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DT7I
To my dear family and all my friends: thanks for your good company in these years…
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List of papers
This thesis is based on the following papers:
I. Juan Jia, Marco Maccarana, Xiao Zhang, Maxim Bespalov, Ulf Lindahl and Jin-ping Li. (2009) Lack of L-iduronic acid in heparan sulfate affects interaction with growth factors and cell signaling. J Biol Chem 284(23), 15942-15950
II. Martha L Escobar Galvis*, Juan Jia*, Xiao Zhang, Nadja Jastre- bova, Dorothe Spillmann, Eva Gottfridsson, Toin H van Kuppevelt, Eyal Zcharia, Israel Vlodavsky, Ulf Lindahl and Jin-ping Li. (2007) Transgenic or tumor-induced expression of heparanase upregulates sulfation of heparan sulfate. Nat Chem Biol 3(12), 773-778 * Shared first co-authorship
III. Eyal Zcharia, Juan Jia, Xiao Zhang, Lea Baraz, Ulf Lindahl, Tamar Peretz, Israel Vlodavsky and Jin-ping Li. (2009) Newly generated heparanase knock-out mice unravel co-regulation of heparanase and matrix metalloproteinases. PLoS ONE 4(4), e5181
IV. Juan Jia, Xiao Zhang, Eyal Zcharia, Israel Vlodavsky, Ulf Lindahl, Gunnar Pejler and Jin-ping Li. (2009) Heparanase cleavage of hepa- rin regulates protease storage in mast cells. Preliminary manuscript
Reprints were made with permission from the publishers.
Contents
Introduction...... 9 Background ...... 10 Proteoglycans and glycosaminoglycans...... 10 HS/heparin proteoglycans ...... 11 Classification...... 11 HS/heparin biosynthesis and structure ...... 13 Regulation of HS structure...... 17 Functions of HSPGs ...... 17 Interactions of HS and proteins...... 17 HSPGs in developmental processes...... 19 HSPGs in diseases ...... 20 Functions of heparin ...... 21 Interaction of heparin and antithrombin - AT...... 21 Heparin in mast cells ...... 22 Heparanase ...... 22 Activity and processing ...... 22 Functions ...... 23 Mast cells...... 24 Present Investigations...... 26 Aims of study...... 26 Results ...... 27 Paper I...... 27 Paper II ...... 27 Paper III ...... 28 Paper IV...... 29 Discussion and Future Perspectives ...... 30 Populärvetenskaplig Sammanfattning...... 33 ...... 34 Acknowledgements ...... 35 References...... 37
Abbreviations
AT Antithrombin bFGF Basic fibroblast growth factor CS Chondroitin sulfate ECM Extracellular matrix EXT Exostosin EXTL Exostosin-like FGF Fibroblast growth factor FGFR Fibroblast growth factor receptor FSMC Fetal skin-derived mast cell GAG Glycosaminoglycan GDNF Glia-derived neurotrophic factor GlcA Glucuronic acid GlcN Glucosamine GlcNAc N-acetylglucosamine GlcNS N-sulfated glucosamine Hpse Heparanase HS Heparan sulfate Hsepi Glucuronyl C5-epimerase HSPG Heparan sulfate proteoglycan Hs2st 2-O-sulfotransferase Hs3st 3-O-sulfotransferase Hs6st 6-O-sulfotransferase IdoA Iduronic acid KS Keratan sulfate MEF Mouse embryonic fibroblast MMP Matrix metalloproteinase mMCP Mouse mast cell protease Ndst N-deacetylase/N-sulfotransferase PG Proteoglycan Sulf Endo-6-O-sulfatase VEGF Vascular endothelial growth factor
Introduction
Proteoglycans are macromolecules ubiquitously found on the surface and in the extracellular matrix of mammalian cells. Proteoglycans generally consist of a core protein to which one or more glycosaminoglycan chains are covalently attached. These glycosaminoglycan chains are long, linear carbo- hydrate polymers that are negatively charged due to the occurrence of sulfate and carboxyl groups. Heparan sulfate proteoglycans form a subgroup of proteoglycans that carry one or more heparan sulfate polysaccharide chains. Heparan sulfate proteoglycans are found in all mammalian tissues. Heparan sulfate, the side chain, binds to a variety of protein ligands and regulates a broad range of biological activities, including developmental processes, angiogenesis and tumor metastasis. The fine structure of heparan sulfate varies in different tissues, developmental stages and pathological conditions, but overall structure is conserved in the same tissue from differ- ent species. The regulation mechanisms that determine the fine structure are not fully understood. Heparin, an intracellular glycosaminoglycan, is synthesized and stored in mast cells. Heparin shares high similarity with heparan sulfate in structure, but is more sulfated. The biological functions of heparin in mast cells are not entirely clear, however it is known that heparin is crucial for expression and storage of proteases. This thesis summarizes the studies on the correlation of structure and functions of heparan sulfate and heparin, focusing on two enzymes, glucuro- nyl C5-epimerase and heparanase. Glucuronyl C5-epimerase is a key en- zyme in heparan sulfate/heparin biosynthesis catalyzing the conversion of D- glucuronic acid into L-iduronic acid. Heparanase is an endo-glucuronidase known as a specific heparan sulfate-degrading enzyme.
9 Background
Proteoglycans and glycosaminoglycans Proteoglycans (PGs) are important components in the extracellular matrix (ECM) and on the cell surface. They are composed of a core protein with one or more covalently O-linked glycosaminoglycan (GAG) chain(s) (Kjellén and Lindahl, 1991; Bernfield et al., 1999). GAGs are linear poly- saccharide composed of repeating disaccharide units. The GAGs are divided into four classes: 1. Heparan sulfate (HS)/heparin; 2. Chondroitin sulfate (CS) and dermatan sulfate (DS); 3. Keratan sulfate (KS); 4. Hyaluronan (Kjellén and Lindahl, 1991) (Fig. 1). HS/heparin are composed of repeating disaccharide units of hexuronic acid (HexA) and glucosamine (GlcN) in -HexA / 1,4-GlcN 1,4- structure with sulfation at various positions. The HexA is either D-glucuronic acid (GlcA) or L-iduronic acid (IdoA), and the GlcN residues can be N-acetylated (GlcNAc), N-sulfated (GlcNS) or, in rare cases, N-unsubstituted (GlcNH2) (Fig. 1).
COO- COO- CH OR CH2OR 2 O OOO O COO- COOCOO- - RO O O O OH OR O O OHOR OR HNR’ OR HNAc Heparan sulfate/Heparin Chondroitin sulfate/Dermatan sulfate
COO- CH OR CH OR CH2OH 2 O 2 O HO O OO O O OH O OH O OH O HO OH HNAc OH HNAc Keratan sulfate Hyaluronan
- Figure 1. Disaccharide structure of GAGs. R represents H or SO3 , R’ repre- - sents H, COCH3 or SO3 ; the GlcA of HS/heparin and CS can be epimerized to IdoA (indicated in grey).
10 CS and DS contain repeating disaccharide units of HexA and N- acetylgalactosamine (GalNAc) in -HexA / 1,3-GalNAc 1,4- structure, CS only contains GlcA, while some of the GlcA can be epimerized to IdoA in DS. Both HexA and GalNAc can be sulfated (Fig. 1). KS is composed of alternating partially sulfated galactose (Gal) and GlcNAc residues (-Gal 1,4-GlcNAc 1,3-) (Fig. 1). Hyaluronan is a non-sulfated GAG composed of repeating -GlcA 1,3- GlcNAc 1,4- disaccharide units (Fig. 1).
