N-Linked Glycosylation of Protease-activated Receptor-1 Second Extracellular Loop: A CRITICAL DETERMINANT FOR LIGAND-INDUCED RECEPTOR ACTIVATION AND INTERNALIZATION*

Protease-activated receptor-1 (PAR1) contains five N-linked glycosylation consensus sites as follows: three residing in the N terminus and two localized on the surface of the second extracellular loop (ECL2). To study the effect of N-linked glycosylation in the regulation of PAR1 signaling and trafficking, we generated mutants in which the critical asparagines of the consensus sites were mutated. Here, we report that both the PAR1 N terminus and ECL2 serve as sites for N-linked glycosylation but have different functions in the regulation of receptor signaling and trafficking. N-Linked glycosylation of the PAR1 N terminus is important for transport to the cell surface, whereas the PAR1 mutant lacking glycosylation at ECL2 (NA ECL2) trafficked to the cell surface like the wild-type receptor. However, activated PAR1 NA ECL2 mutant internalization was impaired compared with wild-type receptor, whereas constitutive internalization of unactivated receptor remained intact. Remarkably, thrombin-activated PAR1 NA ECL2 mutant displayed an enhanced maximal signaling response compared with wild-type receptor. The increased PAR1 NA ECL2 mutant signaling was not due to defects in the ability of thrombin to cleave the receptor or signal termination mechanisms. Rather, the PAR1 NA ECL2 mutant displayed a greater efficacy in thrombin-stimulated G protein signaling. Thus, N-linked glycosylation of the PAR1 extracellular surface likely influences ligand docking interactions and the stability of the active receptor conformation. Together, these studies strongly suggest that N-linked glycosylation of PAR1 at the N terminus versus the surface of ECL2 serves distinct functions critical for proper regulation of receptor trafficking and the fidelity of thrombin signaling.     

N-Glycosylation Regulates Fibroblast Growth Factor Receptor/EGL-15 Activity in Caenorhabditis elegans in Vivo

The regulation of cell function by fibroblast growth factors (FGFs) classically occurs through a dual receptor system of a tyrosine kinase receptor (FGFR) and a heparan sulfate proteoglycan co-receptor. Mutations in some consensus N-glycosylation sites in human FGFR result in skeletal disorders and craniosynostosis syndromes, and biophysical studies in vitro suggest that N-glycosylation of FGFR alters ligand and heparan sulfate binding properties. The evolutionarily conserved FGFR signaling system of Caenorhabditis elegans has been used to assess the role of N-glycosylation in the regulation of FGFR signaling in vivo. The C. elegans FGF receptor, EGL-15, is N-glycosylated in vivo, and genetic substitution of specific consensus N-glycosylation sites leads to defects in the maintenance of fluid homeostasis and differentiation of sex muscles, both of which are phenotypes previously associated with hyperactive EGL-15 signaling. These phenotypes are suppressed by hypoactive mutations in EGL-15 downstream signaling components or activating mutations in the phosphatidylinositol 3-kinase pathway, respectively. The results show that N-glycans negatively regulate FGFR activity in vivo supporting the notion that mutation of N-glycosylation sites in human FGFR may lead to inappropriate activation of the receptor.     

The C-terminal Peptide of Chondroadherin Modulates Cellular Activity by Selectively Binding to Heparan Sulfate Chains

Chondroadherin, a leucine-rich repeat family member, contains a very C-terminal sequence CKFPTKRSKKAGRH³⁵⁹, now shown to bind to heparin with a K_D of 13 μm. This observation led us to investigate whether chondroadherin interacts via this C-terminal heparin-binding domain with glycosaminoglycan chains of proteoglycans at the cell surface. Cells were shown to bind this heparin-binding peptide in FACS analysis, and the interaction was shown to be with glycosaminoglycans because it was abolished when sulfation was inhibited by chlorate treatment of the cells. In separate experiments, heparin and heparan sulfate inhibited the peptide interaction in a dose-dependent manner. Using a human chondrosarcoma and a murine osteoblast cell line, heparan sulfate proteoglycans were identified as the cell surface receptors involved in the binding. Different binding syndecans were identified in the two different cell lines, indicating that the same protein core of a proteoglycan may have structural and functional differences in the attached heparan sulfate chains. Upon binding to coated peptide, cells spread, demonstrating engagement of the cytoskeleton, but no focal adhesion complex was formed. The number of cells adhering via their β₁ integrin receptor to collagen type II or chondroadherin was profoundly and rapidly enhanced by the addition of the heparin-binding peptide. The peptide added to the cells caused ERK phosphorylation, showing that it triggered intracellular signaling. The results show that heparan sulfate chains differ between various members of the proteoglycan families on a given cell, but also differ between the same proteoglycan on different cells with a potential for differential regulation of cellular activities.     

