Journal of Cell Science 113, 193-205 (2000) 193 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0690

COMMENTARY Synthesis and sorting of proteoglycans

Kristian Prydz1,* and Knut Tomas Dalen2 1Department of Biochemistry and 2Institute for Nutrition Research, University of Oslo, Norway *Author for correspondence (e-mail: [email protected])

Published on WWW 13 January 2000

SUMMARY

Proteoglycans are widely expressed in animal cells. type, which is acquired by use of particular Ser-Gly sites in Interactions between negatively charged the protein core, might therefore be important for post- chains and molecules such as growth factors are essential for synthesis sorting. This links regulation of glycosaminoglycan differentiation of cells during development and maintenance synthesis to the post-Golgi fate of proteoglycans. of tissue organisation. We propose that glycosaminoglycan chains play a role in targeting of proteoglycans to their proper cellular or extracellular location. The variability seen Key words: Proteoglycan, Glycosaminoglycan, Sorting, Polarised in glycosaminoglycan chain structure from cell type to cell cell, Epithelial cell, Glycan

INTRODUCTION VARIATION IN PROTEOGLYCAN COMPOSITION

Proteoglycans (PGs) consist of a protein portion and long, PGs were initially grouped together because of the high unbranched ( or GAGs). negative charges of their GAG chains, which make separation The latter have a high negative charge, owing to the presence from other molecules by ion-exchange chromatography easy. of acidic sugar residues and/or modification by sulphate PGs are, however, not that similar. The core protein size ranges groups. The acidic sugar alternates with an amino sugar in from 10 kDa to >500 kDa, and the number of GAG chains repeated units. The GAGs adopt an extended attached varies from one to >100 (for review, see Poole, 1986; conformation, attract cations, and bind water. Hydrated GAG Rouslahti, 1988; Kjellén and Lindahl, 1991; Silbert and gels enable joints and tissues to absorb large pressure Sugumaran, 1995). In addition, several PGs carry GAG chains changes. of more than one type (hybrid PGs; Rapraeger et al., 1985; In addition to buffering pressure changes, PGs play Sugahara et al., 1992a) and/or have additional N-linked or O- important roles in control of growth and differentiation. linked sugar modifications. Particular sulphation patterns in the GAG chains allow Not all PGs are ‘full-time’ PGs. Some proteins, such as interactions, normally of ionic nature, with growth factors, for MHC class II invariant chain, thrombomodulin and the example. Recent studies have identified ~30 PG protein cores. transferrin receptor are ‘part-time’ PGs (Fransson, 1987), These cores are not just scaffolds for GAGs: they contain alternatively spliced variants having GAG-initiation sites. PGs domains that have particular biological activities (Iozzo, 1998). such as versican and CD44 also occur as alternatively spliced Many PGs are thus multifunctional molecules that engage in forms (Greenfield et al., 1999; Dours-Zimmermann and several different specific interactions at the same time. Zimmermann, 1994; Naso et al., 1994) whose sugar After synthesis PGs are transported from the Golgi to their modifications vary. A variant of versican without CS- destinations: the extracellular matrix (ECM), the cell surface attachment sites has been discovered, and thus the PG versican or intracellular organelles. Such vectorial transport requires could also be regarded as a part-time PG (Iozzo, 1998). mechanisms for recognition, sorting and delivery, which are GAG (except for in keratan sulphate (KS); Baker et al., especially important in cells such as epithelial cells and 1975; Stein et al., 1982) synthesis is initiated by sequential neurons, where the cell membrane comprises separate addition of four ( (Xyl), domains. Recognition and sorting must require determinants in (Gal), galactose and glucuronic acid (GlcA); see Fig. 1). From the GAG chains and/or in the PG protein cores. Here we this linker , the sugar chains are extended by discuss how and where chondroitin sulphate (CS)/dermatan addition of two alternating monosaccharides, an aminosugar sulphate (DS) and heparan sulphate (HS)/heparin GAGs are and GlcA. In heparin and HS, the aminosugar is N-acetyl- synthesised, and how these GAGs influence the sorting of PGs glucosamine (GlcNAc) and in CS/DS it is N-acetyl- to the sites at which they act. galactosamine (GalNAc; see Fig. 2). The extent of 194 K. Prydz and K. T. Dalen epimerisation of GlcA to iduronic acid (IdoA) and the are initiated as N-linked or O-linked and sulphation pattern of the disaccharide units distinguish heparin extended by addition of GlcNAc and Gal. from HS, and DS from CS (see Figs 1 and 2). In KS, the GAGs There is also regional variability to the epimerisation and Diverging point UDP-GlcA transferase I UDP-Gal transferase II UDP-Gal transferase I UDP-Xyl transferase

COO - CH2OH CH2OH H H O HO O HO O H O NH H H H H O O O H H H OH H O CH2 CH HO H H H H CO H OH H OH H OH H OH GlcA β(1→3) Gal β(1→3) Gal β(1→4) Xyl β O−Serine