HS/heparin proteoglycans HS proteoglycans (HSPGs) are macromolecules associated with cell surface and ECM of a wide range of cells of vertebrate and invertebrate tissues ( Kjellén and Lindahl, 1991; Iozzo and San Antonio, 2001). They participate in many developmental and pathological processes by interactions with a variety of ligands in ECM, such as growth factors and adhesion molecules (Tumova et al., 2000). The interactions between HS and ligands are gener- ally believed to depend on the number and relative positions of carboxyl and in particular sulfate groups within the HS chains.
Classification A HSPG is composed of a core protein to which several linear HS chains are covalently O-linked (Fransson et al., 2000). According to the distribution, HSPGs are divided into three classes: intracellular heparin PGs (serglycin); cell surface associated HSPGs (syndecan and glypican); and ECM HSPGs (perlecan, agrin and collagen XVIII) (Halfter et al., 1998; Bernfield et al., 1999; Iozzo et al., 2005) (Fig. 2). Syndecans are components of plasma cell membrane (Fig. 2). The syndecan family consists of four different members. Each syndecan has a short cyto- plasmic domain, a transmembrane domain and an extracellular domain with attachment sites for three to five HS or CS chains near the N-terminus (Tkachenko et al., 2005). The molecular size of syndecan core proteins ranges from 22 to 45 kDa because of the distinct extracellular domains (Oh and Couchman, 2004). Apart from the functions of cell-cell and cell-ECM interac- tions, syndecan is involved in interactions inside of cells, such as cytoskeletal organization which is involved in cell adhesion and signal transduction (Tkachenko et al., 2005). Many cell types express more than one type of syn- decans and the expression levels may change during development and differ- entiation (Elenius et al., 1991). Mice lacking syndecan-1 (Alexander et al., 2000), -3 (Reizes et al., 2001) or -4 (Echtermeyer et al., 2001) are all viable but exhibit relatively mild defects, such as abnormal feeding behavior (synde- can-3) or delayed wound healing (syndecan-4).
11 Glypicans are attached to the plasma membrane via a glycosylphospatidy- linositol (GPI) anchor (Fig. 2), and generally carry three to four HS chains (Lindahl and Li, 2009). Glypican, with a molecular size of around 60 kDa, constitutes a family with six members that are all highly expressed during embryonic development (Veugelers et al., 1999). The glypican-3 deficient mice are perinatal death with abnormal kidney and lung development (Cano- Gauci et al., 1999).
3 4
1
2 ECM
Cell membrane
Cytoplasm 5 Secretory granule
Core protein HS/heparin chain Proteases Histamine
Figure 2. HS/heparin PGs Syndecans (1) and glypicans (2) are cell membrane HSPGs. Agrin (3) and perlecan (4) are ECM HSPGs. Heparin PG (5) is packed into secretory granules of mast cells.
Agrin, perlecan and collagen XVIII are typical secreted ECM PGs (Fig. 2). Agrin, with molecular size 220 kDa, is mainly found in kidney and brain, while perlecan (400 kDa) is expressed in nearly all basement membrane and connective tissue. Collagen XVIII is part of most epithelial and endothe- lial basement membrane and is also found in cartilage and fibrocartilage (Rehn and Pihlajaniemi, 1995; Pufe et al., 2004). Deletion of perlecan (Arikawa-Hirasawa et al., 1999) and agrin (Gautam et al., 1996) lead to perinatal lethality due to general severe developmental defects (perlecan) and disruption of neuromuscular junctions (agrin). Mice lacking collagen XVIII are viable but with eye developmental defects (Fukai et al., 2002).
12 Heparin is produced and stored in the secretory granules of connective tissue mast cells (Fig. 2). Heparin chains are covalently O-linked to serine residues within serglycin (Kolset and Gallagher, 1990). These negatively charged chains are known to be important for storage of histamine, proteases and other inflammatory mediators within the granules (Kolset and Zer- nichow, 2008).
HS/heparin biosynthesis and structure Initiation of HS/heparin biosynthesis The HS/heparin chains are assembled on core proteins by enzymes in the Golgi compartment, using nucleotide sugars imported from the cytoplasma. Briefly, the synthesis initiates with the transfer of xylose (Xyl) from uridine- 5’-diphosphate (UDP)-Xyl by xylosyltransferase-1/2 to specific serine resi- dues (Ser) within the core protein. Attachment of two Gal residues by galac- tosyltransferases-1/2 and GlcA by glucuronyltransferase-1 completes the ‘‘linkage tetrasaccharide’’, -GlcA-1,3-Gal-1,3-Gal-1,4-Xyl-Ser.
Polymerization of HS/heparin chains After attachment of the first GlcNAc residue to the tetrasaccharide linker by exostosin-like-2/3 (EXTL-2/3), the HS/heparin chain is elongated by the alternating addition of GlcA and GlcNAc residues catalyzed by polymerases (EXT-1/EXT-2 complex) (Busse and Kusche-Gullberg, 2003). The nascent HS/heparin chains generally contain 50-100 disaccharide units.
Modification of HS/heparin chains As the chains are polymerized, they undergo a series of modification reac- tions involving N-deacetylation and N-sulfation of the GlcNAc, epimeriza- tion of GlcA to IdoA, and O-sulfation at various positions (Lindahl and Li, 2009) (Fig. 3). The first polymer modification is the N-deacetylation/N-sulfation of GlcNAc residues into GlcNS. This is a prerequisite for all subsequent modi- fication reactions and is catalyzed by N-deacetylase/N-sulfotransferase (Ndst), of which four members have been identified. The N-deacetylase ac- tivity of Ndst removes acetyl-groups from GlcNAc residues, generating free amino-groups, which are then sulfated through the N-sulfotransferase activ- ity. A majority of cells express mRNA for at least two Ndst isoforms, usu- ally Ndst-1 and -2 (Grobe et al., 2002). Mice deficient in Ndst-1, producing under-sulfated HS, are neonatally lethal due to lung defect (Fan et al., 2000; Ringvall et al., 2000); whereas disruption of Ndst-2 showed a phenotype restricted to connective tissue-type mast cells resulting in heparin deficiency, alteration of mouse mast cell proteases (mMCPs) and histamine in the secre- tory granules of mast cells (Forsberg et al., 1999).
