Heparan Sulfate Proteoglycans Research Articles

Lactoferrin exhibits PEDV antiviral activity by interfering with spike-heparan sulfate proteoglycans binding and activating mucosal immune response.

Neonatal piglets infected with Porcine Epidemic Diarrhea Virus (PEDV) experience a mortality rate of up to 90%, resulting in significant economic losses to the swine industry in China. Current strategies using specific antibodies in sow milk to prevent Porcine Epidemic Diarrhea (PED) in these piglets through specific antibodies in sow milk is unsatisfactory. Preliminary studies have shown limited success. Emerging evidence suggests that general immune factors in sow milk provide protection to neonatal piglets, particularly, lactoferrin, play a crucial role in protecting piglets by inhibiting PEDV replication. However, the precise mechanism by which lactoferrin exerts its antiviral effects remains unclear. This study sought to clarify these mechanisms through both in vitro and in vivo approaches, proposing that higher concentrations of lactoferrin lead to greater antiviral activity. It was hypothesized that lactoferrin can impede PEDV by blocking its binding to heparan sulfate proteoglycans (HSPG) on the surface of target cells, and molecular docking experiments was conducted to identify the binding sites between lactoferrin and HSPG. Additionally, the findings indicated that lactoferrin can effectively trigger the maturation of porcine dendritic cells and boosts their antigen-presenting functions, thereby improving intestinal mucosal immunity in neonatal piglets against PEDV. Overall, these findings aid to elucidate the antiviral actions and mechanisms of lactoferrin in sow colostrum, offering new insights for the effective prevention and control of PED in neonatal piglets.

Endothelial lipase-binding peptides similar to netrin-1 inhibit hepatitis B virus infection.

Hepatitis B virus (HBV) infects cells by attaching to heparan sulfate proteoglycans (HSPG) and Na+/taurocholate cotransporting polypeptide (NTCP). The endothelial lipase LIPG bridges HSPG and HBV, facilitating HBV attachment. From a randomized peptide expression library, we identified a short sequence binding to LIPG. This identified sequence closely resembled a sequence in the V domain of netrin-1, a protein known to bind heparin through its V domain. We designed two synthetic peptides based on this sequence and found that both synthetic peptides and netrin-1 suppressed HBV infection in chimeric mice with humanized livers and in primary hepatocytes isolated from them. The data reveal an antiviral function of the peptides and netrin-1 in HBV infection that is independent of LIPG lipase activity.

Effects of Heparan Sulfate Trisaccharide Containing Oleanolic Acid in Attenuating Hyperphosphorylated Tau-Induced Cell Dysfunction Associated with Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia, marked by progressive brain degeneration and cognitive decline. A major pathological feature of AD is the accumulation of hyperphosphorylated tau (p-tau) in the form of neurofibrillary tangles (NFTs), which leads to neuronal death and neurodegeneration. P-tau also induces endoplasmic reticulum (ER) stress and activates the unfolded protein response, causing inflammation and apoptosis. Additionally, p-tau spreads in the brain through interactions with heparan sulfate (HS) proteoglycans, promoting aggregation and internalization. Targeting the tau-HS interaction offers a potential therapeutic strategy for AD. We present a novel HS mimetic with a lipophilic oleanolic acid linker and a sulfated trisaccharide, which shows strong cytoprotective effects against p-tau. Moreover, this compound alleviates p-tau-induced ER stress and inflammation. Molecular docking studies indicate that the conjugation of oleanolic acid enhances binding between the ligand and tau protofilament cores, facilitating protective interactions. These findings provide a foundation for the development of novel HS mimetics, enabling further investigation of tau-HS interactions in AD and other tauopathies.

HSPG-binding peptide Pep19-2.5 is a potent inhibitor of HPV16 infection.

