Heparan sulfate proteoglycans in healthy and diseased systems

Focus Article Heparan sulfate proteoglycans in healthy and diseased systems John Whitelock1∗ and James Melrose2 Heparin and heparan sulfate (HS) are glycosaminoglycans (GAGs) that are synthesized in the tissues and organs of mammals. They are synthesized and attached to a core protein as proteoglycans through serine–glycine concensus motifs along the core protein. These GAGs are linear polysaccharides composed of repeating disaccharide saccharide units that are variously modified along their length. As a consequence of these modifications naturally occurring heparin and HS are extremely heterogeneous in their structures. A diverse range of proteins bind heparin and HS. The types of proteins that bind are dictated by the structure of the HS or heparin chains with which they are interacting. Heparan sulfates play major roles in tissue development and in maintaining homeostasis within healthy individuals. Recent genetic studies illustrate that alterations in their structural organization can have important consequences often giving rise to, or directly causing, a disease situation. A greater understanding of the repertoire of proteins with which heparin and HS interact and the diseases that can be caused by perturbations in the structures of heparin and HS proteoglycan may provide insights into possible therapeutic interventions. These issues are discussed with a focus on musculoskeletal phenotypes and diseases.  2011 John Wiley & Sons, Inc. WIREs Syst Biol Med 2011 3 739–751 DOI: 10.1002/wsbm.149 INTRODUCTION H eparin and heparan sulfate (HS) are members of the glycosaminoglycan (GAG) family of polysaccharides that are produced by many mammalian cell types. HS is found attached to the core proteins of a diverse range of cell membrane associated, pericellular and extracellular matrix (ECM) and in the case of serglycin, intracellular proteoglycans. HS is critical to many biological processes in embryonic development, skeletogenesis and tissue homeostasis, matrix-remodeling, and wound healing. A lack of HS is a lethal condition while misassembly of the HS monosaccharide backbone or of the sulfation motifs of its constituent monosaccharides either through mutation or knock down of the enzymes responsible for this process has profound effects on tissue function (Table 1). HS is also involved in a number ∗ Correspondence to: [email protected] 1 Graduate School of Biomedical Engineering, The University of New South Wales, Kensington, New South Wales, Australia 2 Raymond Purves Laboratory, Institute of Bone and Joint Research, Kolling Institute of Medical Research, The University of Sydney at the Royal North Shore Hospital, St. Leonards, New South Wales, Australia DOI: 10.1002/wsbm.149 Vo lu me 3, No vember/December 2011 of disease processes including tumor angiogenesis, pathogen adhesion, and neurodegenerative disorders. The widespread influence of HS on the above processes stems from its assembly into a diverse range of structural permutations which allow it to adopt an extended range of structural conformations. This equips heparin and HS with the ability to bind to many different proteins to regulate their functions. This interactivity is provided by variable modifications of the monomeric units along the HS chain, including N- and O-sulfation of N-acetyl glucosamine, epimerization of d-glucuronic acid at C5 to l-iduronic acid, sulfation of l-iduronic acid, and variable single and multisulfation of d-glucuronic acid. Glucosaminyl N-deacetylase/N-sulfotransferase (NDST) is a key enzyme which acts early in the biosynthesis of HS chains in the Golgi apparatus defining the overall sulfation pattern along the emerging HS chain which will determine downstream effects on it’s ability to interact with target molecules.1 NDST also deacetylates N-acetyl glucosamine replacing them with sulfate groups.2 These N-sulfate groups are essential for further modifications during HS biosynthesis, thus without N-sulfation, no O-sulfation or epimerization of d-glucuronic acid to l-Iduronic acid can occur in  2011 Jo h n Wiley & So n s, In c. 739 wires.wiley.com/sysbio Focus Article TABLE 1 Mouse Models Demonstrating Skeletal, Vascular, Nervous, Urinary, Muscular, and Respiratory Defects Due to (A) Deletion of HS-Proteoglycan, (B) Deletion, Abnormal Assembly, or In Situ Modification of the HS Chains of HS Proteoglycans, and (C) Degradation of HS Chains Gene/mutation System Phenotype (A) Deletion of HS proteoglycans Hspg2 −/− Skeletal Cartilage and growth plate defects9,10 Gpc3 −/− Skeletal