The role of vascular-derived perlecan in modulating cell adhesion,proliferation and growth factor signaling

Smooth muscle cell proliferation can be inhibited by heparan sulfate proteoglycans whereas the removal or digestion of heparan sulfate from perlecan promotes their proliferation. In this study we characterized the glycosaminoglycan side chains of perlecan isolated from either primary human coronary artery smooth muscle or endothelial cells and determined their roles in mediating cell adhesion and proliferation, and in fibroblast growth factor (FGF) binding and signaling. Smooth muscle cell perlecan was decorated with both heparan sulfate and chondroitin sulfate, whereas endothelial perlecan contained exclusively heparan sulfate chains. Smooth muscle cells bound to the protein core of perlecan only when the glycosaminoglycans were removed, and this binding involved a novel site in domain III as well as domain V/endorepellin and the α₂β₁ integrin. In contrast, endothelial cells adhered to the protein core of perlecan in the presence of glycosaminoglycans. Smooth muscle cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains and promoted the signaling of FGF2 but not FGF1. Also endothelial cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains, but in contrast, promoted the signaling of both growth factors. Based on this differential bioactivity, we propose that perlecan synthesized by smooth muscle cells differs from that synthesized by endothelial cells by possessing different signaling capabilities, primarily, but not exclusively, due to a differential glycanation. The end result is a differential modulation of cell adhesion, proliferation and growth factor signaling in these two key cellular constituents of blood vessels.     

UDP-N-acetylglucosamine Transporter (SLC35A3) Regulates Biosynthesis of Highly Branched N-Glycans and Keratan Sulfate

SLC35A3 is considered the main UDP-N-acetylglucosamine transporter (NGT) in mammals. Detailed analysis of NGT is restricted because mammalian mutant cells defective in this activity have not been isolated. Therefore, using the siRNA approach, we developed and characterized several NGT-deficient mammalian cell lines. CHO, CHO-Lec8, and HeLa cells deficient in NGT activity displayed a decrease in the amount of highly branched tri- and tetraantennary N-glycans, whereas monoantennary and diantennary ones remained unchanged or even were accumulated. Silencing the expression of NGT in Madin-Darby canine kidney II cells resulted in a dramatic decrease in the keratan sulfate content, whereas no changes in biosynthesis of heparan sulfate were observed. We also demonstrated for the first time close proximity between NGT and mannosyl (α-1,6-)-glycoprotein β-1,6-N-acetylglucosaminyltransferase (Mgat5) in the Golgi membrane. We conclude that NGT may be important for the biosynthesis of highly branched, multiantennary complex N-glycans and keratan sulfate. We hypothesize that NGT may specifically supply β-1,3-N-acetylglucosaminyl-transferase 7 (β3GnT7), Mgat5, and possibly mannosyl (α-1,3-)-glycoprotein β-1,4-N-acetylglucosaminyltransferase (Mgat4) with UDP-GlcNAc.     

Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases,in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains

