Heparan sulfate regulates fibrillin-1 N- and C-terminal interactions

Fibrillin-1 N- and C-terminal heparin binding sites have been characterized. An unprocessed monomeric N-terminal fragment (PF1) induced a very high heparin binding response, indicating heparin-mediated multimerization. Using PF1 deletion and short fragments, a heparin binding site was localized within the domain encoded by exon 7 after the first hybrid domain. Rodent embryonic fibroblasts adhered to PF1 and deletion fragments, and, when cells were plated on fibrillin-1 or fibronectin Arg-Gly-Asp cell-binding fragments, cells showed heparin-dependent spreading and focal contact formation in response to soluble PF1. Within domains encoded by exons 59-62 near the fibrillin-1 C terminus are novel conformation-dependent high affinity heparin and tropoelastin binding sites. Heparin disrupted tropoelastin binding but did not disrupt N- and C-terminal fibrillin-1 interactions. Thus, fibrillin-1 N-terminal interactions with heparin/heparan sulfate directly influence cell behavior, whereas C-terminal interactions with heparin/heparan sulfate regulate elastin deposition. These data highlight how heparin/heparan sulfate controls fibrillin-1 interactions.     

Heparan sulfate regulates self-renewal and pluripotency of embryonic stem cells

Embryonic stem (ES) cell self-renewal and pluripotency are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct3/4 and Nanog. The signaling cascades are activated by extrinsic factors, such as leukemia inhibitory factor, bone morphogenic protein, and Wnt. However, the mechanism that regulates extrinsic signaling in ES cells is unknown. Heparan sulfate (HS) chains are ubiquitously present as the cell surface proteoglycans and are known to play crucial roles in regulating several signaling pathways. Here we investigated whether HS chains on ES cells are involved in regulating signaling pathways that are important for the maintenance of ES cells. RNA interference-mediated knockdown of HS chain elongation inhibited mouse ES cell self-renewal and induced spontaneous differentiation of the cells into extraembryonic endoderm. Furthermore, autocrine/paracrine Wnt/beta-catenin signaling through HS chains was found to be required for the regulation of Nanog expression. We propose that HS chains are important for the extrinsic signaling required for mouse ES cell self-renewal and pluripotency.     

Rac regulates cardiovascular superoxide through diverse molecular interactions: more than a binary GTP switch

The small G protein Rac has been implicated in multiple cardiovascular processes. Rac has two major functions: 1) it regulates the organization of the actin cytoskeleton, and 2) it controls the activity of the key enzyme complex NADPH oxidase to control superoxide production in both phagocytes and nonphagocytic cells. In phagocytes, superoxide derived from NADPH has a bactericidal function, whereas Rac-derived superoxide in the cardiovascular system has a diverse array of functions that have recently been a subject of intense interest. Rac is differentially activated by cellular receptors coupled to distinct Rac-activating adapter molecules, with each leading to pathway-specific arrays of downstream effects. Thus it may be important to investigate not just whether Rac is activated but also where, how, and for what effector. An understanding of the biochemical functions of Rac and its effectors lays the groundwork for a dissection of the exact array of effects produced by Rac in common cardiovascular processes, including cardiac and vascular hypertrophy, hypertension, leukocyte migration, platelet biology, and atherosclerosis. In addition, investigation of the spatiotemporal regulation of both Rac activation and consequent superoxide generation may produce new insights into the development of targeted antioxidant therapies for cardiovascular disease and enhance our understanding of important cardiovascular drugs, including angiotensin II antagonists and statins, that may depend on Rac modulation for their effect. small G proteins; antioxidants; atherosclerosis; NADPH oxidase     

Heparan sulfate regulates VEGF165- and VEGF121-mediated vascular hyperpermeability

