Sulfate Glycosaminoglycan Chains Research Articles

Astrocyte extracellular matrix modulates neuronal dendritic development.

Major developmental events occurring in the hippocampus during the third trimester of human gestation and neonatally in altricial rodents include rapid and synchronized dendritic arborization and astrocyte proliferation and maturation. We tested the hypothesis that signals sent by developing astrocytes to developing neurons modulate dendritic development in vivo. We altered neuronal development by neonatal (third trimester-equivalent) ethanol exposure in mice; this treatment increased dendritic arborization in hippocampal pyramidal neurons. We next assessed concurrent changes in the mouse astrocyte translatome by translating ribosomal affinity purification (TRAP)-seq. We followed up on ethanol-inhibition of astrocyte Chpf2 and Chsy1 gene translation because these genes encode for biosynthetic enzymes of chondroitin sulfate glycosaminoglycan (CS-GAG) chains (extracellular matrix components that inhibit neuronal development and plasticity) and have not been explored before for their roles in dendritic arborization. We report that Chpf2 and Chsy1 are enriched in astrocytes and their translation is inhibited by ethanol, which also reduces the levels of CS-GAGs measured by Liquid Chromatography/Mass Spectrometry. Finally, astrocyte-conditioned medium derived from Chfp2-silenced astrocytes increased neurite branching of hippocampal neurons in vitro. These results demonstrate that CS-GAG biosynthetic enzymes in astrocytes regulates dendritic arborization in developing neurons.

Glycosaminoglycan modifications of betaglycan regulate ectodomain shedding to fine-tune TGF-β signaling responses in ovarian cancer

In pathologies including cancer, aberrant Transforming Growth Factor-β (TGF-β) signaling exerts profound tumor intrinsic and extrinsic consequences. Intense clinical endeavors are underway to target this pathway. Central to the success of these interventions is pinpointing factors that decisively modulate the TGF-β responses. Betaglycan/type III TGF-β receptor (TβRIII), is an established co-receptor for the TGF-β superfamily known to bind directly to TGF-βs 1–3 and inhibin A/B. Betaglycan can be membrane-bound and also undergo ectodomain cleavage to produce soluble-betaglycan that can sequester its ligands. Its extracellular domain undergoes heparan sulfate and chondroitin sulfate glycosaminoglycan modifications, transforming betaglycan into a proteoglycan. We report the unexpected discovery that the heparan sulfate glycosaminoglycan chains on betaglycan are critical for the ectodomain shedding. In the absence of such glycosaminoglycan chains betaglycan is not shed, a feature indispensable for the ability of betaglycan to suppress TGF-β signaling and the cells' responses to exogenous TGF-β ligands. Using unbiased transcriptomics, we identified TIMP3 as a key inhibitor of betaglycan shedding thereby influencing TGF-β signaling. Our results bear significant clinical relevance as modified betaglycan is present in the ascites of patients with ovarian cancer and can serve as a marker for predicting patient outcomes and TGF-β signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan chains of betaglycan for shedding and influence on TGF-β signaling responses. Dysregulated shedding of TGF-β receptors plays a vital role in determining the response and availability of TGF-βs’, which is crucial for prognostic predictions and understanding of TGF-β signaling dynamics.

V3: an enigmatic isoform of the proteoglycan versican.

V3 is an isoform of the extracellular matrix (ECM) proteoglycan (PG) versican generated through alternative splicing of the versican gene such that the two major exons coding for sequences in the protein core that support chondroitin sulfate (CS) glycosaminoglycan (GAG) chain attachment are excluded. Thus, versican V3 isoform carries no GAGs. A survey of PubMed reveals only 50 publications specifically on V3 versican, so it is a very understudied member of the versican family, partly because to date there are no antibodies that can distinguish V3 from the CS-carrying isoforms of versican, that is, to facilitate functional and mechanistic studies. However, a number of in vitro and in vivo studies have identified the expression of the V3 transcript during different phases of development and in disease, and selective overexpression of V3 has shown dramatic phenotypic effects in "gain and loss of function" studies in experimental models. Thus, we thought it would be useful and instructive to discuss the discovery, characterization, and the putative biological importance of the enigmatic V3 isoform of versican.

