Structural basis for FGF hormone signalling.

α/ßKlotho coreceptors simultaneously engage fibroblast growth factor (FGF) hormones (FGF19, FGF21 and FGF23)1,2 and their cognate cell-surface FGF receptors (FGFR1-4) thereby stabilizing the endocrine FGF-FGFR complex3-6. However, these hormones still require heparan sulfate (HS) proteoglycan as an additional coreceptor to induce FGFR dimerization/activation and hence elicit their essential metabolic activities6. To reveal the molecular mechanism underpinning the coreceptor role of HS, we solved cryo-electron microscopy structures of three distinct 1:2:1:1 FGF23-FGFR-αKlotho-HS quaternary complexes featuring the 'c' splice isoforms of FGFR1 (FGFR1c), FGFR3 (FGFR3c) or FGFR4 as the receptor component. These structures, supported by cell-based receptor complementation and heterodimerization experiments, reveal that a single HS chain enables FGF23 and its primary FGFR within a 1:1:1 FGF23-FGFR-αKlotho ternary complex to jointly recruit a lone secondary FGFR molecule leading to asymmetric receptor dimerization and activation. However, αKlotho does not directly participate in recruiting the secondary receptor/dimerization. We also show that the asymmetric mode of receptor dimerization is applicable to paracrine FGFs that signal solely in an HS-dependent fashion. Our structural and biochemical data overturn the current symmetric FGFR dimerization paradigm and provide blueprints for rational discovery of modulators of FGF signalling2 as therapeutics for human metabolic diseases and cancer.

APRIL limits atherosclerosis by binding to heparan sulfate proteoglycans.

Atherosclerotic cardiovascular disease causes heart attacks and strokes, which are the leading causes of mortality worldwide1. The formation of atherosclerotic plaques is initiated when low-density lipoproteins bind to heparan-sulfate proteoglycans (HSPGs)2 and become trapped in the subendothelial space of large and medium size arteries, which leads to chronic inflammation and remodelling of the artery wall2. A proliferation-inducing ligand (APRIL) is a cytokine that binds to HSPGs3, but the physiology of this interaction is largely unknown. Here we show that genetic ablation or antibody-mediated depletion of APRIL aggravates atherosclerosis in mice. Mechanistically, we demonstrate that APRIL confers atheroprotection by binding to heparan sulfate chains of heparan-sulfate proteoglycan 2 (HSPG2), which limits the retention of low-density lipoproteins, accumulation of macrophages and formation of necrotic cores. Indeed, antibody-mediated depletion of APRIL in mice expressing heparan sulfate-deficient HSPG2 had no effect on the development of atherosclerosis. Treatment with a specific anti-APRIL antibody that promotes the binding of APRIL to HSPGs reduced experimental atherosclerosis. Furthermore, the serum levels of a form of human APRIL protein that binds to HSPGs, which we termed non-canonical APRIL (nc-APRIL), are associated independently of traditional risk factors with long-term cardiovascular mortality in patients with atherosclerosis. Our data reveal properties of APRIL that have broad pathophysiological implications for vascular homeostasis.

The exostosin glycosyltransferase 1/STAT3 axis is a driver of breast cancer aggressiveness.

The epithelial-mesenchymal transition (EMT) program is crucial for transforming carcinoma cells into a partially mesenchymal state, enhancing their chemoresistance, migration, and metastasis. This shift in cell state is tightly regulated by cellular mechanisms that are not yet fully characterized. One intriguing EMT aspect is the rewiring of the proteoglycan landscape, particularly the induction of heparan sulfate proteoglycan (HSPG) biosynthesis. This proteoglycan functions as a co-receptor that accelerates cancer-associated signaling pathways through its negatively-charged residues. However, the precise mechanisms through which EMT governs HSPG biosynthesis and its role in cancer cell plasticity remain elusive. Here, we identified exostosin glycosyltransferase 1 (EXT1), a central enzyme in HSPG biosynthesis, to be selectively upregulated in aggressive tumor subtypes and cancer cell lines, and to function as a key player in breast cancer aggressiveness. Notably, ectopic expression of EXT1 in epithelial cells is sufficient to induce HSPG levels and the expression of known mesenchymal markers, subsequently enhancing EMT features, including cell migration, invasion, and tumor formation. Additionally, EXT1 loss in MDA-MB-231 cells inhibits their aggressiveness-associated traits such as migration, chemoresistance, tumor formation, and metastasis. Our findings reveal that EXT1, through its role in HSPG biosynthesis, governs signal transducer and activator of transcription 3 (STAT3) signaling, a known regulator of cancer cell aggressiveness. Collectively, we present the EXT1/HSPG/STAT3 axis as a central regulator of cancer cell plasticity that directly links proteoglycan synthesis to oncogenic signaling pathways.