VEGF was first described as vascular permeability factor, a potent inducer of vascular leakage. Genetic evidence indicates that VEGF-stimulated endothelial proliferation in vitro and angiogenesis in vivo depend on heparan sulfate, but a requirement for heparan sulfate in vascular hyperpermeability has not been explored. Here we show that altering endothelial cell heparan sulfate biosynthesis in vivo decreases hyperpermeability induced by both VEGF(165) and VEGF(121). Because VEGF(121) does not bind heparan sulfate, the requirement for heparan sulfate suggested that it interacted with VEGF receptors rather than the ligand. By applying proximity ligation assays to primary brain endothelial cells, we show a direct interaction in situ between heparan sulfate and the VEGF receptor, VEGFR2. Furthermore, the number of heparan sulfate-VEGFR2 complexes increased in response to both VEGF(165) and VEGF(121). Genetic or heparin lyase-mediated alteration of endothelial heparan sulfate attenuated phosphorylation of VEGFR2 in response to VEGF(165) and VEGF(121), suggesting that the functional VEGF receptor complex contains heparan sulfate. Pharmacological blockade of heparan sulfate-protein interactions inhibited hyperpermeability in vivo, suggesting heparan sulfate as a potential target for treating hyperpermeability associated with ischemic disease.     

Heparan sulfate regulates targeting of syndecan-1 to a functional domain on the cell surface

In polarized B lymphoid cells, syndecan-1 is targeted specifically to a discrete membrane domain termed the uropod that is located at the cell's trailing edge. Within this functional domain, syndecan-1 promotes cell-cell adhesion and concentration of heparin binding growth factors. The present study reveals the surprising finding that targeting of syndecan-1 to uropods is mediated by its heparan sulfate chains and that targeting is regulated by cell surface events rather than solely by intracellular mechanisms. The addition of exogenous heparin or the treatment of polarized cells with heparitinase initiates a rapid and dramatic redistribution of uropod syndecan-1 over the entire cell surface, and a mutated syndecan-1 lacking heparan sulfate chains fails to concentrate within uropods. Interestingly, the heparan sulfate-bearing proteoglycans glypican-1 and beta glycan fail to concentrate in uropods, indicating that targeting may require heparan sulfate structural motifs unique to syndecan-1 or that the core protein of syndecan-1 participates in specific interactions that promote heparan sulfate-mediated targeting. These findings suggest functional specificity for syndecan-1 within uropods and, in addition, reveal a novel mechanism for the targeting of molecules to discrete membrane subcellular domains via heparan sulfate.     

Leucine-rich glioma inactivated 3 regulates adipogenesis through ADAM23

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein and a member of LGI/epitempin family. We previously showed that LGI3 was highly expressed in brain and played regulatory roles in neuronal exocytosis and differentiation. Besides the nervous system, LGI3 was shown to be expressed in diverse tissues. In this study, we found that LGI3 and its receptor candidate ADAM23 were expressed in adipose tissues and 3T3-L1 cells. 3T3-L1 preadipocytes secreted a 60-kDa protein, a major secreted form of LGI3, which declined with adipocyte differentiation. LGI3 was also expressed in adipose tissue macrophages in the ob/ob mice and in macrophage cell line. The 60-kDa LGI3 protein was selectively increased in the ob/ob adipose tissues comparing with the lean mice. Pull-down experiments, coimmunoprecipitation and immunocytochemistry indicated that LGI3 associated with ADAM23 in adipose tissues and 3T3-L1 cells. Knockdown of LGI3 or ADAM23 by siRNA increased adipogenesis in 3T3-L1 cells. Treatment with LGI3 protein did not affect preadipocyte proliferation but attenuated adipogenesis and this effect was reversed by siRNA-mediated knockdown of ADAM23. Taken together, we propose that LGI3 may be a candidate adipokine that is perturbed in obesity and suppresses adipogenesis through its receptor, ADAM23.     

