Sulfation Patterns Research Articles

Chondroitin Sulfate-Coated Heteroduplex-Molecular Spherical Nucleic Acids.

Molecular Spherical Nucleic Acids (MSNAs) are atomically uniform dendritic nanostructures and potential delivery vehicles for oligonucleotides. The radial formulation combined with covalent conjugation may hide the oligonucleotide content and simultaneously enhance the role of appropriate conjugate groups on the outer sphere. The conjugate halo may be modulated to affect the delivery properties of the MSNAs. In the present study, [60]fullerene-based molecular spherical nucleic acids, consisting of a 2'-deoxyribonucleotide and a ribonucleotide sequence, were used as hybridization-mediated carriers (''DNA and RNA-carriers'') for an antisense oligonucleotide, suppressing Tau protein, (i.e. Tau-ASO) and its conjugates with chondroitin sulfate tetrasaccharides (CS) with different sulfation patterns. The impact of the MSNA carriers, CS-moieties on the conjugates and the CS-decorations on the MSNAs on cellular uptake and - activity (Tau-suppression) of the Tau-ASO was studied with hippocampal neurons in vitro. The formation and stability of these heteroduplex ASO-MSNAs were evaluated by UV melting profile analysis, polyacrylamide gel electrophoresis (PAGE), dynamic light scattering (DLS) and size exclusion chromatography equipped with a multi angle light scattering detector (SEC-MALS). The cellular uptake and - activity were studied by confocal microscopy and Western blot analysis, respectively.

Microgels With Electrostatically Controlled Molecular Affinity to Direct Morphogenesis.

Concentration gradients of soluble signaling molecules-morphogens-determine the cellular organization in tissue development. Morphogen-releasing microgels have shown potential to recapitulate this principle in engineered tissue constructs, however, with limited control over the molecular cues in space and time. Inspired by the functionality of sulfated glycosaminoglycans (sGAGs) in morphogen signaling in vivo, a library of sGAG-based microgels is developed and designated as µGel Units to Instruct Development (µGUIDEs). Adjustment of the microgel's sGAG sulfation patterns and concentration enabled the programming of electrostatic affinities that control the release of morphogens. Based on computational analyses of molecular transport processes, µGUIDEs provided unprecedented precision in the spatiotemporal modulation of vascular endothelial growth factor (VEGF) gradients in a microgel-in-gel vasculogenesis model and kidney organoid cultures. The versatile approach offers new options for creating morphogen signaling centers to advance the understanding of tissue and organ development.

Enzyme-Sialylation-Controlled Chemical Sulfation of Glycan Epitopes for Decoding the Binding of Siglec Ligands.

Widely distributed in nature, sulfated glycan epitopes play important roles in diverse pathophysiological processes. However, due to their structural complexity, the preparation of glycan epitopes with structurally defined sulfation patterns is challenging, which significantly hampers the detailed elucidation of their biological functions at the molecular level. Here, we introduce a strategy for site-specific chemical sulfation of glycan epitopes, leveraging enzymatic sialylation and desialylation processes to precisely control the regio-specificity of sulfation of disaccharide or trisaccharide glycan backbones. Using this method, a sulfated glycan library covering the most common sialylated glycan epitopes was prepared in high yield and efficiency. By screening a microarray prepared with this glycan library, we systematically probed their binding specificity with human Siglecs (sialic acid-binding immunoglobulin-type lectins), many of which function as glyco-immune checkpoints to suppress immune system activation. Our investigation revealed that sulfation and sialylation patterns serve as important determinants of Siglec binding affinity and specificity. Thus, these findings offer new insights for the development of research tools and potential therapeutic agents targeting glyco-immune checkpoints by modulating the Siglec signaling pathway.

Time-course remodeling and pathology intervention of α-synuclein amyloid fibril by heparin and heparin-like oligosaccharides.

Amyloid fibrils represent a pathological state of protein polymer that is closely associated with various neurodegenerative diseases. Polysaccharides have a prominent role in recognizing amyloid fibrils and mediating their pathogenicity. However, the mechanism underlying the amyloid-polysaccharide interaction remains elusive. We also do not know its impact on the structure and pathology of formed fibrils. Here, we used cryo-electron microscopy to analyze the atomic structures of mature α-synuclein (α-syn) fibrils upon binding with polymeric heparin and heparin-like oligosaccharides. The fibril structure, including the helical twist and conformation of α-syn, changed over time upon the binding of heparin but not oligosaccharides. The sulfation pattern and numbers of saccharide units are important for the binding. Similarly, negatively charged biopolymers typically interact with amyloid fibrils, including tau and various α-syn polymorphs, leading to alterations in their conformation. Moreover, we show that heparin-like oligosaccharides can not only block neuronal uptake and propagation of formed α-syn fibrils but also inhibit α-syn fibrillation. This work demonstrates a distinctive activity of heparin and biopolymers in remodeling amyloid fibrils and suggests the pharmaceutical potential of heparin-like oligosaccharides.

