Heparan Sulfate Attachment Research Articles

A precise design strategy for a cell-derived extracellular matrix based on CRISPR/Cas9 for regulating neural stem cell function.

The extracellular matrix (ECM) is a natural microenvironment pivotal for stem cell survival, as well as proliferation, differentiation and metastasis, composed of a variety of biological molecular complexes secreted by resident cells in tissues and organs. Heparan sulfate proteoglycan (HSPG) is a type of ECM protein that contains one or more covalently attached heparan sulfate chains. Heparan sulphate chains have high affinity with growth factors, chemokines and morphogens, acting as cytokine-binding domains of great importance in development and normal physiology. Herein, we constructed endogenous HSPG2 overexpression in mouse embryonic fibroblasts based on the CRISPR/Cas9 synergistic activation mediator system and then fabricated a cell-derived HSPG2 functional ECM (ECMHSPG2). The ECMHSPG2 is capable of enriching basic fibroblast growth factor (bFGF), which binds more strongly than the negative control ECM. With a growing bFGF concentration, ECMHSPG2 could better maintain neural stem cell (NSCs) stemness and promote NSC proliferation and differentiation in culture. These findings provide a precise design strategy for producing a specific cell-derived ECM for biomaterials in research and regenerative medicine.

Heparan sulfate proteoglycans in cancer: Pathogenesis and therapeutic potential.

The heparan sulfate proteoglycans (HSPGs) are glycoproteins that consist of a proteoglycan "core" protein and covalently attached heparan sulfate (HS) chain. HSPGs are ubiquitously expressed in mammalian cells on the cell surface and in the extracellular matrix (ECM) and secretory vesicles. Within HSPGs, the protein cores determine when and where HSPG expression takes place, and the HS chains mediate most of HSPG's biological roles through binding various protein ligands, including cytokines, chemokines, growth factors and receptors, morphogens, proteases, protease inhibitors, and ECM proteins. Through these interactions, HSPGs modulate cell proliferation, adhesion, migration, invasion, and angiogenesis to display essential functions in physiology and pathology. Under physiological conditions, the expression and localization of HSPGs are finely regulated to orchestrate their physiological functions, and this is disrupted in cancer. The HSPG dysregulation elicits multiple oncogenic signaling, including growth factor signaling, ECM and Integrin signaling, chemokine and immune signaling, cancer stem cell, cell differentiation, apoptosis, and senescence, to prompt cell transformation, proliferation, tumor invasion and metastasis, tumor angiogenesis and inflammation, and immunotolerance. These oncogenic roles make HSPGs an attractive pharmacological target for anti-cancer therapy. Several therapeutic strategies have been under development, including anti-HSPG antibodies, peptides and HS mimetics, synthetic xylosides, and heparinase inhibitors, and shown promising anti-cancer efficacy. Therefore, much progress has been made in this line of study. However, it needs to bear in mind that the roles of HSPGs in cancer can be either oncogenic or tumor-suppressive, depending on the HSPG and the cancer cell type with the underlying mechanisms that remain obscure. Further studies need to address these to fill the knowledge gap and rationalize more efficient therapeutic targeting.

Transmembrane Protein 184A and Syndecan‐4 function in the same pathway to promote angiogenesis