Heparan sulfate proteoglycan as a plasma membrane carrier

The plasma membrane defines the border of living cells and provides a barrier to extracellular components. Advances in molecular biology have resulted in the development of novel therapeutic strategies (e.g. gene therapy and cellular protein delivery) which rely on the entry of charged macromolecules into the intracellular compartment. Recent reports demonstrate an intriguing role for heparan sulfate proteoglycans in cellular internalization of viruses, basic peptides and polycation-nucleic-acid complexes and the possibility that they have important implications for gene transfer and protein delivery to mammalian cells. This review focuses on heparan sulfate proteoglycan as a plasma membrane carrier.     

Heparan sulfate proteoglycan as a cell-surface endocytosis receptor

How various macromolecules are exchanged between cells and how they gain entry into recipient cells are fundamental questions in cell biology with important implications e.g. non-viral drug delivery, infectious disease, metabolic disorders, and cancer. The role of heparan sulfate proteoglycan (HSPG) as a cell-surface receptor of diverse macromolecular cargo has recently been manifested. Exosomes, cell penetrating peptides, polycation–nucleic acid complexes, viruses, lipoproteins, growth factors and morphogens among other ligands enter cells through HSPG-mediated endocytosis. Key questions that partially have been unraveled over recent years include the respective roles of HSPG core protein and HS chain structure specificity for macromolecular cargo endocytosis, the down-stream intracellular signaling events involved in HSPG-dependent membrane invagination and vesicle formation, and the biological significance of the HSPG transport pathway. Here, we discuss the intriguing role of HSPGs as a major entry pathway of macromolecules in mammalian cells with emphasis on recent in vitro and in vivo data that provide compelling evidence of HSPG as an autonomous endocytosis receptor.     

Heparan sulfate provides a mechanism to respond to FGFR2b and control regenerative expansion

Fast varying 3-O-sulfated groups in epithelial KIT+ progenitors provides a cellular mechanism to respond to FGFR2b-mediated signaling and expansion of fetal and adult progenitors. This is allowed by the synthesis of 3-O-sulfated heparan sulfate that up-regulate KIT and 3-O-sulfotransferase enzymes, augmenting 3-O-sulfated heparan sulfate.     

Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's beta-secretase

Cleavage of amyloid precursor protein (APP) by the Alzheimer's beta-secretase (BACE1) is a key step in generating amyloid beta-peptide, the main component of amyloid plaques. Here we report evidence that heparan sulfate (HS) interacts with beta-site APP-cleaving enzyme (BACE) 1 and regulates its cleavage of APP. We show that HS and heparin interact directly with BACE1 and inhibit in vitro processing of peptide and APP substrates. Inhibitory activity is dependent on saccharide size and specific structural characteristics, and the mechanism of action involves blocking access of substrate to the active site. In cellular assays, HS specifically inhibits BACE1 cleavage of APP but not alternative cleavage by alpha-secretase. Endogenous HS immunoprecipitates with BACE1 and colocalizes with BACE1 in the Golgi complex and at the cell surface, two of its putative sites of action. Furthermore, inhibition of cellular HS synthesis results in enhanced BACE1 activity. Our findings identify HS as a natural regulator of BACE1 and suggest a novel mechanism for control of APP processing.     

Heparan sulfate regulates the number and centrosome positioning of Drosophila male germline stem cells

Stem cell division is tightly controlled via secreted signaling factors and cell adhesion molecules provided from local niche structures. Molecular mechanisms by which each niche component regulates stem cell behaviors remain to be elucidated. Here we show that heparan sulfate (HS), a class of glycosaminoglycan chains, regulates the number and asymmetric division of germline stem cells (GSCs) in the Drosophila testis. We found that GSC number is sensitive to the levels of 6-O sulfate groups on HS. Loss of 6-O sulfation also disrupted normal positioning of centrosomes, a process required for asymmetric division of GSCs. Blocking HS sulfation specifically in the niche, termed the hub, led to increased GSC numbers and mispositioning of centrosomes. The same treatment also perturbed the enrichment of Apc2, a component of the centrosome-anchoring machinery, at the hub–GSC interface. This perturbation of the centrosome-anchoring process ultimately led to an increase in the rate of spindle misorientation and symmetric GSC division. This study shows that specific HS modifications provide a novel regulatory mechanism for stem cell asymmetric division. The results also suggest that HS-mediated niche signaling acts upstream of GSC division orientation control.     

