PC bilayer as a result of electrostatic interactions between the positively charged quaternary ammonium groups of choline and the negatively charged sulfate groups of CS. Compared to an aqueous medium, the adsorbed polysaccharide chains adopt more elongated conformations, which facilitates the electrostatic interactions with the membrane, and have a high degree of freedom to change their conformations and to adhere to and detach from the membrane surface. Penetrating slightly between the polar groups of the bilayer, they form a loosely anchored layer, but do not intrude into the hydrophobic region of the PC bilayer. The CS adsorption spread the PC headgroups apart, which is manifested by an increase in the value of the area pre lipid. The expansion of the lipid polar groups weakens the dispersion interactions between the lipid acyl chains. As a result, the lipid membrane in the membrane-polysaccharide contact areas becomes more fluid. Our outcomes may help to understand in detail the interaction of chondroitin sulfate with zwitterionic membranes at the molecular level, which is of biological interest since many biological processes depend on lipid-CS interactions.

Keywords:  Chondroitin sulfate; Liposomes; Molecular dynamics simulations; Phosphatidylcholine membranes

DOI:  https://doi.org/10.1016/j.chemphyslip.2024.105417
Elife. 2024 Jul 01. pii: RP93871. [Epub ahead of print]13

Heparan sulfate-dependent phase separation of CCL5 and its chemotactic activity.

Xiaolin Yu, Guangfei Duan, Pengfei Pei, Long Chen, Renji Gu, Wenrui Hu, Hongli Zhang, Yan-Dong Wang, Lili Gong, Lihong Liu, Ting-Ting Chu, Jin-Ping Li, Shi-Zhong Luo.

  Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid-liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.

Keywords:  E. coli; biochemistry; cell biology; chemical biology; chemotaxis; diffusion; gradient; human; mouse; phase separation

DOI:  https://doi.org/10.7554/eLife.93871
ACS Appl Mater Interfaces. 2024 Jul 03.

Chondroitin Sulfate Derivative Cross-Linking of Decellularized Heart Valve for the Improvement of Mechanical Properties, Hemocompatibility, and Endothelialization.

Ge Yan, Min Fan, Ying Zhou, Minghui Xie, Jiawei Shi, Nianguo Dong, Qin Wang, Weihua Qiao.

  Tissue-engineered heart valve (TEHV) has emerged as a prospective alternative to conventional valve prostheses. The decellularized heart valve (DHV) represents a promising TEHV scaffold that preserves the natural three-dimensional structure and retains essential biological activity. However, the limited mechanical strength, fast degradation, poor hemocompatibility, and lack of endothelialization of DHV restrict its clinical use, which is necessary for ensuring its long-term durability. Herein, we used oxidized chondroitin sulfate (ChS), one of the main components of the extracellular matrix with various biological activities, to cross-link DHV to overcome the above problems. In addition, the ChS-adipic dihydrazide was used to react with residual aldehyde groups, thus preventing potential calcification. The results indicated notable enhancements in mechanical properties and resilience against elastase and collagenase degradation in vitro as well as the ability to withstand extended periods of storage without compromising the structural integrity of valve scaffolds. Additionally, the newly cross-linked valves exhibited favorable hemocompatibility in vitro and in vivo, thereby demonstrating exceptional biocompatibility. Furthermore, the scaffolds exhibited traits of gradual degradation and resistance to calcification through a rat subcutaneous implantation model. In the rat abdominal aorta implantation model, the scaffolds demonstrated favorable endothelialization, commendable patency, and a diminished pro-inflammatory response. As a result, the newly constructed DHV scaffold offers a compelling alternative to traditional valve prostheses, which potentially advances the field of TEHV.

Keywords:  calcification; chondroitin sulfate; decellularized heart valve; endothelialization; mechanical properties; tissue-engineered heart valves

DOI:  https://doi.org/10.1021/acsami.4c03171
Essays Biochem. 2024 Jul 03. pii: EBC20240001. [Epub ahead of print]

Chemical approaches to the sulfation of small molecules: current progress and future directions.

Jaber A Alshehri, Alan M Jones.

