Temporal Alterations in Cerebrovascular Glycocalyx and Cerebral Blood Flow after Exposure to a High-Intensity Blast in Rats.

The glycocalyx is a proteoglycan-glycoprotein structure lining the luminal surface of the vascular endothelium and is susceptible to damage due to blast overpressure (BOP) exposure. The glycocalyx is essential in maintaining the structural and functional integrity of the vasculature and regulation of cerebral blood flow (CBF). Assessment of alterations in the density of the glycocalyx; its components (heparan sulphate proteoglycan (HSPG/syndecan-2), heparan sulphate (HS), and chondroitin sulphate (CS)); CBF; and the effect of hypercapnia on CBF was conducted at 2-3 h, 1, 3, 14, and 28 days after a high-intensity (18.9 PSI/131 kPa peak pressure, 10.95 ms duration, and 70.26 PSI·ms/484.42 kPa·ms impulse) BOP exposure in rats. A significant reduction in the density of the glycocalyx was observed 2-3 h, 1-, and 3 days after the blast exposure. The glycocalyx recovered by 28 days after exposure and was associated with an increase in HS (14 and 28 days) and in HSPG/syndecan-2 and CS (28 days) in the frontal cortex. In separate experiments, we observed significant decreases in CBF and a diminished response to hypercapnia at all time points with some recovery at 3 days. Given the role of the glycocalyx in regulating physiological function of the cerebral vasculature, damage to the glycocalyx after BOP exposure may result in the onset of pathogenesis and progression of cerebrovascular dysfunction leading to neuropathology.

Vascular Heparan Sulfate and Amyloid-ß in Alzheimer's Disease Patients.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease characterized by the accumulation of extracellular amyloid-ß peptides (Aß) within the cerebral parenchyma and vasculature, which is known as cerebral amyloid angiopathy (CAA). This study utilized confocal imaging to investigate heparan sulfate (HS) expression within the cerebrovasculature and its associations with Aß, gender, and ApoE4 genotype in AD. Our investigation revealed elevated levels of HS in the cerebrovasculature of AD patients with severe CAA. Additionally, these patients exhibited higher HS colocalization with Aß in the cerebrovasculature, including both endothelial and vascular smooth muscle cell compartments. Intriguingly, a reversal in the polarized expression of HS within the cerebrovasculature was detected in AD patients with severe CAA. Furthermore, male patients exhibited lower levels of both parenchymal and cerebrovascular HS. Additionally, ApoE4 carriers displayed heightened cerebrovascular Aß expression and a tendency of elevated cerebrovascular HS levels in AD patients with severe CAA. Overall, these findings reveal potential intricate interplay between HS, Aß, ApoE, and vascular pathology in AD, thereby underscoring the potential roles of cerebrovascular HS in CAA development and AD pathology. Further study of the underlying mechanisms may present novel therapeutic avenues for AD treatment.

Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis.

The essence of difference between hemostasis and thrombosis is that the clotting reaction is a highly fine-tuned process. Vascular protein disulfide isomerase (PDI) represents a critical mechanism regulating the functions of hemostatic proteins. Herein we show that histidine-rich glycoprotein (HRG) is a substrate of PDI. Reduction of HRG by PDI enhances the procoagulant and anticoagulant activities of HRG by neutralization of endothelial heparan sulfate (HS) and inhibition of factor XII (FXIIa) activity, respectively. Murine HRG deficiency (Hrg-/-) leads to delayed onset but enhanced formation of thrombus compared to WT. However, in the combined FXII deficiency (F12-/-) and HRG deficiency (by siRNA or Hrg-/-), there is further thrombosis reduction compared to F12-/- alone, confirming HRG's procoagulant activity independent of FXIIa. Mutation of target disulfides of PDI leads to a gain-of-function mutant of HRG that promotes its activities during coagulation. Thus, PDI-HRG pathway fine-tunes thrombosis by promoting its rapid initiation via neutralization of HS and preventing excessive propagation via inhibition of FXIIa.

Molecular recognition and proteoglycan mimic arrangement: modulating cisplatin toxicity.