Regulation of human ADAM 12 protease by the prodomain. Evidence for a functional cysteine switch

The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins that are believed to play key roles in cell-cell and cell-matrix interactions. We have shown recently that human ADAM 12-S (meltrin alpha) is an active metalloprotease. It is synthesized as a zymogen, with the prodomain maintaining the protease in a latent form. We now provide evidence that the latency mechanism of ADAM 12 can be explained by the cysteine switch model, in which coordination of Zn2+ in the active site of the catalytic domain by a cysteine residue in the prodomain is critical for inhibition of the protease. Replacing Cys179 with other amino acids results in an ADAM 12 proform that is proteolytically active, but latency can be restored by placing cysteine at other positions in the propeptide. None of the amino acids adjacent to the crucial cysteine residue is essential for blocking activity of the protease domain. In addition to its latency function, the prodomain is required for exit of ADAM 12 protease from the endoplasmic reticulum. Tissue inhibitor of metalloprotease-1, -2, and -3 were not found to block proteolytic activity of ADAM 12, hence a physiological inhibitor of ADAM 12 protease in the extracellular environment remains to be identified.     

Alpha-catenin is a molecular switch that binds E-cadherin-beta-catenin and regulates actin-filament assembly

Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that alpha-catenin links the adhesion protein E-cadherin through beta-catenin to the actin cytoskeleton, in the accompanying paper we report that alpha-catenin does not bind simultaneously to both E-cadherin-beta-catenin and actin filaments. Here we demonstrate that alpha-catenin exists as a monomer or a homodimer with different binding properties. Monomeric alpha-catenin binds more strongly to E-cadherin-beta-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that alpha-catenin is an allosteric protein. Significantly, alpha-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for alpha-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion.     

Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I

Human beta1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis. GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Gal beta 1-3Gal beta 1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. We have now determined the crystal structure of GlcAT-1 at 2.3 A in the presence of the donor substrate product UDP, the catalytic Mn(2+) ion, and the acceptor substrate analog Gal beta 1-3Gal beta 1-4Xyl. The enzyme is a alpha/beta protein with two subdomains that constitute the donor and acceptor substrate binding site. The active site residues lie in a cleft extending across both subdomains in which the trisaccharide molecule is oriented perpendicular to the UDP. Residues Glu(227), Asp(252), and Glu(281) dictate the binding orientation of the terminal Gal-2 moiety. Residue Glu(281) is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor. The conserved DXD motif (Asp(194), Asp(195), Asp(196)) has direct interaction with the ribose of the UDP molecule as well as with the Mn(2+) ion. The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.     

Heparan sulfate: decoding a dynamic multifunctional cell regulator

The heparan sulfates are a family of cell-surface and matrix polysaccharides with an incredible degree of structural diversity that are distributed widely in virtually all metazoan organisms. Recent genetic, biochemical and cell-biological studies have led to increased understanding of the biosynthetic mechanisms that produce these complex molecules, as well as their functional versatility in regulating protein activities. The dynamic expression of heparan sulfates with differing sugar sequences suggests a new concept in which the repertoire of sequences produced by a particular cell or tissue is designated its 'heparanome'. This review discusses recent developments and surveys emerging experimental strategies that hold promise for revealing the functional specificity and mechanisms of action of heparan sulfates as multifunctional cell regulators.     