13 COO- CH2OH O O O O OROH O OH N-deacetylation/N-sulfation HNAc OH (N-deacetylase/N-sulfotransferase 1-4) COO- CH2OH O O O OH O OROH O Epimerization HNSO - OH 3 (Glucuronyl C5-epimerase)
CH2OH O O COO- O OH O OROH O 2-O-sulfation HNSO - OH 3 (2-O-sulfotransferase)
CH2OH O O COO- O 6-O-sulfation O OROH O OH
- OSO - (6-O-sulfotransferase 1-3) HNSO3 3
- CH2OSO3 O O COO- O OH O OROH O 3-O-sulfation
- - HNSO OSO3 3 (3-O-sulfotransferase 1-6)
- CH2OSO3 O O COO- O - OH O OROSO3 O
- - HNSO OSO3 3 Figure 3. Scheme of HS/heparin chain modifications. Biosynthesis of HS/heparin takes place in the Golgi compartment, the chain is elongated by alternat- ing additions of GlcA and GlcNAc residues and modified by various enzymes. (The numbers after the name of enzymes represent isoforms of the enzyme)
Epimerization is catalyzed by glucuronyl C5-epimerase (Hsepi) at the polymer level. It is a single gene (Hsepi) encoded enzyme in mammals (Crawford et al., 2001; Li et al., 2001). This enzyme catalyzes the conver- sion of GlcA to IdoA that occurs in the N-sulfated regions of HS/heparin chains. The reaction involves two steps (Fig. 4): the first step is an abstrac- tion of the C5 proton, which results in formation of a carbanion intermediate; the second step is an addition of a proton from the water medium to the in- termediate that will be stabilized in either L-Ido or D-Gluco configuration and to terminate the reaction (Hagner-McWhirter et al., 2004). Substrate recognition requires N-sulfate groups which are at the non-reducing side of the target HexA and will not accept 2-O-sulfated GlcA or adjacent 6-O- sulfated GlcN units (Jacobsson et al., 1984). The epimerization reaction is reversible in vitro but not in vivo presumably due to coupled 2-O-sulfation of IdoA product units (Hagner-McWhirter et al., 2004). Targeted disruption of
14 the mouse Hsepi genes results in a structurally altered HS/heparin that lacks IdoA (Li et al., 2003; Feyerabend et al., 2006). The Hsepi mutant mice are neonatally lethal with renal agenesis, lung defects and skeletal malforma- tions. Unexpectedly, major organ systems, including the brain, liver, gastro- intestinal tract and heart appear normal (Li et al., 2003).
(Hagner-Mcwhirter et al., 2000) Figure 4. Glucuronyl C5-epimerase reaction in HS/heparin biosynthesis. The first step of this reaction is the abstraction of a proton at C-5 of D-GlcA residue, leading to the formation of a carbanion intermediate, and the second step is an addi- tion of a proton to the intermediate resulting in L-IdoA.
Following the epimerization, 2-O-sulfation is catalyzed by 2-O- sulfotransferase (Hs2st) that is encoded by a single gene (Bullock et al., 1998). Deletion of Hs2st results in neonatal lethality, lack of kidneys and skeletal malformations (Bullock et al., 1998). 2-O-sulfation occurs mostly on IdoA, although 2-O-sulfated-GlcA residues can be found in HS/heparin chains. 6-O-sulfation is catalyzed by 6-O-sulfotransferase (Hs6st). At least three isoforms of Hs6st have been identified. Hs6st transfers sulfate groups to both GlcNAc and GlcNSO3 residues, with a preference for N-sulfated target re- gions. The Hs6st-1and Hs6st-2 are strongly expressed in mouse liver and brain respectively, whereas Hs6st-3 is ubiquitously expressed (Habuchi et al., 2000). Mice deficient in Hs6st-1 exhibit defective HS biosynthesis and late embryonic lethality (Habuchi et al., 2007). 3-O-sulfation of HS/heparin by 3-O-sulfotransferase (Hs3st) is the rarest modification within the biosynthesis system (Shworak et al., 1999); however,
15 Hs3st activity is required to generate functional antithrombin (AT) binding region within heparin chains (Kusche et al., 1988). Currently six isoforms of Hs3st have been identified in mammals (Shworak et al., 1999; Xia et al., 2002; Kusche-Gullberg and Kjellén, 2003). Mice lacking Hs3st-1 (Shworak et al., 2002) showed only mild phenotypes, such as growth retardation, which suggests overlapping functions or compensatory mechanisms of the isoforms. In HS biosynthesis, a number of GlcNAc residues escape N-deacetylation /N-sulfation and are not further modified. Thus three types of domain struc- ture are generated during modification reactions: unmodified N-acetylated regions with GlcA units (NA domains), contiguous N-sulfated regions that are 2-O- and 6-O-sulfated and contain both IdoA and GlcA residues (NS domains) and mixed regions of alternating N-sulfated and N-acetylated dis- accharides (NA/NS domains) (Fig. 5).
NS domain NS domain
Heparin O-sulfation C5-epimerization N-deacetylation/N-sulfation Ser N-deacetylation/N-sulfation C5-epimerization O-sulfation Heparan sulfate
NS domain NA/NS domain NA domain
GlcNAc GlcA Gal O-sulfate group
GlcNSO - IdoA Xyl Ser HSPG core protein 3 Figure 5. Scheme of domain structure within HS/heparin chains.
Although heparin shares the common biosynthetic pathway with HS, heparin shows higher N- and O-sulfate contents than HS. More than half of the HexA in heparin is IdoA; whereas generally HexA in HS is largely GlcA (Casu and Lindahl, 2001). Therefore the predominant disaccharide unit in heparin is -IdoA2S-GlcNS6S-, which generate abundant NS domains within heparin chains (Fig. 5). HS/heparin biosynthetic reactions occur consecutively, such that the prod- ucts of early events provide the substrates for later steps. Some of the en- zymes are known to function as a complex, for instance, EXT-1/EXT-2 ( McCormick et al., 2000; Senay et al., 2000; Busse et al., 2007), EXT-2/Ndst- 1 (Esko and Selleck, 2002; Presto et al., 2008) and Hsepi/Hs2st (Pinhal et al., 2001; Hagner-McWhirter et al., 2004).
16 Regulation of HS structure How the HS biosynthesis is regulated and why HS from various sources are distinct from each other are not well known so far. The biosynthetic process involves many different enzymes that recognize their specific or preferred substrates. Many sites in the HS chains escape certain modifications. Varia- tions in concentration of enzymes/isoforms and the random binding to their substrates are possible reasons for HS structural variability. In addition, the modification of HS/heparin chains is partly regulated by the substrate specificities of the enzymes involved in the process. For instance, the Hsepi and O-sulfotransferases require the presence of preformed N-sulfate groups. The recently proposed “GAGosome” concept implies that the biosynthetic enzymes are organized and tightly packed in a complex. Previous studies suggested that regulation in HS biosynthesis depends critically on the mode of selection and assembly of the enzymes (Ledin et al., 2006). Moreover, “compensatory” mechanisms have been implicated, such as the up- regulation of N- and 6-O-sulfation in mice depleted of Hsepi or Hs2st (Merry et al., 2001; Li et al., 2003). In addition to the regulation during HS biosynthesis, endo-6-O-sulfatases (Sulf) that selective release 6-O-sulfates, is regarded to regulate HS structure post-biosynthetically. Targeted disruption of Sulf-1 and Sulf-2 resulted in increase of -IdoA2S-GlcNS6S- and loss of -IdoA2S-GlcNS- units, hence affecting several different signaling pathways (Ai et al., 2007; Lamanna et al., 2007).
Functions of HSPGs Interactions of HS and proteins General properties HSPGs contribute significantly to the integrity of the ECM by binding and assembling ECM proteins (i.e., laminin, fibronectin, collagen type IV), thereby playing important roles in cell–cell and cell–ECM interactions. These interactions may play important roles in control of normal and patho- logical processes, such as morphogenesis, tissue repair, inflammation, vascu- larization and cancer metastasis (Kjellén and Lindahl, 1991; Bernfield et al., 1999; Iozzo and San Antonio, 2001). The classic view of HSPGs is that they serve as co-receptors for growth factors/morphogens on the cell surface, thus facilitating the formation of ligand-receptor complexes. Basic-fibroblast growth factor (bFGF) was the first growth factor shown to depend on HS for interaction with its receptor (Rapraeger et al., 1991; Yayon et al., 1991). This general model has been extended to many other signaling pathways, including Hedgehog (Hh), bone
17 morphogenic protein (BMP) and Wnt signaling, all of which are affected by deprivation of endogenous HS (Bornemann et al., 2004; Takei et al., 2004). However, several lines of evidence suggest that signaling by some growth factors relies on contributions from the protein core in addition to the HS chains (Kramer and Yost, 2003), for instance, the non-glycanated glypican-3 core protein retain some capacity to promote Wnt signaling (Ohkawara et al., 2003; Capurro et al., 2005; Kirkpatrick et al., 2006). In addition to growth factors, a large number of proteins, including en- zymes, enzyme inhibitors, morphogens and ECM components with a wide variety of functions are known to bind HS/heparin (Bernfield et al., 1999). Although it is known that sulfate and carboxyl groups in the HS chains play a key role by interacting with basic residues on the proteins, little is under- stood about how these ionic interactions provide the necessary specificity for protein binding (Akerstrom et al., 1994; Kreuger et al., 2006).