Heparan sulfates and heparan sulfate proteoglycans in hematopoiesis.

ABSTRACT: From signaling mediators in stem cells to markers of differentiation and lineage commitment to facilitators for the entry of viruses, such as HIV-1, cell surface heparan sulfate (HS) glycans with distinct modification patterns play important roles in hematopoietic biology. In this review, we provide an overview of the importance of HS and the proteoglycans (HSPGs) to which they are attached within the major cellular subtypes of the hematopoietic system. We summarize the roles of HSPGs, HS, and HS modifications within each main hematopoietic cell lineage of both myeloid and lymphoid arms. Lastly, we discuss the biological advances in the detection of HS modifications and their potential to further discriminate cell types within hematopoietic tissue.

Galectin-3-Binding Protein Inhibits Extracellular Heparan 6-O-Endosulfatase Sulf-2.

Human extracellular 6-O-endosulfatases Sulf-1 and Sulf-2 are the only enzymes that post-synthetically alter the 6-O sulfation of heparan sulfate proteoglycans (HSPG), which regulates interactions of HSPG with many proteins. Oncogenicity of Sulf-2 in different cancers has been documented, and we have shown that Sulf-2 is associated with poor survival outcomes in head and neck squamous cell carcinoma (HNSCC). Despite its importance, limited information is available on direct protein-protein interactions of the Sulf-2 protein in the tumor microenvironment. In this study, we used monoclonal antibody (mAb) affinity purification and mass spectrometry to identify galectin-3-binding protein (LG3BP) as a highly specific binding partner of Sulf-2 in the conditioned media of HNSCC cell lines. We validated their direct interaction in vitro using recombinant proteins and have shown that the chondroitin sulfate (CS) covalently bound to the Sulf-2 influences the binding to LG3BP. We confirmed the importance of the CS chain for the interaction by generating a mutant Sulf-2 protein that lacks the CS. Importantly, we have shown that the LG3BP inhibits Sulf-2 activity in vitro in a concentration-dependent manner. As a consequence, the addition of LG3BP to a spheroid cell culture inhibited the invasion of the HNSCC cells into Matrigel. Thus, Sulf-2 interaction with LG3BP may regulate the physiological activity of the Sulf-2 enzyme as well as its activity in the tumor microenvironment.

Heparin is essential for optimal cell signaling by FGF21 and for regulation of ßKlotho cellular stability.

While important insights were gained about how FGF21 and other endocrine fibroblast growth factors (FGFs) bind to Klotho proteins, the exact mechanism of Klotho/FGF receptor assembly that drives receptor dimerization and activation has not been elucidated. The prevailing dogma is that Klotho proteins substitute for the loss of heparan sulfate proteoglycan (HSPG) binding to endocrine FGFs by high-affinity binding of endocrine FGF molecules to Klotho receptors. To explore a potential role of HSPG in FGF21 signaling, we have analyzed the dynamic properties of FGF21-induced FGF21-ßKlotho-FGFR1c complexes on the surface of living wild-type (WT) or HSPG-deficient Chinese hamster ovary (CHO) cells by employing quantitative single-molecule fluorescence imaging analyses. Moreover, detailed analyses of FGF21 and FGF1 stimulation of cellular signaling pathways activated in WT or in HSPG-deficient CHO cells are also analyzed and compared. These experiments demonstrate that heparin is required for the formation of FGF21-ßKlotho-FGFR1c complexes on the cell membrane and that binding of heparin or HSPG to FGFR1c is essential for optimal FGF21 stimulation of FGFR1c activation, mitogen-activated protein kinase responses, and intracellular Ca2+ release. It is also shown that FGF1 binding stimulates assembly of ßKlotho and FGFR1c on cell membranes, resulting in endocytosis and degradation of ßKlotho. We conclude that heparin or HSPG is essential for FGF21 signaling and for regulation of ßKlotho cellular stability by acting as a coligand of FGFR1c.