Abstract 6312: Targeting HSPG is more efficient than using soluble heparin for interfering with cancer cell adhesion and migration

Abstract Given the relevance of heparan sulfate proteoglycans (HSPG) and of heparin binding ligands in the regulation of cancer cell adhesion, migration and invasiveness, drugs that may interfere with HSPG functions have been studied for decades for their possible use in oncology. In this view heparin analogues have been tested for their effectiveness not only in cancer associated thromboembolism but also for their potential effects on cancer growth and metastasis. Despite a number of promising preclinical results and clinical hypothesis, many different clinical trials on the use of heparin analogues in different cancers reported no significant benefits in tumor progression and overall survival, indicating that using heparin as a soluble decoy for the many HSPG ligands does not appear to be the best possible approach (1,2). Taking advantage of the tetra-branched peptide NT4, which is a specific ligand of HSPG sulfated glycosaminoglycan (GAG) chains (3, 4), we tested the effectiveness of targeting cell membrane HSPG, as a potential alternative approach for interfering with HSPG functions in cancer cell invasiveness. We had demonstrated that NT4 can either inhibit or increase oriented migration in respectively PANC-1 pancreas adenocarcinoma and TE671 rhabdomyosarcoma human cancer cells, indicating a crucial but diverse role of HSPGs in oriented cell migration in different cancer cells. NT4 and heparin were here compared for their effect on adhesion and migration of different cancer cell lines on extracellular matrix (ECM) supports, as well as for their effect on proliferation and in vitro invasiveness of the same cell lines. We found that inhibition of cancer cells adhesion to ECM proteins by NT4 varies in different cancer cell lines, ranging from 90% inhibition to as low as 20%, whereas heparin has no effect. Analogous results were obtained when testing cancer cell migration. NT4 inhibited migration and in vitro invasiveness of different human cancer cell lines, with the sole exception of TE671 cells. Heparin did not modify cell migration in any of the tested cancer cell lines. NT4 had no effect on cell proliferation, whereas, notably, heparin resulted to stimulate cell proliferation in some cancer cell lines. Differently from using heparin as an unspecific soluble decoy, targeting HSPG with well-defined ligands can result in an efficient tumor-specific treatment. Nevertheless, as for most cancer cellular targets, a detailed definition of cellular mechanisms and a related personalized approach is mandatory. 1) Schünemann HJ et al. Lancet Haematol. 2020;7(10):e746. 2) Posch F et al. ibid:e703. 3) Brunetti J et al. Sci. Rep. 2016,6,27174. 4) Depau L et al. J Med Chem 2020;63(24):15997. Citation Format: Lorenzo Depau, Elisabetta Mandarini, Jlenia Brunetti, Marta Zanchi, Chiara Falciani, Luisa Bracci. Targeting HSPG is more efficient than using soluble heparin for interfering with cancer cell adhesion and migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6312.

Inhibition of Cytomegalovirus by Pentacta pygmaea Fucosylated Chondroitin Sulfate Depends on Its Molecular Weight.

Many viruses attach to host cells by first interacting with cell surface proteoglycans containing heparan sulfate (HS) glycosaminoglycan chains and then by engaging with specific receptor, resulting in virus entry. In this project, HS-virus interactions were targeted by a new fucosylated chondroitin sulfate from the sea cucumber Pentacta pygmaea (PpFucCS) in order to block human cytomegalovirus (HCMV) entry into cells. Human foreskin fibroblasts were infected with HCMV in the presence of PpFucCS and its low molecular weight (LMW) fractions and the virus yield at five days post-infection was assessed. The virus attachment and entry into the cells were visualized by labeling the purified virus particles with a self-quenching fluorophore octadecyl rhodamine B (R18). The native PpFucCS exhibited potent inhibitory activity against HCMV specifically blocking virus entry into the cell and the inhibitory activities of the LMW PpFucCS derivatives were proportional to their chain lengths. PpFucCS and the derived oligosaccharides did not exhibit any significant cytotoxicity; moreover, they protected the infected cells from virus-induced lytic cell death. In conclusion, PpFucCS inhibits the entry of HCMV into cells and the high MW of this carbohydrate is a key structural element to achieve the maximal anti-viral effect. This new marine sulfated glycan can be developed into a potential prophylactic and therapeutic antiviral agent against HCMV infection.

