Cultured monolayers of NMuMG mouse mammary epithelial cells have augmented amounts of cell surface chondroitin sulfate glycosaminoglycan (GAG) when cultured in transforming growth factor-beta (TGF-beta), presumably because of increased synthesis on their cell surface proteoglycan (named syndecan), previously shown to contain chondroitin sulfate and heparan sulfate GAG. This increase occurs throughout the monolayer as shown using soluble thrombospondin as a binding probe. However, comparison of staining intensity of the GAG chains and syndecan core protein suggests variability among cells in the attachment of GAG chains to the core protein. Characterization of purified syndecan confirms the enhanced addition of chondroitin sulfate in TGF-beta: (a) radiosulfate incorporation into chondroitin sulfate is increased 6.2-fold in this proteoglycan fraction and heparan sulfate is increased 1.8-fold, despite no apparent increase in amount of core protein per cell, and (b) the size and density of the proteoglycan are increased, but reduced by removal of chondroitin sulfate. This is shown in part by treatment of the cells with 0.5 mM xyloside that blocks the chondroitin sulfate addition without affecting heparan sulfate. Higher xyloside concentrations block heparan sulfate as well and syndecan appears at the cell surface as core protein without GAG chains. The enhanced amount of GAG on syndecan is partly attributed to an increase in chain length. Whereas this accounts for the additional heparan sulfate synthesis, it is insufficient to explain the total increase in chondroitin sulfate; an approximately threefold increase in chondroitin sulfate chain addition occurs as well, confirmed by assessing chondroitin sulfate ABC lyase (ABCase)-generated chondroitin sulfate linkage stubs on the core protein. One of the effects of TGF-beta during embryonic tissue interactions is likely to be the enhanced synthesis of chondroitin sulfate chains on this cell surface proteoglycan.
Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of this pervasive cell recognition is not understood. We demonstrate here that binding to the substratum and to host cells is partially mediated by interaction with sulfated glycosaminoglycans (GAGs). Addition of excess soluble GAGs blocked parasite attachment to serum-coated glass, thereby preventing gliding motility of extracellular parasites. Similarly, excess soluble GAGs decreased the attachment of parasites to human host cells from a variety of lineages, including monocytic, fibroblast, endothelial, epithelial, and macrophage cells. The inhibition of parasite attachment by GAGs was observed with heparin and heparan sulfate and also with chondroitin sulfates, indicating that the ligands for attachment are capable of recognizing a broad range of GAGs. The importance of sulfated proteoglycan recognition was further supported by the demonstration that GAG-deficient mutant host cells, and wild-type cells treated enzymatically to remove GAGs, were partially resistant to parasite invasion. Collectively, these studies reveal that sulfated proteoglycans are one determinant used for substrate and cell recognition by Toxoplasma. The widespread distribution of these receptors may contribute to the broad host and tissue ranges of this highly successful intracellular parasite.
The authors provide a detailed analysis of the compartmentalization in the human retina of glycosaminoglycans (GAGs), by both chain type and sulfation pattern. This reference map provides a basis for understanding the functional regulation of GAG-binding proteins in health and disease processes.
To map the distribution of different classes of glycosaminoglycans (GAGs) in the healthy human retina, choroid, and sclera.
Frozen tissue sections were made from adult human donor eyes. The GAG chains of proteoglycans (PGs) were detected with antibodies directed against various GAG structures (either directly or after pretreatment with GAG-degrading enzymes); hyaluronan (HA) was detected using biotinylated recombinant G1-domain of human versican. The primary detection reagents were identified with FITC-labeled probes and analyzed by fluorescence microscopy.
Heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and HA were present throughout the retina and choroid, but keratan sulfate (KS) was detected only in the sclera. HS labeling was particularly strong in basement membrane–containing structures, the nerve fiber layer (NFL), and retinal pigment epithelium (RPE)—for example, intense staining was seen with an antibody that binds strongly to sequences containing 3-O-sulfation in the internal limiting membrane (ILM) and in the basement membrane of blood vessels. Unsulfated CS was seen throughout the retina, particularly in the ILM and interphotoreceptor matrix (IPM) with 6-O-sulfated CS also prominent in the IPM. There was labeling for DS throughout the retina and choroid, especially in the NFL, ganglion cell layer, and blood vessels.