Myosin 9 and N-glycans jointly regulate human papillomavirus entry.

Persistent high-risk HPV infection is closely associated with cervical cancer development, and there is no drug targeting HPV on the market at present, so it is particularly important to understand the interaction mechanism between HPV and the host which may provide the novel strategies for treating HPV diseases. HPV can hijack cell surface heparan sulfate proteoglycans (HSPGs) as primary receptors. However, the secondary entry receptors for HPV remain elusive. We identify myosin-9 (NMHC-IIA) as a host factor that interacts with HPV L1 protein and mediates HPV internalization. Efficient HPV entry required myosin-9 redistribution to the cell surface regulated by HPV-hijacked MEK-MLCK signaling. Myosin-9 maldistribution by ML-7 or ML-9 significantly inhibited HPV pseudoviruses infection in vitro and in vivo. Meanwhile, N-glycans, especially the galactose chains, may act as the decoy receptors for HPV, which can block the interaction of HPV to myosin-9 and influence the way of HPV infection. Taken together, we identify myosin-9 as a novel functional entry receptor for high-risk HPV both in vitro and in vivo, and unravel the new roles of myosin-9 and N-glycans in HPV entry, which provides the possibilities for host targets of antiviral drugs.

Differential heparan sulfate dependency of the Drosophila glypicans.

Heparan sulfate proteoglycans (HSPGs) are composed of a core protein and glycosaminoglycan (GAG) chains and serve as coreceptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the proteoglycan function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment serine residues are substituted to alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dallyΔGAG allele defined Dally functions that do not require heparan sulfate (HS) chains and that need both core protein and HS chains. We found a new, dallyΔGAG-specific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dallyΔGAG, dlpΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation.

Infection and transmission of SARS-CoV-2 depend on heparan sulfate proteoglycans.

The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and outbreaks of new variants highlight the need for preventive treatments. Here, we identified heparan sulfate proteoglycans as attachment receptors for SARS-CoV-2. Notably, neutralizing antibodies against SARS-CoV-2 isolated from COVID-19 patients interfered with SARS-CoV-2 binding to heparan sulfate proteoglycans, which might be an additional mechanism of antibodies to neutralize infection. SARS-CoV-2 binding to and infection of epithelial cells was blocked by low molecular weight heparins (LMWH). Although dendritic cells (DCs) and mucosal Langerhans cells (LCs) were not infected by SARS-CoV-2, both DC subsets efficiently captured SARS-CoV-2 via heparan sulfate proteoglycans and transmitted the virus to ACE2-positive cells. Notably, human primary nasal cells were infected by SARS-CoV-2, and infection was blocked by pre-treatment with LMWH. These data strongly suggest that heparan sulfate proteoglycans are important attachment receptors facilitating infection and transmission, and support the use of LMWH as prophylaxis against SARS-CoV-2 infection.

Regulation of morphogen pathways by a Drosophila chondroitin sulfate proteoglycan Windpipe.

Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. We found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. Our study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways.

Chondroitin sulfate enhances the barrier function of basement membrane assembled by heparan sulfate.