Investigation of Glypican-4 and -6 by Infrared Spectral Imaging during the Hair Growth Cycle

The expression of glypicans in different hair follicle (HF) compartments is still poorly understood. Heparan sulfate proteoglycans (HSPGs) distribution in HF is classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. Our previous study proposed a novel approach to assess hair histology and glypican-1 (GPC1) distribution changes in the HF at different phases of the hair growth cycle using infrared spectral imaging (IRSI). We show in the present manuscript for the first time complementary data on the distribution of glypican-4 (GPC4) and glypican-6 (GPC6) in HF at different phases of the hair growth cycle using IR imaging. Findings were supported by Western blot assays focusing on the GPC4 and GPC6 expression in HFs. Like all proteoglycan features, the glypicans are characterized by a core protein to which sulfated and/or unsulfated glycosaminoglycan (GAG) chains are covalently linked. Our study demonstrates the capacity of IRSI to identify the different HF tissue structures and to highlight protein, proteoglycan (PG), GAG, and sulfated GAG distribution in these structures. The comparison between anagen, catagen, and telogen phases shows the qualitative and/or quantitative evolution of GAGs, as supported by Western blot. Thus, in one analysis, IRSI can simultaneously reveal the location of proteins, PGs, GAGs and sulfated GAGs in HFs in a chemical and label-free manner. From a dermatological point of view, IRSI may constitute a promising technique to study alopecia.

Chondroitin Sulfate Targeting Nanodrug Achieves Near-Infrared Fluorescence-Guided Chemotherapy Against Triple-Negative Breast Primary and Lung Metastatic Cancer.

Lack of highly expressed tumor target and ligands limits application of nano-medicine against triple-negative breast cancer (TNBC). Previous study reported that placenta-derived oncofetal chondroitin sulfate glycosaminoglycan chain (CSA) expressed on 90% of stage I-III invasive ductal breast carcinomas. Our study found the CSA anchor protein VAR2CSA derived small peptide plCSA had strong binding activity with TNBC cell lines and tumor tissue. Here, we combined the AIEgens TBZ-DPNA and therapy drug paclitaxel (PTX) to fabricate near-infrared fluorescence-guided nanodrug (plCSA-NP) to investigate its targeting and anti-tumor effect on TNBC. We synthesized and purified TBZ-DPNA with one step, measured optical properties and photoluminescence (PL) spectra. We prepared nanodrug plCSA-NP by encapsulating TBZ-DPNA and PTX and conjugating them with peptide plCSA. We evaluated plCSA-NP targeting activity by examining AIEdots fluorescence signal on TNBC cell lines and subcutaneous and lung metastatic mouse model. We assessed PTX delivery effect by cytotoxicity assay on TNBC line and tumor growth of subcutaneous and lung metastatic mouse models. PL spectra and TEM imaging results showed plCSA-NP had maximum emission feature at 718 nm and nearly monodispersed nanosphere with an average diameter of 70 nm. In vitro studies showed plCSA-NPs had high affinity and cytotoxicity with TNBC cell lines. In vivo subcutaneous and lung metastasis mouse studies showed plCSA-NPs accumulated on TNBC tumor tissue, and significantly prevented TNBC subcutaneous and lung metastasis tumor growth. In conclusion, we provide solid evidence for chondroitin sulfate targeting peptide plCSA guided nanodrug, exhibit good targeting efficiency and therapeutic effect against TNBC primary and lung metastatic tumor growth.

Alterations in glycosaminoglycan biosynthesis associated with the Ehlers-Danlos syndromes.