Morpholino (MO) knockdown (KD) of the novel vascular heparin receptor, Transmembrane protein 184A (TMEM184A), increases endothelial cell (EC) proliferation rates, leading to disorganized and stunted vessel outgrowth in the regenerating zebrafish caudal fin. A similar vascular phenotype is observed in the developing zebrafish embryo, where TMEM184A KD reduces the number and length of intersegmental vessels (ISVs), as well as total Vascular Endothelial Cadherin (VEC) protein in ISV ECs. The Heparan Sulfate Proteoglycan Syndecan‐4 (Sdcn‐4) is required for VEC internalization in angiogenesis and interacts with membrane proteins to promote cellular signaling and molecular processes through interactions with its attached heparan sulfate (HS) chains. Since HS is nearly identical to heparin in structure and both TMEM184A and Sdcn‐4 appear to be required for stable angiogenesis, we asked whether TMEM184A and Sdcn‐4 interact and function in the same pathway to promote vascular sprouting and regeneration. Using MO knockdown of TMEM184A and Sdcn‐4 in the regenerating zebrafish caudal fin, we demonstrate that co‐injected subthreshold concentrations of both MOs synergize to produce the same vascular defect. In cells, we find that immunostaining of TMEM184A, Sdcn‐4, and VEC show trimeric colocalization at the cell surface, that in vesicle puncta they colocalize with the recycling marker Rab5a, and that TMEM184A precipitates Sdcn‐4 in whole cell lysate. In TMEM184A overexpression (OE) backgrounds and in ECs treated with heparin, immunostaining of VEC show increased expression levels in adherens junctions. Collectively, these results suggest that TMEM184A and Sdcn‐4 function in the same pathway to promote stable angiogenic repair, that TMEM184A interacts with both Syndecan‐4 and VEC in ECs, that they form a complex that is potentially recycled at the membrane surface, and that TMEM184A overexpression or activation stabilizes VEC in adherens junctions. Further study is required to determine whether TMEM184A and Syndecan‐4 function together to regulate VEC trafficking and promote angiogenesis or whether TMEM184A expression impacts on VEC membrane stability are distinct from the TMEM184A–Sdcn‐4 molecular mechanism that regulates angiogenic repair.

The SARS-CoV-2 Entry Inhibition Mechanisms of Serine Protease Inhibitors, OM-85, Heparin and Soluble HS Might Be Linked to HS Attachment Sites.

This article discusses the importance of D-xylose for fighting viruses (especially SARS-CoV-2) that use core proteins as receptors at the cell surface, by providing additional supporting facts that these viruses probably bind at HS/CS attachment sites (i.e., the hydroxyl groups of Ser/Thr residues of the core proteins intended to receive the D-xylose molecules to initiate the HS/CS chains). Essentially, the additional supporting facts, are: some anterior studies on the binding sites of exogenous heparin and soluble HS on the core proteins, the inhibition of the viral entry by pre-incubation of cells with heparin, and additionally, corroborating studies about the mechanism leading to type 2 diabetes during viral infection. We then discuss the mechanism by which serine protease inhibitors inhibit SARS-CoV-2 entry. The biosynthesis of heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and heparin (Hep) is initiated not only by D-xylose derived from uridine diphosphate (UDP)-xylose, but also bioactive D-xylose molecules, even in situations where cells were previously treated with GAG inhibitors. This property of D-xylose shown by previous anterior studies helped in the explanation of the mechanism leading to type 2 diabetes during SARS-CoV-2 infection. This explanation is completed here by a preliminary estimation of xyloside GAGs (HS/CS/DS/Hep) in the body, and with other previous studies helping to corroborate the mechanism by which the D-xylose exhibits its antiglycaemic properties and the mechanism leading to type 2 diabetes during SARS-CoV-2 infection. This paper also discusses the confirmatory studies of regarding the correlation between D-xylose and COVID-19 severity.

Analyzing the Role of Heparan Sulfate Proteoglycans in Axon Guidance In Vivo in Zebrafish.

One of the most fascinating questions in the field of neurobiology is to understand how neuronal connections are properly wired to form functional circuits. During development, neurons extend axons that are guided along defined paths by attractive and repulsive cues to reach their brain target. Most of these guidance factors are regulated by heparan sulfate proteoglycans (HSPGs), a family of cell surface and extracellular core proteins with attached heparan sulfate (HS) glycosaminoglycans. The unique diversity and structural complexity of HS sugar chains, as well as the variety of core proteins, have been proposed to generate a complex "sugar code" essential for brain wiring. While the functions of HSPGs have been well characterized in C. elegans or Drosophila, less is known about their roles in nervous system development in vertebrates. In this chapter, we describe the advantages and the different methods available to study the roles of HSPGs in axon guidance directly in vivo in zebrafish. We provide protocols for visualizing axons in vivo, including precise dye labeling and time-lapse imaging, and for disturbing the functions of HS-modifying enzymes and core proteins.

A Systems View of the Heparan Sulfate Interactome.