Reduced trabecular bone, delayed osteoclastogenesis, BMP-4 interactions, postaxial polydactyly, malformations in ribs and skull sutures11,12 Glp3 −/− Skeletal Defects in cardiac and coronary vascular development13 Hspg2 −/− Vascular Early vascular and cardiovascular insufficiency, transposition of major vessels9 Col18a1−/− Vascular Increased microvasculature, increased angiogenesis associated with atherosclerotic plaque formation14,15 Sdc1−/− Vascular Increased inflammation-mediated corneal angiogenesis16 Sdc4 −/− Vascular Delayed cutaneous wound healing, impaired placental blood vessel development, delayed angiogenesis in granulation tissue of wounds17,18 Sdc3 −/− Nervous Enhanced long-term nervous potentiation, impaired hippocampal function, altered binding of the heparin-binding growth-associated molecule (HB-CAM)19 Col18a1−/− Nervous Defective basement membrane formation—leading to hydrocephalus and anterior ocular defects20,21 Agrn−/− Muscular Defective acetylcholine receptor clustering at neuromuscular junction altered synapse formation22,23 Muscular Perturbed acetylcholinesterase localization at synaptic junctions24 , muscle hypertrophy25 Muscular Satellite cell defects26 Urinary Thickened basement membrane with reduced filtrative capacity21 Respiratory Airway hyper-responsiveness, hypersecretion, eosinophilia, lung inflammation27,28 Haemopoietic, vascular Defective platelet aggregation and thrombus formation,29,30 defective localization of neutrophil elastase in azurophilic ganules of neutrophils,31 defective storage of histamine and serotonin in mast cells,32 defective storage of granzyme B in cytotoxic T lymphocytes,33 defective mast cell dependant activation of proMMP-2 by mast cell serine proteases34 Hspg2 −/− Sdc3 −/− , Sdc4 −/− Col18a1−/− Sdc1 −/− Sgrn−/− (B) Deletion, abnormal assembly, or in situ modification of the HS chains in HS proteoglycans Hspg2
3−/
3− Urinary Loss of Hspg2 HS chains reduces renal filtration, proteinuria after protein loading35,36 Skeletal Absence of HS results in corneal defect, impaired FGF2 dependant angiogenesis, delayed wound repair and tendon maturational changes leading to altered function35 Ext1Gt /Gt Skeletal Growth plate abnormalities in ihh signaling and spatial distribution of ihh37 Ext2 +/− Skeletal Rib exostoses, chondrocyte differentiation38,39 Skeletal Differentiation-induced loss of HS in growth plate40 Ndst1−/− Skeletal Defective craniofacial development, lack of or delay in ossification, mandibular and temporomandibular joint development41–44 Hsglce −/− Skeletal Shortened body length, excessive mineralization, lack of proximal phalanges and tarsal bones, postaxial polydactyly, malformations in rib cage and sternum45 Hsglce −/− Vascular Impaired lympoid organ development46 Nervous Cerebral hypoplasia, defective neural-tube closure, eye and lens defects42,44,47 Nervous Defective eye development45,48 Hspg2 Ext1,
3−/
3− 2 −/− Ndst1−/− Hs2st −/− , Hsglce −/− Hs2st −/− , Hsglce −/− Urinary Renal agenesis45,48 −/− Respiratory Blocks lobuloalveolar development49 Ndst1−/− Respiratory Lung hypoplasia, surfactant insufficiency49,50 Respiratory Impaired lung inflation, lung walls thickened, cell-enriched alveolar walls45 Ndst1 Hsglce 740 −/−  2011 Jo h n Wiley & So n s, In c. Vo lu me 3, No vember/December 2011 WIREs Systems Biology and Medicine HS in healthy and diseased systems TABLE 1 Continued Gene/mutation System Phenotype (C) Degradation of HS chains Hpa −/− Skeletal No apparent skeletal abnormalities. Upregulation of MMP-2, 14 in the liver and kidney compensates for Hpa knock down8 Hpa tg Skeletal Enhanced osteogenic differentiation of bone marrow stromal cells, increased trabecular bone mass, increased corticol bone thickness and rate of bone formation6 Sulf1−/− Sulf2 −/− Skeletal Spontaneous cartilage degeneration, increased expression Mmp-13, Adamts-5, noggin, decreased expression Col2a1 and Acan51,52 Hpa tg Urinary Elevation in urinary protein and creatinine levels, decreased renal amyloid deposits53,54 Ext, exostosin copolymerase; Hspg2, perlecan; Hspg2
3− /