The detection of GAG chains with specific probes and fluorescence microscopy provides for the first time a detailed analysis of their compartmentalization in the human retina, by both GAG chain type and sulfation pattern. This reference map provides a basis for understanding the functional regulation of GAG-binding proteins in health and disease processes.
Leptospirosis is a global public health problem, primarily in the tropical developing world. The pathogenic mechanisms of the causative agents, several members of the genus Leptospira, have been underinvestigated. The exception to this trend has been the demonstration of the binding of pathogenic leptospires to the extracellular matrix (ECM) and its components. In this work, interactions of Leptospira interrogans bacteria with mammalian cells, rather than the ECM, were examined. The bacteria bound more efficiently to the cells than to the ECM, and a portion of this cell-binding activity was attributable to attachment to glycosaminoglycan (GAG) chains of proteoglycans (PGs). Chondroitin sulfate B PGs appeared to be the primary targets of L. interrogans attachment, while heparan sulfate PGs were much less important. Inhibition of GAG/PG-mediated attachment resulted in partial inhibition of bacterial attachment, suggesting that additional receptors for L. interrogans await identification. GAG binding may participate in the pathogenesis of leptospirosis within the host animal. In addition, because GAGs are expressed on the luminal aspects of epithelial cells in the proximal tubules of the kidneys, this activity may play a role in targeting the bacteria to this critical site. Because GAGs are shed in the urine, GAG binding may also be important for transmission to new hosts through the environment.
We have proposed a model in which fibroblast growth factor (FGF) signalling requires the interaction of FGF with at least two FGF receptors, a heparan sulfate proteoglycan (HSPG) and a tyrosine kinase. Since FGF may be a key mediator of skeletal muscle differentiation, we examined the synthesis of glycosaminoglycans in MM14 skeletal muscle myoblasts and their participation in FGF signalling. Proliferating and differentiated MM14 cells exhibit similar levels of HSPG, while differentiated cells exhibit reduced levels of chondroitin sulfate proteoglycans and heparan sulfate chains. HSPGs, including syndecan, present in proliferating cells bind bFGF, while the majority of chondroitin sulfate and heparan sulfate chains do not. Treatment of skeletal muscle cells with chlorate, a reversible inhibitor of glycosaminoglycan sulfation, was used to examine the requirement of sulfated proteoglycans for FGF signalling. Chlorate treatment reduced glycosaminoglycan sulfation by 90% and binding of FGF to high affinity sites by 80%. Chlorate treatment of MM14 myoblasts abrogated the biological activity of acidic, basic, and Kaposi's sarcoma FGFs resulting in terminal differentiation. Chlorate inhibition of FGF signalling was reversed by the simultaneous addition of sodium sulfate or heparin. Further support for a direct role of heparan sulfate proteoglycans in fibroblast growth factor signal transduction was demonstrated by the ability of heparitinase to inhibit basic FGF binding and biological activity. These results suggest that activation of FGF receptors by acidic, basic or Kaposi's sarcoma FGF requires simultaneous binding to a HSPG and the tyrosine kinase receptor. Skeletal muscle differentiation in vivo may be dependent on FGFs, FGF tyrosine kinase receptors, and HSPGs. The regulation of these molecules may then be expected to have important implications for skeletal muscle development and regeneration.
Proteoglycans are important components of the extracellular matrix of all tissues. Proteoglycans are comprised of a core protein and one or more covalently attached glycosaminoglycan (GAG) chains. The major chondroitin sulfate (CS) and dermatan sulfate (DS) proteoglycans are aggrecan, versican, biglycan and decorin. Cells synthesize GAGs of natural or basal lengths and the GAG chains are subject to considerable growth factor, hormonal and metabolic regulation to yield longer GAG chains with altered structure and function. The mechanism by which the CS/DS GAG chains are polymerized is unknown. Recent work has identified several monosaccharide transferases which when co-expressed yield GAG polymers and the length of the polymers depends upon the pair of enzymes coexpressed. The further extension of these chains is regulated by signaling pathways. Inhibition of these latter pathways may be a therapeutic target to prevent the elongation which is associated with increased binding of atherogenic lipids and the disease process of atherosclerosis.
Proteoglycans; chondroitin sulfate; glycosaminoglycans; enzymology; polymerases; signaling.