Interactions of HS and fibroblast growth factors (FGFs) FGFs are a family of growth factors essential for embryonic development, wound healing and angiogenesis. The FGFs are HS-binding proteins and their interactions with cell-surface associated HSPGs have been shown to be essential for signal transduction. Binding of FGFs to their cognate cell sur- face tyrosine kinase FGF-receptors (FGFRs) requires HS chains acting as a template that bridges FGFs and the FGFRs (Forsten-Williams et al., 2005). So far, 22 FGF members and four types of FGFRs (FGFR1-4) have been identified (Williams, 1993; Faham et al., 1998; Johnson and Szebenyi and Fallon, 1999; Friedl et al., 2001). FGFR contains three extracellular immu- noglobulin (Ig)-like domains (I, II and III), an acidic region (between I and II), a transmembrane helix and two intracellular tyrosine kinase domains (Fig. 6) (Lee et al., 1989; Johnson and Williams, 1993). Alternative RNA splicing results in two different Ig-like domain III (b and c) in FGFR 1-3. The splicing is an essential determinant of ligand binding specificity (John- son and Williams, 1993; Yeh et al., 2003). Activation of FGFRs results in stimulation of various signal transduction cascades that have been implicated in multiple aspects, such as embryonic development, angiogenesis, wound healing and tumor growth (Powers et al., 2000; Ornitz and Itoh, 2001). Three main pathways stimulated by FGFs have been extensively studied: Ras- mitogen activated protein kinase (MAPK) pathway, phoshatidylinositol-3- kinase-protein kinase B (PI3K-Akt) and phospholipase C (PLC) pathway. The MAPK pathway is the most studied pathway since it is activated in all cell types in response to FGFs and associated with mitogenic response (cell migration and proliferation), whereas the other two are cell type specific and crucial for other responses (apoptosis) (Boilly et al., 2000; Schlessinger et al., 2000; Dailey et al., 2005). Recent studies on FGF1 and FGF2 have demonstrated that the HS- dependent signaling relies on stabilization of the FGF-HS-FGFR ternary
18 complex (Fig. 6) and that the overall level of O-sulfation in HS appears to be more important than the precise distribution of sulfate groups (Jastrebova et al., 2006). In addition, in vitro biochemical studies have pointed to the im- portance of IdoA residue in binding to FGFs (Turnbull et al., 1992; Macca- rana et al., 1993)
I Ig-like ECM II domains III Cell membrane I Tyrosine Cytoplasm II kinase domains
signaling
FGFR HSPG FGF
Figure 6. FGF-HS-FGFR complex. HS chains promote the formation of FGF- FGFR complex, leading to dimerization of FGFRs.
Two crystal structures of FGF-HS-FGFR ternary complex have been pub- lished: the symmetrical 2:2:2 complex involves two components of each kind and heparin chains functioning as a stabilizer of ligand-receptor and receptor- receptor contacts (Schlessinger et al., 2000); the asymmetrical structure 2:2:1 complex is formed around a single heparin chain that function as a bridge be- tween ligand and receptor molecules (Pellegrini et al., 2000). A recent study showed that both of the interaction modes can occur depending on the length and structure of the HS/heparin oligosaccharide used. For instance, heparin fragments larger than octasaccharide dimerize FGF2 on heparin (FGF2-heparin 2:1) in a cooperative manner and lead to a very rapid and efficient dimerization of FGFR1c on FGF2 (FGF2-FGFR-heparin 2:2:1) (Goodger et al., 2008); whereas shorter heparin fragments cannot dimerize FGF2, but form 1:1:1 com- plex (FGF2-Heparin-FGFR) and then the association of the 1:1:1 complexes form symmetrical 2:2:2 complexes (Goodger et al., 2008).
HSPGs in developmental processes Animal development involves differentiation of individual cell types and their assembly into specific tissues. Mutational studies in Drosophila and targeted disruption of murine genes involved in HS biosynthesis or encoding HSPG core proteins, have demonstrated a critical role for HSPGs in various developmental processes. For instance, elimination of murine EXT-1 and 2, resulted in early embryonic lethality (Lin et al., 2000; Stickens et al., 2005); targeted disruption of murine Ndst-1, Hs2st and Hsepi resulted in perinatal
19 lethality (Bullock et al., 1998; Fan et al., 2000; Ringvall et al., 2000; Li et al., 2003; Grobe et al., 2005); however, mutant animals deficient in Ndst-2 and Hs3st-1 show only mild phenotypes (Forsberg et al., 1999; Humphries et al., 1999; HajMohammadi et al., 2003). These deficiencies might due to failure to assemble active signaling complexes or relate to the morphogen gradients involved in many developmental contexts. HS facilitates the accu- mulation and stabilization of different morphogens such as members of the Wnt, BMP, Hh, transforming growth factor- (TGF-) and FGF families. Since HSPGs influence both the stability of extracellular signaling and the ability to spread through tissues, the net effects of HSPGs on a morphogen gradient depend on the balance between these activities in a particular con- text.
HSPGs in diseases Inflammation HS expressed on endothelial cells is involved in multiple stages of inflam- matory response. In the case of inflammation, leukocytes attach to activated endothelial cells and migrate into areas of damaged tissue. In this process, binding of L-selectin expressed by leukocytes to endothelial HS initiates the attachment and rolling of leukocytes into blood vessels. Chemokines, small pro-inflammatory signaling proteins, are produced under stimuli and trans- ported from the ablumenal to the lumenal side of endothelial cells (Parish, 2005). Chemokines bound to HS are presented to leukocytes, resulting in activation of integrin and firm adhesion of rolling cells (Middleton et al., 1997; Gotte, 2003). In addition, HS retains chemokines on the endothelial cell surface, allowing gradient formation, which facilitates the leukocytes following the gradients to enter the target tissue. The precise roles of HS during in vivo inflammatory responses are not yet clear. However, recent studies on mutant mice with a targeted disruption of Ndst-1 in endothelial cells demonstrated at least three functions of endothelial HS in inflamma- tion: HS acts as a ligand for L-selectin during neutrophil rolling; HS trans- ports chemokines across endothelial barrier; HS binds and presents chemoki- nes to leukocytes (Wang et al., 2005).