Glycosaminoglycans are ubiquitously expressed polysaccharides that are attached to proteoglycans. Here, we showed that ablation of the heparan sulfate (HS) polymerase Ext1 in retinal progenitor cells did not affect initial progression of retinal angiogenesis, but it disrupted the pruning of blood vessels and establishment of arterioles and venules. In the absence of retinal HS, blood vessels were also vulnerable to high oxygen tension in early postnatal stages, which could be rescued by exogenous vascular endothelial growth factor (VEGF), consistent with the role of retinal HS in the fine-tuning of VEGF signaling. Furthermore, we observed that the retinal inner limiting membrane (ILM) was disrupted by deletion of Ext1 in a timing-specific manner, suggesting that retinal HS is required for the assembly but not the maintenance of the basement membrane. Lastly, we showed that further deletion of C4st1, a chondroitin sulfate (CS) sulfation enzyme, did not affect the assembly of the ILM but, when combined with Ext1 deletion, it aggravated the retinal permeability by disrupting the retinal glycocalyx. These results demonstrate an important role of CS and HS in establishing the barrier function of the extracellular matrix.

Novel insights into the host cell glycan binding profile of human metapneumovirus.

Numerous viruses have been found to exploit glycoconjugates expressed on human cells as their initial attachment factor for viral entry and infection. The virus-cell glycointeractome, when characterized, may serve as a template for antiviral drug design. Heparan sulfate proteoglycans extensively decorate the human cell surface and were previously described as a primary receptor for human metapneumovirus (HMPV). After respiratory syncytial virus, HMPV is the second most prevalent respiratory pathogen causing respiratory tract infection in young children. To date, there is neither vaccine nor drug available to prevent or treat HMPV infection. Using a multidisciplinary approach, we report for the first time the glycointeractome of the HMPV fusion (F) protein, a viral surface glycoprotein that is essential for target-cell recognition, attachment, and entry. Our glycan microarray and surface plasmon resonance results suggest that Galß1-3/4GlcNAc moieties that may be sialylated or fucosylated are readily recognized by HMPV F. The bound motifs are highly similar to the N-linked and O-linked glycans primarily expressed on the human lung epithelium. We demonstrate that the identified glycans have the potential to compete with the cellular receptors used for HMPV entry and consequently block HMPV infection. We found that lacto-N-neotetraose demonstrated the strongest HMPV binding inhibition in a cell infection assay. Our current findings offer an encouraging and novel avenue for the design of anti-HMPV drug candidates using oligosaccharide templates.IMPORTANCEAll cells are decorated with a dense coat of sugars that makes a sugar code. Many respiratory viruses exploit this sugar code by binding to these sugars to cause infection. Human metapneumovirus is a leading cause for acute respiratory tract infections. Despite its medical importance, there is no vaccine or antiviral drug available to prevent or treat human metapneumovirus infection. This study investigates how human metapneumovirus binds to sugars in order to more efficiently infect the human host. We found that human metapneumovirus binds to a diverse range of sugars and demonstrated that these sugars can ultimately block viral infection. Understanding how viruses can take advantage of the sugar code on our cells could identify new intervention and treatment strategies to combat viral disease.

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.

Heparan sulfate proteoglycans: Mediators of cellular and molecular Alzheimer's disease pathogenic factors via tunnelling nanotubes?

Neurological disorders impact around one billion individuals globally (15 % approx.), with significant implications for disability and mortality with their impact in Australia currently amounts to 6.8 million deaths annually. Heparan sulfate proteoglycans (HSPGs) are complex extracellular molecules implicated in promoting Tau fibril formation resulting in Tau tangles, a hallmark of Alzheimer's disease (AD). HSPG-Tau protein interactions contribute to various AD stages via aggregation, toxicity, and clearance, largely via interactions with the glypican 1 and syndecan 3 core proteins. The tunnelling nanotubes (TNTs) pathway is emerging as a facilitator of intercellular molecule transport, including Tau and Amyloid ß proteins, across extensive distances. While current TNT-associated evidence primarily stems from cancer models, their role in Tau propagation and its effects on recipient cells remain unclear. This review explores the interplay of TNTs, HSPGs, and AD-related factors and proposes that HSPGs influence TNT formation in neurodegenerative conditions such as AD.