Fibroblast growth factor-18 (FGF-18) has been shown to regulate the growth plate chondrocyte proliferation, hypertrophy and cartilage vascularization necessary for endochondral ossification. The heparan sulfate proteoglycan perlecan is also critical for growth platechondrocyte proliferation. FGF-18 null mice exhibit a skeletal dwarfism similar to that of perlecan null mice. Growth plate perlecan contains chondroitin sulfate (CS) and heparan sulfate (HS) chains and FGF-18 is known to bind to heparin and to heparan sulfate from some sources. We used cationic filtration and immunoprecipitation assays to investigate the binding of FGF-18 to perlecan purified from the growth plate and to recombinant perlecan domains expressed in COS-7 cells. FGF-18 bound to perlecan with a kd of 145 nM. Near saturation, ~103 molecules of FGF-18 bound per molecule of perlecan. At the lower concentrations used, FGF-18 bound with a kd of 27.8 nM. This binding was not significantly altered by chondroitinase nor heparitinase digestion of perlecan, but was substantially and significantly reduced by reduction and alkylation of the perlecan core protein. This indicates that the perlecan core protein (and not the CS nor HS chains) is involved in FGF-18 binding. FGF-18 bound equally to full length perlecan purified from the growth plate and to recombinant domains I-III and III of perlecan. These data indicate that low affinity binding sites for FGF-18 are present in cysteine-rich regions of domain III of perlecan. FGF-18 stimulated 3H-thymidine incorporation in growth plate chondrocyte cultures derived from the lower and upper proliferating zones by 9- and 14-fold, respectively. The addition of perlecan reversed this increased incorporation in the lower proliferating chondrocytes by 74% and in the upper proliferating cells by 37%. These results suggest that perlecan can bind FGF-18 and alter the mitogenic effect of FGF-18 on growth plate chondrocytes.
Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and at the cell-surface where they modulate the binding and activity of a variety of growth factors and other molecules. Most of the functions of HSPGs are mediated by the variable sulfated glycosaminoglycan (GAG) chains attached to a core protein. Sulfation of the GAG chain is key as evidenced by the renal agenesis phenotype in mice deficient in the HS biosynthetic enzyme, heparan sulfate 2-O sulfotransferase (Hs2st; an enzyme which catalyzes the 2-O-sulfation of uronic acids in heparan sulfate). We have recently demonstrated that this phenotype is likely due to a defect in induction of the metanephric mesenchyme (MM), which along with the ureteric bud (UB), is responsible for the mutually inductive interactions in the developing kidney (Shah et al., 2010). Here, we sought to elucidate the role of variable HS sulfation in UB branching morphogenesis, particularly the role of 6-O sulfation. Endogenous HS was localized along the length of the UB suggesting a role in limiting growth factors and other molecules to specific regions of the UB. Treatment of cultures of whole embryonic kidney with variably desulfated heparin compounds indicated a requirement of 6O-sulfation in the growth and branching of the UB. In support of this notion, branching morphogenesis of the isolated UB was found to be more sensitive to the HS 6-O sulfation modification when compared to the 2-O sulfation modification. In addition, a variety of known UB branching morphogens (i.e., pleiotrophin, heregulin, FGF1 and GDNF) were found to have a higher affinity for 6-O sulfated heparin providing additional support for the notion that this HS modification is important for robust UB branching morphogenesis. Taken together with earlier studies, these findings suggest a general mechanism for spatio-temporal HS regulation of growth factor activity along the branching UB and in the developing MM.
kidney development; ureteric bud; branching morphogenesis; heparan sulfate; growth factor
Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology.
By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary structure analysis suggested helices and sheets in both recombinant species. rhPln.D1 demonstrated binding to rhFGF-2 with an apparent kD of 2 ± 0.2 nM with almost complete susceptibility to digestion by heparinase III in ligand blot analysis but not to chondroitinase digestion. Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs. Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor.
With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.