Amyloid diseases Amyloid diseases, including several serious conditions such as Parkinson's disease, Alzheimer's disease and type II diabetes, are characterized by the formation of insoluble fibrillar aggregates of misfolded proteins in multiple organs and tissues (Temussi et al., 2003). There are at least 22 polypeptides known to refold and assemble into highly organized fibrils, which associate with HSPGs to form tissue deposits termed amyloid (Elimova et al., 2004). Many studies have demonstrated that HSPGs enhance fibrillization of the peptides and stabilize already formed aggregates against proteolytic degrada-
20 tion (Kisilevsky, 2000; van Horssen et al., 2003). The precise roles of HSPGs in amyloidosis are not fully elucidated; however, recent studies on heparanase transgenic mice showed that amyloid deposition is prevented by shortened HS chains due to high activity of heparanase (Li et al., 2005). These findings provide direct in vivo evidence that HSPGs play important roles in amyloid diseases. Recently, HS has been identified as the first naturally occurring inhibitor of -secretase that processes the precursor protein, -amyloid polypeptide (-APP). This inhibitory activity is dependent on the structure and size of the HS chains, with emphasis on specific sulfation pattern (Scholefield et al., 2003; Patey, 2006). These findings have led to the investigation of HS ana- logues as potential therapeutic approaches to treat Alzheimer’s disease (Patey, 2006).
Cancer Cancer is a group of diseases in which cells grow and divide without respect to normal limits; the abnormal cells invading and destroying adjacent tissues, often followed by spreading to other locations in the body. HS chains, functioning as a barrier of the basement membrane, are destroyed by heparanase released from tumor cells to facilitate entry of tumor cells into blood circulation and further dissemination. Angiogenesis is important in tumor progression, since tumor growth is dependent on vascularization and new blood supply. Several angiogenic factors, such as vascular endothelial growth factor (VEGF), FGFs and hepatocyte growth factor (HGF), have been shown to interact with HSPGs (Bogenrieder and Herlyn, 2003; Jiang and Couchman, 2003).
Functions of heparin Interaction of heparin and antithrombin - AT Heparin is best known for its anticoagulant activity, and has been used clini- cally as an anticoagulant for over 60 years (Rabenstein, 2002). The antico- agulant effect is exerted by the interaction of heparin with AT, which is the best-studied example of heparin-ligand binding. The binding mode, referred to as "lock-and-key" which depends on a specific structure of a pentasaccha- ride, containing a central 3-O-sulfated GlcNSO3 residue (Fig. 7) (Bourin and Lindahl, 1993). The binding of AT to heparin induces a conformational change of the protein, which increases the rate of inactivation of proteases involved in coagulation (such as thrombin) (Jordan et al., 1980). Approxi- mately only one-third of the polysaccharide chains in heparin preparations contain the pentasaccharide sequence (Andersson et al., 1976; Höök et al., 1976; Lam et al., 1976).
21 CH OSO - COO- CH OR - 2 3 2 CH2OSO3 O O O O O COO- OORH OH O OORSO - OH O OORH O 3 O O HNR’ OH HNSO - OSO - HNSO - 3 3 3
Figure 7. Antithrombin binding sequence in heparin. The 3-O-sulfate group - is a marker for the antithrombin-binding sequence (R represents H or SO3 , R’ repre- - sents COCH3 or SO3 ).
Heparin in mast cells In spite of the extensive clinical use of heparin as an antithrombotic drug, its physiological functions remain unclear. Recent studies suggest that heparin is essential for the storage of specific granule proteases in mast cells (Forsberg et al., 1999; Humphries et al., 1999; Åbrink et al., 2004). The de- tails of the interaction between heparin and positively charged mast cell pro- teases are not known but presumably involve electrostatic interactions of the positively charged proteases with the highly negatively charged heparin (Rabenstein, 2002).
Apart from anticoagulation and protease binding, heparin is potentially ex- ploitable in therapy due to its ability to modulate angiogenesis, tumor metas- tasis, inflammation and microbial invasion. These effects have been ascribed to inhibition of heparanase and as well as interactions with HS-binding pro- teins (Folkman and Shing, 1992; Vlodavsky et al., 1994; Borsig et al., 2001; Rabenstein, 2002).
Heparanase Activity and processing Heparanase is an endo--D-glucuronidase that catalyzes the hydrolytic cleavage of the -1,4-glycosidic bond between a D-glucuronate and a D- glucosamine in HS/heparin chains (Fig. 8), yielding fragments of variable size (Freeman and Parish, 1998; Pikas et al., 1998; Vlodavsky and Fried- mann, 2001). Heparanase seems to recognize various sequences in HS/heparin chains, but may prefer some specific sequences that are not fully elucidated. Previous studies indicate that a 2-O-sulfate group on a HexA residue located two monosaccharide units away from the cleavage site (Fig. 8) appears essential for the recognition (Pikas et al., 1998).
22 Heparanase
CH OSO - COO - - - - 2 3 CH2OSO3 COO CH OSO 2 3 CH2OH O O O O O O O COO- O OORH O OH OROH OH O OORH OH O O O OORH O
- OH HNAc - - - HNSO3 OH HNSO OSO HNSO 3 3 3 Figure 8. Heparanase cleavage site. Heparanase cleaves the -1,4-glycosidic bonds between GlcA and GlcN.
Heparanase is synthesized as a latent 65 kDa precursor whose processing involves proteolytic cleavage and formation of an active enzyme composed of a 50 kDa subunit associated non-covalently with an 8 kDa peptide. (Cohen et al., 2005). Heparanase exhibits maximal endoglycosidase activity between pH 5.0 and 6.0, and is inactivated at pH>8.0 (Gilat et al., 1995). Heparanase is expressed at a low level in essentially all tissues, but it is often elevated in tumor tissues in which malignant tumor cells may provide an acidic environment required for degradation of HS by heparanase.
Functions Normal development and tissue remodeling The role of heparanse in normal development is still not well characterized. As the predominant degradation enzyme of HS, heparanase plays important roles in maintaining the matrix integrity and balancing soluble/insoluble molecules of the ECM. Heparanase appears to be involved in embryonic implantation and development (Dempsey et al., 2000; Goldshmidt et al., 2001), wound repair, tissue remodeling, immune surveillance and hair growth (Zcharia et al., 2005a; Zcharia et al., 2005b).
Pathophysiology Overexpression of heparanase in tumor cells correlates with increased metas- tatic potential (Vlodavsky et al., 1999; Parish et al., 2001; Vlodavsky and Friedmann, 2001). It is generally believed that heparanase is involved in tumor metastasis by playing several major roles: it induces angiogenesis by paving the way for new blood vessels to grow and releasing angiogenic fac- tors, such as FGF2 and VEGF (Folkman et al., 1988); it enables invasion of the tumor cells by digesting the ECM of the surrounding tissue ( Gohji et al., 2001; Tang et al., 2002; Ohkawa et al., 2004; Beckhove et al., 2005); it fa- cilitates metastasis and the distribution of secondary tumors, enabling tumor cells to extravasate by digesting HS on the basement membrane of blood vessels (Vlodavsky et al., 2006); moreover, HS fragments released by heparanase potentiate FGF2 receptor binding, dimerization and signaling (Vlodavsky et al., 1996; Elkin et al., 2001; Escobar Galvis et al., 2007).
23 In addition to tumor metastasis, heparanase is found to be up-regulated in inflammation, wound healing and diabetic nephropathy, effects being attrib- uted to enhanced cell dissemination as a consequence of HS cleavage and remodeling of the ECM barrier (Dempsey et al., 2000; Parish et al., 2001; Vlodavsky and Friedmann, 2001). Involvement of heparanase in various pathological conditions points to heparanase as a target for therapy. Several inhibitors of heparanase have been studied and tested in animal models. Heparin was shown to inhibit HS degradation by heparanase (Vlodavsky et al., 1999), owing to the higher affinity of heparin for heparanase compared to HS. On a general note, hepa- rin has higher affinity for essentially all HS-binding proteins due to its high sulfation degree; therefore heparin can inhibit the interactions between growth factors and HS (Fuster and Esko, 2005).