Glycosaminoglycan Concentration and Sulfation Patterns of Biohybrid Polymer Matrices Direct Microvascular Network Formation and Stability

AbstractComplexation with sulfated glycosaminoglycans (GAGs) regulates the signaling of pro‐angiogenic growth factors in vivo. To use this principle in modulating microvascular network formation and stability in vitro, a library of biohybrid hydrogels containing heparin, a highly sulfated GAG, or its selectively desulfated derivatives, is prepared. The hydrogels are applied to systematically investigate the effects of GAG concentration and sulfation patterns on embedded cultures of human umbilical vein endothelial cells (HUVECs) and cocultures of HUVECs with mesenchymal stromal cells (MSCs). Formation and morphology of microvascular networks, stromal cell expansion, and extracellular matrix (ECM) protein deposition are found to be significantly influenced by the hydrogel's GAG concentrations and sulfation patterns, presumably through differences in the resulting availability and bioactivity of GAG‐binding growth factors. In particular, cocultures in hydrogels with either low heparin concentration (500 µm) or higher concentrations of heparin desulfated at the 6O or N position resulted in the formation of dense, stable microvascular networks. The respective conditions also displayed enhanced stromal cell proliferation and ECM protein deposition, leading to increased matrix stiffness. Therefore, precisely tuning the affinity of biohybrid materials for pro‐angiogenic factors effectively enhances the vascularization of engineered tissue constructs in vitro.

Site-specific sulfations regulate the physicochemical properties of papillomavirus-heparan sulfate interactions for entry.

Certain human papillomaviruses (HPVs) are etiological agents for several anogenital and oropharyngeal cancers. During initial infection, HPV16, the most prevalent cancer-causing type, specifically interacts with heparan sulfates (HSs), not only enabling initial cell attachment but also triggering a crucial conformational change in viral capsids termed structural activation. It is unknown, whether these HPV16-HS interactions depend on HS sulfation patterns. Thus, we probed potential roles of HS sulfations using cell-based functional and physicochemical assays, including single-molecule force spectroscopy. Our results demonstrate that N-sulfation of HS is crucial for virus binding and structural activation by providing high-affinity sites, and that additional 6O-sulfation is required to mechanically stabilize the interaction, whereas 2O-sulfation and 3O-sulfation are mostly dispensable. Together, our findings identify the contribution of HS sulfation patterns to HPV16 binding and structural activation and reveal how distinct sulfation groups of HS synergize to facilitate HPV16 entry, which, in turn, likely influences the tropism of HPVs.

Branched oncolytic peptides target HSPGs, inhibit metastasis, and trigger the release of molecular determinants of immunogenic cell death in pancreatic cancer.

Immunogenic cell death (ICD) can be exploited to treat non-immunoreactive tumors that do not respond to current standard and innovative therapies. Not all chemotherapeutics trigger ICD, among those that do exert this effect, there are anthracyclines, irinotecan, some platinum derivatives and oncolytic peptides. We studied two new branched oncolytic peptides, BOP7 and BOP9 that proved to elicit the release of damage-associated molecular patterns DAMPS, mediators of ICD, in pancreatic cancer cells. The two BOPs selectively bound and killed tumor cells, particularly PANC-1 and Mia PaCa-2, but not cells of non-tumor origin such as RAW 264.7, CHO-K1 and pgsA-745. The cancer selectivity of the two BOPs may be attributed to their repeated cationic sequences, which enable multivalent binding to heparan sulfate glycosaminoglycans (HSPGs), bearing multiple anionic sulfation patterns on cancer cells. This interaction of BOPs with HSPGs not only fosters an anti-metastatic effect in vitro, as demonstrated by reduced adhesion and migration of PANC-1 cancer cells, but also shows promising tumor-specific cytotoxicity and low hemolytic activity. Remarkably, the cytotoxicity induced by BOPs triggers the release of DAMPs, particularly HMGB1, IFN-β and ATP, by dying cells, persisting longer than the cytotoxicity of conventional chemotherapeutic agents such as irinotecan and daunorubicin. An in vivo assay in nude mice showed an encouraging 20% inhibition of tumor grafting and growth in a pancreatic cancer model by BOP9.