We have demonstrated that cisplatin (CP), an anticancer drug, showed a preference for binding the sulfated-L-iduronic acid (S-L-IdoA) unit over the sulfated-D-glucuronic acid unit of heparan sulfate. The multivalency of S-L-IdoA, such as in the proteoglycan mimic, resulted in distinct modes of cell-surface engineering in normal and cancer cells, with these disparities having a significant impact on CP-mediated toxicity.

Cancer-associated point mutations within the extracellular domain of PTPRD affect protein stability and HSPG interaction.

PTPRD, a well-established tumor suppressor gene, encodes the protein tyrosine phosphatase-type D. This protein consists of three immunoglobulin-like (Ig) domains, four to eight fibronectin type 3 (FN) domains, a single transmembrane segment, and two cytoplasmic tandem tyrosine phosphatase domains. PTPRD is known to harbor various cancer-associated point mutations. While it is assumed that PTPRD regulates cellular functions as a tumor suppressor through the tyrosine phosphatase activity in the intracellular region, the function of its extracellular domain (ECD) in cancer is not well understood. In this study, we systematically examined the impact of 92 cancer-associated point mutations within the ECD. We found that 69.6% (64 out of 92) of these mutations suppressed total protein expression and/or plasma membrane localization. Notably, almost all mutations (20 out of 21) within the region between the last FN domain and transmembrane segment affected protein expression and/or localization, highlighting the importance of this region for protein stability. We further found that some mutations within the Ig domains adjacent to the glycosaminoglycan-binding pocket enhanced PTPRD's binding ability to heparan sulfate proteoglycans (HSPGs). This interaction is proposed to suppress phosphatase activity. Our findings therefore suggest that HSPG-mediated attenuation of phosphatase activity may be involved in tumorigenic processes through PTPRD dysregulation.

Rational synthesis of a heparan sulfate saccharide that promotes the activity of BMP2.

Heparan sulfate (HS) is a glycosaminoglycan (GAG) found throughout nature and is involved in a wide range of functions including modulation of cell signalling via sequestration of growth factors. Current consensus is that the specificity of HS motifs for protein binding are individual for each protein. Given the structural complexity of HS the synthesis of libraries of these compounds to probe this is not trivial. Herein we present the synthesis of an HS decamer, the design of which was undertaken rationally from previously published data for HS binding to the growth factor BMP-2. The biological activity of this HS decamer was assessed in vitro, showing that it had the ability to both bind BMP-2 and increase its thermal stability as well as enhancing the bioactivity of BMP-2 in vitro in C2C12 cells. At the same time no undesired anticoagulant effect was observed. This decamer was then analysed in vivo in a rabbit model where higher bone formation, bone mineral density (BMD) and trabecular thickness were observed over an empty defect or collagen implant alone. This indicated that the HS decamer was effective in promoting bone regeneration in vivo.

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.

Chemical Synthesis of Δ-4,5 Unsaturated Heparan Sulfate Oligosaccharides for Biomarker Discovery.

A methodology is described that can provide heparan sulfate oligosaccharides having a Δ4,5-double bond, which are needed as analytical standards and biomarkers for mucopolysaccharidoses. It is based on chemical oligosaccharide synthesis followed by modification of the C-4 hydroxyl of the terminal uronic acid moiety as methanesulfonate. This leaving group is stable under conditions used to remove temporary protecting groups, O-sulfation, and hydrogenolysis. Treatment with NaOH results in elimination of the methanesulfonate and formation of a Δ4,5-double bond.