Circadian control of heparan sulfate levels times phagocytosis of amyloid beta aggregates.

Alzheimer's Disease (AD) is a neuroinflammatory disease characterized partly by the inability to clear, and subsequent build-up, of amyloid-beta (Aß). AD has a bi-directional relationship with circadian disruption (CD) with sleep disturbances starting years before disease onset. However, the molecular mechanism underlying the relationship of CD and AD has not been elucidated. Myeloid-based phagocytosis, a key component in the metabolism of Aß, is circadianly-regulated, presenting a potential link between CD and AD. In this work, we revealed that the phagocytosis of Aß42 undergoes a daily circadian oscillation. We found the circadian timing of global heparan sulfate proteoglycan (HSPG) biosynthesis was the molecular timer for the clock-controlled phagocytosis of Aß and that both HSPG binding and aggregation may play a role in this oscillation. These data highlight that circadian regulation in immune cells may play a role in the intricate relationship between the circadian clock and AD.

Mechanism-based heparanase inhibitors reduce cancer metastasis in vivo.

Heparan sulfate proteoglycans (HSPGs) mediate essential interactions throughout the extracellular matrix (ECM), providing signals that regulate cellular growth and development. Altered HSPG composition during tumorigenesis strongly aids cancer progression. Heparanase (HPSE) is the principal enzyme responsible for extracellular heparan sulfate catabolism and is markedly up-regulated in aggressive cancers. HPSE overactivity degrades HSPGs within the ECM, facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Reducing extracellular HPSE activity reduces cancer growth, but few effective inhibitors are known, and none are clinically approved. Inspired by the natural glycosidase inhibitor cyclophellitol, we developed nanomolar mechanism-based, irreversible HPSE inhibitors that are effective within physiological environments. Application of cyclophellitol-derived HPSE inhibitors reduces cancer aggression in cellulo and significantly ameliorates murine metastasis. Mechanism-based irreversible HPSE inhibition is an unexplored anticancer strategy. We demonstrate the feasibility of such compounds to control pathological HPSE-driven malignancies.

Hippo cell signaling and HS-proteoglycans regulate tissue form and function, age-dependent maturation, extracellular matrix remodeling, and repair.

This review examined how Hippo cell signaling and heparan sulfate (HS)-proteoglycans (HSPGs) regulate tissue form and function. Despite being a nonweight-bearing tissue, the brain is regulated by Hippo mechanoresponsive cell signaling pathways during embryonic development. HS-proteoglycans interact with growth factors, morphogens, and extracellular matrix components to regulate development and pathology. Pikachurin and Eyes shut (Eys) interact with dystroglycan to stabilize the photoreceptor axoneme primary cilium and ribbon synapse facilitating phototransduction and neurotransduction with bipolar retinal neuronal networks in ocular vision, the primary human sense. Another HSPG, Neurexin interacts with structural and adaptor proteins to stabilize synapses and ensure specificity of neural interactions, and aids in synaptic potentiation and plasticity in neurotransduction. HSPGs also stabilize the blood-brain barrier and motor neuron basal structures in the neuromuscular junction. Agrin and perlecan localize acetylcholinesterase and its receptors in the neuromuscular junction essential for neuromuscular control. The primary cilium is a mechanosensory hub on neurons, utilized by YES associated protein (YAP)-transcriptional coactivator with PDZ-binding motif (TAZ) Hippo, Hh, Wnt, transforming growth factor (TGF)-ß/bone matrix protein (BMP) receptor tyrosine kinase cell signaling. Members of the glypican HSPG proteoglycan family interact with Smoothened and Patched G-protein coupled receptors on the cilium to regulate Hh and Wnt signaling during neuronal development. Control of glycosyl sulfotransferases and endogenous protease expression by Hippo TAZ YAP represents a mechanism whereby the fine structure of HS-proteoglycans can be potentially modulated spatiotemporally to regulate tissue morphogenesis in a similar manner to how Hippo signaling controls sialyltransferase expression and mediation of cell-cell recognition, dysfunctional sialic acid expression is a feature of many tumors.