Heparan sulfate has been shown to be an important mediator of Plasmodium sporozoite homing and invasion of the liver, but the role of this glycosaminoglycan in mosquito vector host-sporozoite interactions is unknown. We have biochemically characterized the function of Anopheles gambiae peptide-O-xylosyltransferase (AgOXT1) and confirmed that AgOXT1 can modify peptides representing model heparan and chondroitin sulfate proteoglycans in vitro. Moreover, we also demonstrated that the mosquito salivary gland basal lamina proteoglycans are modified by heparan sulfate. We used RNAi-mediated knockdown of heparan sulfate biosynthesis in An. gambiae salivary glands to determine if Plasmodium falciparum sporozoites that are released from mosquito midgut oocysts use salivary gland heparan sulfate as a receptor for tissue invasion. Our data suggests that salivary gland basal lamina heparan sulfate glycosaminoglycans only partially mediate midgut sporozoite invasion of this tissue, and that in the absence of heparan sulfate, the presence of other surface co-receptors is sufficient to facilitate parasite entry.
Anopheles; malaria; glycobiology; glycosaminoglycan; cell invasion
Glycosaminoglycans (GAGs) are linear polysaccharides that are found in the extracellular matrix and biological fluids of animals where they interact with hundreds of proteins and perform a variety of critical roles. There are five classes of animal GAGs: heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronan (HA). Many biological functions can be monitored directly by their impact on GAG quantity. Thus, simple, sensitive, and robust GAG quantification methods are needed for the development of biomarkers. We have systematically compared three available GAG quantification assays including an HPLC-based assay, a simplified Alcian Blue assay, and a miniaturized carbazole assay. The carbazole and Alcian Blue assays were reproducible and simple to perform in general lab settings, but had important limitations: The carbazole assay could not detect KS and it overestimated GAGs that were contaminated with salts or dissolved in PBS. The Alcian Blue assay detected only those GAGs that were sulfated. In contrast, while the HPLC method was time-consuming, it was a robust and sensitive assay that not only detected all GAGs but also quantified glucosamine-GAGs and galactosamine-GAGs simultaneously. The HPLC assay was not affected by salt or level of GAG sulfation and it yielded reproducible values for all types of GAGs tested. These results suggest that an automated HPLC assay would be generally useful for the routine measurement of a panel of GAG-based biomarkers while the carbazole assay and the Alcian Blue assays could prove valuable for more specific purposes.
The interactions between glycosaminoglycans (GAGs), important components of the extracellular matrix, and proteins such as growth factors and chemokines play critical roles in cellular regulation processes. Therefore, the design of GAG derivatives for the development of innovative materials with bio-like properties in terms of their interaction with regulatory proteins is of great interest for tissue engineering and regenerative medicine. Previous work on the chemokine interleukin-8 (IL-8) has focused on its interaction with heparin and heparan sulfate, which regulate chemokine function. However, the extracellular matrix contains other GAGs, such as hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS), which have so far not been characterized in terms of their distinct molecular recognition properties towards IL-8 in relation to their length and sulfation patterns. NMR and molecular modeling have been in great part the methods of choice to study the structural and recognition properties of GAGs and their protein complexes. However, separately these methods have challenges to cope with the high degree of similarity and flexibility that GAGs exhibit. In this work, we combine fluorescence spectroscopy, NMR experiments, docking and molecular dynamics simulations to study the configurational and recognition properties of IL-8 towards a series of HA and CS derivatives and DS. We analyze the effects of GAG length and sulfation patterns in binding strength and specificity, and the influence of GAG binding on IL-8 dimer formation. Our results highlight the importance of combining experimental and theoretical approaches to obtain a better understanding of the molecular recognition properties of GAG–protein systems.
GAG–protein interaction; glycosaminglycans (GAGs); interleukin-8 (IL-8); molecular modeling; NMR
Perlecan, a heparan sulfate proteoglycan, is widely distributed in developing and adult tissues and plays multiple, important physiological roles. Studies with knockout mouse models indicate that expression of perlecan and heparan sulfate is critical for proper skeletal morphogenesis. Heparan sulfate chains bind and potentiate the activities of various growth factors such as fibroblast growth factor 2 (FGF-2). Previous studies indicate that important biological activities are associated with the heparan sulfate-bearing domain I of perlecan (PlnDI; French et al. J. Bone Miner. Res. 17, 48, 2002). In the present study, we have used recombinant, glycosaminoglycan-bearing PlnDI to reconstitute three-dimensional scaffolds of collagen I. Collagen I fibrils bound PlnDI much better than native collagen I monomers or heat-denatured collagen I preparations. Heparitinase digestion demonstrated that recombinant PlnDI was substituted with heparan sulfate and that these heparan sulfate chains were critically important not only for efficient integration of PlnDI into scaffolds, but also for FGF-2 binding and retention. PlnDI-containing collagen I scaffolds to which FGF-2 was bound sustained growth of both MG63, an osteoblastic cell line, and human bone marrow stromal cells (hBMSCs) significantly better than scaffolds lacking either PlnDI or FGF-2. Collectively, these studies demonstrate the utility of PlnDI in creating scaffolds that better mimic natural extracellular matrices and better support key biological activities.