Functional dissection of the spike glycoprotein S1 subunit and identification of cellular cofactors for regulation of swine acute diarrhea syndrome coronavirus entry.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus, and the broad interspecies infection of SADS-CoV poses a potential threat to human health. This study provides experimental evidence to dissect the roles of distinct domains within the SADS-CoV spike S1 subunit in cellular entry. Specifically, we expressed the S1 and its subdomains, S1A and S1B. Cell binding and invasion inhibition assays revealed a preference for the S1B subdomain in binding to the receptors on the cell surface, and this unknown receptor is not utilized by the porcine epidemic diarrhea virus. Nanoparticle display demonstrated hemagglutination of erythrocytes from pigs, humans, and mice, linking the S1A subdomain to the binding of sialic acid (Sia) involved in virus attachment. We successfully rescued GFP-labeled SADS-CoV (rSADS-GFP) from a recombinant cDNA clone to track viral infection. Antisera raised against S1, S1A, or S1B contained highly potent neutralizing antibodies, with anti-S1B showing better efficiency in neutralizing rSADS-GFP infection compared to anti-S1A. Furthermore, depletion of heparan sulfate (HS) by heparinase treatment or pre-incubation of rSADS-GFP with HS or constituent monosaccharides could inhibit SADS-CoV entry. Finally, we demonstrated that active furin cleavage of S glycoprotein and the presence of type II transmembrane serine protease (TMPRSS2) are essential for SADS-CoV infection. These combined observations suggest that the wide cell tropism of SADS-CoV may be related to the distribution of Sia or HS on the cell surface, whereas the S1B contains the main protein receptor binding site. Specific host proteases also play important roles in facilitating SADS-CoV entry.IMPORTANCESwine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel pathogen infecting piglet, and its unique genetic evolution characteristics and broad species tropism suggest the potential for cross-species transmission. The virus enters cells through its spike (S) glycoprotein. In this study, we identify the receptor binding domain on the C-terminal part of the S1 subunit (S1B) of SADS-CoV, whereas the sugar-binding domain located at the S1 N-terminal part of S1 (S1A). Sialic acid, heparan sulfate, and specific host proteases play essential roles in viral attachment and entry. The dissection of SADS-CoV S1 subunit's functional domains and identification of cellular entry cofactors will help to explore the receptors used by SADS-CoV, which may contribute to exploring the mechanisms behind cross-species transmission and host tropism.

Structure and function of Semaphorin-5A glycosaminoglycan interactions.

Integration of extracellular signals by neurons is pivotal for brain development, plasticity, and repair. Axon guidance relies on receptor-ligand interactions crosstalking with extracellular matrix components. Semaphorin-5A (Sema5A) is a bifunctional guidance cue exerting attractive and inhibitory effects on neuronal growth through the interaction with heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAGs), respectively. Sema5A harbors seven thrombospondin type-1 repeats (TSR1-7) important for GAG binding, however the underlying molecular basis and functions in vivo remain enigmatic. Here we dissect the structural basis for Sema5A:GAG specificity and demonstrate the functional significance of this interaction in vivo. Using x-ray crystallography, we reveal a dimeric fold variation for TSR4 that accommodates GAG interactions. TSR4 co-crystal structures identify binding residues validated by site-directed mutagenesis. In vitro and cell-based assays uncover specific GAG epitopes necessary for TSR association. We demonstrate that HS-GAG binding is preferred over CS-GAG and mediates Sema5A oligomerization. In vivo, Sema5A:GAG interactions are necessary for Sema5A function and regulate Plexin-A2 dependent dentate progenitor cell migration. Our study rationalizes Sema5A associated developmental and neurological disorders and provides mechanistic insights into how multifaceted guidance functions of a single transmembrane cue are regulated by proteoglycans.

Metabolism mechanism of glycosaminoglycans by the gut microbiota: Bacteroides and lactic acid bacteria: A review.

Glycosaminoglycans (GAGs), as a class of biopolymers, play pivotal roles in various biological metabolisms such as cell signaling, tissue development, cell apoptosis, immune modulation, and growth factor activity. They are mainly present in the colon in free forms, which are essential for maintaining the host's health by regulating the colonization and proliferation of gut microbiota. Therefore, it is important to explain the specific members of the gut microbiota for GAGs' degradation and their enzymatic machinery in vivo. This review provides an outline of GAGs-utilizing entities in the Bacteroides, highlighting their polysaccharide utilization loci (PULs) and the enzymatic machinery involved in chondroitin sulfate (CS) and heparin (Hep)/heparan sulfate (HS). While there are some variations in GAGs' degradation among different genera, we analyze the reputed GAGs' utilization clusters in lactic acid bacteria (LAB), based on recent studies on GAGs' degradation. The enzymatic machinery involved in Hep/HS and CS metabolism within LAB is also discussed. Thus, to elucidate the precise mechanisms utilizing GAGs by diverse gut microbiota will augment our understanding of their effects on human health and contribute to potential therapeutic strategies for diseases.

Exploring Heparanase Levels in Tears: Insights From Herpes Simplex Virus-1 Keratitis Patients and Animal Studies.