Heparan sulfate proteoglycans consist of a small family of proteins decorated with one or more covalently attached heparan sulfate glycosaminoglycan chains. These chains have intricate structural patterns based on the position of sulfate groups and uronic acid epimers, which dictate their ability to engage a large repertoire of heparan sulfate-binding proteins, including extracellular matrix proteins, growth factors and morphogens, cytokines and chemokines, apolipoproteins and lipases, adhesion and growth factor receptors, and components of the complement and coagulation system. This review highlights recent progress in the characterization of the so-called "heparan sulfate interactome," with a major focus on systems-wide strategies as a tool for discovery and characterization of this subproteome. In addition, we compiled all heparan sulfate-binding proteins reported in the literature to date and grouped them into a few major functional classes by applying a networking approach.

Precise Design Strategy Of  Smart Extracellular Matrix Based on CRISPR/Cas9 for Regulating Neural Stem Cell Function

The extracellular matrix (ECM) is the natural microenvironment of stem cells survival, as well as proliferation, differentiation and metastasis, containing a variety of biological molecular complexes secreted by the resident cells in tissues and organs. Heparan sulfate proteoglycans (HSPG) is a major component of ECM, containing one or more covalently attached heparan sulfate chains. The heparan sulphate chains have high affinity with growth factors, chemokines and morphogens, acting as cytokine-binding domains with great importance in development and normal physiology. Herein, lentiviral single-guide RNA vectors were constructed to activate the endogenous HSPG2 expression in mouse embryonic fibroblasts based on CRISPR/Cas9 synergistic activation mediator system, and then HSPG2 functional cell-derived ECM (ECM HSPG2 ) was fabricated. The ECM HSPG2 is capable to enrich cytokines effectively, such as basic fibroblast growth factor (bFGF), which was about 3-flod stronger than binding with negative control ECM. With encapsulating abundant bFGF, ECM HSPG2 could maintain the neural stem cells (NSCs) stemness and promote the NSCs proliferation and differentiation in culture. These findings provide a novel and precise design strategy of smart and special functional cell-derived ECM for the biomaterials research and regenerative medicine.

Heparanase-enhanced Shedding of Syndecan-1 and Its Role in Driving Disease Pathogenesis and Progression.

Both heparanase and syndecan-1 are known to be present and active in disease pathobiology. An important feature of syndecan-1 related to its role in pathologies is that it can be shed from the surface of cells as an intact ectodomain composed of the extracellular core protein and attached heparan sulfate and chondroitin sulfate chains. Shed syndecan-1 remains functional and impacts cell behavior both locally and distally from its cell of origin. Shedding of syndecan-1 is initiated by a variety of stimuli and accomplished predominantly by the action of matrix metalloproteinases. The accessibility of these proteases to the core protein of syndecan-1 is enhanced, and shedding facilitated, when the heparan sulfate chains of syndecan-1 have been shortened by the enzymatic activity of heparanase. Interestingly, heparanase also enhances shedding by upregulating the expression of matrix metalloproteinases. Recent studies have revealed that heparanase-induced syndecan-1 shedding contributes to the pathogenesis and progression of cancer and viral infection, as well as other septic and non-septic inflammatory states. This review discusses the heparanase/shed syndecan-1 axis in disease pathogenesis and progression, the potential of targeting this axis therapeutically, and the possibility that this axis is widespread and of influence in many diseases.

Heparan sulfate attachment receptor is a major selection factor for attenuated enterovirus 71 mutants during cell culture adaptation.