3− perlecan exon 3 HS attachment deletion; Ndst, GlcNAc N-deacetylase; Gpc3, Glypican-3; Hsglce, HS glucuronyl C5 epimerase; Hpa, heparanase; Sdc, syndecan; Col18a1, Collagen XVIII; Gt, gene trap allele; tg, trans gene; Sulf-1, 2, HS 6Oendosulfatases-1, 2; Hs2st, HS 2-O-sulfotransferase; ihh, Indian hedgehog; Acan, aggrecan; Agrn Agrin; Hs2st, Hs 2-O-sulfotransferase; Sgrn, serglycin; MMP, matrix metalloprotease. the nascent HS chain. Extracellular modification of the regions of the HS chain by cell membrane bound endosulfatases (Sulf-1, 2) active at neutral pH, further modulates the HS motifs and increases the level of structural diversity and complexity displayed by HS in situ. Focal removal of the HS chains from the HS proteoglycans by heparanase further modulates the regulatory functions of these proteoglycans in situ.3–8 Knock out of NDST-1, 2 in mice has severe effects on connective tissue development.2 NDST-2−/− mice have defective mast cells lacking sulfated heparin which effects protease storage and activity.1,2 THE HEPARAN SULFATE PROTEOGLYCANS As already noted, HS does not occur as free GAG chains in tissues but is attached to protein cores of six classes of HS proteoglycans.55,56 These include the cell-surface glycosylphosphatidylinositol (GPI) anchored glypican family (glypicans 1–6) and transmembrane syndecan family (syndecans 1–4), perlecan, type XVIII collagen, agrin, and the part-time HS co-receptor proteoglycans betaglycan, neuropilin1, 2 and CD-44 variants (epican). The Glypican Proteoglycan Family Six glypican family members have been identified; HS chains are attached to a 50 amino acid C-terminal region near their GPI anchor at the cell membrane. The HS chains on the glypicans can modify cellsignaling pathways,57 morphogen gradient formation, and contribute to cellular proliferation and tissue growth.58,59 The Syndecan Proteoglycan Family The syndecans have roles to play in wound healing, inflammation, cell adhesion, and vascular biology.60,61 Vo lu me 3, No vember/December 2011 The syndecan proteoglycan family (syndecans 1–4) are single span transmembrane proteoglycans which carry 3–5 HS and chondroitin sulfate (CS) chains which facilitate interactions with a large variety of ligands. The syndecans act as co-receptors for a number of growth factors including members of the fibroblast growth factor (FGF), vascular endothelial cell growth factor (VEGF), and transforming growth factor (TGF)-β families.62 Syndecans 1 and 3 and syndecans 2 and 4 make up separate subfamilies having arisen by gene duplication and divergent evolution from a single ancestral gene. All syndecans have an N-terminal signal peptide, an ectodomain, a single hydrophobic transmembrane domain, and a short C-terminal cytoplasmic domain.61 Perlecan Perlecan is a large modular proteoglycan that binds to and cross-links many ECM components and cellsurface molecules.63,64 Perlecan is synthesized by a wide selection of vascular endothelial and smooth muscle cells and cells of tensional and weightbearing connective tissues.63,65 The human perlecan core protein has a molecular weight of 467 kDa which is encoded by 96 exons. In endothelial cell perlecan, three HS chains are attached to domain I; however, in the perlecan, synthesized by smooth muscle cells, chondrocytes, and keratinocytes, some of these HS chains are replaced with CS or keratan sulfate (KS).63,66,67 The perlecan core protein consists of five distinct structural domains. The N-terminal domain I (aa ∼1–195) contains the GAG attachment sites. Domain II comprises four amino acid repeat regions homologous to the ligand-binding portion of the low-density lipoprotein receptor (LDLR) with six conserved cysteine residues and a pentapeptide (AspGly-Ser-Asp-Glu) which mediates ligand binding by the LDLR. Perlecan domain III has homology to the  2011 Jo h n Wiley & So n s, In c. 741 wires.wiley.com/sysbio Focus Article domain IVa and IVb of laminin. Perlecan domain IV consists of a series of immunoglobulin repeats, in mouse perlecan this domain is truncated by ∼20 kDa compared to the human perlecan core protein. The Cterminal domain V of perlecan, bearing homology to the G domain of the long arm of laminin, is responsible for self-assembly and may be important for basement membrane formation in vivo and spatiotemporal organization of the neuromuscular junction (NMJ) during synaptogenesis. Type XVIII Collagen Type XVIII collagen is expressed in a number of basement membranes, cartilage, and fibrocartilage.68 Type XVIII collagen is the only known collagen which carries HS chains.69 Three Ser–Gly concensus sequences have been identified in the middle and Nterminal regions of the core protein of recombinant type XVIII collagen and of these sites one was found to be occupied by HS, the remaining two had mixed CS/HS side chains, type XVIII collagen isolated from chick tissues is exclusively substituted with HS. Agrin Agrin is a large HS proteoglycan with nine domains homologous to protease inhibitors with three potential HS attachment sites of which only two actually carry HS chains when the protein