GalNAc- EXTL2 transferase I GlcNAc- transferase I α → GalNAc (1 4) GlcNAc α(1→4) GalNAc β(1→4) EXT1 and EXT 2 (a hetero-oligmeric complex GlcA- C-5 GlcA GlcA β(1→4) that contains both GlcNAc- epimerase transferase and GlcA- transferase II ? transferase activity) GlcA → IdoA β → GlcA (1 3) epimerisation [GlcA β(1→4) GlcNAc α(1→4)] GalNAc- n transferase II GlcNAc N-deacetylase/ N-sulfotransferase N-Deacetylation β → α → β → β → GlcA (1 4) GlcN (1 4) (NDST) Alterating activity of GalNAc (1 4) GalNAc (1 4) (Until now, three different GlcA-transferase II and NDSTs have been identified). GalNAc-transferase II N-Sulphation 2- GlcA β(1→4) GlcNSO4 α(1→4) [GlcA β(1→3) GalNAc β(1→4)]n followed by GlcA →IdoA [GlcA β(1→3) GalNAc β(1→4)]n epimerisation C5 GlcA - 2-0-GlcA/IdoA 6-0-GalNAc sulpho- epimerase sulpho- 6-0-GalNAc sulpho- transferase C5 GlcA - transferase transferase C-5 GlcA 4-0-GalNAc sulpho- epimerisation 4-0-GalNAc sulpho- epimerase transferase transferase 2-0-GlcA sulpho- 2-0-IdoA- 2-0-GlcA- transferase sulphation sulphation 6-0-GlcN COO CH2OH H CH2OH sulpho- O H HO O H O H HO O transferase O H COO - OH H O O H H OH H H O 6-0-GlcN- 6-0-GlcN- 6-0-GlcN- 6-0-GlcN- H H H H H H H OH sulphation sulphation sulphation sulphation H NHCOCH 3 H OH H NHCOCH 3 Chondroitin Dermatan 3-0-GlcN sulphotransferases (Several are known, with different substrate specificities). sulphate sulphate 3-0-GlcN- sulphation

_ COO CH2OH H CH2OH O H O H H H _ O H O H H H COO H O OH H OH H OH H O OH H O H O H O

H OH H NHCOCH 3 H OH H NHCOCH COCH 3 3 Heparin and Heparan sulphate

Figure 1: The different steps in the synthesis of CS, DS, HS and heparin glycosaminoglycan chains of the GlcA-Gal Xyl-linker region. Synthesis and sorting of proteoglycans 195 sulphation in each GAG chain. GAG Hexuronic or Galactose Hexosamine Disaccharide composition Studies of these patterns have Iduronic acid defined the motifs required for _ specific interactions with growth COO CH2OH H O H O H factors, cytokines, matrix H H OH H O OH H components, enzymes and other H O proteins (Salmivirta et al., 1996). H H D-glucuronic D-glucosamine OH NHCOCH3 The minimal requirement for Heparan sulphate/ acid (GlcNAc) GlcA β(1→4) GlcNAc α( (1→4) binding to GAGs may differ from (GlcA) - Heparin H CH OH protein to protein Ð for instance L-iduronic acid 2 H _ O H O H fibroblast growth factors FGF-1 and (IdoA) COO H OH H O OH H FGF-2 are recognised by different H O H OH H HS structures expressed in discrete NHCOCH3 domains of the HS polymers IdoA α(1 →4) GlcNAc α(1→4) (Kreuger et al., 1999). Chlorate treatment (5-20 mM) of MDCK CH2OH CH2OH cells reduces 6-O-sulphation; higher HO O HO O Galactose D-glucosamine H H concentrations (50 mM) also reduce Keratan O O - (Gal) (GlcNAc) H OH H 2-O-sulfation, but N-sulphation of sulphate H H H OH H HSPG is not affected (Safaiyan et H NHCOCH3 al., 1999). In parallel with the dose- Gal β(1→4) GlcNAc β(1→3) dependent loss of 6-O-sulphation COO- there is a reduction in binding to CH2OH H O HO O FGF-1, whereas binding to FGF-2 is D-glucuronic D-galactosamine H Chondroitin O H OH H O essentially unchanged (Kreuger et sulphate acid - (GalNAc) H (GlcA) H H H al., 1999). H OH H NHCOCH3 GlcA β(1→3) GalNAc β(1→4)