Selenite negatively regulates caspase-3 through a redox mechanism

Selenium, an essential biological trace element, exerts its modulatory effects in a variety of cellular events including cell survival and death. In our study we observed that selenite protects HEK293 cells from cell death induced by ultraviolet B radiation (UVB). Exposure of HEK293 cells to UVB radiation resulted in the activation of caspase-3-like protease activity, and pretreatment of the cells with z-DEVD-fmk (N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone), a caspase-3 inhibitor, prevented UVB-induced cell death. Interestingly, enzymatic activity of caspase-3-like protease in cell lysates of UVB-exposed cells was repressed in vitro by the presence of selenite. Selenite also inhibited the in vitro activity of purified recombinant caspase-3 in cleaving Ac-DEVD-pNA (N-acetyl-Asp-Glu-Asp-p-nitroanilide) or ICAD(L) (inhibitor of a caspase-activated deoxyribonuclease) and in the induction of DNA fragmentation. The inhibitory action of selenite on a recombinant active caspase-3 could be reversed by sulfhydryl reducing agents, such as dithiothreitol and beta-mercaptoethanol. Furthermore, pretreatment of cells with selenite suppressed the stimulation of the caspase-3-like protease activity in UVB-exposed cells, whereas dithiothreitol and beta-mercaptoethanol reversed this suppression of the enzymatic activity. Taken together, our data suggest that selenite inhibits caspase-3-like protease activity through a redox mechanism and that inhibition of caspase-3-like protease activity may be the mechanism by which selenite exerts its protective effect against UVB-induced cell death.     

RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism

In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as a Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85alpha and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1.     

Heparan sulfate is a cellular receptor for purified infectious prions

Prions replicate in the host cell by the self-propagating refolding of the normal cell surface protein, PrP(C), into a beta-sheet-rich conformer, PrP(Sc). Exposure of cells to prion-infected material and subsequent endocytosis can sometimes result in the establishment of an infected culture. However, the relevant cell surface receptors have remained unknown. We have previously shown that cellular heparan sulfates (HS) are involved in the ongoing formation of scrapie prion protein (PrP(Sc)) in chronically infected cells. Here we studied the initial steps in the internalization of prions and in the infection of cells. Purified prion "rods" are arguably the purest prion preparation available. The only proteinaceous component of rods is PrP(Sc). Mouse neuroblastoma N2a, hypothalamus GT1-1, and Chinese hamster ovary cells efficiently bound both hamster and mouse prion rods (at 4 degrees C) and internalized them (at 37 degrees C). Treating cells with bacterial heparinase III or chlorate (a general inhibitor of sulfation) strongly reduced both binding and uptake of rods, whereas chondroitinase ABC was inactive. These results suggested that the cell surface receptor of prion rods involves sulfated HS chains. Sulfated glycans inhibited both binding and uptake of rods, probably by competing with the binding of rods to cellular HS. Treatments that prevented endocytosis of rods also prevented the de novo infection of GT1-1 cells when applied during their initial exposure to prions. These results indicate that HS are an essential part of the cellular receptor used both for prion uptake and for cell infection. Cellular HS thus play a dual role in prion propagation, both as a cofactor for PrP(Sc) synthesis and as a receptor for productive prion uptake.     

Heparan sulfate modulates kinin release by Trypanosoma cruzi through the activity of cruzipain.

Trypanosoma cruzi activates the kinin pathway through the activity of its major cysteine proteinase, cruzipain. Because kininogen molecules may be displayed on cell surfaces by binding to glycosaminoglycans, we examined whether the ability of cruzipain to release kinins from high molecular weight kininogen (HK) is modulated by heparan sulfate (HS). Kinetic assays show that HS reduces the cysteine proteinase inhibitory activity (K(i app)) of HK about 10-fold. Conversely, the catalytic efficiency of cruzipain on kinin-related synthetic fluorogenic substrates is enhanced up to 6-fold in the presence of HS. Analysis of the HK breakdown products generated by cruzipain indicated that HS changes the pattern of HK cleavage products. Direct measurements of bradykinin demonstrated an up to 35-fold increase in cruzipain-mediated kinin liberation in the presence of HS. Similarly, kinin release by living trypomastigotes increased up to 10-fold in the presence of HS. These studies suggest that the efficiency of T. cruzi to initiate kinin release is potently enhanced by the mutual interactions between cruzipain, HK, and heparan sulfate proteoglycans.