is expressed.70,71 It has roles in the embryonic development of the NMJ, aggregation of acetylcholine receptors, and binds bone morphogenetic proteins (BMP)-2, 4 and TGF-β during synaptogenesis.72,73 Agrin is highly expressed by chondrocytes during normal development.74 Cell-Surface HS-Co-Receptors and Part Time-HS Proteoglycans A number of cell-surface co-receptors have been identified as part-time CS–HS proteoglycans. These include betaglycan and neuropilin-1, 2,62 the CD-44 variants, V3, 3–10, and 8–10, while keratinocytes synthesise a HS substituted form of CD-44 (epican).75 Serglycin Serglycin localizes to the α-secretory granules of platelets and mast cells, where it is thought to bind and control the activity of various mediators including factor 4 in platelets or proteases like tryptase and chymase in mast cells,76,77 Serglycin is decorated with CS chains in the secretory granules of circulating basophils, whereas it is decorated with highly sulfated heparin when it is present in the granules of the tissue resident mast cells.76 742 Mouse Models: HS and Tissue Development and Remodeling As already discussed, HS can display a diverse range of structural permutations in situ supporting the concept of HS as an information module78 with these different structural permutations and the conformations they provide equipping HS with the ability to interact with a diverse array of ligands. Knock-out mouse models have demonstrated that disruption in the normal assembly, elongation, and posttranslational modification of HS in the matrix can have severe consequences on skeletal development. Disease processes may also be affected and this may have a genetic basis involving a number of HS-modifying enzymes, HS proteoglycans, and HS degradative enzymes (Table 1). HS does not act in isolation in musculoskeletal tissues, the proteoglycan core protein to which the HS is attached acts as a molecular scaffold, which, through its own interactive properties with matrix components, localizes the HS chains within tissues where it can exert it’s biological activity. This biological activity is a function of the specific modifications and distributions of the saccharide components along the HS side chain. Specific motifs in the HS side chains act as docking modules for growth factors and morphogens and interact with a diverse range of matrix components. Table 1 shows a list of a number of mouse studies where specific HS-proteoglycan genes, HSmodifying enzymes, or HS-degradative enzymes have been knocked out or over-expressed to deduce the contribution that these components make to the normal organization and function of musculoskeletal tissues. Musculoskeletal Phenotypes Attributable to Knock Out or Partial Deletion of a Specific HS Proteoglycan Knock out of perlecan (Hspg 2) is a lethal condition and severely impacts on cartilage and growth plate development,9 vasculogenesis, respiratory development, and defective acetylcholinesterase receptor (AChR) and acetylcholinesterase (AChE) clustering at the NMJ, impaired synapse formation,24 and muscle hypertrophy25 impacting on normal muscle function.79 While this clearly demonstrates the essential role of perlecan in a number of musculoskeletal systems, the lethality of it’s deletion makes the Hspg2−/− mouse model unsuitable for the postnatal assessment of perlecan’s roles in musculoskeletal tissues. A further mouse model has been developed where deletion of exon 3 (which encodes a portion of the perlecan domain-I core protein containing the HS attachment site) results in ablation of the HS  2011 Jo h n Wiley & So n s, In c. Vo lu me 3, No vember/December 2011 WIREs Systems Biology and Medicine HS in healthy and diseased systems chains from perlecan. Thus, the specific contribution of these HS chains to the pathobiology of perlecan can be independently assessed by comparison of the Hspg2 exon 3 null mutant and wild type mice. The exon 3 null Hspg2 mouse displays a milder phenotype than the Hspg2−/− mouse and mouse pups apparently develop normally. A mild phenotype is evident in these mice affecting corneal development,80 impaired FGF2 dependant angiogenesis leading to delayed wound repair,80 impaired renal filtration and proteinuria after protein loading,35 and a decreased number of excitatory neurons is also evident in the cerebral cortex.81 Knock out of glypican-3 (Gpc 3) results in reduced trabecular bone, delayed osteoclastogenesis, impaired BMP-4 interactions, postaxial polydactyly and malformations in rib development and the skull sutures,11,12 and growth factor dependent defects in cardiac