The significant role of glycosaminoglycans in tooth development.

This review delves into the roles of glycosaminoglycans (GAGs), integral components of proteoglycans, in tooth development. Proteoglycans consist of a core protein linked to GAG chains, comprised of repeating disaccharide units. GAGs are classified into several types, such as hyaluronic acid, heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate. Functioning as critical macromolecular components within the dental basement membrane, these GAGs facilitate cell adhesion and aggregation, and play key roles in regulating cell proliferation and differentiation, thereby significantly influencing tooth morphogenesis. Notably, our recent research has identified the hyaluronan-degrading enzyme Transmembrane protein 2 (Tmem2) and we have conducted functional analyses using mouse models. These studies have unveiled the essential role of Tmem2-mediated hyaluronan degradation and its involvement in hyaluronan-mediated cell adhesion during tooth formation. This review provides a comprehensive summary of the current understanding of GAG functions in tooth development, integrating insights from recent research, and discusses future directions in this field.

Structure-guided design of VAR2CSA-based immunogens and a cocktail strategy for a placental malaria vaccine.

Placental accumulation of Plasmodium falciparum infected erythrocytes results in maternal anemia, low birth weight, and pregnancy loss. The parasite protein VAR2CSA facilitates the accumulation of infected erythrocytes in the placenta through interaction with the host receptor chondroitin sulfate A (CSA). Antibodies that prevent the VAR2CSA-CSA interaction correlate with protection from placental malaria, and VAR2CSA is a high-priority placental malaria vaccine antigen. Here, structure-guided design leveraging the full-length structures of VAR2CSA produced a stable immunogen that retains the critical conserved functional elements of VAR2CSA. The design expressed with a six-fold greater yield than the full-length protein and elicited antibodies that prevent adhesion of infected erythrocytes to CSA. The reduced size and adaptability of the designed immunogen enable efficient production of multiple variants of VAR2CSA for use in a cocktail vaccination strategy to increase the breadth of protection. These designs form strong foundations for the development of potent broadly protective placental malaria vaccines.

Identification of the growth cone as a probe and driver of neuronal migration in the injured brain.

Axonal growth cones mediate axonal guidance and growth regulation. We show that migrating neurons in mice possess a growth cone at the tip of their leading process, similar to that of axons, in terms of the cytoskeletal dynamics and functional responsivity through protein tyrosine phosphatase receptor type sigma (PTPσ). Migrating-neuron growth cones respond to chondroitin sulfate (CS) through PTPσ and collapse, which leads to inhibition of neuronal migration. In the presence of CS, the growth cones can revert to their extended morphology when their leading filopodia interact with heparan sulfate (HS), thus re-enabling neuronal migration. Implantation of an HS-containing biomaterial in the CS-rich injured cortex promotes the extension of the growth cone and improve the migration and regeneration of neurons, thereby enabling functional recovery. Thus, the growth cone of migrating neurons is responsive to extracellular environments and acts as a primary regulator of neuronal migration.

Chondroitin sulfate-modified antiangiogenic peptide conjugate induces cell apoptosis via the mitochondria-mediated pathway to perform antitumor activity.

Tumor growth and metastasis heavily rely on angiogenesis, crucial for solid tumor development. Inhibiting angiogenesis associated with tumors emerges as a potent therapeutic approach. Our previous work synthesized the chondroitin sulfate-modified antiangiogenic peptide CS-ES2-AF (CS-EA), which exhibited better antiangiogenic activity, longer half-life, and more robust targeting. In this work, we further evaluated the stability in vitro, cellular uptake mechanism, cell apoptosis mechanism, antitumor activity in vivo, and safety of CS-EA. The stability of CS-EA was consistently superior to that of EA at different temperatures and in different pH ranges. Furthermore, CS-EA mainly entered EAhy926 cells through the clathrin-mediated endocytosis pathway. CS-EA inhibited endothelial cell proliferation, and induced cell apoptosis through downregulating the Bcl-2, reducing mitochondria membrane potential, upregulating cytochrome c, Caspase 3, and reactive oxygen species levels. CS-EA showed better antitumor activity in the B16 xenografted tumor model, with a tumor inhibition rate 1.92 times higher than EA. Simultaneously, it was observed that CS-EA did not cause any harmful effects on the vital organs of the mice. These findings indicate that CS-EA holds significant promise for the treatment of tumors.