Heparan sulfate proteoglycans are ubiquitously located on cell surfaces and in the extracellular matrices. The negatively charged heparan sulfate chains interact with a multitude of different proteins, thereby influencing a variety of cellular and developmental processes, for example cell adhesion, migration, tissue morphogenesis, and differentiation. The human exostosin (EXT) family of genes contains five members: the heparan sulfate polymerizing enzymes, EXT1 and EXT2, and three EXT-like genes, EXTL1, EXTL2, and EXTL3. EXTL2 has been ascribed activities related to the initiation and termination of heparan sulfate chains. Here we further investigated the role of EXTL2 in heparan sulfate chain elongation by gene silencing and overexpression strategies. We found that siRNA-mediated knockdown of EXTL2 in human embryonic kidney 293 cells resulted in increased chain length, whereas overexpression of EXTL2 in the same cell line had little or no effect on heparan sulfate chain length. To study in more detail the role of EXTL2 in heparan sulfate chain elongation, we tested the ability of the overexpressed protein to catalyze the in vitro incorporation of N-acetylglucosamine and N-acetylgalactosamine to oligosaccharide acceptors resembling unmodified heparan sulfate and chondroitin sulfate precursor molecules. Analysis of the generated products revealed that recombinant EXTL2 showed weak ability to transfer N-acetylgalactosamine to heparan sulfate precursor molecules but also, that EXTL2 exhibited much stronger in vitro N-acetylglucosamine-transferase activity related to elongation of heparan sulfate chains.     

Distinct Roles of N-Glycosylation at Different Sites of Corin in Cell Membrane Targeting and Ectodomain Shedding

Corin is a membrane-bound protease essential for activating natriuretic peptides and regulating blood pressure. Human corin has 19 predicted N-glycosylation sites in its extracellular domains. It has been shown that N-glycans are required for corin cell surface expression and zymogen activation. It remains unknown, however, how N-glycans at different sites may regulate corin biosynthesis and processing. In this study, we examined corin mutants, in which each of the 19 predicted N-glycosylation sites was mutated individually. By Western analysis of corin proteins in cell lysate and conditioned medium from transfected HEK293 cells and HL-1 cardiomyocytes, we found that N-glycosylation at Asn-80 inhibited corin shedding in the juxtamembrane domain. Similarly, N-glycosylation at Asn-231 protected corin from autocleavage in the frizzled-1 domain. Moreover, N-glycosylation at Asn-697 in the scavenger receptor domain and at Asn-1022 in the protease domain is important for corin cell surface targeting and zymogen activation. We also found that the location of the N-glycosylation site in the protease domain was not critical. N-Glycosylation at Asn-1022 may be switched to different sites to promote corin zymogen activation. Together, our results show that N-glycans at different sites may play distinct roles in regulating the cell membrane targeting, zymogen activation, and ectodomain shedding of corin.     

HpSulf,a heparan sulfate 6-O-endosulfatase,is involved in the regulation of VEGF signaling during sea urchin development

Cell surface heparan sulfate proteoglycans (HSPGs) play significant roles in the regulation of developmental signaling, including vascular endothelial growth factor (VEGF), fibroblast growth factor, Wnt and bone morphogenetic protein signaling, through modification of their sulfation patterns. Recent studies have revealed that one of the functions of heparan sulfate 6-O-endosulfatase (Sulf) is to remove the sulfate from the 6-O position of HSPGs at the cell surface, thereby regulating the binding activities of heparan sulfate (HS) chains to numerous ligands and receptors in animal species. In this study, we focused on the sea urchin Hemicentrotus pulcherrimus homolog of Sulf (HpSulf), and analyzed its expression pattern and functions during development. HpSulf protein was present throughout development and localized at cell surface of all blastomeres. In addition, the HS-specific epitope 10E4 was detected at the cell surface and partially colocalized with HpSulf. Knockdown of HpSulf using morpholino antisense oligonucleotides (MO) caused abnormal morphogenesis, and the development of MO-injected embryos was arrested before the hatched blastula stage, indicating that HpSulf is necessary for the early developmental process of sea urchin embryos. Furthermore, we found that injection of HpSulf mRNA suppressed the abnormal skeleton induced by overexpression of HpVEGF mRNA, whereas injection of an inactive form of HpSulf mRNA, containing mutated cysteines in the sulfatase domain, did not have this effect. Taken together, these results suggest that HpSulf is involved in the regulation of various signal transductions, including VEGF signaling, during sea urchin development.     