CIRCULATING HEPARAN SULFATE PROFILES IN PEDIATRIC ACUTE RESPIRATORY DISTRESS SYNDROME.

Introduction: Sepsis-induced degradation of endothelial glycocalyx heparan sulfate (HS) contributes to the pulmonary microvascular endothelial injury characteristic of acute respiratory distress syndrome (ARDS) pathogenesis. Our objectives were to (1) examine relationships between plasma indices of HS degradation and protein biomarkers of endothelial injury and (2) identify patient subgroups characterized by distinct profiles of HS degradation in children with ARDS. Methods: We analyzed prospectively collected plasma (2018-2020) from a cohort of invasively mechanically ventilated children (aged >1 month to <18 years) with ARDS. Mass spectrometry characterized and quantified patterns of HS disaccharide sulfation. Protein biomarkers reflective of endothelial injury (e.g., angiopoietin-2, vascular cell adhesion molecule-1, soluble thrombomodulin) were measured with a multiplex immunoassay. Pearson correlation coefficients were used to construct a biomarker correlation network. Centrality metrics detected influential biomarkers (i.e., network hubs). K-means clustering identified unique patient subgroups based on HS disaccharide profiles. Results: We evaluated 36 patients with pediatric ARDS. HS disaccharide sulfation patterns, 6S, NS, and NS2S, positively correlated with all biomarkers of endothelial injury (all P < 0.05) and were classified as network hubs. We identified three patient subgroups, with cluster 3 (n = 5) demonstrating elevated levels of 6S and N-sulfated HS disaccharides. In cluster 3, 60% of children were female and nonpulmonary sepsis accounted for 60% of cases. Relative to cluster 1 (n = 12), cluster 3 was associated with higher oxygen saturation index (P = 0.029) and fewer 28-day ventilator-free days (P = 0.016). Conclusions: Circulating highly sulfated HS fragments may represent emerging mechanistic biomarkers of endothelial injury and disease severity in pediatric ARDS.

Chemoenzymatic Synthesis of Keratan Sulfate Oligosaccharides Using UDP-Galactose-6-aldehyde To Control Sulfation at Galactosides.

Keratan sulfate (KS) is a highly complex proteoglycan that has a poly-LacNAc chain that can be modified by diverse patterns of sulfate esters at C-6 positions of galactoside (Gal) and N-acetylglucosamine (GlcNAc) residues. Here, a chemo-enzymatic methodology is described that can control the pattern of sulfation at Gal using UDP-Gal-aldehyde as a donor for poly-LacNAc assembly to temporarily block specific sites from sulfation by galactose 6-sulfotransferase (CHST1).

Sulfated polysaccharide from Apostichopus japonicus viscera exhibits anti-inflammatory properties in vitro and in vivo

Polysaccharides from sea cucumbers are known for their biological activities, but little is known about those from sea cucumber viscera. The present study isolated a sulfated polysaccharide (SCVP-2) from the viscera of Apostichopus japonicas, which had a molecular weight of 209.1 kDa. SCVP-2 comprised 66.3 % total sugars, 2.1 % uronic acid, 4.5 % proteins, and 25.5 % sulfate groups, containing glucosamine, galactosamine, glucose, galactose, and fucose. FT-IR and NMR analyses identified SCVP-2 as a fucoidan sulfate with sulfation patterns of the fucose branches as Fuc2S, Fuc4S, and Fuc0S. SEM and AFM analyses showed irregular clusters and linear conformations. SCVP-2 demonstrated strong anti-inflammatory properties both in vitro and in vivo. In lipopolysaccharide (LPS)-induced inflammation in macrophage RAW264.7 cells, SCVP-2 significantly reduced nitric oxide (NO) and cytokine secretion (IL-1β, IL-6, TNF-α). Additionally, it downregulated the expression of these cytokine genes. Furthermore, the anti-inflammatory mechanism of SCVP-2 was related to the inhibition of the MAPKs and NF-κB pathways. SCVP-2's anti-inflammatory capacity was confirmed in acute inflammation models, including xylene-induced ear swelling and acetic acid-induced peritoneal capillary permeability, and in high-fat diet-induced systemic low-grade chronic inflammation. In conclusion, SCVP-2 exhibits significant anti-inflammatory activity, suggesting its potential for development as a functional food ingredient or therapeutic agent for inflammation-related diseases.