WHERE ARE H CH2OH PROTEOGLYCANS H O HO O - D-glucuronic D-galactosamine COO O H SYNTHESISED? Dermatan OH H O acid - (GalNAc) H sulphate H H H (GlcA) H OH H NHCOCH The cell takes up the building L-iduronic acid 3 blocks for GAG synthesis, (IdoA) IdoA β(1→3) GalNAc β(1→4) monosaccharides and sulphate, COO- through specialised transporter CH2OH H O H O complexes in the plasma membrane. D-glucuronic D-glucosamine H O H Hyaluronic O Sugars (with a few exceptions) and acid - (GlcNAc) OH H H acid H HO H sulphate are then activated by (GlcA) H OH H NHCOCH3 nucleotide consumption in the β → β → cytosol to form UDP-sugars and GlcA (1 3) GlcNAc (1 4) 3′-phosphoadenosine 5′-phospho- sulphate (PAPS), respectively (Fig. Figure 2: Structure of the different glycosaminoglycan chains. 3). Specific transporters then The structure of the repeating in the different types of glycosaminoglycan chains is translocate UDP-sugars and PAPS drawn without sulphation. The different sulphation positions in each GAG are marked by encircling (Mandon et al., 1994) into the with a dashed red line ( ). endoplasmic reticulum (ER) and Golgi lumens (Hirschberg and Snider, 1987; Hirschberg et have also put forward kinetic arguments for Golgi localisation al., 1998). and glycolipids are also often of xylosyl transferase (XT) in rat chondrosarcoma cells. CHO sulphated. PAPS is the universal donor of sulphate to all cells lacking either XT (Esko et al., 1985), galactosyl sulphotransferases, both in the Golgi and the cytosol. transferase I (GT I; Esko et al., 1987) or glucuronic acid transferase I (GlcAT I; Bai et al., 1999) can synthesise neither The linker tetrasaccharide HS nor CS, which indicates that the pathways for synthesis of Although the lumen of the Golgi apparatus is the main site for the linker of these GAG types share enzymes. GAG synthesis, the formation of the linker tetrasaccharide Cells lacking GT I still synthesise GAGs on xylosides*, might start earlier in the secretory pathway (Fig. 4). In chicken however (Esko et al., 1987), which indicates that an alternative chondrocytes, xylosylation clearly takes place in a pre-Golgi GT activity can synthesise xyloside-based GAGs. Actually, compartment (Kearns et al., 1991, 1993; Vertel et al., 1993). Xylosylation was more efficiently catalysed by detergent- *Xylosides: compounds in which xylose is coupled to a hydrophobic group. Xylosides treated Golgi fractions than ER fractions from rat liver cross membranes and initiate GAG synthesis, bypassing the need for xylosylated core (Nuwayhid et al., 1986), and Lohmander et al. (1980, 1989) proteins. The GAGs initiated on xylosides are in almost all cases of the CS type. 196 K. Prydz and K. T. Dalen decorin protein cores contain xylose and one or two Gal able to add GalNAc to the linker tetrasaccharide to produce residues in the presence of xylosides (Moses et al., 1999). The structures that have ‘unproductive’ pentasaccharides because a competition between synthesis of xyloside-based GAGs and β-linkage is required for further elongation of CS chains. The endogenous CSPG might thus be mainly at the level of chain same group has recently shown that this enzyme also catalyses polymerisation. Recently, a cDNA encoding a novel GT was the addition of α-GlcNAc (Kitagawa et al., 1998, 1999), which transfected into GT-I-deficient CHO cells, restoring the PG is the proper sugar in HS chain synthesis. This indicates that synthesis (Okajima et al., 1999). Patients with Ehlers-Danlos the enzyme is the GlcNAcT I active in HS and heparin syndrome have previously been shown to exhibit reduced GT synthesis. This enzyme could be important for regulation, I activity (Quentin et al., 1990), and two missense substitutions capping some linker tetrasaccharides with α-GalNAc and have now been identified in the GT I gene (Almeida et al., preventing their elongation. Several other factors might also 1999). regulate GAG-type synthesis (see below). A mutant MDCK cell line that exhibits reduced import of UDP-Gal into the Golgi lumen, has dramatically reduced The amino acid sequence flanking the serine synthesis of KS, but essentially normal CS and HS production residue (Toma et al., 1996). Gal is incorporated into the polymerising Some differences in the consensus amino acid sequences for GAG chain in KS, whereas in CS/DS and HS/heparin Gal is attachment of CS and HS have been found. Most HSPGs found only in the linker tetrasaccharide (see Fig. 1). GTs contain repetitive (Ser-Gly)n segments (n >1) and a nearby involved in KS chain polymerisation (GT III and IV) and linker cluster of acidic residues (Zhang et al., 1995), whose position tetrasaccharide synthesis (GT I and II) might have different and composition are quite flexible. Substitution of these acidic Kms for UDP-Gal, but another possibility is that linker residues in betaglycan gave more CS and less HS (Zhang and tetrasaccharide synthesis is localised to a compartment Esko, 1994). Three Ser-Gly-Asp motifs accepted mainly HS if excluded from the MDCK cell Golgi fraction initially an acidic motif was present N-terminally. Changing acidic characterised by Brändli et al. (1988). A pre-Golgi UDP-Gal residues to increased the proportion of CS as did transporter activity has not been demonstrated (Kawakita et al., deletion of one or two GAG sites (Dolan et al., 1997). Acidic 1998), but a functional GT has been localised to the ER clusters also occur in some CSPGs (Bourdon et al., 1987; (Sprong et al., 1998). UDP-Gal therefore must also be available Brinkmann et al., 1997) and are thus necessary, but not in the lumen of this compartment. sufficient, for HS modification. Tryptophan residues also Enzyme studies and a study using xylosides to prime GAG influence the choice of GAG for a particular site. In betaglycan, synthesis indicated that GT I and II localise to different sub- a Trp ⇒ Ala mutation near the Ser-Gly pair causes a shift from regions of rat liver Golgi (Sugumaran et al., 1992; Etchison et HS to CS production, whereas introduction of a tryptophan al., 1995). Also, the last enzyme needed for synthesis of the residue near a CS site caused a shift to HS production (Zhang linker tetrasaccharide, GlcAT I, has a dual density distribution and Esko, 1994). Removal of 110 residues from the basement after gradient fractionation. The distribution resembles that of membrane PG perlecan reduced the HS content slightly (Dolan GlcAT II, which is involved in CS polymerisation, but is clearly et al., 1997). different from those of GT I and II (Sugumaran et al., 1998). Many isoforms of CD44 are modified by addition of GAGs. Dual localisation of enzymes involved in GAG synthesis could Most sites accept CS only, but a Ser-Gly-Ser-Gly motif in exon be interpreted in the context of the recently revived Golgi V3 also accepts HS. Substituting an acidic eight residue cisternal maturation model, in which a fraction of the sequence downstream of this motif with eight residues from transferases is constantly being transported retrogradely in downstream of a CS site in exon E5 changes the modification vesicles from maturing Golgi cisternae (Bonfanti et al., 1998; correspondingly (Greenfield et al., 1999). Incubation of Mironov et al., 1998; Glick and Malhotra, 1999). different cell types with xylosides has indicated that generation of CS-GAGs is a default modification: HS-GAGs on xylosides are rare. However, there are indications that xylosides are WHAT DETERMINES WHETHER THE segregated from CSPG core proteins early during biosynthesis GLYCOSAMINOGLYCAN CHAIN BECOMES (Moses et al., 1999) and that different CSPG core proteins CHONDROITIN SULPHATE/DERMATAN SULPHATE might even be segregated from each other (Vertel et al., 1989; OR HEPARAN SULPHATE/HEPARIN? Wong-Palms and Plaas, 1995). It is thus not clear whether some protein cores have more stringent requirements for After completion of the linker tetrasaccharide, the addition of modification by CS-GAG than others. the fifth saccharide determines whether the GAG chain becomes CS/DS or HS/heparin. This sugar is GlcNAc in the Access of UDP-sugars and the presence of case of HS/heparin and GalNAc in the case of CS/DS. glycosaminoglycan-synthesising enzymes in the GlcNAcT I (Fritz et al., 1997) and GalNAcT I mediate addition Golgi of these sugars (Sugumaran et al., 1998) and are postulated to Formation of CS/DS and HS/heparin requires UDP-Xyl, UDP- be distinct from those enzymes used in elongation of the GAG Gal, UDP-GlcA, UDP-GlcNAc and UDP-GalNAc in the Golgi chains of both CS (Rohrmann et al., 1985) and HS (Fritz et al., and/or ER lumens. Uptake of UDP-sugars, ATP and PAPS into 1994). Nadanaka et al. (1999), however, provide evidence that Golgi vesicles in vitro increases the concentration 50- to 100- the same enzyme catalyses the addition of the fifth sugar fold in comparison with the incubation medium (Hirschberg (GalNAc) to the linker tetrasaccharide and GalNAc addition and Snider, 1987). The UDP-Gal transporter (Ishida et al., during CS poymerisation. 