SARS-CoV-2 spike protein-ACE2 interaction increases carbohydrate sulfotransferases and reduces N-acetylgalactosamine-4-sulfatase by p38 MAPK.

Immunostaining in lungs of patients who died with COVID-19 infection showed increased intensity and distribution of chondroitin sulfate and decline in N-acetylgalactostamine-4-sulfatase (Arylsulfatase B; ARSB). To explain these findings, human small airway epithelial cells were exposed to the SARS-CoV-2 spike protein receptor binding domain (SPRBD) and transcriptional mechanisms were investigated. Phospho-p38 MAPK and phospho-SMAD3 increased following exposure to the SPRBD, and their inhibition suppressed the promoter activation of the carbohydrate sulfotransferases CHST15 and CHST11, which contributed to chondroitin sulfate biosynthesis. Decline in ARSB was mediated by phospho-38 MAPK-induced N-terminal Rb phosphorylation and an associated increase in Rb-E2F1 binding and decline in E2F1 binding to the ARSB promoter. The increases in chondroitin sulfotransferases were inhibited when treated with phospho-p38-MAPK inhibitors, SMAD3 (SIS3) inhibitors, as well as antihistamine desloratadine and antibiotic monensin. In the mouse model of carrageenan-induced systemic inflammation, increases in phospho-p38 MAPK and expression of CHST15 and CHST11 and declines in DNA-E2F binding and ARSB expression occurred in the lung, similar to the observed effects in this SPRBD model of COVID-19 infection. Since accumulation of chondroitin sulfates is associated with fibrotic lung conditions and diffuse alveolar damage, increased attention to p38-MAPK inhibition may be beneficial in ameliorating Covid-19 infections.

Chondroitin sulfate modification of CSPG4 regulates the maintenance and differentiation of glioma-initiating cells via integrin-associated signaling.

Glioma stem cell/glioma-initiating cell (GIC) and their niches are considered responsible for the therapeutic resistance and recurrence of malignant glioma. To clarify the molecular mechanisms of GIC maintenance/differentiation, we performed a unique integrated proteogenomics utilizing GIC clones established from patient tumors having the potential to develop glioblastoma. After the integration and extraction of the transcriptomics/proteomics data, we found that chondroitin sulfate proteoglycan 4 (CSPG4) and its glycobiosynthetic enzymes were significantly upregulated in GICs. Glyco-quantitative PCR array revealed that chondroitin sulfate (CS) biosynthetic enzymes, such as xylosyltransferase 1 (XYLT1) and carbohydrate sulfotransferase 11, were significantly downregulated during serum-induced GIC differentiation. Simultaneously, the CS modification on CSPG4 was characteristically decreased during the differentiation and also downregulated by XYLT1 knockdown. Notably, the CS degradation on CSPG4 by ChondroitinaseABC treatment dramatically induced GIC differentiation, which was significantly inhibited by the addition of CS. GIC growth and differentiation ability were significantly suppressed by CSPG4 knockdown, suggesting that CS-CSPG4 is an important factor in GIC maintenance/differentiation. To understand the molecular function of CS-CSPG4, we analyzed its associating proteins in GICs and found that CSPG4, but not CS-CSPG4, interacts with integrin αV during GIC differentiation. This event sequentially upregulates integrin-extracellular signal-regulated kinase signaling, which can be inhibited by cyclic-RGD (Arg-Gly-Asp) integrin αV inhibitor. These results indicate that CS-CSPG4 regulates the GIC microenvironment for GIC maintenance/differentiation via the CS moiety, which controls integrin signaling. This study demonstrates a novel function of CS on CSPG4 as a niche factor, so-called "glyco-niche" for GICs, and suggests that CS-CSPG4 could be a potential target for malignant glioma.

Bacteroides salyersiae is a potent chondroitin sulfate-degrading species in the human gut microbiota.