Dystroglycan-HSPG interactions provide synaptic plasticity and specificity.

AIM: This study examined the roles of the laminin and proteoglycan receptor dystroglycan (DG) in extracellular matrix stabilization and cellular mechanosensory processes conveyed through communication between the extracellular matrix (ECM) and cytoskeleton facilitated by DG. Specific functional attributes of HS-proteoglycans (HSPGs) are conveyed through interactions with DG and provide synaptic specificity through diverse interactions with an extensive range of cell attachment and adaptor proteins which convey synaptic plasticity. HSPG-DG interactions are important in phototransduction and neurotransduction and facilitate retinal bipolar-photoreceptor neuronal signaling in vision. Besides synaptic stabilization, HSPG-DG interactions also stabilize basement membranes and the ECM and have specific roles in the assembly and function of the neuromuscular junction. This provides neuromuscular control of muscle systems that control conscious body movement as well as essential autonomic control of diaphragm, intercostal and abdominal muscles and muscle systems in the face, mouth and pharynx which assist in breathing processes. DG is thus a multifunctional cell regulatory glycoprotein receptor and regulates a diverse range of biological and physiological processes throughout the human body. The unique glycosylation of the αDG domain is responsible for its diverse interactions with ECM components in cell-ECM signaling. Cytoskeletal cell regulatory switches assembled by the ßDG domain in its role as a nuclear scaffolding protein respond to such ECM cues to regulate cellular behavior and tissue homeostasis thus DG has fascinating and diverse roles in health and disease.

Hedgehog pathway modulation by glypican 3-conjugated heparan sulfate.

Glypicans are a family of cell surface heparan sulfate proteoglycans that play critical roles in multiple cell signaling pathways. Glypicans consist of a globular core, an unstructured stalk modified with sulfated glycosaminoglycan chains, and a glycosylphosphatidylinositol anchor. Though these structural features are conserved, their individual contribution to glypican function remains obscure. Here, we investigate how glypican 3 (GPC3), which is mutated in Simpson-Golabi-Behmel tissue overgrowth syndrome, regulates Hedgehog signaling. We find that GPC3 is necessary for the Hedgehog response, surprisingly controlling a downstream signal transduction step. Purified GPC3 ectodomain rescues signaling when artificially recruited to the surface of GPC3-deficient cells but has dominant-negative activity when unattached. Strikingly, the purified stalk, modified with heparan sulfate but not chondroitin sulfate, is necessary and sufficient for activity. Our results demonstrate a novel function for GPC3-associated heparan sulfate and provide a framework for the functional dissection of glycosaminoglycans by in vivo biochemical complementation. This article has an associated First Person interview with the first author of the paper.

Glypican 4 regulates planar cell polarity of endoderm cells by controlling the localization of Cadherin 2.

Noncanonical Wnt/planar cell polarity (Wnt/PCP) signaling has been implicated in endoderm morphogenesis. However, the underlying cellular and molecular mechanisms of this process are unclear. We found that, during convergence and extension (C&E) in zebrafish, gut endodermal cells are polarized mediolaterally, with GFP-Vangl2 enriched at the anterior edges. Endoderm cell polarity is lost and intercalation is impaired in the absence of glypican 4 (gpc4), a heparan-sulfate proteoglycan that promotes Wnt/PCP signaling, suggesting that this signaling is required for endodermal cell polarity. Live imaging revealed that endoderm C&E is accomplished by polarized cell protrusions and junction remodeling, which are impaired in gpc4-deficient endodermal cells. Furthermore, in the absence of gpc4, Cadherin 2 expression on the endodermal cell surface is increased as a result of impaired Rab5c-mediated endocytosis, which partially accounts for the endodermal defects in these mutants. These findings indicate that Gpc4 regulates endodermal planar cell polarity during endoderm C&E by influencing the localization of Cadherin 2. Thus, our study uncovers a new mechanism by which Gpc4 regulates planar cell polarity and reveals the role of Wnt/PCP signaling in endoderm morphogenesis.