1996; Miura et al., 1996) and the UDP-GlcNAc transporter Kitagawa et al. (1995) have shown that an α-GalNAcT is (Guillen et al., 1998) from mammalian species have Synthesis and sorting of proteoglycans 197 been characterised and cloned, whereas the UDP-GalNAc Distinguishing a CS site from a DS site, and an HS site from transporter from rat liver has been characterised but not yet a heparin site, probably involves regulation of enzymes cloned (Puglielli et al., 1999a). involved in GAG elongation in the Golgi complex. The Reduced levels of UDP-Gal in the Golgi lumen in intact regulation of HS synthesis versus heparin synthesis is complex MDCK cells give reduced levels of KS (Toma et al., 1996). In and probably determined by the proportions of the different an in vitro system, the chain length of heparin GAGs produced sulphotransferases and epimerases (Aikawa and Esko, 1999). is determined by the ratio of UDP-GlcNAc to UDP-GlcA CS and DS differ in the critical C5 epimerisation of GlcA to (Lidholt et al., 1988). In a similar way, the ratio of UDP- IdoA in DS. Once the first IdoA is formed, the C5 epimerase GalNAc and UDP-GlcNAc could influence the extent of seems to make more IdoA (Malmstroem et al., 1993). In some CS/DS versus HS/heparin synthesis. Normally, UDP-GalNAc PGs isolated from bovine aorta, this epimerisation starts with is formed by epimerisation of UDP-GlcNAc, which is the GlcA of the linker region (Sugahara et al., 1995a,b). Thus, catalysed in the cytosol by UDP-GlcNAc-4-epimerase, an the first epimerisation reaction might be a trigger for DS enzyme that also catalyses epimerisation of UDP- to synthesis and at the same time prevent the synthesis of CS and UDP-Gal (Piller et al., 1983). A kidney cell line deficient in HS. this enzyme has defects in the synthesis of glycolipids and the N-linked and O-linked chains of glycoproteins. Synthesis of heparan sulphate/heparin The defect can be corrected by exogenous Gal and GalNAc Lidholt et al. suggested that addition of the sixth sugar and of (Kingsley et al., 1986). Interestingly, a UDP-GlcNAc all the following sugars, to HS and heparin precursors is pyrophosphorylase isolated from kidney can use GalNAc-1-P catalysed by a bifunctional 70-kDa enzyme (i.e. it catalyses and UTP to catalyse the formation of UDP-GalNAc (Szumilo addition of alternating GlcA and GlcNAc units; Lidholt et al., et al., 1996). Thus, alternative pathways for formation of UDP- 1992; Lind et al., 1993). More recently, it has become evident Gal and UDP-GlcNAc seem to exist. The enzymatic systems that two different proteins are involved in the catalysis of HS producing the different UDP-sugars, and the translocators, are and heparin polymerisation (McCormick et al., 1998; Lind et thus likely to influence the concentration of UDP-sugars in the al., 1998). Both of these are products of tumor suppressor Golgi lumen and therefore also GAG-chain synthesis. genes (EXT1 and EXT2) of the hereditary multiple exostoses gene family Ð as is GlcNAc transferase I (EXTL2). The EXT1 Modifications of the linker region and EXT2 gene products are both needed for normal HSPG Several modifications of the linker region have been synthesis in CHO cells, but the exact role of each protein is not discovered: phosphorylation of C-2 of xylose, sulphation at clear. EXT1 and EXT2 must form larger hetero-oligomeric various positions and epimerisation of the last sugar of the complexes to aquire proper Golgi localisation. Such complexes linker tetrasaccharide. The C-2 of xylose is a major have previously been described for other Golgi enzymes phosphorylation site in both CSPG (Oegma et al., 1984; (Nilsson et al., 1993, 1994). Overexpression of only one of Sugahara et al., 1992a,b,c) and HSPG (Fransson et al., 1985) EXT1 or EXT2 resulted in ER localisation of the monomeric in certain tissues (Fig. 5). However, Xyl is generally not protein (McCormick et al., 2000). Overexpressed EXTL2 also phosphorylated in PGs derived from tissues that have abundant localised to the ER and might also require a ‘partner’ protein extracellular matrix (Sugahara et al., 1995a,b; Cheng et al., to localise to the Golgi. If the transferases involved in linker 1996). Phosphorylation of C-2 in Xyl is most prominent after tetrasaccharide synthesis also exist in ER and Golgi forms, this addition of the two Gal residues (Moses et al., 1999), whereas would explain some of the difficulties investigators have had addition of the first GlcA residue is followed by rapid in unequivocally localising these enzymes to a particular region dephosphorylation (Moses et al., 1997). In PGs produced by of the Golgi. melanomas, this dephosphorylation is complete (Spiro et The growing GAG chains are modified at various positions. al., 1991), whereas chondrosarcoma aggrecan contains The modifications include the following: (1) deacetylation/N- phosphate even in secreted PGs (Oegma et al., 1984). This is sulphation of GlcNAc units in HS and heparin; (2) epimeri- also the case when high concentrations of xylosides are added sation of GlcA to IdoA in HS and heparin (and also DS); (3) to cells (Greve and Kresse, 1988; Moses et al., 1999). O-sulphation in various positions of the disaccharides of HS, Dephosphorylation might be inefficient in some tumour cells heparin (and CS/DS). and at high production rates and, in some way, cause the altered As already mentioned, heparin is modified more extensively PG synthesis seen in these cells. The role of C-2 than HS but, whereas HS is expressed in virtually all cell types phosphorylation is thus not clear, but it might provide a signal of the body, heparin is synthesised mainly by connective tissue for secretory transport of PGs or for further modifications of mast cells. However, heparin-like structures have been found the growing glycan chain (Moses et al., 1997, 1999). The in glial cells (Stringer et al., 1999), and highly sulphated HS phosphorylation of the linker residues might occur in the ER, regions with heparin-like segments have also been found in the Golgi or in both locations, since functional ATP in thymic epithelial cells (Werneck et al., 1999). Three transporters are present in both membrane systems (Guillen bifunctional N-deacetylase/N-sulphotransferases (DASTs or and Hirschberg, 1995; Puglielli et al., 1999b). NDSTs; Wei et al., 1993) have been cloned (Hashimoto et al., Sulphation of the linker region has been observed only in 1992; Eriksson et al., 1993; Orellana et al., 1994; Humphries DS/CS and not in heparin/HS. Both CS and DS may have C- et al., 1997, 1998; Aikawa and Esko, 1999), but the specificity 4 sulphation of the second Gal (Sugahara et al., 1988) and C- and function of these enzymes are not established. A putative 4 or C-6 sulfation in the first GalNAc after the linker region heparin-specific NDST is also expressed in cells that do not (Sugahara et al., 1991). Modification by sulphate might produce heparin (Toma et al., 1998). influence the degree of synthesis of CS/DS versus HS/heparin. The bifunctional nature of the enzymes operating in 198 K. Prydz and K. T. Dalen