Developing and Benchmarking Sulfate and Sulfamate Force Field Parameters via Ab Initio Molecular Dynamics Simulations To Accurately Model Glycosaminoglycan Electrostatic Interactions.

Glycosaminoglycans (GAGs) are negatively charged polysaccharides found on cell surfaces, where they regulate transport pathways of foreign molecules toward the cell. The structural and functional diversity of GAGs is largely attributed to varied sulfation patterns along the polymer chains, which makes understanding their molecular recognition mechanisms crucial. Molecular dynamics (MD) simulations, thanks to their unmatched microscopic resolution, have the potential to be a reference tool for exploring the patterns responsible for biologically relevant interactions. However, the capability of molecular dynamics force fields used in biosimulations to accurately capture sulfation-specific interactions is not well established, partly due to the intrinsic properties of GAGs that pose challenges for most experimental techniques. In this work, we evaluate the performance of molecular dynamics force fields for sulfated GAGs by studying ion pairing of Ca2+ to sulfated moieties─N-methylsulfamate and methylsulfate─that resemble N- and O-sulfation found in GAGs, respectively. We tested available nonpolarizable (CHARMM36 and GLYCAM06) and explicitly polarizable (Drude and AMOEBA) force fields, and derived new implicitly polarizable models through charge scaling (prosECCo75 and GLYCAM-ECC75) that are consistent with our developed "charge-scaling" framework. The calcium-sulfamate/sulfate interaction free energy profiles obtained with the tested force fields were compared against reference ab initio molecular dynamics (AIMD) simulations, which serve as a robust alternative to experiments. AIMD simulations indicate that the preferential Ca2+ binding mode to sulfated GAG groups is solvent-shared pairing. Only our scaled-charge models agree satisfactorily with the AIMD data, while all other force fields exhibit poorer agreement, sometimes even qualitatively. Surprisingly, even explicitly polarizable force fields display a notable disagreement with the AIMD data, likely attributed to difficulties in their optimization and possible inherent limitations in depicting high-charge-density ion interactions accurately. Finally, the underperforming force fields lead to unrealistic aggregation of sulfated saccharides, which qualitatively disagrees with our understanding of the soft glycocalyx environment. Our results highlight the importance of accurately treating electronic polarization in MD simulations of sulfated GAGs and caution against over-reliance on currently available models without thorough validation and optimization.

Introducing Ion Mobility Mass Spectrometry in Brain Glycosaminoglycomics: Application to Chondroitin/Dermatan Sulfate Octasaccharide Domains.

Glycosaminoglycans (GAGs) are sulfated linear O-glycan chains abundantly expressed in the extracellular matrix (ECM). Among GAGs, chondroitin sulfate (CS) and dermatan sulfate (DS) play important roles at the brain level, where the distribution and location of the sulfates within the CS/DS chains are responsible for numerous biological events. The diversity of the neural hybrid CS/DS expressed in the brain and the need to elucidate their structure gave rise to considerable efforts toward the development of analytical methods able to discover novel regularly and irregularly sulfated domains. In this context, we report here the introduction of ion mobility separation (IMS) mass spectrometry (MS) in brain glycosaminoglycomics. Based on IMS MS and tandem MS (MS/MS) by collision-induced dissociation (CID), we have developed a powerful approach for the screening and structural analysis of neural CS/DS and optimized and validated the method for the structural analysis of octasaccharides that were released from brain proteoglycans by β-elimination and pooled after chain depolymerization using chondroitin AC lyase. The IMS MS data revealed the separation of CS/DS octamers into mobility families based on the amount of sulfation. Among the discovered oversulfated domains, of major biological importance is the pentasulfated-[4,5-Δ-GlcAGalNAc(IdoAGalNAc)3], for which the (-) nanoESI IMS CID MS/MS analysis disclosed through the presence of distinct drift times, the incidence of two isomers. Moreover, the generated fragment ions revealed an uncommon trisulfated motif and an uncommon pentasulfated motif. Hence, using IMS MS and CID MS/MS, novel brain-related CS/DS domains of atypical sulfation patterns were discovered and structurally characterized in detail.

Chemoenzymatic Synthesis of Sulfated N-Glycans Recognized by Siglecs and Other Glycan-Binding Proteins.