Peptide-based therapeutics are gaining attention for their potential to target various viral and host cell factors. One notable example is Pep19-2.5 (Aspidasept), a synthetic anti-lipopolysaccharide peptide that binds to heparan sulfate proteoglycans (HSPGs) and has demonstrated inhibitory effects against certain bacteria and enveloped viruses. This study explores, for the first time, the effectiveness of Pep19-2.5 against a non-enveloped virus, using pseudoviruses of the oncogenic human papillomavirus type 16 (HPV16) as a model. HPV16 infects epithelial cells of the skin and mucosa by using multiple cell surface receptors with initial attachment to HSPGs. Pharmacological inhibition with Pep19-2.5 in HeLa and HaCaT cells resulted in a concentration-dependent reduction of HPV16 PsV infection, with near-complete blockade observed at higher concentrations. The half-maximal inhibitory concentration (IC50) was determined to be 116 nM in HeLa cells and 183 nM in HaCaT cells, highlighting its potent antiviral activity. Our results demonstrate that Pep19-2.5 not only inhibits HPV16 PsV binding to the cell surface but also significantly reduces infection when administered post-binding. Imaging analyses revealed Pep19-2.5-dependent release of large cell-associated crowds of viral particles, suggesting interference with the transfer to secondary receptor molecules. This was corroborated by the effectiveness of Pep19-2.5 in an HSPG-negative cell line, indicating that the peptide disrupts virus binding to both primary and secondary interaction partners. Based on these findings, we propose that the antimicrobial effect of Pep19-2.5 is not limited to HSPG-dependent infections. Additionally, Pep19-2.5 may be a valuable tool for dissecting specific steps in the viral entry process.

Heparan sulfate proteoglycan affinity of adeno-associated virus vectors: Implications for retinal gene delivery.

Adeno-associated virus (AAV)-based vectors have emerged as an effective and widely used technology for somatic gene therapy approaches, including those targeting the retina. A major advantage of the AAV technology is the availability of a large number of serotypes that have either been isolated from nature or produced in the laboratory. These serotypes have different properties in terms of sensitivity to neutralizing antibodies, cellular transduction profile and efficiency. The infectivity of AAV vectors depends on the affinity to certain molecules on the cell surface, in particular to cellular glycosaminoglycans (GAGs) such as heparan sulfate proteoglycans (HSPGs). Here, we tested how altering HSPG affinity in AAV vectors affects cellular tropism and transduction efficiency. The previously developed AAV2.GL variant was used as a starting variant to alter or disrupt HSPG affinity. The HSPG-independent AAV9 serotype was used to introduce different HSPG-binding sites. As an indicator of HSPG affinity, we measured the binding strength of the vector variant on a heparin chromatography column. We show that modification of capsid-exposed residues has a strong impact on HSPG affinity, cellular tropism and transduction efficiency in HeLa cells and in vivo in mouse retina. Our study shows that key properties of AAV vectors can be tailored in different directions and used to improve tropism and efficiency.

Extracellular matrix components in preeclampsia.

Preeclampsia (PE) is a gestational complication affecting 5% to 10% of all pregnancies. PE is characterized by hypertension and endothelial dysfunction, whose etiology involves, among other factors, alterations in the extracellular matrix (ECM) that can compromise vascular remodeling and trophoblast invasion, ie, processes essential for placental development. Endothelial dysfunction is caused by release of antiangiogenic factors, mainly a soluble fms-like tyrosine kinase-1 (sFlt-1), which antagonizes two endothelial angiogenic factors, the vascular endothelial growth factor (VEGF) and placental growth factor (PLGF). This angiogenic imbalance contributes to clinical symptoms including hypertension and multisystem dysfunction. This review aims to summarize recent advances in understanding PE, particularly with altered ECM components such as heparan sulfate proteoglycans, the glycosidase heparanase, fibronectin, collagen XVIII (endostatin), and metalloproteases. This comprehensive narrative review was conducted on PubMed from 1994 to 2024, focusing on articles on the pathophysiology of PE, particularly endothelial dysfunction caused by ECM modifications. The data shows a reduced expression of matrix metalloproteinases, increased collagen fragment XVIII, and significant changes in fibronectin associated with PE. Furthermore, endothelial dysfunction was associated with increased degradation of heparan sulfate chains from proteoglycans and increased sFlt-1. Understanding these ECM modifications is crucial for developing potential new therapeutic interventions that improve maternal and fetal outcome in PE.

Competitive displacement of lipoprotein lipase from heparan sulfate is orchestrated by a disordered acidic cluster in GPIHBP1.