HS/heparin synthesis is likely to support a high speed of (Wong-Palms and Plaas, 1995; Kreuger et al., 1999; Safaiyan synthesis. The HS/heparin polymerisation rate in the Golgi et al., 1999). A low level of sulphation can still be sufficient to apparatus has been estimated to be at least 80 monosaccharides promote chain elongation, as is seen in patients that have per minute (Lidholt et al., 1988). The sulfotransferase activity achondrogenesis type 1B: sulphation of PGs may be reduced resides in the C-terminal half of NDSTs (Berninsone and to about 25% of the control level, without any effect on GAG- Hirschberg, 1998), with a critical lysine at position 614 chain length (Rossi et al., 1996). Still, it is unclear how closely (Sueyoshi et al., 1998). The crystal structure of the coupled the polymerisation and modification reactions are, and sulphotransferase (ST) domain of NDST 1 was recently what factors determine how long GAG chains are allowed to determined (Kakuta et al., 1999). grow before termination of synthesis. Whereas only one epimerase that converts GlcA into IdoA (Li et al., 1997) and one each of the STs that catalyse 2-O- (Bai Synthesis of chondroitin sulfate/dermatan sulfate and Esko, 1996; Kobayashi et al., 1999) and 6-O-sulfation In the case of the CSPG decorin, which has a single GAG (Habuchi et al., 1998) have been cloned, multiple isoforms that chain, N-terminal deletions in the propeptide result in synthesis have 3-O-ST activity have been cloned and demonstrated to have quite different substrate Glc specificities and to be expressed at different levels in different human ATP tissues (Shworak et al., 1999). As soon as modification Gln Glu UTP ADP reactions are initiated by an NDST, Acetyl-CoA the other enzymes are able to Glc-1-P Glc-6-P GlcN-6-P act. Most of the disaccharides PPi CoA-SH in heparin undergo ‘default’ modification by addition of three UDP-Gal sulphate groups per disaccharide, UDP-Glc GlcNAc-6-P whereas only a minor fraction of 2 NAD+ HS disaccharides acquire this H2O structure (Salmivirta et al., 1996). Gal-1-P ADP Successful GAG elongation has 2 NADH been postulated to depend on efficient sulphation of the polymer UDP-GlcA GlcNAc-1-P (Lidholt et al., 1989; Lidholt and ATP UTP Lindahl, 1992). In this context, Gal Golgi apparatus apparatus sulphation is a measure of completion of all the modification PPi steps, but the modification UDP-GlcNAc generating the possible message to the bifunctional polymerase UDP-Xyl could also be the epimerase, which introduces a more flexible ATP ADP conformation to the GAG chain. UDP-GalNAc The concept of a coupling APS PAPS between the polymerisation and ERGIC modification steps has arisen from PPi experiments with microsomal ? fractions, in which the organisation + of the synthesis machinery may be H 2- suboptimal. Alternative studies of ATP + SO4 PG synthesis have also been carried out in intact cells in Endoplasmic reticulum the presence of inhibitors of Abbreviations:Abbreviations: Gl cA: Glucuronic acid sulphation. One such inhibitor, ′ Ê APS:APS: Adenosine-5 Adenisone-5’-phosphosulphate-phosphosulphate GlÊ GlcA: cN: Glucuronic N-Glucosamine acid selenate, blocks synthesis of GAG ERGIC:ERGIC: ER-Golgi ER-Golgi Intermediate intermediate GlcN: GlcNAc: N-Glucosamine N-acetyl-Glucosamine Compartment GlcNAc: N-acetyl-Glucosamine chains, both short and long Compartment IdoA: Iduronic acid Gal: Galactose IdoA: Iduronic acid (Dietrich et al., 1988). Incubations GalNAc:Gal: N-acetyl-Galactosamine Galactose P PAPS: APS: 3′-Phosphoadenosine-5 3’-Phosphoadenosine-5’-phosphosulphate′-phosphosulphate in the presence of other inhibitors, Glc:GalNAc: Glucose N-acetyl-Galactosamine X Xyl: yl: Xylose Xylose such as chlorate and brefeldin A, Figure 3: Synthesis pathways for the formation of UDP-sugars and PAPS. produces undersulphated GAG Synthesis pathways for the formation of UDP-sugars and PAPS needed for synthesis of proteoglycans. Each UDP-sugar chains of similar length to or and PAPS is actively transported from the cytosol into the Golgi lumen, and into the lumen of ER (in the case of UDP-Xyl), longer than those in control cells by a corresponding transporter. Synthesis and sorting of proteoglycans 199