Chondroitin sulfate (CS) has widely been used as a symptomatic slow-acting drug or a dietary supplement for the treatment and prevention of osteoarthritis. However, CS could not be absorbed after oral intake due to its polyanionic nature and large molecular weight. Gut microbiota has recently been proposed to play a pivotal role in the metabolism of drugs and nutrients. Nonetheless, how CS is degraded by the human gut microbiota has not been fully characterized. In the present study, we demonstrated that each human gut microbiota was characterized with a unique capability for CS degradation. Degradation and fermentation of CS by the human gut microbiota produced significant amounts of unsaturated CS oligosaccharides (CSOSs) and short-chain fatty acids. To uncover which microbes were responsible for CS degradation, we isolated a total of 586 bacterial strains with a potential CS-degrading capability from 23 human fecal samples. Bacteroides salyersiae was a potent species for CS degradation in the human gut microbiota and produced the highest amount of CSOSs as compared to other well-recognized CS-degraders, including Bacteroides finegoldii, Bacteroides thetaiotaomicron, Bacteroides xylanisolvens, and Bacteroides ovatus. Genomic analysis suggested that B. salyersiae was armed with multiple carbohydrate-active enzymes that could potentially degrade CS into CSOSs. By using a spent medium assay, we further demonstrated that the unsaturated tetrasaccharide (udp4) produced by the primary degrader B. salyersiae could serve as a "public goods" molecule for the growth of Bacteroides stercoris, a secondary CS-degrader that was proficient at fermenting CSOSs but not CS. Taken together, our study provides insights into the metabolism of CS by the human gut microbiota, which has promising implications for the development of medical and nutritional therapies for osteoarthritis. Video Abstract.

Inter-alpha-trypsin inhibitor (IαI) and hyaluronan modifications enhance the innate immune response to influenza virus in the lung.

The inter-alpha-trypsin inhibitor (IαI) complex is composed of the bikunin core protein with a single chondroitin sulfate (CS) attached and one or two heavy chains (HCs) covalently linked to the CS chain. The HCs from IαI can be transferred to hyaluronan (HA) through a TNFα-stimulated gene-6 (TSG-6) dependent process to form an HC•HA matrix. Previous studies reported increased IαI, HA, and HC•HA complexes in mouse bronchoalveolar lavage fluid (BALF) post-influenza infection. However, the expression and incorporation of HCs into the HA matrix of the lungs during the clinical course of influenza A virus (IAV) infection and the biological significance of the HC•HA matrix are poorly understood. The present study aimed to better understand the composition of HC•HA matrices in mice infected with IAV and how these matrices regulate the host pulmonary immune response. In IAV infected mice bikunin, HC1-3, TSG-6, and HAS1-3 all show increased gene expression at various times during a 12-day clinical course. The increased accumulation of IαI and HA was confirmed in the lungs of infected mice using immunohistochemistry and quantitative digital pathology. Western blots confirmed increases in the IαI components in BALF and lung tissue at 6 days post-infection (dpi). Interestingly, HCs and bikunin recovered from BALF and plasma from mice 6 dpi with IAV, displayed differences in the HC composition by Western blot analysis and differences in bikunin's CS chain sulfation patterns by mass spectrometry analysis. This strongly suggests that the IαI components were synthesized in the lungs rather than translocated from the vascular compartment. HA was significantly increased in BALF at 6 dpi, and the HA recovered in BALF and lung tissues were modified with HCs indicating the presence of an HC•HA matrix. In vitro experiments using polyinosinic-polycytidylic acid (poly(I:C)) treated mouse lung fibroblasts (MLF) showed that modification of HA with HCs increased cell-associated HA, and that this increase was due to the retention of HA in the MLF glycocalyx. In vitro studies of leukocyte adhesion showed differential binding of lymphoid (Hut78), monocyte (U937), and neutrophil (dHL60) cell lines to HA and HC•HA matrices. Hut78 cells adhered to immobilized HA in a size and concentration-dependent manner. In contrast, the binding of dHL60 and U937 cells depended on generating a HC•HA matrix by MLF. Our in vivo findings, using multiple bronchoalveolar lavages, correlated with our in vitro findings in that lymphoid cells bound more tightly to the HA-glycocalyx in the lungs of influenza-infected mice than neutrophils and mononuclear phagocytes (MNPs). The neutrophils and MNPs were associated with a HC•HA matrix and were more readily lavaged from the lungs. In conclusion, this work shows increased IαI and HA accumulation and the formation of a HC•HA matrix in mouse lungs post-IAV infection. The formation of HA and HC•HA matrices could potentially create specific microenvironments in the lungs for immune cell recruitment and activation during IAV infection.