  Sulfation is one of the most important modifications that occur to a wide range of bioactive small molecules including polysaccharides, proteins, flavonoids, and steroids. In turn, these sulfated molecules have significant biological and pharmacological roles in diverse processes including cell signalling, modulation of immune and inflammation response, anti-coagulation, anti-atherosclerosis, and anti-adhesive properties. This Essay summarises the most encountered chemical sulfation methods of small molecules. Sulfation reactions using sulfur trioxide amine/amide complexes are the most used method for alcohol and phenol groups in carbohydrates, steroids, proteins, and related scaffolds. Despite the effectiveness of these methods, they suffer from issues including multiple-purification steps, toxicity issues (e.g., pyridine contamination), purification challenges, stoichiometric excess of reagents which leads to an increase in reaction cost, and intrinsic stability issues of both the reagent and product. Recent advances including SuFEx, the in situ reagent approach, and TBSAB show the widespread appeal of novel sulfating approaches that will enable a larger exploration of the field in the years to come by simplifying the purification and isolation process to access bespoke sulfated small molecules.

Keywords:  Metabolism; Sulfation; Synthesis

DOI:  https://doi.org/10.1042/EBC20240001
Turk J Phys Med Rehabil. 2024 Jun;70(2): 259-268

The effect of combined hydrolyzed type 2 collagen, methylsulfonylmethane, glucosamine sulfate and chondroitin sulfate supplementation on knee osteoarthritis symptoms.

Fikriye Figen Ayhan, Ayşegül Demirci Çoban, Ayça Utkan Karasu, Belgin Karaoğlan, Ece Çınar, Sibel Eyigör, Öznur Uzun, Pınar Borman, Seçil Vural, Ayşegül Yaman, Songül Keskin Kavak, Lale Aktekin, Burcu Duyur Çakıt, Habibe Kandaşoğlu, Başak Mansız Kaplan, Hüma Bölük Şenlikçi, Meltem Dalyan.

   Objectives: This study aimed to evaluate the effects of the combined hydrolyzed type 2 collagen, methylsulfonylmethane (MSM), glucosamine sulfate (GS), and chondroitin sulfate (CS) supplement on knee pain intensity in patients with knee osteoarthritis (OA).
Patients and methods: This multicenter, observational, noninterventional study included 98 patients (78 females, 20 males; mean age: 52.8±6.5 years; range, 40 to 64 years) who had Grade 1-3 knee OA between May 2022 and November 2022. The patients were prescribed the combination of hydrolyzed type 2 collagen, MSM, GS, and CS as a supplement for knee OA. The sachet form of the combined supplement containing 1250 mg hydrolyzed type 2 collagen, 750 mg MSM, 750 mg GS, and 400 mg CS was used once daily for two consecutive months. Patients were evaluated according to the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Visual Analog Scale (VAS)-pain, and Health Assessment Questionnaire (HAQ). Patients were scheduled to visit for follow-up four weeks (Visit 2) and eight weeks (Visit 3) after Visit 1 (baseline; day 0 of the study).
Results: For the VAS-pain, WOMAC, WOMAC-subscale, and HAQ scores, the differences in improvement between the three visits were significant (p<0.001 for all). The patient compliance with the supplement was a median of 96.77%, both for Visit 2 and Visit 3.
Conclusion: The combination of hydrolyzed type 2 collagen, MSM, GS, and CS for eight weeks in knee OA was considered an effective and safe nutritional supplement.

Keywords:  Chondroitin sulfate; function; glucosamine sulfate; osteoarthritis of the knee; pain; type 2 collagen.

DOI:  https://doi.org/10.5606/tftrd.2024.13735
Biochem Biophys Res Commun. 2024 Jun 27. pii: S0006-291X(24)00857-X. [Epub ahead of print]727 150321

A method for rapid and reliable quantification of VEGF-cell binding activity.

Prabuddha Waduge, Avinash Kaur, Wei Li.

  Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor that binds a broad spectrum of cell types and regulates diverse cellular processes, including angiogenesis, growth and survival. However, it is technically difficult to quantify VEGF-cell binding activity because of reversible nature of ligand-receptor interactions. Here we used T7 bacteriophage display to quantify and compare binding activity of three human VEGF-A (hVEGF) isoforms, including hVEGF111, 165 and 206. All three isoforms bound equally well to immobilized aflibercept, a decoy VEGF receptor. hVEGF111-Phage exhibited minimal binding to immobilized heparan sulfate, whereas hVEGF206-Phage and hVEGF165-Phage had the highest and intermediate binding to heparan, respectively. In vitro studies revealed that all three isoforms bound to human umbilical vein endothelial cells (HUVECs), HEK293 epithelial and SK-N-AS neuronal cells. hVEGF111-Phage has the lowest binding activity, while hVEGF206-Phage has the highest binding. hVEGF206-Phage was the most sensitive to detect VEGF-cell binding, albeit with the highest background binding to SK-N-AS cells. These results suggest that hVEGF206-Phage is the best-suited isoform to quantify VEGF-cell binding even though VEGF165 is the most biologically active. Furthermore, this study demonstrates the utility of T7 phage display as a platform for rapid and convenient ligand-cell binding quantification with pros and cons discussed.

Keywords:  Heparin sulfate; Phage display; VEGF; VEGF isoforms; VEGF-Cell binding assay; Vascular endothelial growth factor

DOI:  https://doi.org/10.1016/j.bbrc.2024.150321
Nat Commun. 2024 Jul 04. 15(1): 5619

Insight into binding of endogenous neurosteroid ligands to the sigma-1 receptor.

Chunting Fu, Yang Xiao, Xiaoming Zhou, Ziyi Sun.

The sigma-1 receptor (σ1R) is a non-opioid membrane receptor, which responds to a diverse array of synthetic ligands to exert various pharmacological effects. Meanwhile, candidates for endogenous ligands of σ1R have also been identified. However, how endogenous ligands bind to σ1R remains unknown. Here, we present crystal structures of σ1R from Xenopus laevis (xlσ1R) bound to two endogenous neurosteroid ligands, progesterone (a putative antagonist) and dehydroepiandrosterone sulfate (DHEAS) (a putative agonist), at 2.15-3.09 Å resolutions. Both neurosteroids bind to a similar location in xlσ1R mainly through hydrophobic interactions, but surprisingly, with opposite binding orientations. DHEAS also forms hydrogen bonds with xlσ1R, whereas progesterone interacts indirectly with the receptor through water molecules near the binding site. Binding analyses are consistent with the xlσ1R-neurosteroid complex structures. Furthermore, molecular dynamics simulations and structural data reveal a potential water entry pathway. Our results provide insight into binding of two endogenous neurosteroid ligands to σ1R.

DOI: https://doi.org/10.1038/s41467-024-49894-7
J Neuropathol Exp Neurol. 2024 Jul 01. pii: nlae057. [Epub ahead of print]

Keratan sulfate proteoglycan: putative template for neuroblast migratory and axonal fascicular pathways and fetal expression in globus pallidus, thalamus, and olfactory bulb.

Harvey B Sarnat, Weiming Yu.

  Keratan sulfate (KS) is a proteoglycan secreted in the fetal brain astrocytes and radial glia into extracellular parenchyma as granulofilamentous deposits. KS surrounds neurons except dendritic spines, repelling glutamatergic and facilitating GABAergic axons. The same genes are expressed in both neuroblast migration and axonal growth. This study examines timing of KS during morphogenesis of some normally developing human fetal forebrain structures. Twenty normal human fetal brains from 9-41 weeks gestational age were studied at autopsy. KS was examined by immunoreactivity in formalin-fixed paraffin sections, plus other markers including synaptophysin, S-100β protein, vimentin and nestin. Radial and tangential neuroblast migratory pathways from subventricular zone to cortical plate were marked by KS deposits as early as 9wk GA, shortly after neuroblast migration initiated. During later gestation this reactivity gradually diminished and disappeared by term. Long axonal fascicles of the internal capsule and short fascicles of intrinsic bundles of globus pallidus and corpus striatum also appeared as early as 9-12wk, as fascicular sleeves before axons even entered. Intense KS occurs in astrocytic cytoplasm and extracellular parenchyma at 9wk in globus pallidus, 15wk thalamus, 18wk corpus striatum, 22wk cortical plate, and hippocampus postnatally. Corpus callosum and anterior commissure do not exhibit KS at any age. Optic chiasm shows reactivity at the periphery but not around intrinsic subfasciculi. We postulate that KS forms a chemical template for many long and short axonal fascicles before axons enter and neuroblast migratory pathways at initiation of migration. Cross-immunoreactivity with aggrecan may render difficult molecular distinction.