HSPG modulation of BMP signaling in fibrodysplasia ossificans progressiva cells

Cell surface heparan sulfate proteoglycans (HSPGs) play important roles in morphogen gradient formation and cell signaling. Bone morphogenetic protein (BMP) signaling is dysregulated in fibrodysplasia ossificans progressiva (FOP), a disabling disorder of progressive heterotopic bone formation. Here, we investigated the role of HSPG glycosaminoglycan (GAG) side chains on BMP signaling and found increased total and HSPG-specific GAG chain levels and dysregulation in HSPG modulation of BMP signaling in FOP lymphoblastoid cells (LCLs). Specifically, HSPG profiling demonstrated abundant mRNA and protein levels of glypican 1 and syndecan 4 on control and FOP LCLs, with elevated core protein levels on FOP cells. Targeted downregulation of glypican 1 core protein synthesis by siRNA enhanced BMP signaling in control and FOP cells, while reduction of syndecan 4-core protein synthesis decreased BMP signaling in control, but not FOP cells. These results suggest that FOP cells are resistant to the stimulatory effects of cell surface HSPG GAG chains, but are susceptible to the inhibitory effects, as shown by downregulation of glypican 1. These data support that HSPG modulation of BMP signaling is altered in cells from patients with FOP and that altered HSPG-related BMP signaling may play a role in the pathogenesis of the disease.     

Crystal structure of N-glycosylated human glypican-1 core protein: structure of two loops evolutionarily conserved in vertebrate glypican-1

Glypicans are a family of cell-surface proteoglycans that regulate Wnt, hedgehog, bone morphogenetic protein, and fibroblast growth factor signaling. Loss-of-function mutations in glypican core proteins and in glycosaminoglycan-synthesizing enzymes have revealed that glypican core proteins and their glycosaminoglycan chains are important in shaping animal development. Glypican core proteins consist of a stable α-helical domain containing 14 conserved Cys residues followed by a glycosaminoglycan attachment domain that becomes exclusively substituted with heparan sulfate (HS) and presumably adopts a random coil conformation. Removal of the α-helical domain results in almost exclusive addition of the glycosaminoglycan chondroitin sulfate, suggesting that factors in the α-helical domain promote assembly of HS. Glypican-1 is involved in brain development and is one of six members of the vertebrate family of glypicans. We expressed and crystallized N-glycosylated human glypican-1 lacking HS and N-glycosylated glypican-1 lacking the HS attachment domain. The crystal structure of glypican-1 was solved using crystals of selenomethionine-labeled glypican-1 core protein lacking the HS domain. No additional electron density was observed for crystals of glypican-1 containing the HS attachment domain, and CD spectra of the two protein species were highly similar. The crystal structure of N-glycosylated human glypican-1 core protein at 2.5 Å, the first crystal structure of a vertebrate glypican, reveals the complete disulfide bond arrangement of the conserved Cys residues, and it also extends the structural knowledge of glypicans for one α-helix and two long loops. Importantly, the loops are evolutionarily conserved in vertebrate glypican-1, and one of them is involved in glycosaminoglycan class determination.     

Modulatory roles of NHERF1 and NHERF2 in cell surface expression of the glutamate transporter GLAST

The PDZ (PSD-95/Drosophila discs-large protein/zonula occludens protein) domain-containing proteins Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) and NHERF2 interact with the glutamate transporter GLAST. To characterize the roles of these NHERF proteins in the plasma membrane targeting of GLAST, we examined the interaction of green fluorescent protein (EGFP)-tagged GLAST with epitope-tagged NHERF proteins in human embryonic kidney (HEK) 293T cells. Co-expression of either NHERF protein increased the cell surface expression of EGFP-GLAST. Deletion of the C-terminal PDZ domain-binding motif caused an increase in EGFP-GLAST with immature endoglycosidase H-sensitive N-linked oligosaccharides, suggesting impaired exit of EGFP-GLAST from the endoplasmic reticulum (ER). Immunoprecipitation experiments revealed that NHERF1 predominantly bound EGFP-GLAST containing immature N-glycans, whereas NHERF2 co-precipitated EGFP-GLAST with mature N-glycans. Expression of a dominant-negative mutant of the GTPase Sar1 increased the interaction of EGFP-GLAST with NHERF1 in the ER. By contrast, immunofluorescence microscopy showed that NHERF2 co-localized with EGFP-GLAST in ER–Golgi intermediate compartments (ERGICs), at the plasma membrane and in early endosomes, but not in the ER. These results suggest that NHERF1 interacts with GLAST during ER export, while NHERF2 interacts with GLAST in the secretory pathway from the ERGIC to the plasma membrane, thereby modulating the cell surface expression of GLAST.     