A chondroitin sulfate purified from shark cartilage and bovine serum albumin interaction activity.

Chondroitin sulfate (CS) was extracted and purified from shark cartilage, and its interaction with bovine serum albumin (BSA) were studied. The content of chondroitin sulfate in shark cartilage was 29.97 % using the 1,9-dimethyl-methylene blue method. The molecular weight of CS was determined to be 62.464 kDa by high-performance gel permeation chromatography. UV and FT-IR spectroscopy identified the characteristics of CS and its functional group information. NMR spectroscopy and disaccharide derivatization revealed that CS was predominantly composed of disulfated disaccharides, specifically ΔDi4,6S. Fluorescence quenching experiments indicated that the interaction between CS and BSA exhibited static quenching, with a binding site number of 1. The binding process was primarily mediated by van der Waals forces and hydrogen bonds. Furthermore, synchronous and 3D fluorescence spectroscopy demonstrated that CS had minimal impact on the polarity and hydrophobicity of the microenvironment surrounding Tyr and Trp residues. UV-vis absorption and circular dichroism (CD) spectroscopy demonstrated the altered structure of BSA. The molecular docking analysis revealed that CS formed hydrogen bonds and salt bridges with BSA, predominantly binding to the IIA substructure domain of BSA. Investigating the interaction between CS and BSA holds the potential for enhancing its applications in drug delivery and tissue engineering endeavors.

Disaccharide compositional analysis of chondroitin sulphate using WAX HILIC-MS with pre-column procainamide labelling; application to the placenta in pre-eclampsia.

Chondroitin sulphate (CS) and dermatan sulphate are negatively charged linear heteropolysaccharides. These glycosaminoglycans (GAG) are involved in cellular signalling via binding to growth factors. CS is expressed in a range of tissue and biological fluids and is highly expressed in the placenta. There is evidence that decorin; a CS proteoglycan is significantly decreased in pre-eclampsia and fetal growth restriction. It is considered that GAG chain composition may influence cellular processes that are altered in pre-eclampsia. The goal of the present study was to develop an LC-MS method with precolumn procainamide labelling for the disaccharide compositional analysis of CS. The method was used to investigate whether the disaccharide composition of placenta-extracted CS is altered in pre-eclampsia. The study revealed differential disaccharide compositions of placental chondroitin sulphate between pre-eclampsia and other pregnancy conditions. This suggests that the method may have diagnostic potential for pregnancy disorders. Furthermore, the findings suggest that CS sulphation might play a significant role in maternal labour.

Effect of Oral Chondroitin Sulfate Supplementation on Acute Brain Injury in a Murine Necrotizing Enterocolitis Model.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating condition where inflammatory changes and necrosis in the gut results in activation of brain microglia and subsequent neurodevelopmental impairment. Chondroitin sulfate (CS) is a glycosaminoglycan in human breast milk that is absent in conventional formulas. We hypothesized that oral formula supplementation with CS during a murine model of experimental NEC would not only attenuate intestinal injury, but also brain injury. STUDY DESIGN: NEC was induced in mouse pups on postnatal days (PNDs) 5 to 8. Three conditions were studied: (1) breastfed controls, (2) NEC, and (3) NEC+enteral CS (formula+200 mg/kg/d of CS). Pups were euthanized on PND 9 or reunited with dams by the evening of PND 8. Intestinal segments were H&E stained, and immunohistochemistry was performed on brain tissue for Iba-1 to assess for microglial morphology and cortical changes. Neurodevelopmental assays were performed on mice reunited with foster dams on PND 9. Single-cell RNA-sequencing analysis was performed on human intestinal epithelial cells exposed to (1) nothing, (2) hydrogen peroxide (H 2 O 2 ) alone, or (3) H 2 O 2 + CS to look at the differential gene expression between groups. Groups were compared with ANOVA or Kruskal-Wallis tests as appropriate with p < 0.05 considered significant. RESULTS: Compared with NEC, mice treated with oral CS showed improved clinical outcomes, decreased intestinal injury, and attenuated microglial activation and deleterious cortical change. Mice with CS performed better on early neurodevelopmental assays when compared with NEC alone. Single-cell analysis of HIEC-6 cells demonstrated that CS treatment down regulated several inflammatory pathways including nuclear factor κB-suggesting an explanation for the improved Th17 intestinal cytokine profile. CONCLUSIONS: Oral CS supplementation improved both physiological, clinical, and developmental outcomes. These data suggest that CS is a safe compound for formula supplementation for the prevention of NEC.