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease (HFMD). However, this infection is sometimes associated with severe neurological complications. Identification of neurovirulence determinants is important to understand the pathogenesis of EV71. One of the problems in evaluating EV71 virulence is that its genome sequence changes rapidly during replication in cultured cells. The factors that induce rapid mutations in the EV71 genome in cultured cells are unclear. Here, we illustrate the population dynamics during adaptation to RD-A cells using EV71 strains isolated from HFMD patients. We identified a reproducible amino acid substitution from glutamic acid (E) to glycine (G) or glutamine (Q) in residue 145 of the VP1 protein (VP1-145) after adaptation to RD-A cells, which was associated with attenuation in human scavenger receptor B2 transgenic (hSCARB2 tg) mice. Because previous reports demonstrated that VP1-145G and Q mutants efficiently infect cultured cells by binding to heparan sulfate (HS), we hypothesized that HS expressed on the cell surface is a major factor for this selection. Supporting this hypothesis, selection of the VP1-145 mutant was prevented by depletion of HS and overexpression of hSCARB2 in RD-A cells. In addition, this mutation promotes the acquisition of secondary amino acid substitutions at various positions of the EV71 capsid to increase its fitness in cultured cells. These results indicate that attachment receptors, especially HS, are important factors for selection of VP1-145 mutants and subsequent capsid mutations. Moreover, we offer an efficient method for isolation and propagation of EV71 virulent strains with minimal selection pressure for attenuation.

Molecular insights and immune responses of big belly seahorse syndecan-2 (CD362): Involvement of ectodomain in regulating cell survival, proliferation, and wound healing

Syndecan-2, also known as CD362, is a transmembrane heparan sulfate proteoglycan which regulates cell growth, proliferation, cell adhesion, wound healing, and recruits immune cells. In the present study, we performed bioinformatics, spatial and temporal expression analyses of Hippocampus abdominalis syndecan-2 (HaSDC-2). Additionally, functional assays were conducted. HaSDC-2 has five major domains; an extracellular heparan sulfate attachment domain, a co-receptor binding domain, a transmembrane domain, two conserved domains (C1 domain, C2 domain), and a variable (V) domain. The ectodomain contained a signal peptide and GAG attachment sites. In-silico analysis revealed that HaSDC-2 contained a 798 bp long ORF and protein sequence of 265 amino acid residues. Further analysis of the amino acid sequence predicted a 28.9 kDa molecular weight and a 4.13 theoretical isoelectric point. The spatial expression of HaSDC-2 was ubiquitous in all tested tissues. HaSDC-2 expression in the liver was upregulated 24 h post-injection in response to all stimuli. Further, HaSDC-2 expression in blood cells was upregulated at 12 and 72 h post-injection in response to all the stimuli. HaSDC-2 + pcDNA™3.1(+) transfected cells exhibited significant survival in response to cell stressors such as H2O2 and HED. The ectodomain of recombinant HaSDC-2 treated cells showed significant cell proliferation in a concentration-dependent manner. The scratch wound healing assay showed significant Δ gap closures with increasing concentrations of HaSDC-2. Collectively, these results indicated that syndecan-2 was involved in regulating immune responses and cell stress conditions.

Point-of-Care Electrochemical Detection of Sulf Marker Activity for Lung Cancer Diagnosis in Human Body Fluids

Sulf, as the 6-O endosulfatase of heparan sulfate proteoglycans (HSPGs), has been reported in many previous works that it plays the significent role in the pathogenesis of a variety of human cancers. Here we proposed that instead of concentration, the actived Sulf will be more critical in this process. However, there are very few reports about point-of-care method to evaluate sulf 1 activity currently. In this work, an electrochemical method using HSPGs as a substrate was employed to detect the concentration of sulf 1. HSPGs, a glycoprotein with the common characteristic of containing one or more covalently attached heparan sulfate (HS) chains, is a type of glycosaminogly can mediate the activation of a variety of cell growth factor signal pathway to promote the progression of cancers. The disaccharide units of HS chains have four different sulfation modification sites, which are substituted on the N-, 3-O, and 6-O positions of glucosamine and the 2-O position of uronic acid residues. Considering sulf 1 can transform the 6-O sulfation of HSPGs to OH group, as shown in figure 1, the structure and electrical property of HSPGs could take some changes. Here, electrochemical impedance spectroscopy (EIS) was used to detect the changes of electrical property of HSPGs before and after reacting with sulf standard solution. Through analyzing and calculating the electrical parameters obtained by EIS, such as charge, capacity and degree of phase, we established the calibration curve of sulf concentration, which could be further used to detect the sulf concentration of human blood samples. This work offers a new method for sulf detection and has great potential in the early detection of lung cancers.