Insulin/IGF-I Signaling Pathways Enhances Tumor Cell Invasion through Bisecting GlcNAc N-glycans Modulation. An Interplay with E-Cadherin

Changes in glycosylation are considered a hallmark of cancer, and one of the key targets of glycosylation modifications is E-cadherin. We and others have previously demonstrated that E-cadherin has a role in the regulation of bisecting GlcNAc N-glycans expression, remaining to be determined the E-cadherin-dependent signaling pathway involved in this N-glycans expression regulation. In this study, we analysed the impact of E-cadherin expression in the activation profile of receptor tyrosine kinases such as insulin receptor (IR) and IGF-I receptor (IGF-IR). We demonstrated that exogenous E-cadherin expression inhibits IR, IGF-IR and ERK 1/2 phosphorylation. Stimulation with insulin and IGF-I in MDA-MD-435 cancer cells overexpressing E-cadherin induces a decrease of bisecting GlcNAc N-glycans that was accompanied with alterations on E-cadherin cellular localization. Concomitantly, IR/IGF-IR signaling activation induced a mesenchymal-like phenotype of cancer cells together with an increased tumor cell invasion capability. Altogether, these results demonstrate an interplay between E-cadherin and IR/IGF-IR signaling as major networking players in the regulation of bisecting N-glycans expression, with important effects in the modulation of epithelial characteristics and tumor cell invasion. Here we provide new insights into the role that Insulin/IGF-I signaling play during cancer progression through glycosylation modifications.     

Several N-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission

The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1_NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three N-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission.     

Pattern Recognition Receptors Require N-Glycosylation to Mediate Plant Immunity

N-Glycans attached to the ectodomains of plasma membrane pattern recognition receptors constitute likely initial contact sites between plant cells and invading pathogens. To assess the role of N-glycans in receptor-mediated immune responses, we investigated the functionality of Arabidopsis receptor kinases EFR and FLS2, sensing bacterial translation elongation factor Tu (elf18) and flagellin (flg22), respectively, in N-glycosylation mutants. As revealed by binding and responses to elf18 or flg22, both receptors tolerated immature N-glycans induced by mutations in various Golgi modification steps. EFR was specifically impaired by loss-of-function mutations in STT3A, a subunit of the endoplasmic reticulum resident oligosaccharyltransferase complex. FLS2 tolerated mild underglycosylation occurring in stt3a but was sensitive to severe underglycosylation induced by tunicamycin treatment. EFR accumulation was significantly reduced when synthesized without N-glycans but to lesser extent when underglycosylated in stt3a or mutated in single amino acid positions. Interestingly, EFR^N143Q lacking a single conserved N-glycosylation site from the EFR ectodomain accumulated to reduced levels and lost the ability to bind its ligand and to mediate elf18-elicited oxidative burst. However, EFR-YFP protein localization and peptide:N-glycosidase F digestion assays support that both EFR produced in stt3a and EFR^N143Q in wild type cells correctly targeted to the plasma membrane via the Golgi apparatus. These results indicate that a single N-glycan plays a critical role for receptor abundance and ligand recognition during plant-pathogen interactions at the cell surface.     

Heparin releasable and nonreleasable forms of heparan sulfate proteoglycan are found on the surfaces of cultured porcine aortic endothelial cells

Evidence suggests that endothelial cell layer heparan sulfate proteoglycans include a variety of different sized molecules which most likely contain different protein cores. In the present report, approximately half of endothelial cell surface associated heparan sulfate proteoglycan is shown to be releasable with soluble heparin. The remaining cell surface heparan sulfate proteoglycan, as well as extracellular matrix heparan sulfate proteoglycan, cannot be removed from the cells with heparin. The heparin nonreleasable cell surface proteoglycan can be released by membrane disrupting agents and is able to intercalate into liposomes. When the heparin releasable and nonreleasable cell surface heparan sulfate proteoglycans are compared, differences in proteoglycan size are also evident. Furthermore, the intact heparin releasable heparan sulfate proteoglycan is closer in size to proteoglycans isolated from the extracellular matrix and from growth medium than to that which is heparin nonreleasable. These data indicate that cultured porcine aortic endothelial cells contain at least two distinct types of cell surface heparan sulfate proteoglycans, one of which appears to be associated with the cells through its glycosaminoglycan chains. The other (which is more tightly associated) is probably linked via a membrane intercalated protein core.Abbreviations ECM extracellular matrix - HSPG heparan sulfate proteoglycan - PAE porcine aortic endothelial - PBS phosphate buffered saline     