BrefeldinBrefeldin A treatment

trans-Golgi network STs CS/DS- trans-Golgi Polymerising enzymes

GlcAT medial-Golgi HS/heparin- All or some enzymes necessary for CS/DS Polymerising Some enzymes are elongation are retained in retained in TGN, which enzymes the TGN, and CS/DS in some cells results in cis-Golgi synthesis does not take place. reduced synthesis of HS polymers. GT I Cis-Golgi GT II network

Enzymes necessary for HS/ heparin elongation are trans- ported to the ER, where they catalyse the elongation of HS polymers. ERGIC XT

ER

Figure 4: The localisation of different proteoglycan-synthesising enzymes in the ER and Golgi apparatus with BFA treatment. The enzymes responsible for the synthesis of the linker region and GAG polymer isation are found at different locations in the endoplasmic reticulum (ER) and the Golgi apparatus. With Brefeldin A (BFA) treatment, the anterograde transport through the Golgi apparatus is blocked, whereas retrograde transport is intact, which results in transport of cis-, medial- and trans-Golgi cisternae components to the ER. Golgi enzymes relocated to the ER retain their activity and can still modify their natural substrates, which can no longer exit the ER. of a form of decorin that has a shorter GAG chain (Oldberg et 1991). Golgi enzymes relocated to the ER retain their activity al., 1996). The reason for this early termination is not known. and can still modify their natural substrates, which can no Elongation of CS chains is mediated by two distinct longer exit the ER. Components of the most distal part of the transferases that operate by alternate transfer of GalNAc and Golgi complex, the trans-Golgi network (TGN), are not GlcA (Richmond et al., 1973). The enzymatic activity that directed to the ER by BFA treatment. This makes enzymes transfers GlcA to the growing GAG chain, glucuronosyl localised in the TGN inaccessible to their newly synthesised transferase II (Sugumaran et al., 1997), is different from the activity that transfers GlcA to Gal to complete the linker region (Sugumaran et al., 1998). Two CS/DS STs have been cloned, the Sulphate is isnot not yet yetidentified identified in in 6-O-ST (Uchimura et al., 1998) and the 2-O-ST HS or or heparin heparin linker linker regions. regions. 2- 2- Phosphate has never been found (Kobayashi et al., 1999), whereas a 4-O-ST has SO4 SO4 Phosphate has never been found 2- together with sulphate groups in been purified from the culture medium of a rat SO4 togetherthe linker regionwith sulphate of CS/DS. groups in chondrosarchoma cell line (Yamauchi et al., the linker region of CS/DS. 1999).