Electrochemical Quantification of Sulfatase Activity for Point-of-Care Diagnosis

Sulf 1&2, as the 6-O endosulfatase of heparan sulfate proteoglycans (HSPGs), has been reported in many previous works that it plays the critical role in the pathogenesis of a variety of human cancers, especially lung and breast cancer. However, there are very few reports about point-of-care detection of Sulf 1&2 currently. In this work, an electrochemical method using HSPGs as an anchor was employed to quantify the activity of Sulf 1&2. HSPGs, a glycoprotein with the common characteristic of containing one or more covalently attached heparan sulfate (HS) chains and is also a type of glycosaminoglycan, can mediate the activation of a variety of cell growth factor signal pathways to promote the progression of cancers. Here, electrochemical impedance spectroscopy (EIS) was used to monitor the changes of electrical properties of HSPGs with Sulf 1&2 variation. Through analyzing and calculating the electrical parameters obtained by EIS, such as charge, capacity and degree of phase, we established the calibration curve of sulf 1&2 concentration, which could be further used to evaluate the sulf 1&2 activity within human blood samples. This work offers a new method for sulf 1&2 detection and has great potential in the early detection of cancers.

A VP1 mutation acquired during an enterovirus 71 disseminated infection confers heparan sulfate binding ability and modulates ex vivo tropism.

Enterovirus 71 (EV71) causes hand, foot and mouth disease, a mild and self-limited illness that is sometimes associated with severe neurological complications. EV71 neurotropic determinants remain ill-defined to date. We previously identified a mutation in the VP1 capsid protein (L97R) that was acquired over the course of a disseminated infection in an immunocompromised host. The mutation was absent in the respiratory tract but was present in the gut (as a mixed population) and in blood and cerebrospinal fluid (as a dominant species). In this study, we demonstrated that this mutation does not alter the dependence of EV71 on the human scavenger receptor class B2 (SCARB2), while it enables the virus to bind to the heparan sulfate (HS) attachment receptor and modifies viral tropism in cell lines and in respiratory, intestinal and neural tissues. Variants with VP197L or VP197R were able to replicate to high levels in intestinal and neural tissues and, to a lesser extent, in respiratory tissues, but their preferred entry site (from the luminal or basal tissue side) differed in respiratory and intestinal tissues and correlated with HS expression levels. These data account for the viral populations sequenced from the patient’s respiratory and intestinal samples and suggest that improved dissemination, resulting from an acquired ability to bind HS, rather than specific neurotropism determinants, enabled the virus to reach and infect the central nervous system. Finally, we showed that iota-carrageenan, a highly sulfated polysaccharide, efficiently blocks the replication of HS-dependent variants in cells and 2D neural cultures. Overall, the results of this study emphasize the importance of HS binding in EV71 pathogenesis and open new avenues for the development of antiviral molecules that may prevent this virus’s dissemination.

Regulation of eosinophil recruitment and allergic airway inflammation by heparan sulfate proteoglycan (HSPG) modifying enzymes