Enrichment of hydrophobic membrane proteins using Triton X-114 and subsequent analysis of their N-glycosylation

BackgroundNumerous proteins depend on correct glycosylation for their proper function and nearly all membrane, as well as secreted, proteins are glycosylated. Glycosylation of membrane proteins plays a crucial role in many processes including the intercellular recognition and intermolecular interactions on the cell surface. The composition of N-glycans attached to membrane proteins has not been sufficiently studied due to the lack of efficient and reproducible analytical methods.MethodsThe aim of this study was to optimise cloud-point extraction (CPE) of membrane proteins with the non-ionic detergent Triton X-114 and analyse their N-glycosylation using hydrophilic interaction liquid chromatography (HILIC-UPLC). Purification of isolated proteins from the excess of detergent proved to be the key step. Therefore, several purification procedures were tested to efficiently remove detergent, while retaining maximum protein recoveries.ResultsCPE showed to be an efficient method to simultaneously extract membrane and soluble proteins, which subsequently resulted in different N-glycan profiles of the aforementioned protein groups. The resulting protocol showed satisfactory reproducibility and potential for N-glycan analysis of both membrane and intracellular (soluble) proteins from different kinds of biological material.ConclusionsThis method can be used as a new analytical tool for reliable detection and quantification of oligomannose and complex type N-glycans attached to membrane proteins, thus serving to distinguish between differences in cell types and states.General significanceThe simple method was successfully optimised to generate reliable HILIC-UPLC profiles of N-glycans released from membrane proteins. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

Involvement of Galectin-3 with Vascular Cell Adhesion Molecule-1 in Growth Regulation of Mouse BALB/3T3 Cells

β-Galactose residues on N-glycans have been implicated to be involved in growth regulation of cells. In the present study we compared the galactosylation of cell surface N-glycans of mouse Balb/3T3 cells between 30 and 100% densities and found the β-1,4-galactosylation of N-glycans increases predominantly in a 100-kDa protein band on lectin blot analysis in combination with digestions by diplococcal β-galactosidase and N-glycanase. When cells at 100% density were treated with jack bean β-galactosidase, the incorporation of 5-bromodeoxyuridine into the cells was stimulated in a dose-dependent manner, suggesting the involvement of the galactose residues in growth regulation of cells. A galactose-binding protein was isolated from the plasma membranes of cells at 100% density by affinity chromatography using an asialo-transferrin-Sepharose column and found to be galectin-3 as revealed by mass spectrometric analysis. The addition of recombinant galectin-3 into cells at 50% density inhibited the incorporation of 5-bromodeoxyuridine in a dose-dependent manner, but the inhibition was prevented with haptenic sugar. An immunocytochemical study showed that galectin-3 is present at the surface of cells at 100% density but not at 30% density where it locates inside the cells. Several glycoproteins bind to a galectin-3-immobilized column, a major of which was identified as vascular cell adhesion molecule (VCAM)-1. Immunocytochemical studies showed that some galectin-3 and VCAM-1 co-localize at the surface of cells at 100% density, indicating that the binding of galectin-3 secreted from cells to VCAM-1 is one of the pathways involved in the growth regulation of Balb/3T3 cells.     