Non-enzymatic activities In addition to the catalytic feature of the enzyme, heparanase was noted to exert biological functions apparently independent of its enzymatic activity (Levy-Adam et al., 2008). For instance, adhesion of lymphoma and T cells was enhanced by overexpression of non-enzymatic heparanase in the cells through activation of Akt signaling pathway (Goldshmidt et al., 2003; Gingis-Velitski et al., 2004). A recent study revealed that noncatalytic heparanase facilitates cell adhesion and spreading by clustering of syndecan- 1 and -4 (Levy-Adam et al., 2008).
Mast cells Mast cells are specialized secretory cells that play an important role in host defense by discharging a variety of pro-inflammatory mediators and im- munoregulatory cytokines (Metcalfe et al., 1997). Mast cells are derived from hematopoietic stem cells, but they do not or- dinarily circulate in mature form; instead, they undergo the terminal stages of their differentiation and maturation after the migration of their precursors into those vascularized tissues or serosal cavities in which they ultimately will reside (Kawakami and Galli, 2002). In mammals, mast cells are widely distributed throughout vascularized tissues, particularly beneath, and in some cases within, epithelia, and around blood vessels, nerves, smooth muscle cells, mucus-producing glands and hair follicles. Mast cells are important specially because they locate near surfaces exposed to the environment, in- cluding the skin, airways and gastrointestinal tract, where pathogens, aller- gens and other environmental agents are frequently encountered (Galli et al., 2005). When the host encounters specific antigens, IgE that is produced by the host under stimulation will bind to its high-affinity receptor (FcRI) ex- pressed on mast cells. Crosslinking of FcRI-IgE with antigen initiates the
24 activation of mast cells (Kitamura, 1989; Marshall, 2004). This FcRI- dependent cell-activation process has three outcomes: degranulation, with the secretion of preformed mediators that are stored in the cytoplasmic gran- ules of mast cells; the de novo synthesis of pro-inflammatory lipid media- tors; and the synthesis and secretion of cytokines and chemokines (Kawakami and Galli, 2002). Such mast cell activation can contribute to host resistance to certain parasites or can drive the pathology of allergic disorders such as asthma (Lantz et al., 1998; Williams and Galli, 2000). PGs found in mast cells are generally thought to be the serglycin type, which carries both heparin and CS. It is known that serglycin PGs have an important function in binding to various secretory granule compounds, thus facilitating their storage. For instance, targeting of the genes coding for ser- glycin or Ndst-2 in mice resulted in a similar phenotype in mast cells, with a drastic reduction of the various proteases that are normally found in connec- tive tissue-type mast cells (Forsberg et al., 1999; Humphries et al., 1999; Åbrink et al., 2004).
25 Present Investigations
Aims of study The purpose of this study is to investigate the importance of Hsepi and heparanase on the structure and functions of HS and heparin.
The aims of the individual papers were:
• obtain insight into the mechanism behind the developmental defects in the Hsepi mutant (Hsepi-/-) mice, using murine embryonic fibro- blast (MEF) cell lines (Paper I)
• to analyze the structure of HS isolated from heparanase transgenic (Hpa-tg) mouse livers, tumor cell lines as well as tumor tissues, and elucidate HS functional correlations with its structural changes (Pa- per II)
• to generate heparanase knockout mice (Hpse-KO) and investigate the phenotypes of the mice (Paper III)
• to characterize fetal skin derived mast cells (FSMCs) from Hpse- KO, Hpa-tg and wild-type (WT) mouse embryos and investigate the structure and functions of heparin in mast cells (Paper IV)
26 Results Paper I Lack of L-iduronic acid in heparan sulfate affects interaction with growth factors and cell signaling
The Hsepi mutant mice are neonatally lethal with selective developmental defects, demonstrating the essential roles of this enzyme for animal devel- opment. The aim of the study was to obtain insight into the mechanism be- hind the developmental defects in the Hsepi-/- mice, using MEF cell lines derived from mutant mice. Analysis of HS revealed the structural alterations of HS from mutant MEF cells, which does not contain IdoA residue, but has higher degree of N- sulfation and 6-O-sulfation. However, the molecular mass does not show difference between the mutant and WT MEF cells. To further study the functional significance of IdoA residues in relation to cellular behaviors induced by FGF2, we compared the ability of MEF cells to proliferate and migrate. In the presence of 10% fetal calf serum (FCS), both WT and Hsepi-/- cells performed well showing no significant difference. However, under starvation condition, the proliferation and migration of WT cells were largely restored by stimulation of FGF2, whereas the mutant cells were refractory to FGF2 treatment. Moreover, delay of ERK phosphoryla- tion (an important downstream transduction component in FGF2-induced signaling) following FGF2 stimulation supported the idea that lacking of IdoA in HS affects cellular behavior due to weak signaling response. Study of HS-growth factor interaction revealed an aberrant binding prop- erty of Hsepi-/- HS with FGF2 and GDNF. In summary, Hsepi deficient MEF cells produce defect HS, lacking IdoA but having higher degree of N-sulfation and 6-O-sulfation; the abnormal HS resulted in aberrant binding mode between HS and FGF2, which is corre- lated with the defective response of Hsepi-/- cells to FGF2 stimulation.
Paper II Transgenic or tumor-induced expression of heparanase upregulates sulfation of heparan sulfate
To study the overall effects of heparanase on HS metabolism, we transgenically overexpressed heparanase in mice. HS structure analysis after metabolic labeling of the mice showed that fragmentated and highly N- and O-sulfated HS was produced in Hpa-tg liver that express high levels of heparanase. Further studies revealed that HSPG turnover rate was accelerated along with the structural changes.
27 In addition, we examined subcellular compartments of hepatocytes for expression of heparanase and the structure of HS. Results showed that heparanase contributes to both extracellular and intracellular HS degradation. Confocal microscopy revealed that a limit amount of transgenic heparanase was colocalized with Golgi markers, suggesting that heparanase maybe accociated with HSPG biosynthesis; however the precise mechanism remains to be elucidated. Moreover, the heavily sulfated HS fragments isolated from Hpa-tg liver strongly promoted formation of ternary complexes of FGF1 or FGF2 with FGFR-1. Similarly, human tumors with up-regulated expression of heparanase also showed increased 6-O-sulfation in HS, however, the N-sulfation degree was conserved. The findings in this work may imply a mechanism in tumor metastasis where heparanase is often up-regulated. The increased sulfation of HS modulated by heparanase may facilitate the formation of signaling complexes, accordingly promoting tumor growth and metastasis. This may also explain the effect of tumor attenuation by inhibiton of heparanase.
Paper III Newly generated heparanase knock-out mice unravel co-regulation of heparanase and matrix metalloproteinases
To further study the functions of heparanase, we generated Hpse-KO mice by targeted disruption of the gene. The complete elimination of the gene expression and heparanase activity were confirmed by RT-PCR and enzy- matic assay. The Hpse-KO mice did not show obvious phenotypes, except enhanced angiogenesis under FGF2 stimulation and abundant mammary glands comparing to WT mice. HS structure analysis showed the unfragmentated HS chains in Hpse-KO mice; however, there was no significant difference in HS fine structure. Further characterization revealed that elimination of heparanase has in- duced expression of matrix metalloproteinases (MMP)-2 and 14, suggesting interplay between the MMP and heparanase to modulate the extracellular structure. This hypothesis is supported by the finding that MMP-2,-9 and 14 were markedly decreased in human carcinoma cells in which heparanase was overexpressed. Additional studies revealed that the interplay of heparanase with MMPs is mediated by increased expression and stabilization of - catenin, a transcription factor targeting MMPs.