Although linker tetrasaccharide synthesis COO- CH2OH CH2OH H seems to be a common pathway for both HS H O HO O HO O H O NH H H H H O O O synthesis and CS synthesis, not only are H H H OH H O CH2 CH elongation and modification of CS/DS and HO H H H H CO HS/heparin GAG chains catalysed by different H OH H OH H OH H OH sets of enzymes, but these events also take place The extent of phosphorylation increases with in different subdomains of the Golgi apparatus. The extent of phosphorylation increases with Localisation of enzymatic activities to thethe attachment of ofeach each Gal, Gal, whereas whereas different regions of the Golgi apparatus has been additionaddition of of the the first first GlcA GlcA is followed is followed by by 2- rapidrapid dephosphorylation.dephosphorylation. PO4 facilitated by use of the fungal isoprenoid metabolite brefeldin A (BFA, see Fig. 4), Figure 5: Modification of the linker region. because treatment with this drug induces During synthesis of proteoglycans, the linker region might be modified at various retrograde transport of components of the cis-, positions. Xyl may be phosphorylated in 2-O-position, whereas each Gal may be medial- and trans-Golgi cisternae to the ER sulphated at 4-O or 6-O-position, as shown in the figure. The effects of these (Lippincott-Schwartz et al., 1990; Sandvig et al., modifications are not known. 200 K. Prydz and K. T. Dalen substrates, which consequently do not undergo TGN attach the basolateral side of the epithelium to the ECM are modifications. In several cell types, synthesis of HSPG is still transported to the correct side of the monolayer. Basolateral detectable in the presence of BFA, whereas CSPG synthesis is distribution of syndecan-1 in MDCK cells requires the terminal blocked, which indicates that separate enzymes in different 12 amino acids of the cytoplasmic tail (Miettinen et al., 1994), subcompartments of the Golgi complex are involved in HSPG while basolateral secretion of the major basement membrane and CSPG synthesis (Spiro et al., 1991; Sugumaran et al., PG occurs by a pH-dependent mechanism (Caplan et al., 1992; Fransson et al., 1992; Uhlin-Hansen and Yanagishita, 1987). The fact that different sets of PGs are secreted apically 1993; Calabro and Hascall, 1994). The enzymes necessary for and basolaterally indicates that these molecules are actively the completion of the synthesis of HSPGs are located in the sorted. The recent discovery that endogenously synthesised cis-, medial- and trans-Golgi cisternae, whereas those needed CSPG, and hexyl-β-D-xyloside that has CS chains, are mainly for CSPG synthesis are localised to the trans-Golgi network. secreted apically in MDCK cells suggests that CS chains might contain apical-sorting information (Kolset et al., 1999). HS chains could have an opposite effect. The GPI-linked PG SORTING OF PROTEOGLYCANS glypican was detected predominantly at the basolateral surface of CaCo-2 and MDCK cells (Mertens et al., 1996). A form of PGs are expressed by virtually all vertebrate cells and are also glypican that lacks sites for HS attachment was transported expressed in Drosophila melanogaster (Campbell et al., 1987; strictly to the apical surface in MDCK cells, presumably Cambiazo and Inestrosa, 1990; Spring et al., 1994; Graner et because of its glycosylphosphatidylinositol (GPI) membrane al., 1994) and C. elegans (Rogalski et al., 1993; Schimpf et al., anchor or N-linked sugars, although the latter question was not 1999). How PGs are sorted and transported to their proper addressed. HS chains thus either promote basolateral sorting of destinations can therefore be studied in many different glypican or interfere with the recognition of the apical sorting organisms. After reaching the plasma membrane, PGs can take information in the molecule. part in binding and uptake of signalling molecules, such as Since many GPI-linked proteins are delivered to the apical growth factors and γ-interferon. Cell surface GAGs are surface in epithelial MDCK cells (Lisanti et al., 1988), it has internalised and have been detected in endocytic organelles and been suggested that GPI anchors function as apical sorting in the nucleus (Fedarko and Conrad, 1986; Ishihara et al., 1986; signals. However, both secreted and GPI-linked rat growth Tumova et al., 1999), co-localised with growth factors, but the hormone required N-glycans to be transported apically pathway taken from the cell surface to the nucleus is largely (Scheiffele et. al., 1995; Benting et al., 1999). O-linked sugars unknown. In rat hepatocytes, HSPG is transported from the might also direct apical sorting in MDCK cells (Yeaman et al., TGN to the cell surface in a population of vesicles different 1997), and both N-linked and O-linked sugars play a role in from that which transports serum albumin, apolipoprotein E apical targeting of bovine enteropeptidase (Zheng et al., 1999). and fibrinogen (Nickel et al., 1994; Barthel et al., 1995). For transmembrane proteins, both N-glycans (Gut et al., 1998) Hepatocytes are polarised cells in vivo, but it is now evident and transmembrane domains (Scheiffele et al., 1997) have been that in non-polarized cells there are also at least two alternative proposed to play a role. carriers from the TGN to the cell surface (Keller and Simons, The mechanisms underlying carbohydrate-mediated 1997). Many neural and endocrine cells possess two secretory transport and sorting are not known. Simons and co-workers pathways from the trans-side of the Golgi apparatus: a have proposed that sorting is mediated by lectin molecules that regulated pathway and a constitutive pathway. Frequently PGs accumulate in sphingolipid-cholesterol rafts within the are sorted to the regulated pathway, being released together exoplasmic leaflet of the TGN membrane (Simons and Ikonen, with the other contents of the storage granules. The negatively 1997; Keller and Simons, 1997). VIP36 has been proposed as charged PGs are engaged in binding of small positively a candidate raft-associated lectin (Fiedler et al., 1994), but charged molecules, such as histamine (Grimes and Kelly, 1992; more recently this protein has been localised to the cis region Brion et al., 1992; Castle and Castle, 1998) and proteases of the Golgi (Füllekrug et al., 1999) and, thus, to date no (Huang et al., 1998; Lützelschwab et al., 1997). Mast cells sorting lectin has been identified. from mice that lack the enzyme NDST 2, which is involved in Proteins may also be transported apically in a glycan- heparin synthesis, fail to store several proteins that are independent manner (Rodriguez-Boulan and Gonzalez, 1999), normally bound to heparin in secretory granules. Mast cells and proteins both with (Zheng et al., 1999) and without from NDST 2 knockout mice contained smaller granules and (Alonso et al., 1997) N-linked sugars are transported to the large empty vacuoles, which indicates that heparin GAGs are apical surface without being integrated into rafts, according to needed for normal granule formation (Humphries et al., 1999; the detergent extraction criteria applied for raft association. Forsberg et al., 1999). Thus, different kinds of determinants might mediate apical sorting. A particular protein may contain more than one sorting The role of glycans in apical sorting in epithelial signal, and glycan signals are often recessive to other sorting cells signals contained in the protein portion of a molecule Similarly to hepatocytes and neurons, epithelial cells have their (Mellman et al., 1993). This could explain why attachment plasma membranes divided into two domains, the apical and of a CS chain to a variant of the amyloid-precursor-like basolateral surfaces. To maintain the polarised organisation, protein 2 influences the basolateral sorting of neither the efficient sorting mechanisms target newly synthesised and transmembrane-anchored form nor the secretory form of this recycling molecules to the proper surface domain or protein (Lo et al., 1995). intracellular location. Epithelial cells must ensure that PG In an alternative model, the sorting lectin is not a components of the extracellular matrix (ECM) and those that transmembrane protein that recycles but a secreted PG. Synthesis and sorting of proteoglycans 201