Sulfated N-glycans are present in many glycoproteins, which are implicated in playing important roles in biological recognition processes. Here, we report the systematic chemoenzymatic synthesis of a library of sulfated and sialylated biantennary N-glycans and assess their binding to Siglecs and glycan-specific antibodies that recognize them as glycan ligands. The combined use of three human sulfotransferases, GlcNAc-6-O-sulfotransferase (CHST2), Gal-3-O-sulfotransferase (Gal3ST1), and keratan sulfate Gal-6-O-sulfotransferase (CHST1), resulted in asymmetric and symmetric branch-selective sulfation of the GlcNAc and/or Gal moieties of N-glycans. The extension of the sugar chain using α-2,3- and α-2,6-sialyltransferases afforded the sulfated and sialylated N-glycans. These synthetic glycans with different patterns of sulfation and sialylation were evaluated for binding to selected Siglecs and sulfoglycan-specific antibodies using glycan microarrays. The results confirm previously documented glycan-recognizing properties and further reveal novel specificities for these glycan-binding proteins, demonstrating the utility of the library for assessing the specificity of glycan-binding proteins recognizing sulfated and sialylated glycans.

Structural Insights into Endostatin-Heparan Sulfate Interactions Using Modeling Approaches.

Glycosaminoglycans (GAGs) play a key role in a variety of biological processes in the extracellular matrix (ECM) via interactions with their protein targets. Due to their high flexibility, periodicity and electrostatics-driven interactions, GAG-containing complexes are very challenging to characterize both experimentally and in silico. In this study, we, for the first time, systematically analyzed the interactions of endostatin, a proteolytic fragment of collagen XVIII known to be anti-angiogenic and anti-tumoral, with heparin (HP) and representative heparan sulfate (HS) oligosaccharides of various lengths, sequences and sulfation patterns. We first used conventional molecular docking and a docking approach based on a repulsive scaling-replica exchange molecular dynamics technique, as well as unbiased molecular dynamic simulations, to obtain dynamically stable GAG binding poses. Then, the corresponding free energies of binding were calculated and the amino acid residues that contribute the most to GAG binding were identified. We also investigated the potential influence of Zn2+ on endostatin-HP complexes using computational approaches. These data provide new atomistic details of the molecular mechanism of HP's binding to endostatin, which will contribute to a better understanding of its interplay with proteoglycans at the cell surface and in the extracellular matrix.

Anti-enterovirus 71 activity of native fucosylated chondroitin sulfates and their derivatives

Enterovirus 71 (EV71) is recognized as a major causative agent of hand, foot, and mouth disease (HFMD), posing a significant global public health concern due to its widespread impact and resulting in a major public health issue worldwide. Despite its prevalence, current clinical therapy lacks effective antiviral agents. Fucosylated chondroitin sulfates (FCS) derived from sea cucumber exhibits a range of biological activities including potent antiviral effects. This study provides compelling evidence of the potent antiviral efficacy of FCS against EV71. To further elucidate the impact of structural variations on the anti-EV71 activity, native FCSs with diverse sulfation patterns and a varity of FCS derivatives were prepared and analyzed. Notably, this study presents the detailed structural characterization of FCSs from the sea cucumbers Holothuria scabra Jaege and Holothuria fuscopunctata. Analysis of the structure-activity relationships revealed that molecular weight, sulfated fucose branches, and sulfation pattern were all crucial factors contributing to the potent inhibitory effects of FCS against EV71. Interestingly, molecular weight emerged as the most significant structural determinant of the antiviral potency. These findings suggest the promising potential of utilizing FCS as an innovative EV71 entry inhibitor for the treatment of HFMD.

Epigenetic Targeting of Heparan Sulfate 3-O- and 6-O-Sulfation in Breast Cancer Cells: Prospects for Attenuating Prothrombotic Tumor Cell Activities.