Heparan sulfate proteoglycans (HSPGs) are extracellular macromolecules found on virtually every cell type in eumetazoans. HSPGs are composed of a core protein covalently linked to glycosaminoglycan (GAG) sugar chains that bind and modulate the signaling efficiency of many ligands including Hedgehog (Hh), Wingless (Wg), and Bone Morphogenetic Proteins (BMPs). Here we show that in Drosophila, loss of HSPGs differentially affects embryonic Hh, Wg and BMP signaling. We find that a stage-specific block to GAG synthesis prevents HSPG expression during establishment of the BMP activity gradient that is critical for dorsal embryonic patterning. Subsequently GAG synthesis is initiated coincident with the onset of Hh and Wg signaling which require HSPGs. This temporal regulation is achieved by translational control of HSPG synthetic enzymes through Internal Ribosome Entry Sites (IRES). IRES-like features are conserved in GAG enzyme transcripts from diverse organisms arguing that this represents a novel evolutionarily conserved mechanism for regulating GAG synthesis and modulating growth factor activity.
Heparan sulfate proteoglycans; HSPG; Glycosaminoglycan; GAG; Bone morphogenetic protein signaling; Drosophila embryonic patterning; Decapentaplegic; Dpp; Hedgehog; Wingless
Cationic antimicrobial peptides (CAPs) with antitumor activity constitute a promising group of novel anticancer agents. These peptides induce lysis of cancer cells through interactions with the plasma membrane. It is not known which cancer cell membrane components influence their susceptibility to CAPs. We have previously shown that CAPs interact with the two glycosaminoglycans (GAGs), heparan sulfate (HS) and chondroitin sulfate (CS), which are present on the surface of most cells. The purpose of this study was to investigate the role of the two GAGs in the cytotoxic activity of CAPs.
Various cell lines, expressing different levels of cell surface GAGs, were exposed to bovine lactoferricin (LfcinB) and the designer peptide, KW5. The cytotoxic effect of the peptides was investigated by use of the colorimetric MTT viability assay. The cytotoxic effect on wild type CHO cells, expressing normal amounts of GAGs on the cell surface, and the mutant pgsA-745, that has no expression of GAGs on the cell surface, was also investigated.
We show that cells not expressing HS were more susceptible to CAPs than cells expressing HS at the cell surface. Further, exogenously added heparin inhibited the cytotoxic effect of the peptides. Chondroitin sulfate had no effect on the cytotoxic activity of KW5 and only minor effects on LfcinB cytotoxicity.
Our results show for the first time that negatively charged molecules at the surface of cancer cells inhibit the cytotoxic activity of CAPs. Our results indicate that HS at the surface of cancer cells sequesters CAPs away from the phospholipid bilayer and thereby impede their ability to induce cytolysis.
Monocyte/macrophage lineage cells are target cells in vivo for porcine circovirus 2 (PCV2) replication. The porcine monocytic cell line 3D4/31 supports PCV2 replication in vitro, and attachment and internalization kinetics of PCV2 have been established in these cells. However, PCV2 receptors remain unknown. Glycosaminoglycans (GAG) are used by several viruses as receptors. The present study examined the role of GAG in attachment and infection of PCV2. Heparin, heparan sulfate (HS), chondroitin sulfate B (CS-B), but not CS-A, and keratan sulfate reduced PCV2 infection when these GAG were incubated with PCV2 prior to and during inoculation of 3D4/31 cells. Enzymatic removal of HS and CS-B prior to PCV2 inoculation of 3D4/31 cells significantly reduced PCV2 infection. Similarly, when PCV2 virus-like particles (VLP) were allowed to bind onto 3D4/31 cells in the presence of heparin and CS-B, attachment was strongly reduced. Titration of field isolates and low- and high-passage laboratory strains of PCV2 in the presence of heparin significantly reduced PCV2 titers, showing that the capacity of PCV2 to bind GAG was not acquired during in vitro cultivation but is an intrinsic feature of wild-type virus. When Chinese hamster ovary (CHO) cells were inoculated with PCV2, relative percentages of PCV2-infected cells were 27% ± 8% for HS-deficient and 12% ± 10% for GAG-deficient cells compared to wild-type cells (100%). Furthermore, it was shown using heparin-Sepharose chromatography that both PCV2 and PCV2 VLP directly interacted with heparin. Together, these results show that HS and CS-B are attachment receptors for PCV2.