Proteoglycans consist of a core protein substituted with one or more glycosaminoglycan (GAG) chains and execute versatile functions during many physiological and pathological processes. The biosynthesis of GAG chains is a complex process that depends on the concerted action of a variety of enzymes. Central to the biosynthesis of heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) GAG chains is the formation of a tetrasaccharide linker region followed by biosynthesis of HS or CS/DS-specific repeating disaccharide units, which then undergo modifications and epimerization. The importance of these biosynthetic enzymes is illustrated by several severe pleiotropic disorders that arise upon their deficiency. The Ehlers-Danlos syndromes (EDS) constitute a special group among these disorders. Although most EDS types are caused by defects in fibrillar types I, III, or V collagen, or their modifying enzymes, a few rare EDS types have recently been linked to defects in GAG biosynthesis. Spondylodysplastic EDS (spEDS) is caused by defective formation of the tetrasaccharide linker region, either due to β4GalT7 or β3GalT6 deficiency, whereas musculocontractural EDS (mcEDS) results from deficiency of D4ST1 or DS-epi1, impairing DS formation. This narrative review highlights the consequences of GAG deficiency in these specific EDS types, summarizes the associated phenotypic features and the molecular spectrum of reported pathogenic variants, and defines the current knowledge on the underlying pathophysiological mechanisms based on studies in patient-derived material, in vitro analyses, and animal models.

Isolation and structural characterization of bioactive glycosaminoglycans from the green-lipped mussel Perna canaliculus

Chondroitin sulfate (CS) and heparan sulfate (HS) are sulfated glycosaminoglycan (GAG) chains that consist of repeating disaccharide units composed of hexosamine and hexuronic acid. GAG chains exhibit diverse bioactivities in a structure-specific manner. Marine invertebrates are a rich source of highly sulfated and rare structures of GAG chains. Here, we isolated GAGs from the green-lipped mussel Perna canaliculus, an aquaculture species that is produced on a large scale. We separated GAGs based on the degree of negative charges and analyzed their disaccharide compositions. CS and HS both exhibited characteristic compositions of differently sulfated disaccharides. CS chains showed a higher degree of sulfation than HS chains and contained a high percentage of the E unit disaccharide GlcA-GalNAc(4,6-O-disulfate). Furthermore, CS chains rich in the E unit stimulated the neurite outgrowth of primary cultured neurons. The present results indicate the potential of P. canaliculus GAGs as biomaterials to study the structure-function relationships of GAGs.

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.

Extracellular endosulfatase Sulf-2 harbors a chondroitin/dermatan sulfate chain that modulates its enzyme activity.

Sulfs represent a class of unconventional sulfatases which provide an original post-synthetic regulatory mechanism for heparan sulfate polysaccharides and are involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their invivo functions. Here we show that human Sulf-2 (HSulf-2) harbors a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate-binding domain. We demonstrate that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity invitro and invivo. In addition, we show that mammalian hyaluronidase acts as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a proteoglycan-related enzyme and its GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute to clarifying the conflicting data on the activities of the Sulfs.

Mutations in the heparan sulfate backbone elongating enzymes EXT1 and EXT2 have no major effect on endothelial glycocalyx and the glomerular filtration barrier

In this study, the effect of heterozygous germline mutations in the heparan sulfate (HS) glycosaminoglycan chain co-polymerases EXT1 and EXT2 on glomerular barrier function and the endothelial glycocalyx in humans is investigated. Heparan sulfate (HS) glycosaminoglycans are deemed essential to the glomerular filtration barrier, including the glomerular endothelial glycocalyx. Animal studies have shown that loss of HS results in a thinner glycocalyx. Also, decreased glomerular HS expression is observed in various proteinuric renal diseases in humans. A case report of a patient with an EXT1 mutation indicated that this could result in a specific renal phenotype. This patient suffered from multiple osteochondromas, an autosomal dominant disease caused by mono-allelic germline mutations in the EXT1 or EXT2 gene. These studies imply that HS is indeed essential to the glomerular filtration barrier. However, loss of HS did not lead to proteinuria in various animal models. We demonstrate that multiple osteochondroma patients do not have more microalbuminuria or altered glycocalyx properties compared to age-matched controls (n = 19). A search for all Dutch patients registered with both osteochondroma and kidney biopsy (n = 39) showed that an EXT1 or EXT2 mutation does not necessarily lead to specific glomerular morphological phenotypic changes. In conclusion, this study shows that a heterozygous mutation in the HS backbone elongating enzymes EXT1 and EXT2 in humans does not result in (micro)albuminuria, a specific renal phenotype or changes to the endothelial glycocalyx, adding to the growing knowledge on the role of EXT1 and EXT2 genes in pathophysiology.