ABSTRACTBackground: HSPGs are glycoproteins containing covalently attached heparan sulfate (HS) chains which bind to growth factors, chemokines, etc., and regulate various aspects of inflammation including cell recruitment. We previously showed that deletion of endothelial N-acetylglucosamine N-deacetylase-N-sulfotransferase-1 (Ndst1), an enzyme responsible for N-sulfation during HS biosynthesis, reduces allergic airway inflammation (AAI). Here, we investigated the importance of O-sulfation mediated by uronyl 2-O-sulfotransferase (Hs2st) in development of AAI relative to N-sulfation. Methods: Mice deficient in endothelial and leukocyte Hs2st (Hs2stf/fTie2Cre+) or Ndst1 (Ndst1f/fTie2Cre+) and WT mice were challenged with Alternaria alternata and evaluated for airway inflammation. Trafficking of murine eosinophils on lung endothelial cells was examined in vitro under conditions of flow. Results: Exposure to Alternaria decreased expression level of Hs2st in WT mice while level of Ndst1 remained unchanged. Compared to WT mice, Alternaria-challenged Hs2stf/fTie2Cre+ mice exhibited significantly increased eosinophils in the bone marrow, bronchoalveolar lavage fluid [BALF] and lung tissue associated with persistent airway hyperresponsiveness, airway mucus hypersecretion and elevated Th2 cytokines. In contrast, Alternaria-challenged Ndst1f/fTie2Cre+ mice exhibited a marked reduction in airway eosinophilia, mucus secretion and smooth muscle mass compared to WT counterparts. While BALF eotaxins were lower in Alternaria-challenged Hs2stf/fTie2Cre+ relative to WT mice, they were not reduced to background levels as in allergen-challenged Ndst1f/fTie2Cre+ mice. Trafficking of murine eosinophils under conditions of flow in vitro was similar on Hs2st-deficient and WT endothelial cells. Expression of ZO-1 in Hs2st-deficient lung blood vessels in control and allergen-challenged mice was significantly lower than in WT counterparts. Conclusions: Our study demonstrates that allergen exposure reduces expression of Hs2st; loss of uronyl 2-O-sulfation in endothelial and leukocyte HSPG amplifies recruitment of eosinophils likely due to a compromised vascular endothelium resulting in persistent inflammation whereas loss of N-sulfation limits eosinophilia and attenuates inflammation underscoring the importance of site-specific sulfation in HSPG to their role in AAI.

Functional Requirements for Heparan Sulfate Biosynthesis in Morphogenesis and Nervous System Development in C. elegans.

The regulation of cell migration is essential to animal development and physiology. Heparan sulfate proteoglycans shape the interactions of morphogens and guidance cues with their respective receptors to elicit appropriate cellular responses. Heparan sulfate proteoglycans consist of a protein core with attached heparan sulfate glycosaminoglycan chains, which are synthesized by glycosyltransferases of the exostosin (EXT) family. Abnormal HS chain synthesis results in pleiotropic consequences, including abnormal development and tumor formation. In humans, mutations in either of the exostosin genes EXT1 and EXT2 lead to osteosarcomas or multiple exostoses. Complete loss of any of the exostosin glycosyltransferases in mouse, fish, flies and worms leads to drastic morphogenetic defects and embryonic lethality. Here we identify and study previously unavailable viable hypomorphic mutations in the two C. elegans exostosin glycosyltransferases genes, rib-1 and rib-2. These partial loss-of-function mutations lead to a severe reduction of HS levels and result in profound but specific developmental defects, including abnormal cell and axonal migrations. We find that the expression pattern of the HS copolymerase is dynamic during embryonic and larval morphogenesis, and is sustained throughout life in specific cell types, consistent with HSPGs playing both developmental and post-developmental roles. Cell-type specific expression of the HS copolymerase shows that HS elongation is required in both the migrating neuron and neighboring cells to coordinate migration guidance. Our findings provide insights into general principles underlying HSPG function in development.

Sphingosine 1-phosphate induced synthesis of glycocalyx on endothelial cells

Sphingosine 1-phosphate (S1P) protects glycocalyx against shedding, playing important roles in endothelial functions. We previously found that glycocalyx on endothelial cells (ECs) was shed after plasma protein depletion. In the present study, we investigated the role of S1P on the recovery of glycocalyx, and tested whether it is mediated by phosphoinositide 3-kinase (PI3K) pathway. After depletion of plasma protein, ECs were treated with S1P for another 6h. And then, the major components of glycocalyx including syndecan-1 with attached heparan sulfate (HS) and chondroitin sulfate (CS) on endothelial cells were detected using confocal fluorescence microscopy. Role of PI3K in the S1P-induced synthesis of glycocalyx was confirmed by using the PI3K inhibitor (LY294002). Syndecan-1 with attached HS and CS were degraded with duration of plasma protein depletion. S1P induced recovery of syndecan-1 with attached HS and CS. The PI3K inhibitor LY294002 abolished the effect of S1P on recovery of glycocalyx. Thus, S1P induced synthesis of glycocalyx on endothelial cells and it is mediated by PI3K pathway.