Thrombospondin is a 420-kD platelet alpha-granule glycoprotein that binds specifically to heparin. We examined adhesion to thrombospondin of CHO K1 cells and three mutant CHO lines with varying deficiencies in glycosaminoglycan (GAG) synthesis. In an experiment in which the parent line (K1) had 78% adherence to thrombospondin adsorbed to tissue culture plastic, CHO S745 cells, with less than 6% normal GAG synthesis had 11% adherence. CHO S677 cells, with decreased heparan sulfate proteoglycan but increased chondroitin sulfate proteoglycan, had 42% adherence. CHO S803 cells, with decreased heparan sulfate proteoglycan and normal chondroitin sulfate proteoglycan, had 31% adherence. Heparin inhibited K1 cell adhesion to thrombospondin, but not fibronectin, in a concentration-dependent manner. Dermatan sulfate but not chondroitin sulfate was also inhibitory. There was markedly decreased K1 cell adhesion to a thrombospondin core fragment that lacked the heparin binding NH2-terminal domain. Purified heparin binding domain, although poorly adhesive when adsorbed to substratum, inhibited cell adhesion to intact thrombospondin. Adhesion was better for all cell lines tested, including three human tumor cell lines, when thrombospondin was adsorbed at pH 4.0 compared with pH 7.4. When adsorption of thrombospondin was done at pH 7.4, cell adhesion was better when thrombospondin was adsorbed in the presence of greater than or equal to 0.6 mM calcium, compared to 0.1 mM calcium or EDTA. These findings suggest that thrombospondin can adsorb to plastic with varying degrees of exposure of a cell adhesion domain. We conclude that the thrombospondin cell adhesion receptor on CHO cells is a heparan sulfate proteoglycan, and that cell adhesion to thrombospondin depends on conformation of adsorbed thrombospondin.
Alveolar formation or alveolarization is orchestrated by a finely regulated and complex interaction between growth factors and extracellular matrix proteins. The lung parenchyma contains various extracellular matrix proteins including proteoglycans, which are composed of glycosaminoglycans (GAGs) linked to a protein core. Although GAGs are known to regulate growth factor distribution and activity according to their degree of sulfation the role of sulfated GAG in the respiratory system is not well understood. The degree of sulfation of GAGs is regulated in part, by sulfatases that remove sulfate groups. In vertebrates, the enzyme Sulfatase-Modifying Factor 1 (Sumf1) activates all sulfatases. Here we utilized mice lacking Sumf1−/− to study the importance of proteoglycan desulfation in lung development. The Sumf1−/− mice have normal lungs up until the onset of alveolarization at post-natal day 5 (P5). We detected increased deposition of sulfated GAG throughout the lung parenchyma and a decrease in alveolar septa formation. Moreover, stereological analysis showed that the alveolar volume is 20% larger in Sumf1−/− as compared to wild type (WT) mice at P10 and P30. Additionally, pulmonary function test were consistent with increased alveolar volume. Genetic experiments demonstrate that in Sumf1−/− mice arrest of alveolarization is independent of fibroblast growth factor signaling. In turn, the Sumf1−/− mice have increased transforming growth factor β (TGFβ) signaling and in vivo injection of TGFβ neutralizing antibody leads to normalization of alveolarization. Thus, absence of sulfatase activity increases sulfated GAG deposition in the lungs causing deregulation of TGFβ signaling and arrest of alveolarization.