Inherited Proteoglycan Biosynthesis Defects—Current Laboratory Tools and Bikunin as a Promising Blood Biomarker

Proteoglycans consist of proteins linked to sulfated glycosaminoglycan chains. They constitute a family of macromolecules mainly involved in the architecture of organs and tissues as major components of extracellular matrices. Some proteoglycans also act as signaling molecules involved in inflammatory response as well as cell proliferation, adhesion, and differentiation. Inborn errors of proteoglycan metabolism are a group of orphan diseases with severe and irreversible skeletal abnormalities associated with multiorgan impairments. Identifying the gene variants that cause these pathologies proves to be difficult because of unspecific clinical symptoms, hardly accessible functional laboratory tests, and a lack of convenient blood biomarkers. In this review, we summarize the molecular pathways of proteoglycan biosynthesis, the associated inherited syndromes, and the related biochemical screening techniques, and we focus especially on a circulating proteoglycan called bikunin and on its potential as a new biomarker of these diseases.

Identification of Cell Autonomous and Non-Cell Autonomous Functions of Heparan Sulfate Glycosaminoglycan Chains by Creating Chimeric Mouse Embryos.

Cell surface-tethered heparan sulfate glycosaminoglycan chains primarily function in a cell autonomous manner, while extracellular matrix-associated heparan sulfate glycosaminoglycan chains function in a non-cell autonomous manner. In addition, the cleaved forms of cell surface-tethered heparan sulfate chains enzymatically released by proteases and heparanases, called shedding, can contribute to non-cell autonomous mechanisms. The movement of heparan sulfate chains to surrounding cells mediated by transcytosis or filopodia also involves another non-cell autonomous mechanism. To determine cell autonomous or non-cell autonomous roles of heparan sulfate glycosaminoglycan chains during early embryogenesis, direct conclusions can be drawn by analyzing chimeric embryos which are composed of wild-type and heparan sulfate glycosaminoglycan chain-deficient cells. Here, we describe methods of production of these chimeric embryos and analysis of their cellular phenotypes with immunohistochemistry at a single-cell level.

Modification of gingival proteoglycans by reactive oxygen species: potential mechanism of proteoglycan degradation during periodontal diseases

Reactive oxygen species (ROS) overproduction and oxidative stress are increasingly being implicated in the extracellular matrix (ECM) degradation associated with chronic inflammatory conditions, such as periodontal diseases. The present study investigated the effects of ROS exposure on the proteoglycans of gingival tissues, utilizing an in vitro model system comprised of supra-physiological oxidant concentrations, to ascertain whether gingival proteoglycan modification and degradation by ROS contributed to the underlying mechanisms of ECM destruction during active gingivitis. Proteoglycans were purified from ovine gingival tissues and exposed to increasing H2O2 concentrations or a hydroxyl radical (·OH) flux for 1 h or 24 h, and ROS effects on proteoglycan core proteins and sulfated glycosaminoglycan (GAG) chains were assessed. ROS were capable of degrading gingival proteoglycans, with ·OH species inducing greater degradative effects than H2O2 alone. Degradative effects were particularly manifested as amino acid modification, core protein cleavage, and GAG chain depolymerization. Proteoglycan core proteins were more susceptible to degradation than GAG chains with H2O2 alone, although core proteins and GAG chains were both extensively degraded by ·OH species. Proteoglycan exposure to ·OH species for 24 h induced significant core protein amino acid modification, with decreases in glutamate, proline, isoleucine, and leucine; and concomitant increases in serine, glycine, and alanine residues. As clinical reports have previously highlighted proteoglycan core protein degradation during chronic gingivitis, whereas their sulfated GAG chains remain relatively intact, these findings potentially provide further evidence to implicate ROS in the pathogenesis of active gingivitis, complementing the enzymic mechanisms of periodontal tissue destruction already established.

Sulfation of Glycosaminoglycans Modulates the Cell Cycle of Embryonic Mouse Spinal Cord Neural Stem Cells.