Versican (Zimmermann and Ruoslahti, 1989) might be the deacetylase/N-sulfotransferase. The N-sulfotransferase activity domain is at CSPG secreted apically by MDCK cells (Svennevig et al., the carboxyl half of the holoenzyme. J. Biol. Chem. 273, 25556-25559. 1995). This PG is a member of a group called lecticans that, Bonfanti, L., Mironov, A. Jr., Martinez-Menarguez, J. A., Martella, O., Fusella, A., Baldassarre, M., Buccione, R., Geuze, H. J., Mironov, A. A. in addition to CS chains, contain several interesting protein and Luini, A. (1998). Procollagen traverses the Golgi stack without leaving core domains (Iozzo, 1998). Among these are two hyaluronic the lumen of cisternae: Evidence for cisternal maturation. Cell 95, 993-1003. acid (HA)-binding modules near the N terminus and a C-lectin Bourdon, M. A., Krusius, T., Campbell, S., Schwartz, N. B. and Ruoslahti, domain at the C terminus (Halberg et al., 1988). Versican has E. (1987). Identification and synthesis of a recognition signal for the attachment of glycosaminoglycans to proteins. Proc. Nat. Acad. Sci. USA been proposed to bind tenascin-R (Aspberg et al., 1995), L- 84, 3194-3198. selectin (Kawashima et al., 1999) and sulphated glycolipids Brändli, A. W., Hansson, G. C., Rodriguez-Boulan, E. and Simons, K. through the C-type lectin domain (Miura et al., 1999). L- (1988). A polarized epithelial cell mutant deficient in translocation of UDP- selectin has been shown to bind to, in addition to versican, galactose into the Golgi complex. J. Biol. Chem. 263, 16283-16290. many of the same kinds of ligands as versican and some Brinkmann, T., Weilke, C. and Kleesiek, K. (1997). Recognition of acceptor proteins by UDP-D-xylose proteoglycan core protein beta-D- additional ones (Needham and Schnaar, 1993; Kimura et al., xylosyltransferase. J. Biol. Chem. 272, 11171-11175. 1999; Li et al., 1999; Watanabe et al., 1999). In the Golgi Brion, C., Miller, S. G. and Moore, H. P. (1992). Regulated and constitutive apparatus, the C-lectin domain of versican could bind to secretion. Differential effects of protein synthesis arrest on transport of sulphated glycolipids. Since HA has been proposed to be glycosaminoglycan chains to the two secretory pathways. J. Biol. Chem. 267, 1477-1483. synthesised at the cell surface, the two HA-binding domains Calabro, A. and Hascall, V. (1994). Differential effects of brefeldin A on may be free to bind other sugars in the Golgi apparatus. The chondroitin sulfate and hyaluronan synthesis in rat chondrosarcoma cells. J. HA-binding motifs are structurally similar to C-type lectins, Biol. Chem. 269, 22764-22770. apart from a possible calcium-binding loop (Kohda et al., Cambiazo, V. and Inestrosa, N. C. (1990). Proteoglycan production in 1996). Lectin domains at both ends of the versican molecule Drosophila egg development: effect of beta-D-xyloside on proteoglycan synthesis and larvae motility. Comp. Biochem. Physiol. 97, 307-314. could thus allow binding of one end to the membrane and Campbell, A. G., Fessler, L. I., Salo, T. and Fessler, J. H. (1987). Papilin: a binding of glycanated molecules, such as glycoproteins, to the Drosophila proteoglycan-like sulfated from basement other end. Cross-linking into larger complexes may be membranes. J. Biol. Chem. 262, 17605-17612. necessary for apical sorting of soluble molecules. Caplan, M. J., Stow, J. L., Newman, A. P., Madri, J., Anderson, H. C., Farquhar, M. G., Palade, G. E. and Jamieson, J. D. (1987). Dependence More studies are needed if we are to determine the extent to on pH of polarized sorting of secreted proteins. Nature 329, 632-635. which glycans contain sorting information and to identify the Castle, A. M. and Castle, J. D. (1998). Enhanced glycosylation and sulfation structural determinants involved. 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