and coronary vascular development.13 Glypican-1 (Gpc 1) controls brain size through the regulation of FGF signaling in early neurogenesis.82 Mutations in glypican-6 (Gpc 6) impairs endochondral ossification and causes recessive omodysplasia, a rare skeletal dysplasia characterized by severe congenital micromelia with shortening and distal tapering of the humeri and femora to give a clublike appearance.83 Glypican-1 (Gpc 1) does not appear to define a specific musculoskeletal phenotype, rather it controls brain size. Glypican-1 is over-expressed in breast and brain cancers (gliomas) while glypican-3 (Gpc 3) is over-expressed in liver cancers, lung squamous cell carcinoma, metastatic melanoma, Merkel cell carcinoma, and ovarian clear cell adenocarcinoma. The syndecan proteoglycan family play roles in defining a number of musculoskeletal phenotypes. Mice lacking syndecan-4 (Sdc 4) display delayed wound repair and impaired angiogenesis.17 Syndecan3 and -4 (Sdc 3, 4) have roles to play in muscle regeneration.26 Syndecan-1 (Sdc 1) is an inhibitor of arterial smooth muscle cell growth and intimal hyperplasia, while syndecan-4 is upregulated in osteoarthritis (OA). Inhibition of syndecan-4 which regulates a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5) activation and cartilage destruction in OA reduces the car84 tilage destruction in the mouse models of OA. Syndecan-1 mRNA is reduced and syndecan-4 mRNA elevated in OA cartilage. Syndecans 1–4 are differentially expressed in chronically inflamed synovium. Syndecan-1 is expressed by synovial mononuclear cells in rheumatoid arthritis (RA) patients and Sdc 2 by endothelial and smooth muscle cells in synovial blood vessels, Sdc 3 is expressed by endothelial cells and macrophages of the synovial lining. Expression of Vo lu me 3, No vember/December 2011 syndecans 1–3 in rheumatoid synovium is higher than in normal or OA synovium, thus could have roles in the migration and retention of inflammatory cells that modulate angiogenic events in RA synovium. Knock out of Sdc 3, 4 expression results in delayed cutaneous wound healing,17 impaired placental blood vessel development,18 delayed clearance of granulation tissue from wounds, enhanced long-term nervous potentiation, impaired hippocampus function,19 and satellite cell defects leading to an impairment in muscle development.85 Ablation of Sdc 1 expression results in increased inflammation-mediated corneal angiogenesis,16 airway hyper-responsiveness, hypersecretion, eosinophilia, and lung inflammation of the lung.27 Knock down of agrin (Agrn) results in defective AChR clustering at the NMJ and effects on synaptogenesis86,22 which impact on muscle function. AChE is associated with the specialized synaptic basal lamina of the NMJ, it’s interactivity with perlecan and agrin localizes AchE with other NMJ stabilizing components such as dystroglycan (DG), rapsyn, laminin, and muscle specific tyrosine kinase receptor (MuSK) to form AChE clusters which are important for synaptic function.87 The C-terminus of perlecan core protein (domain V) contains three globular domain modules which are also found in laminin and agrin. These bind to and localize DG, AChR, AchE in the NMJ and co-localize these with HB-GAM (heparin-binding growth-associated molecule) a heparin-binding growth factor.88 Perlecan (Hspg 2) and Agrin (Agrn) therefore have important roles to play in the spatiotemporal organization of the NMJ. This is a function of the interactive capability of their core proteins with ECM components to localize their HS side chains at strategic locations where they can interact with specific biomolecules (growth factors and morphogens) important for muscle development and function. A deficiency of perlecan in Hspg2−/− mice results in a complete absence of AChE and AChR in the NMJ24 and muscle hypertrophy.25 Structural and functional mutations of the perlecan gene cause Schwartz-Jampel Syndrome (SJS) in humans.89 SJS is a nonlethal autosomal recessive skeletal dysplasia characterized by varying degrees of myotonia and is attributable to the truncated form of perlecan lacking domain V in muscle which is unable to correctly organize DG, ChE, AChR, and HB-GAM in the NMJ with attendant effects on muscle function. Agrin also has important roles to play in the postsynaptic differentiation of the NMJ; however, this proteoglycan is incapable of rescuing muscle function in SJS patients indicating that perlecan plays a dominant functional role in this condition.  