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

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.

Exogenous recombinant N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) inhibits progression of B16F10 cutaneous melanomas and modulates cell signaling.

In the syngeneic, subcutaneous B16F10 mouse model of malignant melanoma, treatment with exogenous ARSB markedly reduced tumor size and extended survival. In vivo experiments showed that local treatment with exogenous N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) led to reduced tumor growth over time (p < 0.0001) and improved the probability of survival up to 21 days (p = 0.0391). Tumor tissue from the treated mice had lower chondroitin 4-sulfate (C4S) content and lower sulfotransferase activity. The free galectin-3 declined, and the SHP2 activity increased, due to altered binding with chondroitin 4-sulfate. These changes induced effects on transcription, which were mediated by Sp1, phospho-ERK1/2, and phospho-p38 MAPK. Reduced mRNA expression of chondroitin sulfate proteoglycan 4 (CSPG4), carbohydrate sulfotransferase 15 (N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase), and matrix metalloproteinases 2 and 9 resulted. Experiments in the human melanoma cell line A375 demonstrated similar responses to exogenous ARSB as in the tumors, and inverse effects followed ARSB siRNA. ARSB, which removes the 4-sulfate group at the non-reducing end of C4S, acts as a tumor suppressor, and treatment with exogenous ARSB impacts on vital cell signaling and reduces the expression of critical genes associated with melanoma progression.

Qualitative and quantitative analyses in sulfated glycosaminoglycans, chondroitin sulfate/dermatan sulfate, during 3 T3-L1 adipocytes differentiation.

Chondroitin sulfate/dermatan sulfate (CS/DS) is a member of glycosaminoglycans (GAGs) found in animal tissues. Major CS/DS subclasses, O, A, C, D, and E units, exist based on the sulfation pattern in d-glucuronic acid (GlcA) and N-acetyl-d-galactosamine repeating units. DS is formed when GlcA is epimerized into l-iduronic acid. Our study aimed to analyze the CS/DS profile in 3 T3-L1 cells before and after adipogenic induction. CS/DS contents, molecular weight (Mw), and sulfation pattern were analyzed by using high-performance liquid chromatography. CS/DS synthesis- and sulfotransferase-related genes were analyzed by reverse transcription real-time PCR. CS/DS amount was significantly decreased in the differentiated (DI) group compared to the non-differentiated (ND) group, along with a lower expression of CS biosynthesis-related genes, chondroitin sulfate N-acetylgalactosaminyltransferase 1 and 2, as well as chondroitin polymerizing factor. GAGs in the DI group also showed lower Mw than those of ND. Furthermore, the A unit was the major CS/DS in both groups, with a proportionally higher CS-A in the DI group. This was consistent with the expression of carbohydrate sulfotransferase 12 that encodes chondroitin 4-O-sulfotransferase, for CS-A formation. These qualitative and quantitative changes in CS/DS and CS/DS-synthases before and after adipocyte differentiation reveal valuable insights into adipocyte development.

Structure and functional impact of glycosaminoglycan modification of HSulf-2 endosulfatase revealed by atomic force microscopy and mass spectrometry.