28 Paper IV Heparanase cleavage of heparin regulates protease storage in mast cells
To study the structure and functions of heparin in vivo, we characterized FSMCs from Hpse-KO, Hpa-tg and their WT mouse embryos. Heparin structure analysis revealed unfragmentated heparin chains in Hpse-KO FSMCs and extensively fragmentated heparin chains in Hpa-tg FSMCs, these findings provided the first in vivo evidence that heparanase is the enzyme degrading mast cell heparin. Moreover, we found that extensive fragmentation of heparin in Hpa-tg FSMC caused loss of proteases in the cells; in contrast, increased storage of proteases was observed in Hpse-KO cells. Conclusively, heparanase is responsible for processing of mast cell hepa- rin, thereby it is proposed to modulate protease storage in mast cells through degradation of heparin.
29 Discussion and Future Perspectives
HS is known to be important in developmental and pathological processes, its unique molecular design and flexibility is essential to its ability to modu- late cellular responses to growth factors and morphogens. The enzymes committed to HS biosynthesis and their specific substrates define HS struc- ture. There are over 20 enzymes (including isoforms) known to be involved in HS biosynthesis, generating a great variety of HS structure. To understand the critical roles of these enzymes in HS structure/function relations, various transgenic model organisms have been established and studied. Depending on the enzymes or their isoforms, the phenotypes are highly variable, from gastrulation failure to subtle disturbance of organ development. The present work was undertaken to examine the importance of IdoA unit, particularly in relation to FGF signaling. This was done by in vitro challenging of MEF cells devoid of Hsepi, and thus unable to convert GlcA to IdoA in HS. In a second approach we attempted to assess the importance of chain length, by modulating the expression level of heparanase, the only hydrolase so far known to internally cleave HS/heparin chains. The Hsepi-/- mice die shortly after birth with selective developmental de- fects, such as renal agenesis, skeletal malformations and lung defects. Pre- vious studies showed that Hsepi-/- mice produce abnormal HS chains, lacking IdoA and with severely distorted sulfation pattern. What is the correlation between the phenotypes and the abnormal HS structure? In Paper I, we showed that HS from both Hsepi-/- embryos and MEF cells failed to properly bind selected growth factors, FGF2 and GDNF. Accordingly, Hsepi-/- MEFs responded poorly to FGF2 in signaling, migration and proliferation experi- ments. Previous studies on Hs2st-/- mice showed that mutant MEFs lacking 2-O-sulfation but with normal level of IdoA were able to mount normal FGF2 signaling (Merry et al., 2001), in support of the notion that lack of IdoA compromises HS-dependent FGF2 signaling. Both Hsepi-/- and Hs2st-/- mice lacked kidneys, whereas Ndst-1-/- mice, generating under-sulfated yet IdoA-containing HS showed normal kidney development. These observa- tions suggest that IdoA2S is critical in kidney development, presumably by promoting GDNF signaling (Bullock et al., 1998; Li et al., 2003). Indeed, our studies showed aberrant binding of GDNF to Hsepi-/- HS. Notably, the Hsepi-/- MEFs proliferated and migrated similarly to WT cells in medium containing 10% FCS. These findings suggest that HSPG-independent signal- ing systems activated by growth factors could be recruited to promote cellu- lar functions, such as proliferation and migration; such redundancy could
30 explain the virtually normal morphology of some organs, for instance brain and heart, in the mutant mice (Li et al., 2003). Alternatively, however, the formation of these organs could be critically dependent on participation of HS chains but not on the fine structure of these chains. In fact, the latter al- ternative is favored by the observation of severely perturbed brain morphol- ogy in mice with conditional EXT-1 ablation, resulting in selective lack of cerebral HS (Inatani et al., 2003). An intriguing question in this context is to what extent the “compensatory” up-regulation of N- and 6-O-sulfation of Hsepi-/- HS contributes to HSPG function. Previous studies showed that Hs2st-/- MEF cells were able to mount nor- mal FGF2 signaling in spite of poor binding of FGF2 to Hs2st-/- HS (Merry et al., 2001). The ability of HS to promote signaling thus is not readily pre- dictable from FGF-HS binding data. Another intriguing issue concerning the mechanism behind the abnormal binding properties of Hsepi-/- HS is if it is directly due to lack of IdoA or does it rather reflect a changed domain pat- tern along the HS chains? In addition to roles in stabilizing growth factor/morphogen gradients and as co-receptors in various signaling pathways, HS is also an essential struc- tural component of the intact ECM. Some effects of heparanase overexpres- sion, resulting in increased cleavage of HS chains, presumably reflect per- turbed integrity of the ECM, hence changes of cellular microenvironment. For instance, tumor cells overexpressing heparanase facilitate metastasis by paving the way to other tissues; growth factors/morphogens bound to HSPGs are mobilized through cleavage of HS chains by heparanase and become accessible for stimulation of tumor growth or angiogenesis. To further un- derstand functions of HS, we performed studies on Hpa-tg (Paper II), and Hpse-KO (Paper III) mouse models. Unexpectedly, structural analysis of free HS chains purified from Hpa-tg liver showed not only extensive formation of HS cleavage products, but also up-regulation of N- and O-sulfation. These products were ‘heparin-like’, with markedly increased proportion of the tri-sulfated (-IdoA2S-GlcNS6S-) disac- charide unit. What did cause the structural changes of HS? Our evidence demonstrated that the overexpression of heparanase affects HSPG biosynthe- sis: First, the newly synthesized HS chains (PG-associated HS) showed simi- lar up-regulated sulfation as the free HS fragments; second, the extensively degraded HS chains were found already in the Golgi compartment; third, confocal images showed overlap of heparanase and Golgi markers. Notably, experiments with FGF1 and FGF2 showed increased ability of Hpa-tg com- pared to WT HS to promote formation of FGF-HS-FGFR ternary complexes, in accord with previous observations of overall sulfation being critical in the formation of the signaling complex (Jastrebova et al., 2006). These findings allowed us to correlate the overexpression of heparanase and the increased HS sulfation observed in several malignant tumors with the augmented FGF signalings that are essential to tumor growth and angiogenesis.