2011 Jo h n Wiley & So n s, In c. 743 wires.wiley.com/sysbio Focus Article Not all proteoglycan knock-out models have clear musculoskeletal phenotypes. Knock out of type XVIII collagen (Col18a1) results in increased development of the microvasculature and angiogenesis associated with atherosclerotic plaque formation,14 as well as nervous malfunction due to defective basement membrane formation leading to hydrocephalus and anterior ocular defects,90 while thickening of renal basement membranes results in reduced filtrative capacity. Mouse corneal epithelial cells express type XVIII collagen where it has roles in incisional corneal wound healing. A lack of type XVIII collagen (Col l18a1) and it’s proteolytically generated C-terminal peptide, endostatin, results in eye abnormalities due to delayed regression of blood vessels in the vitreous and abnormal outgrowth of retinal blood vessels. Type XVIII collagen knock-out mice have anterior ocular defects.20 These include delayed regression of the hyaloid vasculature and poor outgrowth of the retinal vessels, a fragile iris, and these mice develop atrophy of the ciliary body indicating that type XVIII collagen has major roles to play in the assembly of ocular epithelial basement membranes. Knock out of the intracellular proteoglycan sergylcin (Srgn) results in defective storage of neutrophil elastase in the azurophilic granules of neutrophils,31 histamine, and serotonin in the secretory granules of mast cells,32 and defective granzyme B storage in cytotoxic T lymphocytes.33 Serglycin knock out (Srgn−/− ) also impairs platelet aggregation and thrombus formation in mice,29,30 and prevents mast-cell serine protease-dependant activation of pro-matrix metalloprotease-2 (proMMP-2) in situ.34 Knock Down or Partial Deletion of HS-Modifying Enzymes: Influences on the Pathobiology of HS in Musculoskeletal Tissues Knock down of members of the exostosin (EXT) copolymerase enzyme system affects HS assembly and results in growth plate abnormalities resulting from abnormalities in Indian hedgehog (ihh) signaling and the spatial distribution of ihh,37 skeletal abnormalities due to the differentiation-induced loss of HS in the osteochondral junction, and the development of rib exostoses due to the abnormal growth plate chondrocyte differentiation.38 Knock out of N-acetyl glucosamine deacetylase (Ndst)-1 results in effects on skeletal development. This includes abnormal craniofacial development, lack or delay in ossification, mandibular, and temporomandibular joint development.41 Aberations in nervous development including cerebral hypoplasia,42 defective neural-tube closure, and eye and lens defects are also evident in Ndst-1 knock-out mice. Respiratory insufficiencies 744 have also been noted including blockage of lobuloalveolar development49 , lung hypoplasia, and surfactant insufficiency.50 HS glucuronyl C5 epimerase (Glce) is an enzyme which is responsible for the epimerization of d-glucuronic acid at C5 to form l-Iduronic acid. Knock out of Glce results in a structurally altered HS lacking IdoA, skeletal abnormalities including shortened body length, renal agenesis, excessive mineralization of growth plates, lack of proximal phalanges and tarsal bones, postaxial polydactyly, and malformations in the rib-cage and sternum.45 Glce knock down also results in impaired lymphoid organ development,46 respiratory malfunction due to impaired lung inflation, and cell-enriched thickened inelastic lung walls. Heparan sulfate 2O-sulfotransferase (Hs2st) catalyzes the transfer of sulfate from 3′ -phosphoadenosine-5′-phosphosulfate to the C2-position of hexuronic acid residues within the maturing HS chain, 2-O-sulfation within HS, particularly of iduronate residues, is essential for HS’s participation in a variety of high-affinity ligandbinding interactions.91 The HS sulfation motifs generated by Hs2st are important for the guidance of axons in retinal ganglion cells, and in the regulation of cell proliferation during the development of the cerebral cortex. Hs2st knock-out mice die perinatally due to an absence of kidney development.48 Degradation of HS In Situ and Effects on Musculoskeletal Tissues in Health and Disease The recent development of heparanase knock-out mice has shown that heparanase and some members of the MMP family are co-regulated.8 Transgenic overexpression of heparanase also demonstrates important physiological functions of HS in morphogenesis and vascularization and has implications on our understanding of the specific roles that specific HS structures play in the intricacies of connective tissue remodeling. Surprisingly, Heparanase (Hpa−/− ) knock-out mice develop normally, display