Structural and Functional Study of d-Glucuronyl C5-epimerase

Heparan sulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. D-glucuronyl C5-epimerase (Glce) is a crucial enzyme in HS synthesis, converting D-glucuronic acid to L-iduronic acid to increase HS flexibility. This modification of HS is important for protein ligand recognition. We have determined the crystal structures of Glce in apo-form (unliganded) and in complex with heparin hexasaccharide (product of Glce following O-sulfation), both in a stable dimer conformation. A Glce dimer contains two catalytic sites, each at a positively charged cleft in C-terminal α-helical domains binding one negatively charged hexasaccharide. Based on the structural and mutagenesis studies, three tyrosine residues, Tyr(468), Tyr(528), and Tyr(546), in the active site were found to be crucial for the enzymatic activity. The complex structure also reveals the mechanism of product inhibition (i.e. 2-O- and 6-O-sulfation of HS keeps the C5 carbon of L-iduronic acid away from the active-site tyrosine residues). Our structural and functional data advance understanding of the key modification in HS biosynthesis.

Alterations of Heparan Sulfate Proteoglycans in Cancer

Heparan sulfate proteoglycans (HSPGs) are glycoproteins, with the common characteristic of containing covalently attached heparan sulfate glycosaminoglycan chains (GAGs). These molecules are highly modified in cancer, contributing to tumorigenesis. In fact, the expression of HSPGs is markedly altered during malignant transformation and tumor progression, affecting both the PG core proteins and the GAG chains. We discuss here some of the main regulatory points in HSPG formation and modification, all of which are implicated in cancer development and progression. Furthermore, we highlight some examples of these alterations in different tumors. Finally, this review aims to outline improvements in our knowledge of HSPGs and cancer, hopefully in order to promote the design of possible new integrated anti-cancer treatment strategies.

Analyzing the role of heparan sulfate proteoglycans in axon guidance in vivo in zebrafish.

One of the most fascinating questions in the field of neurobiology is to understand how neuronal connections are properly formed. During development, neurons extend axons that are guided along defined paths by attractive and repulsive cues to reach their brain target. Most of these guidance factors are regulated by heparan sulfate proteoglycans (HSPGs), a family of cell-surface and extracellular core proteins with attached heparan sulfate (HS) glycosaminoglycans. The unique diversity and structural complexity of HS sugar chains, as well as the variety of core proteins, have been proposed to generate a complex "sugar code" essential for brain wiring. While the functions of HSPGs have been well characterized in C. elegans or Drosophila, relatively little is known about their roles in nervous system development in vertebrates. In this chapter, we describe the advantages and the different methods available to study the roles of HSPGs in axon guidance directly in vivo in zebrafish. We provide protocols for visualizing axons in vivo, including precise dye labeling and time-lapse imaging, and for disturbing the functions of HS-modifying enzymes and core proteins, including morpholino, DNA, or RNA injections.

Regulation of TGFβ superfamily signaling by two separable domains of glypican LON-2 inC. elegans

Regulated intercellular signaling is critical for the normal development and maintenance of multicellular organisms. Glypicans have been shown to regulate signaling by TGFβs, hedgehogs and Wnts, in several cellular contexts. Glypicans comprise a conserved family of heparan sulfated, glycosylphosphatidylinositol (GPI)-linked extracellular proteins. The structural complexity of glypicans may underlie their functional complexity. In a recent study31, we built on previous findings that one of the two C. elegans glypicans, LON-2, specifically inhibits signaling by the TGFβ superfamily member DBL-1. We tested the functional requirements of LON-2 protein core components and post-translational modifications for LON-2 activity. We provide the first evidence that two parts of a glypican can independently regulate TGFβ superfamily signaling in vivo: the N-terminal furin protease product and a C-terminal region containing heparan sulfate attachment sites. Furthermore, we show a protein-protein interaction motif is crucial for LON-2 activity in the N-terminal protein core, suggesting that LON-2 acts by serving as a scaffold for DBL-1 and an RGD-binding protein. In addition, we demonstrate specificity of glypican function by showing C. elegans GPN-1 does not functionally substitute for LON-2. This work reveals a molecular foundation for understanding the complexity and specificity of glypican function.