Characterization of the interaction between Robo1 and heparin and other glycosaminoglycans

Roundabout 1 (Robo1) is the cognate receptor for secreted axon guidance molecule, Slits, which function to direct cellular migration during neuronal development and angiogenesis. The Slit2–Robo1 signaling is modulated by heparan sulfate, a sulfated linear polysaccharide that is abundantly expressed on the cell surface and in the extracellular matrix. Biochemical studies have further shown that heparan sulfate binds to both Slit2 and Robo1 facilitating the ligand–receptor interaction. The structural requirements for heparan sulfate interaction with Robo1 remain unknown. In this report, surface plasmon resonance (SPR) spectroscopy was used to examine the interaction between Robo1 and heparin and other GAGs and determined that heparin binds to Robo1 with an affinity of ∼650 nM. SPR solution competition studies with chemically modified heparins further determined that although all sulfo groups on heparin are important for the Robo1–heparin interaction, the N-sulfo and 6-O-sulfo groups are essential for the Robo1–heparin binding. Examination of differently sized heparin oligosaccharides and different GAGs also demonstrated that Robo1 prefers to bind full-length heparin chains and that GAGs with higher sulfation levels show increased Robo1 binding affinities.     

Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan

The potent oxidants hypochlorous acid (HOCl) and hypobromous acid (HOBr) are produced extracellularly by myeloperoxidase, following release of this enzyme from activated leukocytes. The subendothelial extracellular matrix is a key site for deposition of myeloperoxidase and damage by myeloperoxidase-derived oxidants, with this damage implicated in the impairment of vascular cell function during acute inflammatory responses and chronic inflammatory diseases such as atherosclerosis. The heparan sulfate proteoglycan perlecan, a key component of the subendothelial extracellular matrix, regulates important cellular processes and is a potential target for HOCl and HOBr. It is shown here that perlecan binds myeloperoxidase via its heparan sulfate side chains and that this enhances oxidative damage by myeloperoxidase-derived HOCl and HOBr. This damage involved selective degradation of the perlecan protein core without detectable alteration of its heparan sulfate side chains, despite the presence of reactive GlcNH₂ residing within this glycosaminoglycan. Modification of the protein core by HOCl and HOBr (measured by loss of immunological recognition of native protein epitopes and the appearance of oxidatively-modified protein epitopes) was associated with an impairment of its ability to support endothelial cell adhesion, with this observed at a pathologically-achievable oxidant dose of 425 nmol oxidant/mg protein. In contrast, the heparan sulfate chains of HOCl/HOBr-modified perlecan retained their ability to bind FGF-2 and collagen V and were able to promote FGF-2-dependent cellular proliferation. Collectively, these data highlight the potential role of perlecan oxidation, and consequent deregulation of cell function, in vascular injuries by myeloperoxidase-derived HOCl and HOBr.     

Glypican-3 modulates inhibitory Bmp2-Smad signaling to control renal development in vivo

Renal branching morphogenesis, defined as growth and branching of the ureteric bud (UB), is a tightly regulated process controlled by growth factor-dependent tissue interactions. Previously, using in vitro models of branching morphogenesis, we demonstrated that BMP2 signals via its intracellular effectors, SMAD1 and SMAD4, to control UB cell proliferation and branching in a manner modulated by Glypican-3 (GPC3), a cell surface heparan sulfate proteoglycan. Here, we used loss-of-function genetic mouse models to investigate the functions of Bmp2 and Gpc3-Bmp2 interactions in vivo. Progressively greater increases in UB cell proliferation were observed in Bmp2+/-, Smad4+/-, and Bmp2+/-; Smad4+/- mice compared to Wt. This increased cell proliferation was accompanied by a significant increase in UB branching in Smad4+/- and Bmp2+/-;Smad4+/- mice compared to Wt. Reduction of Gpc3 gene dosage also increased UB cell proliferation, an effect that was enhanced in Gpc3+/-;Bmp2+/- mice to an extent greater than the sum of that observed in Gpc3+/- and Bmp2+/- mice. Reduction of both Gpc3 and Bmp2 gene dosage enhanced cell proliferation in the metanephric mesenchyme compared to Wt, an effect not observed in either Bmp2+/- or Gpc3+/- mice. Phosphorylation of SMAD1, a measure of SMAD1 activation, was progressively decreased in Gpc3+/- and Gpc3+/-;Bmp2+/- mice compared to Wt, suggesting that Gpc3 stimulates Bmp2-dependent SMAD signaling in vivo. These results demonstrate that BMP2-SMAD signaling, modulated by GPC3, inhibits renal branching morphogenesis in vivo.