Purpose: Heparanase (HPSE) cleaves heparan sulfate proteoglycans during herpes simplex virus-1 (HSV-1) infection, aiding in viral egress and disease progression. Its action has been well established in in vitro and in vivo models, but its relevance in human patients remains unclear. This study aimed to specifically evaluate tear HPSE levels of patients with herpes simplex keratitis (HSK) and to correlate these findings with a commonly used murine model. Methods: Tear samples from patient and mice samples were collected at LV Prasad Eye Institute, Hyderabad, India, and at the University of Illinois, Chicago, IL, respectively. Tears were collected from HSV-1 patients, bacterial/fungal keratitis cases, and healthy individuals. For in vivo study, C57BL/6 mice were infected with HSV-1 (McKrae strain) followed by tear fluid collection at various time points (0-10 days). Results: The HSV-1, bacterial keratitis, fungal keratitis, and healthy control groups each had 30 patients. There was a significant difference in HPSE expression in the HSV-1 infected eyes (1.55 ± 0.19 units/mL) compared to HSV-1 contralateral eyes (1.23 ± 0.13 units/mL; P = 0.82), bacterial keratitis eyes (0.87 ± 0.15 units/mL; P = 0.0078), fungal keratitis eyes (0.64 ± 0.09 units/mL; P < 0.00001), and normal controls (0.53 ± 0.06 units/mL; P < 0.00001). C57BL/6 mice tear HPSE expression in infected eyes was 0.66 to 5.57 ng heparan sulfate (HS) removed per minute when compared to non-infected eye (range, 0.70-3.67 ng HS removed per minute). Conclusions: To the best of our knowledge, this study is the first to report elevated HPSE levels in the tears of patients with different forms of HSV-1 keratitis, and it confirms similar findings in a murine model, providing a valuable basis for future in vivo and clinical research on HSV-1 ocular infection.

Structure-Based Discovery of High-Affinity Small Molecule Ligands and Development of Tool Probes to Study the Role of Chitinase-3-Like Protein 1.

Chitinase-3-like-1 (CHI3L1), also known as YKL-40, is a glycoprotein linked to inflammation, fibrosis, and cancer. This study explored CHI3L1's interactions with various oligosaccharides using microscale thermophoresis (MST) and AlphaScreen (AS). These investigations guided the development of high-throughput screening assays to assess interference of small molecules in binding between CHI3L1 and biotinylated small molecules or heparan sulfate-based probes. Small molecule binders of YKL-40 were identified in our chitotriosidase inhibitors library with MST and confirmed through X-ray crystallography. Based on cocrystal structures of potent hit compounds with CHI3L1, small molecule probes 19 and 20 were designed for an AS assay. Structure-based optimization led to compounds 30 and 31 with nanomolar activities and drug-like properties. Additionally, an orthogonal AS assay using biotinylated heparan sulfate as a probe was developed. The compounds' affinity showed a significant correlation in both assays. These screening tools and compounds offer novel avenues for investigating the role of CHI3L1.

Historia natural de la mucopolisacaridosis III en una serie de pacientes colombianos / Natural history of mucopolysaccharidosis type III in a series of Colombian patients

Introducción: La mucopolisacaridosis de tipo III (MPS III), o síndrome de Sanfilippo, es un trastorno de almacenamiento lisosómico con características neurodegenerativas progresivas, predominante del sistema nervioso central. Su diagnóstico se basa en el cuadro clínico, y priman alteraciones en el neurodesarrollo y neuropsiquiátricas, incluso antes de la presencia de alteraciones fenotípicas. El análisis bioquímico para identificar el tipo de glucosaminoglucanos presente, la determinación enzimática y el estudio de genética molecular confirman la enfermedad. Casos clínicos: Se realiza la descripción clínica de ocho pacientes con diagnóstico de MPS III en Colombia, con síntomas iniciales en relación con retraso del desarrollo y trastornos comportamentales evidenciados entre los 3 y 8 años, asociado a facies toscas, cejas pobladas, hepatomegalia y pérdida auditiva progresiva en todos los casos. Uno de los pacientes presentó anomalías cardíacas; dos de ellos, epilepsia focal; y en uno se evidenció atrofia óptica. Todos presentaron alteraciones en las neuroimágenes con evidencia de pérdida del volumen parenquimatoso, atrofia del cuerpo calloso y adelgazamiento cortical; el diagnostico se realizó a través de estudios bioquímicos de cromatografía de glucosaminoglucanos y todos cuentan con un estudio genético confirmatorio. Conclusiones: La MPS III es un desafío diagnóstico, particularmente en pacientes con un curso atenuado de la enfermedad, debido al curso variable, síntomas neuropsiquiátricos tempranos inespecíficos y falta de características somáticas evidentes en comparación con otros tipos de MPS. Cuando se tiene el diagnóstico definitivo, es fundamental brindar atención interdisciplinaria para el paciente y la familia, y apoyar el tratamiento de los síntomas físicos, garantizando ofrecer el mejor cuidado posible y la mejor calidad de vida para el paciente y su familia, al tratarse de una condición neurodegenerativa.(AU)