Alveolarization; Sumf1; Glycosaminoglycans; Sulfatases; TGFβ
Perlecan (Pln) is a large proteoglycan that can bear HS (heparan sulfate) and chondroitin sulfate glycosaminoglycans. Previous studies have demonstrated that Pln can interact with growth factors and cell surfaces either via its constituent glycosaminoglycan chains or core protein. Herein, we summarize studies demonstrating spatially and temporally regulated expression of Pln mRNA and protein in developing and mature cartilage. Mutations either in the Pln gene or in genes involved in glycosaminoglycan assembly result in severe cartilage phenotypes seen in both human syndromes and mouse model systems. In vitro studies demonstrate that Pln can trigger chondrogenic differentiation of multipotential mouse CH310T1/2 stem cells as well as maintain the phenotype of adult human chondrocytes. Structural mapping indicates that these activities lie entirely within domain I, a region unique to Pln, and that they require glycosaminoglycans. We also discuss data indicating that Pln cooperates with the key chondrogenic growth factor, BMP-2, to promote expression of hypertrophic chondrocyte markers. Collectively, these studies indicate that Pln is an important component of human cartilage and may have useful applications in tissue engineering and cartilage-directed therapeutics.
Perlecan; Heparan Sulfate Proteoglycan; Extracellular Matrix; Cartilage; Mouse
Proteoglycans (PGs) can influence cell behaviors through binding events mediated by their glycosaminoglycan (GAG) chains. This report demonstrates that chondroitin sulfate B, also known as dermatan sulfate (DS), a major GAG released during the inflammatory phase of wound repair, directly activates cells at the physiologic concentrations of DS found in wounds. Cultured human dermal microvascular endothelial cells exposed to DS responded with rapid nuclear translocation of nuclear factor-κB (NF-κB), increased expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, and increased ICAM-1 cell surface protein. Heparan sulfate and chondroitin sulfates A and C had no effect on activation of NF-κB or induction of ICAM-1. Inhibition of NF-κB activation blocked the effect of DS. The increase in cell surface ICAM-1 did not involve TNF-α or IL-1 release by endothelial cells, but it was facilitated by autocrine factors whose release was initiated by DS. The ICAM-1–inductive activity of DS was confirmed in vivo. Injection of DS, but not heparin or other chondroitin sulfates, into mice greatly increased circulating levels of soluble ICAM. These data provide evidence that DS, but not other GAGs, initiates a previously unrecognized cell signaling event that can act during the response to injury.
Heparan sulfate and heparin, two sulfated glycosaminoglycans (GAGs), extracted collagen-tailed acetylcholinesterase (AChE) from the extracellular matrix (ECM) of the electric organ of Discopyge tschudii. The effect of heparan sulfate and heparin was abolished by protamine; other GAGs could not extract the esterase. The solubilization of the asymmetric AChE apparently occurs through the formation of a soluble AChE-GAG complex of 30S. Heparitinase treatment but not chondroitinase ABC treatment of the ECM released asymmetric AChE forms. This provides direct evidence for the vivo interaction between asymmetric AChE and heparan sulfate residues of the ECM. Biochemical analysis of the electric organ ECM showed that sulfated GAGs bound to proteoglycans account for 5% of the total basal lamina. Approximately 20% of the total GAGs were susceptible to heparitinase or nitrous acid oxidation which degrades specifically heparan sulfates, and approximately 80% were susceptible to digestion with chondroitinase ABC, which degrades chondroitin-4 and -6 sulfates and dermatan sulfate. Our experiments provide evidence that asymmetric AChE and carbohydrate components of proteoglycans are associated in the ECM; they also indicate that a heparan sulfate proteoglycan is involved in the anchorage of the collagen-tailed AChE to the synaptic basal lamina.
A method is proposed for the measurement of glycosaminoglycans (GAG) on 5-10 ml of plasma. It is based on the adsorption of GAG on small ECTEOLA columns followed by measurement of the hexuronic acid in the NaCl eluates. Routine use of the method has indicated the presence of a GAG fraction that adsorbs readily on ECTEOLA (“free” GAG) and of another that adsorbs on it only after treatment with papain (“bound” GAG). “Free” and “bound” GAG have been measured in normal adults, normal children, and children affected by mucopolysaccharidosis type I; the results obtained are in good agreement with those previously reported in the literature.
Various analyses performed on purified “free” and “bound” GAG have confirmed that chondroitin-4-sulfate is the main glycosaminoglycan of normal human plasma where it occurs both free and bound to protein and at various levels of sulfation. The presence of small amounts of heparan sulfate and keratan sulfate has also been demonstrated.