The deregulation of cell surface heparan sulfate proteoglycans (HSPGs) is a main issue of cancer cells for increasing their malignancy. In these terms, the sulfation pattern of HS, created by an orchestrated activity of enzymes balancing a site-specific sulfation, is of key importance. These enzymes are often deregulated by epigenetic processes in cancer, e.g., being silenced by DNA hypermethylation. Here, we address this issue in human breast cancer cell lines aiming to target epigenetic processes to reactivate HS sulfation, shifting HS into an antithrombotic phenotype for which 3-O-sulfation is particularly important. Treatment of MCF-7 and MDA-MB-231 cells with nontoxic concentrations of 5-azacytidine (azacytidine) and 5-fluoro-2'-deoxycytidine (FdCyd) as DNMT inhibitors or vorinostat for targeting HDAC increased HS3-O-sulfation remarkably, as confirmed by fluorescence microscopy, by upregulating HS3-O-sulfotransferases, detected by quantitative real-time polymerase chain reaction and Western blot. Flow cytometry and microscopic approaches confirm that upon inhibitor treatment, increased HS3-O-sulfation improves cell binding to antithrombin, leading to an antithrombotic activity. Nevertheless, only azacytidine- and vorinostat-treated cells display anticoagulative properties, represented by attenuated thrombin formation, a lower activation of human platelet aggregation, or ATP release. In contrast, FdCyd additionally upregulated tissue factor expression in both cell lines, overshadowing the anticoagulant effects of HS, leading to an overall prothrombotic phenotype. Our data provide evidence for the first time that targeting epigenetic processes in HS sulfation is a valuable means to foster anticoagulative cell properties for decreasing malignancy and metastatic potency. These data warrant further investigations to fine-tune epigenetic targeting and to search for potential biomarkers attributed to these activities.

Heparin Oligosaccharides as Vasoactive Intestinal Peptide Inhibitors via their Binding Process Characterization.

It has been proven that vasoactive intestinal peptide (VIP) was involved in the pathogenesis of prostate cancer. Cardin et al. found that by an alanine scan, the heparin- binding site on VIP was exactly the same sequence in VIP and its receptor. Therefore, heparin could competitively block the binding of VIP and its receptor. However, the structure-activity relationship between heparin and VIP has not been reported, especially in terms of the sequence and sulfation patterns of heparin oligosaccharides upon binding to VIP. A variety of experiments were designed to study the binding process and structure-activity relationship between heparin oligosaccharides and VIP. Heparin was enzymatically digested and purified to produce heparin oligosaccharides, and the structures were characterized by NMR. The binding capacity between heparin oligosaccharides and VIP was analyzed by GMSA and ITC experiments. The binding between heparin oligosaccharides and VIP was simulated using a molecular docking program to show the complex. ELISA assay was used to investigate the effect of non-anticoagulant heparin oligosaccharides on the VIP-mediated cAMP/PKA signaling pathway in vitro. The results indicated that both the length and the sulfation pattern of heparin oligosaccharides affected its binding to VIP. VIP could induce the expression of cAMP at a higher level in PC3 cells, which could be regulated by the interaction of heparin oligosaccharides and VIP. The binding between heparin oligosaccharides and VIP could block the binding between VIP and its receptor on tumor cells. Downloading the regulation of the expression level of cAMP could possibly further affect the subsequent activation of PKA. These non-anticoagulant heparin oligosaccharides may block the VIP-mediated cAMP/PKA signaling pathway and thus exert their antitumor activity.

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

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 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-glycosaminoglycans 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 impacts these PNN features discovered that although early-life stress increases WFL+ PNN density, the CS-glycosaminoglycan 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.

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

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.

Insights into the nature of ichthyotoxins from the Chrysochromulina leadbeateri blooms in Northern Norwegian fjords

In May–June 2019, the microalga Chrysochromulina leadbeateri caused a massive fish-killing event in several fjords in Northern Norway, resulting in the largest direct impact ever on aquaculture in northern Europe due to toxic algae. Motivated by the fact that no algal toxins have previously been described from C. leadbeateri, we set out to investigate the chemical nature and toxicity of secondary metabolites in extracts of two strains (UIO 393, UIO 394) isolated from the 2019 bloom, as well as one older strain (UIO 035) isolated during a bloom in Northern Norway in 1991. Initial LC–DAD–MS/MS-based molecular networking analysis of the crude MeOH extracts of the cultivated strains showed that their profiles of small organic molecules, including a large number of known lipids, were very similar, suggesting that the same class of toxin(s) were likely the causative agents of the two harmful algal bloom (HAB) events. Next, bioassay-guided fractionation using the RTgill-W1 cell line and metabolomics analysis pointed to a major compound affording [M + H]+ ions at m/z 1399.8333 as a possible toxin, corresponding to a compound with the formula C67H127ClO27. Moreover, our study unveiled a series of minor analogues exhibiting distinct patterns of chlorination and sulfation, together defining a new family of compounds, which we propose to name leadbeaterins. Remarkably, these suspected toxins were detected in situ in samples collected during the 2019 bloom close to Tromsø, thereby consistent with a role in fish kills. The elemental compositions of the putative C. leadbeateri ichthyotoxins strongly indicate them to be long linear polyhydroxylated polyketides, structurally similar to sterolysins reported from a number of dinoflagellates.