Introduction: Mucopolysaccharidosis type III (MPS III), also known as Sanfilippo syndrome, is a lysosomal storage disease with progressive neurodegenerative features, predominantly affecting the central nervous system. Diagnosis is based on clinical features, with neurodevelopmental and neuropsychiatric alterations taking precedence, including over phenotype alterations. The disease is confirmed by biochemical analysis to identify the type of glycosaminoglycans present, enzyme assay and molecular genetic studies. Case reports: A clinical description was performed for eight patients diagnosed with MPS III in Colombia. Their initial symptoms were related to developmental delay and behavioural disorders presenting between 3 and 8 years of age, associated in all cases with coarse facial features, thick eyebrows, hepatomegaly and progressive hearing loss. One of the patients presented cardiac anomalies; two presented focal epilepsy; and one presented optic atrophy. They all presented neuroimaging alterations, with evidence of parenchymal volume loss, corpus callosum atrophy and cortical thinning; the diagnosis was performed by biochemical glycosaminoglycan chromatography studies, and all patients have a confirmatory genetic study. Conclusions: MPS III is a challenge for diagnosis, particularly in its early stages and in patients in which the course of the disease is attenuated. This is due to its variable course, non-specific early neuropsychiatric symptoms, and the absence of obvious somatic features compared to other types of MPS. After a definitive diagnosis has been made, interdisciplinary care must be provided for the patient and their family, and support given for the treatment of physical symptoms, ensuring the best possible care and quality of life for the patient and their family, as the condition is neurodegenerative.(AU)

Chemo-enzymatic synthesis of tetrasaccharide linker peptides to study the divergent step in glycosaminoglycan biosynthesis.

Glycosaminoglycans are extended linear polysaccharides present on cell surfaces and within the extracellular matrix that play crucial roles in various biological processes. Two prominent glycosaminoglycans, heparan sulfate and chondroitin sulfate, are covalently linked to proteoglycan core proteins through a common tetrasaccharide linker comprising glucuronic acid, galactose, galactose, and xylose moities. This tetrasaccharide linker is meticulously assembled step by step by four Golgi-localized glycosyltransferases. The addition of the fifth sugar moiety, either N-acetylglucosamine or N-acetylgalactosamine, initiates further chain elongation, resulting in the formation of heparan sulfate or chondroitin sulfate, respectively. Despite the fundamental significance of this step in glycosaminoglycan biosynthesis, its regulatory mechanisms have remained elusive. In this study, we detail the expression and purification of the four linker-synthesizing glycosyltransferases and their utilization in the production of fluorescent peptides carrying the native tetrasaccharide linker. We generated five tetrasaccharide peptides, mimicking the core proteins of either heparan sulfate or chondroitin sulfate proteoglycans. These peptides were readily accepted as substrates by the EXTL3 enzyme, which adds an N-acetylglucosamine moiety, thereby initiating heparan sulfate biosynthesis. Importantly, EXTL3 showed a preference towards peptides mimicking the core proteins of heparan sulfate proteoglycans over the ones from chondroitin sulfate proteoglycans. This suggests that EXTL3 could play a role in the decision-making step during glycosaminoglycan biosynthesis. The innovative strategy for chemo-enzymatic synthesis of fluorescent-labeled linker-peptides promises to be instrumental in advancing future investigations into the initial steps and the divergent step of glycosaminoglycan biosynthesis.