Metabolic experiments performed in rabbits have indicated that plasma GAG derive from peripheral tissues and increase sharply after papain injection. In young animals the “free” GAG have a faster turnover than the “bound,” possibly a reflection of active processes of remodeling and calcification. The synthesis of the “free” and “bound” GAG, as measured with 85S-sulfate incorporation, seems to proceed at the same rate, and the hypothesis has been advanced that as a result of the action of tissue proteases, part of the “bound” GAG may be transformed into “free” GAG, the latter being immediately extruded from the tissues into the circulatory system.
Transforming growth factors beta 1 and beta 2 bind with high affinity to the core protein of a 250-350-kD cell surface proteoglycan. This proteoglycan (formerly referred to as the type III TGF-beta receptor) coexists in many cells with the receptor implicated in TGF-beta signal transduction (type I TGF-beta receptor), but its function is not known. We report here that soluble TGF-beta-binding proteoglycans are released by several cell types into the culture media, and can be found in serum and extracellular matrices. As has been shown for the membrane-bound form, the soluble proteoglycans have a heterogeneous core protein of 100-120 kD that carries chondroitin sulfate and/or heparan sulfate glycosaminoglycan chains and a small amount of N-linked carbohydrate. The membrane-bound form of this proteoglycan is hydrophobic and associates with liposomes, whereas the soluble forms lack a membrane anchor and do not associate with liposomes. Differences in the electrophoretic migration of the soluble and membrane forms of this proteoglycan suggest additional structural differences in their core proteins and glycosaminoglycan chains. These soluble and membrane-bound proteoglycans, for which we propose the name "betaglycans," might play distinct roles in pericellular retention, delivery, or clearance of activated TGF-beta.
Proteoglycans, key molecular effectors of cell surface and pericellular microenvironments, perform multiple functions in cancer and angiogenesis by virtue of their polyhedric nature and their ability to interact with both ligands and receptors that regulate neoplastic growth and neovascularization. Some proteoglycans such as perlecan, have pro- and anti-angiogenic activities, whereas other proteoglycans, such as syndecan and glypican, can also directly affect cancer growth by modulating key signaling pathways. The bioactivity of these proteoglycans is further modulated by several classes of enzymes within the tumor microenvironment: [a] sheddases that cleave transmembrane or cell-associated syndecans and glypicans, [b] various proteinases that cleave the protein core of pericellular proteoglycans, and [c] heparanases and endosulfatases which modify the structure and bioactivity of various heparan sulfate proteoglycans and their bound growth factors. In contrast, some of the small leucine-rich proteoglycans, such as decorin and lumican, act as tumor repressors by physically antagonizing receptor tyrosine kinases including the epidermal growth factor and the Met receptors or integrin receptors thereby evoking anti-survival and pro-apoptotic pathways. In this review we will critically assess the expanding repertoire of molecular interactions attributed to various proteoglycans and will discuss novel proteoglycan functions modulating cancer progression, invasion and metastasis and how these factors regulate the tumor microenvironment.
Perlecan; endorepellin; LG3; VEGF; syndecan; glypican; heparanase; small leucine-rich proteoglycans; decorin; lumican
Heterogeneous heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) polysaccharides are important components of blood circulation. Changes in GAG quantity and structure in blood have been indicated in cancers and other human diseases. However, GAG quantities and structures have not been fully characterized due to lack of robust and sensitive analytical tools. To develop such tools, we isolated GAGs from serum and plasma. We employed liquid chromatography (LC) for GAG quantification and LC/mass spectrometry (MS) for GAG structural analysis. Twenty-four heparan and chondroitin sulfate motifs were identified, including linkage hexasaccharides, repeating disaccharide compositions, reducing, and non-reducing end mono-, di-, tri-, and tetrasaccharide structures. Disaccharides were detectable at picomolar level without radiolabeling or derivitization, so only a few ml of human and fetal bovine serum was required for this study. The detection of different reducing end structures distinct from GAG linkage hexasaccharides revealed that free GAG chains generated by GAG degradation enzymes co-existed with proteoglycans in serum. In addition, a novel sialic acid-modified linkage hexasaccharide was found conjugated to bikunin, the most abundant serum proteoglycan.
human and fetal bovine serum; heparan sulfate; chondroitin sulfate; mass spectrometry