In the developing spinal cord neural stem and progenitor cells (NSPCs) secrete and are surrounded by extracellular matrix (ECM) molecules that influence their lineage decisions. The chondroitin sulfate proteoglycan (CSPG) DSD-1-PG is an isoform of receptor protein tyrosine phosphatase-beta/zeta (RPTPβ/ζ), a trans-membrane receptor expressed by NSPCs. The chondroitin sulfate glycosaminoglycan chains are sulfated at distinct positions by sulfotransferases, thereby generating the distinct DSD-1-epitope that is recognized by the monoclonal antibody (mAb) 473HD. We detected the epitope, the critical enzymes and RPTPβ/ζ in the developing spinal cord. To obtain insight into potential biological functions, we exposed spinal cord NSPCs to sodium chlorate. The reagent suppresses the sulfation of glycosaminoglycans, thereby erasing any sulfation code expressed by the glycosaminoglycan polymers. When NSPCs were treated with chlorate and cultivated in the presence of FGF2, their proliferation rate was clearly reduced, while NSPCs exposed to EGF were less affected. Time-lapse video microscopy and subsequent single-cell tracking revealed that pedigrees of NSPCs cultivated with FGF2 were strongly disrupted when sulfation was suppressed. Furthermore, the NSPCs displayed a protracted cell cycle length. We conclude that the inhibition of sulfation with sodium chlorate interferes with the FGF2-dependent cell cycle progression in spinal cord NSPCs.

Herpes Simplex Virus Type 2 Mucin-Like Glycoprotein mgG Promotes Virus Release from the Surface of Infected Cells.

The contribution of virus components to liberation of herpes simplex virus type 2 (HSV-2) progeny virions from the surface of infected cells is poorly understood. We report that the HSV-2 mutant deficient in the expression of a mucin-like membrane-associated glycoprotein G (mgG) exhibited defect in the release of progeny virions from infected cells manifested by ~2 orders of magnitude decreased amount of infectious virus in a culture medium as compared to native HSV-2. Electron microscopy revealed that the mgG deficient virions were produced in infected cells and present at the cell surface. These virions could be forcibly liberated to a nearly native HSV-2 level by the treatment of cells with glycosaminoglycan (GAG)-mimicking oligosaccharides. Comparative assessment of the interaction of mutant and native virions with surface-immobilized chondroitin sulfate GAG chains revealed that while the mutant virions associated with GAGs ~fourfold more extensively, the lateral mobility of bound virions was much poorer than that of native virions. These data indicate that the mgG of HSV-2 balances the virus interaction with GAG chains, a feature critical to prevent trapping of the progeny virions at the surface of infected cells.

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.

Extracellular Endosulfatase Sulf-2 Harbours a Chondroitin/Dermatan Sulfate Chain That Modulates Its Enzyme Activity

Sulfs represent a class of unconventional sulfatases, which differ from all other members of the sulfatase family by their structures, catalytic features and biological functions. Through their specific endosulfatase activity in extracellular milieu, Sulfs provide an original post-synthetic regulatory mechanism for heparan sulfate complex polysaccharides and have been involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human Sulf-2 (HSulf-2) features a unique polysaccharide post-translational modification. We identified a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate binding domain. We found that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo using a mouse model of tumorigenesis and metastasis. In addition, we showed that mammalian hyaluronidase acted as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a unique proteoglycan enzyme and its newly-identified GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute in clarifying the conflicting data on the activities of the Sulfs and introduce a new paradigm into the study of these enzymes.

Inhibition of Chondroitin Sulfate Proteoglycans by APRIL.

Chondroitin sulfate proteoglycans (CSPGs) are major constituents of the extracellular matrix and well-established obstacles to regeneration in the central nervous system. As such, they are promising targets for therapy in neurological pathologies where repair is needed, such as spinal cord injuries, and multiple sclerosis. Since CSPGs mediate their inhibitory functions by interacting with signaling protein partners through their variably sulfated chondroitin sulfate glycosaminoglycan (CS-GAG) chains, blocking these epitopes presents a path to promoting repair. A member of the tumor necrosis factor (TNF) superfamily, a proliferation-inducing ligand (APRIL) has been shown to bind to CSPGs. Here we describe in vitro methods to evaluate APRIL's ability to block CSPGs from interacting with their partner proteins and promote neuronal growth.