Heparanase inhibition as a systemic approach to protect the endothelial glycocalyx and prevent microvascular complications in diabetes.

BACKGROUND: Diabetes mellitus is a chronic disease which is detrimental to cardiovascular health, often leading to secondary microvascular complications, with huge global health implications. Therapeutic interventions that can be applied to multiple vascular beds are urgently needed. Diabetic retinopathy (DR) and diabetic kidney disease (DKD) are characterised by early microvascular permeability changes which, if left untreated, lead to visual impairment and renal failure, respectively. The heparan sulphate cleaving enzyme, heparanase, has previously been shown to contribute to diabetic microvascular complications, but the common underlying mechanism which results in microvascular dysfunction in conditions such as DR and DKD has not been determined. METHODS: In this study, two mouse models of heparan sulphate depletion (enzymatic removal and genetic ablation by endothelial specific Exotosin-1 knock down) were utilized to investigate the impact of endothelial cell surface (i.e., endothelial glycocalyx) heparan sulphate loss on microvascular barrier function. Endothelial glycocalyx changes were measured using fluorescence microscopy or transmission electron microscopy. To measure the impact on barrier function, we used sodium fluorescein angiography in the eye and a glomerular albumin permeability assay in the kidney. A type 2 diabetic (T2D, db/db) mouse model was used to determine the therapeutic potential of preventing heparan sulphate damage using treatment with a novel heparanase inhibitor, OVZ/HS-1638. Endothelial glycocalyx changes were measured as above, and microvascular barrier function assessed by albumin extravasation in the eye and a glomerular permeability assay in the kidney. RESULTS: In both models of heparan sulphate depletion, endothelial glycocalyx depth was reduced and retinal solute flux and glomerular albumin permeability was increased. T2D mice treated with OVZ/HS-1638 had improved endothelial glycocalyx measurements compared to vehicle treated T2D mice and were simultaneously protected from microvascular permeability changes associated with DR and DKD. CONCLUSION: We demonstrate that endothelial glycocalyx heparan sulphate plays a common mechanistic role in microvascular barrier function in the eye and kidney. Protecting the endothelial glycocalyx damage in diabetes, using the novel heparanase inhibitor OVZ/HS-1638, effectively prevents microvascular permeability changes associated with DR and DKD, demonstrating a novel systemic approach to address diabetic microvascular complications.

Synthesis and Detection of BODIPY-, Biotin-, and 19 F- Labeled Single-Entity Dendritic Heparan Sulfate Mimetics.

Heparin and heparan sulfate (HS) are naturally occurring mammalian glycosaminoglycans, and their synthetic and semi-synthetic mimetics have attracted significant interest as potential therapeutics. However, understanding the mechanism of action by which HS, heparin, and HS mimetics have a biological effect is difficult due to their highly charged nature, broad protein interactomes, and variable structures. To address this, a library of novel single-entity dendritic mimetics conjugated to BODIPY, Fluorine-19 (19 F), and biotin was synthesized for imaging and localization studies. The novel dendritic scaffold allowed for the conjugation of labeling moieties without reducing the number of sulfated capping groups, thereby better mimicking the multivalent nature of HS-protein interactions. The 19 F labeled mimetics were assessed in phantom studies and were detected at concentrations as low as 5 mM. Flow cytometric studies using a fluorescently labeled mimetic showed that the compound associated with immune cells from tumors more readily than splenic counterparts and was directed to endosomal-lysosomal compartments within immune cells and cancer cells. Furthermore, the fluorescently labeled mimetic entered the central nervous system and was detectable in brain-infiltrating immune cells 24 hours after treatment. Here, we report the enabling methodology for rapidly preparing various labeled HS mimetics and molecular probes with diverse potential therapeutic applications.

Antioxidant and antiproliferative effect of a glycosaminoglycan extract from Rapana venosa marine snail.