a normal life-span and do not have overt musculoskeletal abnormalities; however, MMP-2 and -14 are up-regulated in Hpa−/− mice.8 Transgenic overexpression of heparanase results in enhanced osteogenic differentiation of bone marrow stromal cells, increased trabecular bone mass, increased cortical bone thickness and rate of bone formation.6 An elevation in urinary protein and creatinine levels together with decreased amyloid deposition amounts has also been noted in mice with transgenic overexpression of Hpa. The sulfatase family of enzymes catalyze the hydrolysis of sulfate ester bonds in a wide variety of substrates ranging from sulfated proteoglycans  2011 Jo h n Wiley & So n s, In c. Vo lu me 3, No vember/December 2011 WIREs Systems Biology and Medicine HS in healthy and diseased systems to conjugated steroids and sulfate esters of small aromatic molecules. Seventeen human sulfatase proteins and genes have been identified. Of these, two specific endosulfatases, Sulf-1 and -2, are responsible for the removal of 6-O sulfate groups from HS.51 Sulf-1 and -2 are neutral pH, extracellular membrane bound enzymes which can modify HS in the ECM environment thereby modifying it’s interactive properties in a spatiotemporal manner which may be of importance developmentally and during tissue remodeling. Sulf-1−/− and Sulf-2−/− mice develop spontaneous cartilage degeneration, increased expression of MMP-13, ADAMTS-5, noggin, and decreased expression of aggrecan and type II collagen.51 Initial results showed that Sulf-1 was down-regulated in the majority of cancer cell lines. Forced expression of Sulf-1 decreases cell proliferation, migration, and invasion, promotes druginduced apoptosis of tumor cells in vitro, and inhibits tumorogenesis and angiogenesis in vivo, thus it is of considerable interest as a potential tumor suppressor agent92 ; however, Sulf-2 promotes human lung carcinogenesis.93 Heparin/HS Therapeutics As already discussed, heparin and HS are related GAGs which display similar structural modifications in their saccharide components, N-acetyl glucosamine, l-Iduronic acid, and d-glucuronic acid. The distributions of these modified components along the intact GAG side chain however differ, with heparin containing more extensive stretches of highly modified moieties, while HS contains smaller regions of highly modified saccharides flanked by moderately modified regions interspersed between areas which are minimally modified providing a considerable level of structural complexity, diversity, and heterogeneity. The complete library of heparin/HS structures present in a cell or tissue has been given the term ‘heparanome’ and the study of these structures has been referred to as ‘heparanomics’.94,95 The complete repertoire of HS structures present at any one time in cells, tissues, or organs would include HS decorating the cell surface, pericellular and ECM HS proteoglycans described earlier in this review and in the case of mast cells, neutrophils, and T lymphocytes, heparin which decorates the intracellular proteoglycan and serglycin. Heparin was first isolated from canine liver in 191896 where it was shown to inhibit the coagulation pathways in blood that lead to fibrin generation and thrombosis. After its chemical structure was published in 1935,97 the first intravenous pharmaceutical grade heparin was marketed and used clinically in significant quantities by the end of that decade. The first Vo lu me 3, No vember/December 2011 preparations were isolated from liver, which was how it came to be known as ‘heparin’, but these preparations turned out to be very toxic to humans in early trials. Today, pharmaceutical grade heparins are mostly manufactured using porcine intestinal submucosa, but bovine lung tissue has been used as an alternative source. Low-molecular weight forms of heparin have been produced in attempts to confine the activity to the well-characterized interactions with the blood coagulation serine protease inhibitor, antithrombin-III.98 The precise structure that interacts with antithrombin-III to provide anticoagulant activity is a pentasaccharide sequence, which has a distinctive centrally located tri-sulfated glucosamine that is modified with 3-O-sulfate together with 6-O and N-sulfate groups.99 However, other structures in heparin that do not contain the 3-sulfated glucosamine are hypothesized to interact with heparin cofactor II which can also impart anticoagulation effects.100 More recently, attempts to produce clinically useful amounts of this pentasaccharide synthetically have been successful, which has