Movement of lipoprotein lipase (LPL) from myocytes or adipocytes to the capillary lumen is essential for intravascular lipolysis and plasma triglyceride homeostasis-low LPL activity in the capillary lumen causes hypertriglyceridemia. The trans-endothelial transport of LPL depends on ionic interactions with GPIHBP1's intrinsically disordered N-terminal tail, which harbors two acidic clusters at positions 5-12 and 19-30. This polyanionic tail provides a molecular switch that controls LPL detachment from heparan sulfate proteoglycans (HSPGs) by competitive displacement. When the acidic tail was neutralized in gene-edited mice, LPL remained trapped in the sub-endothelial spaces triggering hypertriglyceridemia. Due to its disordered state, the crystal structure of LPL•GPIHBP1 provided no information on these electrostatic interactions between LPL and GPIHBP1s acidic tail. In the current study, we positioned the acidic tail on LPL using zero-length crosslinking. Acidic residues at positions 19-30 in GPIHBP1 mapped to Lys445, Lys441, Lys414 and Lys407 close to the interface between the C- and N-terminal domains in LPL. Modeling this interface revealed widespread polyelectrolyte interactions spanning both LPL domains, which explains why the acidic tail stabilizes LPL activity and protein conformation. In functional assays, we showed that the acidic cluster at 19-30 also had the greatest impact on preserving LPL activity, mitigating ANGPTL4-catalyzed LPL inactivation, preventing PSCK3-mediated LPL cleavage, and, importantly, displacing LPL from HSPGs. Our current study provides key insights into the biophysical mechanism(s) orchestrating intravascular compartmentalization of LPL activity-an intriguing pathway entailing competitive displacement of HSPG-bound LPL by a disordered acidic tail in GPIHBP1.

A Nanoparticle Comprising the Receptor-Binding Domains of Norovirus and Plasmodium as a Combination Vaccine Candidate.

Noroviruses, which cause epidemic acute gastroenteritis, and Plasmodium parasites, which lead to malaria, are two infectious pathogens that pose threats to public health. The protruding (P) domain of norovirus VP1 and the αTSR domain of the circumsporozoite protein (CSP) of Plasmodium sporozoite are the glycan receptor-binding domains of the two pathogens for host cell attachment, making them excellent targets for vaccine development. Modified norovirus P domains self-assemble into a 24-meric octahedral P nanoparticle (P24 NP). We generated a unique P24-αTSR NP by inserting the αTSR domain into a surface loop of the P domain. The P-αTSR fusion proteins were produced in the Escherichia coli expression system and the fusion protein self-assembled into the P24-αTSR NP. The formation of the P24-αTSR NP was demonstrated through gel filtration, electron microscopy, and dynamic light scattering. A 3D structural model of the P24-αTSR NP was constructed, using the known cryo-EM structure of the previously developed P24 NP and P24-VP8* NP as templates. Each P24-αTSR NP consists of a P24 NP core, with 24 surface-exposed αTSR domains that have retained their general conformations and binding function to heparan sulfate proteoglycans. The P24-αTSR NP is immunogenic, eliciting strong antibody responses in mice toward both the norovirus P domain and the αTSR domain of Plasmodium CSP. Notably, sera from mice immunized with the P24-αTSR NP bound strongly to Plasmodium sporozoites and blocked norovirus VLP attachment to their glycan receptors. These data suggest that the P24-αTSR NP may serve as a combination vaccine against both norovirus and Plasmodium parasites.

Exploring the Chemical Features and Biomedical Relevance of Cell-Penetrating Peptides

Cell-penetrating peptides (CPPs) are a diverse group of peptides, typically composed of 4 to 40 amino acids, known for their unique ability to transport a wide range of substances—such as small molecules, plasmid DNA, small interfering RNA, proteins, viruses, and nanoparticles—across cellular membranes while preserving the integrity of the cargo. CPPs exhibit passive and non-selective behavior, often requiring functionalization or chemical modification to enhance their specificity and efficacy. The precise mechanisms governing the cellular uptake of CPPs remain ambiguous; however, electrostatic interactions between positively charged amino acids and negatively charged glycosaminoglycans on the membrane, particularly heparan sulfate proteoglycans, are considered the initial crucial step for CPP uptake. Clinical trials have highlighted the potential of CPPs in diagnosing and treating various diseases, including cancer, central nervous system disorders, eye disorders, and diabetes. This review provides a comprehensive overview of CPP classifications, potential applications, transduction mechanisms, and the most relevant algorithms to improve the accuracy and reliability of predictions in CPP development.

Microglia internalize tau monomers and fibrils using distinct receptors but similar mechanisms.