Marine glycosaminoglycans (GAG) isolated from different invertebrates, such as molluscs, starfish or jellyfish, have been described as unique molecules with important pharmacological applications. Scarce information is available on GAG extract from Rapana venosa marine snail. The aim of this study was to isolate a GAG extract from R. venosa marine snail and to investigate its physicochemical, antioxidant and antiproliferative properties for further biomedical use. The morphology, chemical and elemental composition of the extract were established as well as the sulfate content and N- to O-sulfation ratio. Fourier transform infrared (FTIR) spectra indicated that GAG extract presented similar structural characteristics to bovine heparan sulfate and chondroitin sulfate. The pattern of extract migration in agarose gel electrophoresis and specific digestion with chondroitinase ABC and heparinase III indicated the presence of a mixture of chondroitin sulfate-type GAG, as main component, and heparan sulfate-type GAG. Free radical scavenging and ferric ion reducing assays showed that GAG extract had high antioxidant activity, which slightly decreased after enzymatic treatment. In vitro MTT and Live/Dead assays showed that GAG extract had the ability to inhibit cell proliferation in human Hep-2 cell cultures, at cytocompatible concentrations in normal NCTC clone L929 fibroblasts. This capacity decreased after enzymatic digestion, in accordance to the antioxidant activity of the products. Tumoral cell migration was also inhibited by GAG extract and its digestion products. Overall, GAG extract from R. venosa marine snail exhibited antioxidant and antiproliferative activities, suggesting its potential use as novel bioactive compound for biomedical applications.

Structural and mechanistic characterization of bifunctional heparan sulfate N-deacetylase-N-sulfotransferase 1.

Heparan sulfate (HS) polysaccharides are major constituents of the extracellular matrix, which are involved in myriad structural and signaling processes. Mature HS polysaccharides contain complex, non-templated patterns of sulfation and epimerization, which mediate interactions with diverse protein partners. Complex HS modifications form around initial clusters of glucosamine-N-sulfate (GlcNS) on nascent polysaccharide chains, but the mechanistic basis underpinning incorporation of GlcNS itself into HS remains unclear. Here, we determine cryo-electron microscopy structures of human N-deacetylase-N-sulfotransferase (NDST)1, the bifunctional enzyme primarily responsible for initial GlcNS modification of HS. Our structures reveal the architecture of both NDST1 deacetylase and sulfotransferase catalytic domains, alongside a non-catalytic N-terminal domain. The two catalytic domains of NDST1 adopt a distinct back-to-back topology that limits direct cooperativity. Binding analyses, aided by activity-modulating nanobodies, suggest that anchoring of the substrate at the sulfotransferase domain initiates the NDST1 catalytic cycle, providing a plausible mechanism for cooperativity despite spatial domain separation. Our data shed light on key determinants of NDST1 activity, and describe tools to probe NDST1 function in vitro and in vivo.

Dissection of N-deacetylase and N-sulfotransferase activities of NDST1 and their effects on Wnt8 distribution and signaling in Xenopus embryos.

Wnt is a family of secreted signaling proteins involved in the regulation of cellular processes, including maintenance of stem cells, carcinogenesis, and cell differentiation. In the context of early vertebrate embryogenesis, graded distribution of Wnt proteins has been thought to regulate positional information along the antero-posterior axis. However, understanding of the molecular basis for Wnt spatial distribution remains poor. Modified states of heparan sulfate (HS) proteoglycans are essential for Wnt8 localization, because depletion of N-deacetylase/N-sulfotransferase 1 (NDST1), a modification enzyme of HS chains, decreases Wnt8 levels and NDST1 overexpression increases Wnt8 levels on the cell surface. Since overexpression of NDST1 increases both deacetylation and N-sulfation of HS chains, it is not clear which function of NDST1 is actually involved in Wnt8 localization. In the present study, we generated an NDST1 mutant that specifically increases deacetylation, but not N-sulfation, of HS chains in Xenopus embryos. Unlike wild-type NDST1, this mutant did not increase Wnt8 accumulation on the cell surface, but it reduced canonical Wnt signaling, as determined with the TOP-Flash reporter assay. These results suggest that N-sulfation of HS chains is responsible for localization of Wnt8 and Wnt8 signaling, whereas deacetylation has an inhibitory effect on canonical Wnt signaling. Consistently, overexpression of wild-type NDST1, but not the mutant, resulted in small eyes in Xenopus embryos. Thus, our NDST1 mutant enables us to dissect the regulation of Wnt8 localization and signaling by HS proteoglycans by specifically manipulating the enzymatic activities of NDST1.