led to it becoming a significant therapeutic in its own right. It is intriguing to note that mice deficient in the enzyme, 3-Osulfotransferase-I, that can add the 3-O-sulfate group do not display a thrombotic phenotype.101 Heparin mimetics such as the highly sulfated phosphomannose sugar, PI-88, have been trialled in the treatment of cancer and demonstrated positive short-term effects in controlling tumor growth, but were not as effective in the longer term.102–104 Several research groups have used the Hspg2, perlecan gene as a way to generate and deliver HS to tissues with the aim of modulating growth factor signaling.105–108 This has mostly focused on the use of the N-terminal domain I as a way to produce a glycosylated protein encompassing the 3 GAG attachment sites that are present in that domain, but other domains have also been expressed and studied.105–107,109–116 This has proved to be challenging because we still do not fully understand the precise mechanisms involved in decorating proteoglycan cores with GAG chains as they transit the Golgi apparatus and endoplasmic reticulum. Some work has indicated that sequences within the protein core are involved,117–119 but other work suggested that the addition of protein domains unrelated to the core protein also had significant effects. Addition of the enhanced green fluorescent protein (EGFP) sequence to the C-terminus of domain I resulted in a proteoglycan that contained significantly more HS than CS.120 In many studies using recombinantly expressed perlecan fragments, it has been shown that the presence of HS and in some  2011 Jo h n Wiley & So n s, In c. 745 wires.wiley.com/sysbio Focus Article cases CS is important to get the correct biological activity.105,107,108,121 These studies have used the expression systems developed by Prof. Rupert Timpl’s laboratory in the mid-1990s.109,112,114–116,122 In these systems, it is apparent that the decoration of the Hspg2 gene product was dependant on the cell type where the gene had been introduced. This was most commonly the HEK-293 cell line, which produced a perlecan decorated with HS, CS, and KS.123 Perlecan domain I has been expressed in the human fibrosacrcoma cell line HT1080 using a cytomegalovirus expression vector,124 and in Chinese hamster ovary cells.125 In the former case, the perlecan domain I was substituted solely with CS chains; however, in the latter study the recombinant perlecan domain I contained CS/DS and HS. However, these were of small molecular weight (12 kDa). Recombinant perlecan domains I and II have been expressed in insect cells using a baculovirus gene delivery system but domain I was substituted solely with CS chains.110 An adenoviral gene delivery system has also been used with human embryonic kidney cells (HEK) 293 and human umbilical vein endothelial cells (HUVEC) cells for the production of the N-terminal domain of perlecan.126 To date, this system remains the only one that enables the researcher to produce a HS with a defined structure similar to the HS chains which occur in situ and given the challenges of the use of the adenoviral systems in the clinic, there still remains much research to be completed before we can achieve the goal of delivering defined HS structures to tissues that will have the correct spatial and temporal activities that result in the desired clinical outcomes. CONCLUSIONS Heparan sulfate exists in most systems attached to different types of core proteins with different and varied roles. 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Similarity of recombinant human perlecan domain 1 by alternative expression systems bioactive heterogenous recombinant human perlecan D1. BMC Biotechnol 2010, 10:66. FURTHER READING Gandhi NS, Mancera RL. The structure of glycosaminoglycans and their interactions with proteins. Chem Biol Drug Des 2008, 72:455–482. Kirn-Safran C, Farach-Carson MC, Carson DD. Multifunctionality of extracellular and cell surface heparan sulfate proteoglycans. Cell Mol Life Sci 2009, 66:3421–3434. Ori A, Wilkinson MC, Fernig DG. The heparanome and regulation of cell function: structures, functions and challenges. Front Biosci 2008, 13:4309–4338. Schultz GS, Wysocki A. Interactions between extracellular matrix and growth factors in wound healing. Wound Repair Regen 2009, 17:153–162. Buono M, Cosma MP. Sulfatase activities towards the regulation of cell metabolism and signaling in mammals. Cell Mol Life Sci 2010, 67:769–780. Fux L, Ilan N, Sanderson RD, Vlodavsky I. Heparanase: busy at the cell surface. 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