Alzheimer's disease (AD) and other tauopathies are characterized by intracellular aggregates of microtubule-associated protein tau that are actively released and promote proteopathic spread. Microglia engulf pathological proteins, but how they endocytose tau is unknown. We measured endocytosis of different tau species by microglia after pharmacological modulation of macropinocytosis or clathrin-mediated endocytosis (CME) or antagonism/genetic depletion of known tau receptors heparan-sulfate proteoglycans (HSPGs) and low-density lipoprotein receptor-related protein 1 (LRP1). Dynamin inhibition decreased microglial endocytosis of all tested tau species. Meanwhile, HSPG antagonism blocked only fibril uptake, and LRP1 antagonism or genetic depletion inconsistently inhibited the endocytosis of fibrils and monomers. Cre recombinase robustly enhanced tau uptake with partial selectivity for fibrils. These data show that microglia take up both tau monomers and aggregates via a dynamin-dependent form of endocytosis (eg, CME) but may differ in using HSPGs for entry depending on species. Microglial endocytosis of tau monomers and fibrils is dynamin-dependent. HSPG antagonism blocks microglial uptake of tau fibrils but not monomers. LRP1 antagonism or knockdown inconsistently inhibits tau uptake. TAT-Cre stimulates semi-selective uptake of fibrils over monomers.

Whole exome sequencing reveals heparan sulfate proteoglycan 2 (HSPG2) as a potential causative gene for kidney stone disease in a Thai family

Kidney stone disease (KSD) is a prevalent and complex condition, with an incidence of 85 cases per 100,000 individuals in Thailand. Notably, over 40% of cases are concentrated in the northeastern region, indicating a potential genetic influence, which is supported by genetic mutations reported in several families by our research group. Despite this, the genetic basis of KSD remains largely unknown for many Thai families. This study aimed to identify the genetic mutation responsible for KSD in a specific Thai family, the UBRS131 family, which includes four affected individuals. Whole exome sequencing was performed, and variant filtering using the VarCards2 program identified 10 potentially causative mutations across 9 genes. These mutations were subjected to segregation analysis among family members and screened in 180 control and 179 case samples using real-time PCR-HRM or PCR-RFLP techniques. Prioritization of these variants using GeneDistiller identified the p.Asp775Glu mutation in the heparan sulfate proteoglycan 2 (HSPG2) gene as the likely causative mutation for KSD in this family. The Asp775 residue is highly conserved across vertebrates, and structural analysis suggests that the Glu775 substitution may disrupt the formation of two crucial hydrogen bonds, potentially altering the mutant protein’s configuration. Immunohistochemistry confirmed the presence of perlecan (HSPG2 protein) in the proximal tubules in nephrons. These findings highlight the significant role of the HSPG2 gene in familial KSD within this study family.

An integral membrane constitutively active heparanase enhances the tumor infiltration capability of NK cells

ABSTRACT Eradication of cancer cells by the immune system requires extravasation, infiltration and progression of immune cells through the tumor extracellular matrix (ECM). These are also critical determinants for successful adoptive cell immunotherapy of solid tumors. Together with structural proteins, such as collagens and fibronectin, heparan sulfate (HS) proteoglycans are major components of the ECM. Heparanase 1 (HPSE) is the only enzyme known to have endoglycosidase activity that degrades HS. HPSE is expressed at high levels in almost all hematopoietic cells, which suggests that it plays a relevant role in immune cell migration through solid tissues. Besides, tumor cells express also HPSE as a way to facilitate tumor cell resettlement and metastasis. Therefore, an increase in HPSE in the tumor ECM would be detrimental. Here, we analyzed the effects of constitutive expression of an active, membrane-bound HPSE on the ability of human natural killer (NK) cells to infiltrate tumors and eliminate tumor cells. We demonstrate that NK cells expressing a chimeric active form of HPSE on the cell surface as an integral membrane protein, display significantly enhanced infiltration capability into spheroids of various cancer cell lines, as well as into xenograft tumors in immunodeficient mice. As a result, tumor growth was significantly suppressed without causing noticeable side effects. Altogether, our results suggest that a constitutively expressed active HSPE on the surface of immune effector cells enhances their capability to access and eliminate tumor cells. This strategy opens new possibilities for improving adoptive immune treatments using NK cells.

Genetic variability in proteoglycan biosynthetic genes reveals new facets of heparan sulfate diversity.

Heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans (PG) consist of a core protein to which the glycosaminoglycan (GAG) chains, HS or CS, are attached through a common linker tetrasaccharide. In the extracellular space, they are involved in the regulation of cell communication, assuring development and homeostasis. The HSPG biosynthetic pathway has documented 51 genes, with many diseases associated to defects in some of them. The phenotypic consequences of this genetic variation in humans, and of genetic ablation in mice, and their expression patterns, led to a phenotypically centered HSPG biosynthetic pathway model. In this model, HS sequences produced by ubiquitous NDST1, HS2ST and HS6ST enzymes are essential for normal development and homeostasis, whereas tissue restricted HS sequences produced by the non-ubiquitous NDST2-4, HS6ST2-3, and HS3ST1-6 enzymes are involved in adaptative behaviors, cognition, tissue responsiveness to stimuli, and vulnerability to disease. The model indicates that the flux through the HSPG/CSPG pathways and its diverse branches is regulated by substrate preferences and protein-protein-interactions. This results in a privileged biosynthesis of HSPG over that of CSPGs, explaining the phenotypes of linkeropathies, disease caused by defects in genes involved in the biosynthesis of the common tetrasaccharide linker. Documented feedback loops whereby cells regulate HS sulfation, and hence the interactions of HS with protein partners, may be similarly implemented, e.g., protein tyrosine sulfation and other posttranslational modifications in enzymes of the HSPG pathway. Together, ubiquitous HS, specialized HS, and their biosynthesis model can facilitate research for a better understanding of HSPG roles in physiology and pathology.

Fine-tuning of Fgf8 morphogen gradient by heparan sulfate proteoglycans in the extracellular matrix.

Embryonic development is orchestrated by the action of morphogens, which spread out from a local source and activate, in a field of target cells, different cellular programs based on their concentration gradient. Fibroblast growth factor 8 (Fgf8) is a morphogen with important functions in embryonic organizing centers. It forms a gradient in the extracellular space by free diffusion, interaction with the extracellular matrix (ECM) and receptor-mediated endocytosis. However, morphogen gradient regulation by ECM is still poorly understood. Here we show that specific Heparan Sulfate Proteoglycans (HSPGs) bind Fgf8 with different affinities directly in the ECM of living zebrafish embryos, thus affecting its diffusion and signaling. Using single-molecule Fluorescence Correlation Spectroscopy, we quantify this binding in vivo, and find two different modes of interaction. First, reducing or increasing the concentration of specific HSPGs in the extracellular space alters Fgf8 diffusion, and thus, its gradient shape. Second, ternary complex formation of Fgf8 ligand with Fgf-receptors and HSPGs at the cell surface requires HSPG attachment to the cell membrane. Together, our results show that graded Fgf8 morphogen distribution is achieved by constraining free Fgf8 diffusion through successive interactions with HSPGs at the cell surface and in ECM space.

The role of heparan sulfate proteoglycans for tau pathology in vivo

AbstractBackgroundThe prion model of tau propagation in Alzheimer’s Disease predicts that tau seeds are released from cells and taken up by neighboring cells, resulting in spreading of the disease. Our previous work revealed that tau aggregates bind to heparan sulfate proteoglycans (HSPGs) on the cell surface, followed by cellular uptake via macropinocytosis. HSPGs are glycoproteins, consisting of a protein core and decorated with linear glycosaminoglycan (GAG) chains called heparan sulfate (HS) with highly variable sulfation patterns. We have previously used a combination of biochemical, pharmacological and genetic tools to demonstrate that N‐sulfation (mediated by the N‐sulfotransferase NDST1) of the HS chain is critical for tau uptake in cell models. This project is a stepwise approach to validate HSPGs and specifically NDST1 as a therapeutic target to block tau spread in vivo.MethodWe induced NDST1 knockout using CRISPR/Cas9 followed by rescue experiments and tau uptake assays in HEK293T cells. Tau uptake assays with heparin were conducted in murine hippocampal neurons and astrocytes. We employed stereotactic adeno‐associated virus 8 (AAV8)‐mediated intracerebral delivery of small hairpin RNA (shRNA) to target NDST1 in PS19 mice, a tauopathy mouse model (collaboration with Merck).ResultOur data demonstrates that reduction of the N‐deacetylase activity with selected point mutations within NDST1 can decrease tau uptake substantially (Figure 1). Our experiments in primary murine neurons and astrocytes suggest that HSPGs are crucial for tau uptake in these cell types (Figure 2). NDST1 knockdown with shRNA in PS19 mice achieved >75% reduction of messenger RNA (mRNA) levels and was non‐toxic (Figure 3).ConclusionOur findings suggest that NDST1 is a potentially druggable target, and that the HSPG pathway plays a role for tau uptake in multiple cell types. We worked with GemPharmatech for the generation of a floxed NDST1 mouse line. We plan to breed the floxed NDST1 mice to PS19 mice and selected neuronal, astrocytic and microglial Cre driver lines. We anticipate that knockout of NDST1 in vivo will reduce tau spread in mouse brain. If successful, our study will inspire multiple lines of follow up work such as the design of enzymatic inhibitors and genetic silencing tools.