The human sulfatase HSulf-2 is one of only two known endosulfatases that play a decisive role in modulating the binding properties of heparan sulfate proteoglycans on the cell surface and in the extracellular matrix. Recently, HSulf-2 was shown to exhibit an unusual post-translational modification consisting of a sulfated glycosaminoglycan chain. This study describes the structural characterization of this glycosaminoglycan (GAG) and provides new data on its impact on the catalytic properties of HSulf-2. The unrevealed nature of this GAG chain is identified as a chondroitin/dermatan sulfate (CS/DS) mixed chain, as shown by mass spectrometry combined with NMR analysis. It consists primarily of 6-O and 4-O monosulfated disaccharide units, with a slight predominance of the 4-O-sulfation. Using atomic force microscopy, we show that this unique post-translational modification dramatically impacts the enzyme hydrodynamic volume. We identified human hyaluronidase-4 as a secreted hydrolase that can digest HSulf-2 GAG chain. We also showed that HSulf-2 is able to efficiently 6-O-desulfate antithrombin III binding pentasaccharide motif, and that this activity was enhanced upon removal of the GAG chain. Finally, we identified five N-glycosylation sites on the protein and showed that, although required, reduced N-glycosylation profiles were sufficient to sustain HSulf-2 integrity.

Mechanical forces control the valency of the malaria adhesin VAR2CSA by exposing cryptic glycan binding sites.

Plasmodium falciparum (Pf) is responsible for the most lethal form of malaria. VAR2CSA is an adhesin protein expressed by this parasite at the membrane of infected erythrocytes for attachment to the placenta, leading to pregnancy-associated malaria. VAR2CSA is a large 355 kDa multidomain protein composed of nine extracellular domains, a transmembrane helix, and an intracellular domain. VAR2CSA binds to Chondroitin Sulphate A (CSA) of the proteoglycan matrix of the placenta. Shear flow, as the one occurring in blood, has been shown to enhance the (VAR2CSA-mediated) adhesion of Pf-infected erythrocytes on the CSA-matrix. However, the underlying molecular mechanism governing this enhancement has remained elusive. Here, we address this question by using equilibrium, force-probe, and docking-based molecular dynamics simulations. We subjected the VAR2CSA protein-CSA sugar complex to a force mimicking the tensile force exerted on this system due to the shear of the flowing blood. We show that upon this force exertion, VAR2CSA undergoes a large opening conformational transition before the CSA sugar chain dissociates from its main binding site. This preferential order of events is caused by the orientation of the molecule during elongation, as well as the strong electrostatic attraction of the sugar to the main protein binding site. Upon opening, two additional cryptic CSA binding sites get exposed and a functional dodecameric CSA molecule can be stably accommodated at these force-exposed positions. Thus, our results suggest that mechanical forces increase the avidity of VAR2CSA by turning it from a monovalent to a multivalent state. We propose this to be the molecular cause of the observed shear-enhanced adherence. Mechanical control of the valency of VAR2CSA is an intriguing hypothesis that can be tested experimentally and which is of relevance for the understanding of the malaria infection and for the development of anti placental-malaria vaccines targeting VAR2CSA.

Minimally Invasive Intradiscal Delivery of BM-MSCs via Fibrous Microscaffold Carriers.

Current treatments of degenerated intervertebral discs often provide only temporary relief or address specific causes, necessitating the exploration of alternative therapies. Cell-based regenerative approaches showed promise in many clinical trials, but limitations such as cell death during injection and a harsh disk environment hinder their effectiveness. Injectable microscaffolds offer a solution by providing a supportive microenvironment for cell delivery and enhancing bioactivity. This study evaluated the safety and feasibility of electrospun nanofibrous microscaffolds modified with chitosan (CH) and chondroitin sulfate (CS) for treating degenerated NP tissue in a large animal model. The microscaffolds facilitated cell attachment and acted as an effective delivery system, preventing cell leakage under a high disc pressure. Combining microscaffolds with bone marrow-derived mesenchymal stromal cells demonstrated no cytotoxic effects and proliferation over the entire microscaffolds. The administration of cells attached to microscaffolds into the NP positively influenced the regeneration process of the intervertebral disc. Injectable poly(l-lactide-co-glycolide) and poly(l-lactide) microscaffolds enriched with CH or CS, having a fibrous structure, showed the potential to promote intervertebral disc regeneration. These features collectively address critical challenges in the fields of tissue engineering and regenerative medicine, particularly in the context of intervertebral disc degeneration.