Keywords:  axonal trajectories; cerebral morphogenesis; corpus striatum; fetus; globus pallidus; keratan sulfate proteoglycan; neuroblast migration; olfactory nerve; thalamus

DOI:  https://doi.org/10.1093/jnen/nlae057
J Agric Food Chem. 2024 Jul 03.

RNA Adduction Resulting from the Metabolic Activation of Myristicin by P450 Enzymes and Sulfotransferases.

Yan He, Zihao Cheng, Jingyu Zhang, Yu Chen, Guode Zhao, Hong Tang, Yufen Liao, Tingmin Ye, Ying Peng, Weiwei Li, Jiang Zheng.

  Myristicin (MYR) mainly occurs in nutmeg and belongs to alkoxy-substituted allylbenzenes, a class of potentially toxic natural chemicals. RNA interaction with MYR metabolites in vitro and in vivo has been investigated in order to gain a better understanding of MYR toxicities. We detected two guanosine adducts (GA1 and GA2), two adenosine adducts (AA1 and AA2), and two cytosine adducts (CA1 and CA2) by LC-MS/MS analysis of total RNA extracts from cultured primary mouse hepatocytes and liver tissues of mice after exposure to MYR. An order of nucleoside adductions was found to be GAs > AAs > CAs, and the result of density functional theory calculations was in agreement with that detected by the LC-MS/MS-based approach. In vitro and in vivo studies have shown that MYR was oxidized by cytochrome P450 enzymes to 1'-hydroxyl and 3'-hydroxyl metabolites, which were then sulfated by sulfotransferases (SULTs) to form sulfate esters. The resulting sulfates would react with the nucleosides by SN1 and/or SN2 reactions, resulting in RNA adduction. The modification may alter the biochemical properties of RNA and disrupt RNA functions, perhaps partially contributing to the toxicities of MYR.

Keywords:  P450s; RNA adduction; SULTs; metabolic activation; myristicin

DOI:  https://doi.org/10.1021/acs.jafc.4c01676
Heliyon. 2024 Jun 30. 10(12): e32555

Novel human recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in a glyco-engineered Escherichia coli strain.

Luisa N Pimentel-Vera, Alexander Rodríguez-López, Angela J Espejo-Mojica, Aura María Ramírez, Carolina Cardona, Luis H Reyes, Shunji Tomatsu, Thapakorn Jaroentomeechai, Matthew P DeLisa, Oscar F Sánchez, Carlos J Alméciga-Díaz.

  Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in the accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). Previously, it was reported the production of an active human recombinant GALNS (rGALNS) in E. coli BL21(DE3). However, this recombinant enzyme was not taken up by HEK293 cells or MPS IVA skin fibroblasts. Here, we leveraged a glyco-engineered E. coli strain to produce a recombinant human GALNS bearing the eukaryotic trimannosyl core N-glycan, Man3GlcNAc2 (rGALNSoptGly). The N-glycosylated GALNS was produced at 100 mL and 1.65 L scales, purified and characterized with respect to pH stability, enzyme kinetic parameters, cell uptake, and KS clearance. The results showed that the addition of trimannosyl core N-glycans enhanced both protein stability and substrate affinity. rGALNSoptGly was capture through a mannose receptor-mediated process. This enzyme was delivered to the lysosome, where it reduced KS storage in human MPS IVA fibroblasts. This study demonstrates the potential of a glyco-engineered E. coli for producing a fully functional GALNS enzyme. It may offer an economic approach for the biosynthesis of a therapeutic glycoprotein that could prove useful for MPS IVA treatment. This strategy could be extended to other lysosomal enzymes that rely on the presence of mannose N-glycans for cell uptake.

Keywords:  Escherichia coli; GALNS; N-linked glycosylation

DOI:  https://doi.org/10.1016/j.heliyon.2024.e32555