Hippocampal CA2 neurons disproportionately express AAV-delivered genetic cargo.

Hippocampal area CA2 is unique in many ways, largely based on the complement of genes expressed there. We and others have observed that CA2 neurons exhibit a uniquely robust tropism for adeno-associated viruses (AAVs) of multiple serotypes and variants. In this study, we aimed to systematically investigate the propensity for AAV tropism toward CA2 across a wide range of AAV serotypes and variants, injected either intrahippocampally or systemically, including AAV1, 2, 5, 6, 8, 9, DJ, PHP.B, PHP.eB, and CAP-B10. We found that most serotypes and variants produced disproportionally high expression of AAV-delivered genetic material in hippocampal area CA2, although two serotypes (AAV6 and DJ) did not. In an effort to understand the mechanism(s) behind this observation, we considered perineuronal nets (PNNs) that ensheathe CA2 pyramidal cells and, among other functions, buffer diffusion of ions and molecules. We hypothesized that PNNs might attract AAV particles and maintain them in close proximity to CA2 neurons, thereby increasing exposure to AAV particles. However, genetic deletion of PNNs from CA2 had no effect on AAV transduction. Next, we next considered the AAV binding factors and receptors known to contribute to AAV transduction. We found that the AAV receptor (AAVR), which is critical to transduction, is abundantly expressed in CA2, and knockout of AAVR nearly abolished expression of AAV-delivered material by all serotypes tested. Additionally, we found CA2 enrichment of several cell-surface glycan receptors that AAV particles attach to before interacting with AAVR, including heparan sulfate proteoglycans, N-linked sialic acid and N-linked galactose. Indeed, CA2 showed the highest expression of AAVR and the investigated glycan receptors within the hippocampus. We conclude that CA2 neurons are endowed with multiple factors that make it highly susceptible to AAV transduction, particularly to the systemically available PHP variants, including CAP-B10. Given the curved structure of hippocampus and the relatively small size of CA2, systemic delivery of engineered PHP or CAP variants could all but eliminate the need for intrahippocampal AAV injections, particularly when injecting recombinase-dependent AAVs into animals that express recombinases in CA2.

The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling.

Wnt/β-catenin signaling directs animal development and tissue renewal in a tightly controlled, cell- and tissue-specific manner. In the mammalian central nervous system, the atypical ligand Norrin controls angiogenesis and maintenance of the blood-brain barrier and blood-retina barrier through the Wnt/β-catenin pathway. Like Wnt, Norrin activates signaling by binding and heterodimerizing the receptors Frizzled (Fzd) and Low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to membrane recruitment of the intracellular transducer Dishevelled (Dvl) and ultimately stabilizing the transcriptional coactivator β-catenin. Unlike Wnt, the cystine-knot ligand Norrin only signals through Fzd4 and additionally requires the co-receptor Tetraspanin12 (Tspan12); however, the mechanism underlying Tspan12-mediated signal enhancement is unclear. It has been proposed that Tspan12 integrates into the Norrin-Fzd4 complex to enhance Norrin-Fzd4 affinity or otherwise allosterically modulate Fzd4 signaling. Here, we measure direct, high-affinity binding between purified Norrin and Tspan12 in a lipid environment and use AlphaFold models to interrogate this interaction interface. We find that Tspan12 and Fzd4 can simultaneously bind Norrin and that a pre-formed Tspan12/Fzd4 heterodimer, as well as cells co-expressing Tspan12 and Fzd4, more efficiently capture low concentrations of Norrin than Fzd4 alone. We also show that Tspan12 competes with both heparan sulfate proteoglycans and LRP6 for Norrin binding and that Tspan12 does not impact Fzd4-Dvl affinity in the presence or absence of Norrin. Our findings suggest that Tspan12 does not allosterically enhance Fzd4 binding to Norrin or Dvl, but instead functions to directly capture Norrin upstream of signaling.