31 Hpse-KO mice, unexpectively, did not show any obvious phenotypic change. Instead, we found that various MMPs occurred in increased amounts in Hpse-KO organs. MMPs are involved in tissue remodeling, and are capa- ble of degrading several kinds of ECM proteins to release/inactivate/activate a number of chemokines/cytokines. We hypothesize that lack of heparanase might induce expression of MMPs capable of degrading HSPG core pro- teins. In Hpse-KO mice, we found that different MMPs were increased in different organs, potentially compensating for defective shortening of HS chains by MMP-induced shedding of whole PG extracellular domains. Whether such a phenomenon could help preserve organ and tissue function should be addressed in future work. The heparin used in the clinic as an anticoagulant/antithrombotic for many decades is largely isolated as fragments of serglycin polysaccharide chains generated within the mast cells. The processing implicated behind this deg- radation is still not fully understood. Previous studies indicated that an en- doglucuronidase detected in a mouse mastocytoma was involved in heparin degradation (Ögren and Lindahl, 1975; Jacobsson and Lindahl, 1987). Sub- sequent work suggested that this heparin-degrading enzyme is in fact identi- cal to the heparanase involved in HSPG metabolism (Gong et al., 2003). However, there has been no direct evidence implicating heparanase with the processing of heparin PG. In Paper IV, we used FSMC from Hpa-tg, Hpse- KO and their controls to investigate the processing of heparin and the func- tional consequences. The results showed that heparin is indeed degraded by heparanase in vivo; moreover, heparanase seems to be the only specific en- zyme that is responsible for processing of heparin. Interestingly, shorter heparin chains resulted in reduced storage of proteases whereas the unfrag- mentated heparin chains promoted protease accumulation. These findings lead to the conclusion that storage of proteases by heparin is correlated not only with polysaccharide charge but also with chain length; in other words, hepararanase seems to regulate the storage of proteases in mast cells. Moreover, the results raise intriguing questions regarding protease storage and release under physiological or pathological conditions and the roles of heparanase in regulation of protease homeostasis.
32 Populärvetenskaplig Sammanfattning
Proteoglykaner (PG) består av glykosaminoglykankedjor som är kovalent bundna till ett protein. Heparansulfat (HS)/heparin tillhör denna familj, de är uppbyggda av disackaridenheter som sitter förenade till aminosyran serine i ett protein. Heparansulfatproteoglykaner (HSPG) återfinns på nästan alla kroppens celler och är viktiga i flera fysiologiska och patologiska processer, så som embryonalutveckling, cancer och amyloidsjukdomar. Heparin där- emot produceras av kroppens mastceller och är mest känd för dess kliniska användning som ett antikoagel. HS och heparin syntetiseras i Golgi av över 20 olika enzymer (inklusive isoformer) och startar med en sekvens av GlcA-Gal-Gal-Xyl och följs av alternerande GlcA och GlcNAc enheter. Under tiden kedjan polymeriseras sker även en mängd modifieringar (Fig. 3) vilket leder till att kedjorna är mycket heterogena med avseende på längd, sulfatering och epimerisering. Modifieringarna under biosyntesen men även 6-O-desulfatering av enzymer- na Sulf1/2 bidrar till denna heterogenitet. Det är fortfarande oklart varför det finns en så stor variation av HS-strukturer. Denna avhandling presenterar studier på hur biosyntesenzymet C5- epimeras och enzymet heparanas påverkar HS och heparinets struktur och funktion. Den första delen av denna avhandling fokuserar på C5-epimeras, enzy- met som omvandlar GlcA till IdoA. Vi har visat att denna modifiering är nödvändig för HS-beroende tillväxtfaktorers, speciellt fibroblasttillväxtfak- torn FGF2, funktioner i celler. Den andra delen av avhandlingen fokuserar på enzymet heparanas. Detta enzym är inblandat i metabolismen av HS och klyver HS-kedjorna vid en specifik sekvens, mellan GlcA och glukosamin. Enzymet uttrycks i låga nivåer i de flesta celler men är uppreglerat i många metastaserande tumörer. De studier som presenteras här har gett inblick i hur heparanas påverkar HS- biosyntesen och hur heparanas reglerar matrix metalloproteinaser, MMPs. Vi har dessutom studerat heparanasets funktion i mastceller och kunnat visa att heparanas klyver heparin i mastceller och påverkar lagringen av olika prote- aser.
33 Q
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34 Acknowledgements
I would like to express my sincere gratitude to all the people who have con- tributed to this thesis in different ways:
My supervisor Jin-ping Li, thank you for your excellent scientific supervi- sion, for sharing your profound knowledge in science, for your patience, support and encouragement during the five years. It is a pleasure working in your lab! I would also like to express my appreciation for your care during these years, the wonderful time on weekends with your family and the deli- cious Chinese food, all of these meant a lot to me!
My co-supervisor Ulf Lindahl, thank you for your smart advice on my pro- jects, your encouragement and suggestions for my oral presentations and for supporting me for so many excellent conferences.
My examiner Lena Kjellén, thank you for your nice talk and suggestions on my projects.
Xiao Zhang, thanks for being a great collaborator and for sharing your tips of doing experiments, special thanks for giving me so great family feeling together with Jin-ping.
My roommates: Elina Sandwall thanks for your great friendship, help on my Swedish document papers, and encouragement both on studies and my life. Special thanks for the nice accompany during conferences; Fredrik Noborn and Christoffer Tamm, thanks for the laughs and entertainment, which make the atmosphere in our office is really relax and nice.
Other present colleagues: Dorothe Spillmann thanks for your great sugges- tions on my seminar; Anna Eriksson thanks for your friendship, kindness and being great laughter; Rashmi Ramachandra thanks for your delicious Indian food, the home made cheese is amazing.
Previous colleagues: Anh-tri Do thanks for being a great friend and your encouragement for both my study and life; Eva Gottfridsson thanks for all the laughing, chit-chatting and good company in parties, for sharing so many excellent experiment tips and your help on lab equipment, for being kindness and encouragement; Eva Hjertson, thanks for the fresh vegetables, the sweat Christmas gifts and of course, for taking care of my precious cells;
35 Lena Nylund, thanks for being my great teacher of lab techniques and for- giveness of stealing your buffers; Martha L Escobar Galvis thanks for being a great roommate and collaborator, also for the help on lab experi- ences; Nadja Jastrebova thanks for your kindness and so much great chat- ting; Sabrina Bodevin thanks for your encouragement and sharing so many laughs; Rebecka Hjelm, Pernilla Carlsson, Maarten Vanwildemeersch and Sindhu Kurup thanks for your kindness and laughs.
People in D9:4 corridor: Jimmy Larsson thanks for reminding me to start with the preparation for my dissertation; Irmeli Barkefors thanks for the kindness and laughs; Anders Dagälv, Beata Filipek, Inger Eriksson, Johan Heldin, Johan Kreuger, Karin Forsberg-Nilsson, Katarina Holmborn, Ludvig Petersson, Maud Forsberg, Peter Jonsson, Sébastien Le Jan, Tobias Bergström, Ulrika Wallenquist, Xiao-qun Zhang and Zsolt Kasza thanks for your kindness and creating such a nice atmosphere in our corridor.
The IMBIM administrative staff: Barbro Lowisin, thank you for the nice organization of GLIBS annual meetings and GLIBS courses, from which I have learned a lot about glycobiology; Marianne Wigenius, Rehne Åker- blom, Erika Enström, Kerstin Lidholt and Olav Nordli for helping me with different things.
My friends out of BMC: Paul O’Callaghan, thanks for the nice scientific discussion; Diana Larsson and Niklas Larsson, thanks for all your help and support for my life in Sweden; Louise Halén and Anton Larson, thanks for your encouragement and so many wonderful dinners at William’s.
My Chinese friends: Hao Li () and Wei Sun (), thanks for your great friendship, for all the fun parts and life talks we did together, and also for the fantastic you did for me; Wei Xia ( ) and Miao Wu ( ) thanks for all the scientific discussion, accompany on weekends at BMC and the lunch boxes; Song Sun () thanks for sharing your historical knowledge and funny talks during lunch time.
My family: !"#$%& '()** * +,- ./ 012.3 456 789 $%#)**: 7;<=>?@ABC DE)4 FG H !I#$%J
Zhifen Pan (KLM), thanks for all the care and love during the five years, the happiness we have been together will always a good memory in my mind.
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