Cryo-electron microscopy of IgM-VAR2CSA complex reveals IgM inhibits binding of Plasmodium falciparum to Chondroitin Sulfate A.

Placental malaria is caused by Plasmodium falciparum-infected erythrocytes (IEs) adhering to chondroitin sulfate proteoglycans in placenta via VAR2CSA-type PfEMP1. Human pentameric immunoglobulin M (IgM) binds to several types of PfEMP1, including VAR2CSA via its Fc domain. Here, a 3.6 Å cryo-electron microscopy map of the IgM-VAR2CSA complex reveals that two molecules of VAR2CSA bind to the Cµ4 of IgM through their DBL3X and DBL5ε domains. The clockwise and anti-clockwise rotation of the two VAR2CSA molecules on opposite faces of IgM juxtaposes C-termini of both VAR2CSA near the J chain, where IgM creates a wall between both VAR2CSA molecules and hinders its interaction with its receptor. To support this, we show when VAR2CSA is bound to IgM, its staining on IEs as well as binding of IEs to chondroitin sulfate A in vitro is severely compromised.

Molecular mechanism of decision-making in glycosaminoglycan biosynthesis.

Two major glycosaminoglycan types, heparan sulfate (HS) and chondroitin sulfate (CS), control many aspects of development and physiology in a type-specific manner. HS and CS are attached to core proteins via a common linker tetrasaccharide, but differ in their polymer backbones. How core proteins are specifically modified with HS or CS has been an enduring mystery. By reconstituting glycosaminoglycan biosynthesis in vitro, we establish that the CS-initiating N-acetylgalactosaminyltransferase CSGALNACT2 modifies all glycopeptide substrates equally, whereas the HS-initiating N-acetylglucosaminyltransferase EXTL3 is selective. Structure-function analysis reveals that acidic residues in the glycopeptide substrate and a basic exosite in EXTL3 are critical for specifying HS biosynthesis. Linker phosphorylation by the xylose kinase FAM20B accelerates linker synthesis and initiation of both HS and CS, but has no effect on the subsequent polymerisation of the backbone. Our results demonstrate that modification with CS occurs by default and must be overridden by EXTL3 to produce HS.

A conserved epitope in VAR2CSA is targeted by a cross-reactive antibody originating from Plasmodium vivax Duffy binding protein.

During Plasmodium falciparum infection in pregnancy, VAR2CSA is expressed on the surface of infected erythrocytes (IEs) and mediates their sequestration in the placenta. As a result, antibodies to VAR2CSA are largely restricted to women who were infected during pregnancy. However, we discovered that VAR2CSA antibodies can also be elicited by P. vivax Duffy binding protein (PvDBP). We proposed that infection with P. vivax in non-pregnant individuals can generate antibodies that cross-react with VAR2CSA. To better understand the specificity of these antibodies, we took advantage of a mouse monoclonal antibody (3D10) raised against PvDBP that cross-reacts with VAR2CSA and identified the epitopes targeted by this antibody. We screened two peptide arrays that span the ectodomain of VAR2CSA from the FCR3 and NF54 alleles. Based on the top epitope recognized by 3D10, we designed a 34-amino acid synthetic peptide, which we call CRP1, that maps to a highly conserved region in DBL3X. Specific lysine residues are critical for 3D10 recognition, and these same amino acids are within a previously defined chondroitin sulfate A (CSA) binding site in DBL3X. We showed by isothermal titration calorimetry that the CRP1 peptide can bind directly to CSA, and antibodies to CRP1 raised in rats significantly blocked the binding of IEs to CSA in vitro. In our Colombian cohorts of pregnant and non-pregnant individuals, at least 45% were seroreactive to CRP1. Antibody reactivities to CRP1 and the 3D10 natural epitope in PvDBP region II, subdomain 1 (SD1), were strongly correlated in both cohorts. These findings suggest that antibodies arising from PvDBP may cross-react with VAR2CSA through the epitope in CRP1 and that CRP1 could be a potential vaccine candidate to target a distinct CSA binding site in VAR2CSA.