Feasibility assessment of using cRISPR-Cas9 to improve the infiltration of CAR-T cells in solid tumors

Abstract. Chimeric antigen receptor T-cell immunotherapy (CAR-T) has been developing for decades, CAR-T is playing an increasingly important role in tumor treatment. However, because fibroblasts (CAFs) in solid tumors secrete proteins and glycans to form ECM, CAR-T is faced with the challenge of improving tumor invasion. To this end, a new scheme was put forth to alter CAR T cells such that they release heparinase (HPSE) to break down heparan sulfate proteoglycan (HSPG), which is covered on the outermost layer of the cancerous cells by CAF and released. In order to solve the above problems, CRISPR-Cas9 was proposed to modify CAR T to enhance the secretion of HPSE. Although this method has the advantage of broad spectrum, it still has certain defects. The matrix characteristics of different solid tumors and the subpopulations and regulatory mechanisms of HPSE need to be further studied. The off-target effects of CRISPR-Cas9 gene editing technology and the high cost and time-consuming problems of flow cytometry also limit its application. It is anticipated to enhance cell infiltration in solid tumors, boost CAR-T therapy's effectiveness in treating solid tumors, and advance the field of CAR-T therapy.

The potential of lactoferrin as antiviral and immune-modulating agent in viral infectious diseases.

Emerging infectious diseases are caused by unpredictable viruses with the dangerous potential to trigger global pandemics. These viruses typically initiate infection by utilizing the anionic structures of host cell surface receptors to gain entry. Lactoferrin (Lf) is a multifunctional glycoprotein with multiple properties such as antiviral, anti-inflammatory and antioxidant activities. Due to its cationic structure, Lf naturally interacts with certain host cell receptors, such as heparan sulfate proteoglycans, as well as viral particles and other receptors that are targeted by viruses. Therefore, Lf may interfere with virus-host cell interactions by acting as a receptor competitor for viruses. Herein we summarize studies in which this competition was investigated with SARS-CoV-2, Zika, Dengue, Hepatitis and Influenza viruses in vitro. These studies have demonstrated not only Lf's competitive properties, but also its potential intracellular impact on host cells, such as enhancing cell survival and reducing infection efficiency by inhibiting certain viral enzymes. In addition, the immunomodulatory effect of Lf is highlighted, as it can influence the activity of specific immune cells and regulate cytokine release, thereby enhancing the host's response to viral infections. Collectively, these properties promote the potential of Lf as a promising candidate for research in viral infectious diseases.

Excess Weight Impairs Oocyte Quality, as Reflected by mtDNA and BMP-15.

This prospective, case-control study evaluated the impact of obesity on oocyte quality based on mtDNA expression in cumulus cells (CC), and on bone morphogenetic protein 15 (BMP-15) and heparan sulfate proteoglycan 2 (HSPG2) in follicular fluid (FF). It included women 18 to <40 years of age, divided according to BMI < 24.9 (Group 1, n = 28) and BMI > 25 (Group 2, n = 22). Demographics, treatment, and pregnancy outcomes were compared. The mtDNA in CC, BMP-15, HSPG2, the lipid profile, the hormonal profile, and C-reactive protein were evaluated in FF and in blood samples. The BMP-15 levels in FF and the mitochondrial DNA in CC were higher in Group 1 (38.8 ± 32.5 vs. 14.3 ± 10.8 ng/mL; p = 0.001 and 1.10 ± 0.3 vs. 0.87 ± 0.18-fold change; p = 0.016, respectively) than in Group 2. High-density lipoprotein levels in blood and FF were higher in Group 1 (62 ± 18 vs. 50 ± 12 mg/dL; p = 0.015 and 34 ± 26 vs. 20.9 ± 7.2 mg/dL; p = 0.05, respectively). Group 2 had higher blood C-reactive protein (7.1 ± 5.4 vs. 3.4 ± 4.3 mg/L; p = 0.015), FF (5.2 ± 3.8 vs. 1.5 ± 1.6 mg/L; p = 0.002) and low-density lipoprotein levels (91 ± 27 vs. 71 ± 22 mg/dL; p = 0.008) vs. Group 1. Group 1 demonstrated a trend toward a better clinical pregnancy rate (47.8% vs. 28.6%: p = 0.31) and frozen embryo transfer rate (69.2% vs. 53.8; p = 0.69). Higher BMI resulted in lower BMP-15 levels and reduced mtDNA expression, which reflect decreased oocyte quality in overweight women.