Download Synthesis and sorting of proteoglycans

193
Journal of Cell Science 113, 193-205 (2000)
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.
Interactions between negatively charged glycosaminoglycan
chains and molecules such as growth factors are essential for
differentiation of cells during development and maintenance
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
in glycosaminoglycan chain structure from cell type to cell
type, which is acquired by use of particular Ser-Gly sites in
the protein core, might therefore be important for postsynthesis sorting. This links regulation of glycosaminoglycan
synthesis to the post-Golgi fate of proteoglycans.
INTRODUCTION
VARIATION IN PROTEOGLYCAN COMPOSITION
Proteoglycans (PGs) consist of a protein portion and long,
unbranched polysaccharides (glycosaminoglycans or GAGs).
The latter have a high negative charge, owing to the presence
of acidic sugar residues and/or modification by sulphate
groups. The acidic sugar alternates with an amino sugar in
repeated disaccharide units. The GAGs adopt an extended
conformation, attract cations, and bind water. Hydrated GAG
gels enable joints and tissues to absorb large pressure
changes.
In addition to buffering pressure changes, PGs play
important roles in control of growth and differentiation.
Particular sulphation patterns in the GAG chains allow
interactions, normally of ionic nature, with growth factors, for
example. Recent studies have identified ~30 PG protein cores.
These cores are not just scaffolds for GAGs: they contain
domains that have particular biological activities (Iozzo, 1998).
Many PGs are thus multifunctional molecules that engage in
several different specific interactions at the same time.
After synthesis PGs are transported from the Golgi to their
destinations: the extracellular matrix (ECM), the cell surface
or intracellular organelles. Such vectorial transport requires
mechanisms for recognition, sorting and delivery, which are
especially important in cells such as epithelial cells and
neurons, where the cell membrane comprises separate
domains. Recognition and sorting must require determinants in
the GAG chains and/or in the PG protein cores. Here we
discuss how and where chondroitin sulphate (CS)/dermatan
sulphate (DS) and heparan sulphate (HS)/heparin GAGs are
synthesised, and how these GAGs influence the sorting of PGs
to the sites at which they act.
PGs were initially grouped together because of the high
negative charges of their GAG chains, which make separation
from other molecules by ion-exchange chromatography easy.
PGs are, however, not that similar. The core protein size ranges
from 10 kDa to >500 kDa, and the number of GAG chains
attached varies from one to >100 (for review, see Poole, 1986;
Rouslahti, 1988; Kjellén and Lindahl, 1991; Silbert and
Sugumaran, 1995). In addition, several PGs carry GAG chains
of more than one type (hybrid PGs; Rapraeger et al., 1985;
Sugahara et al., 1992a) and/or have additional N-linked or Olinked sugar modifications.
Not all PGs are ‘full-time’ PGs. Some proteins, such as
MHC class II invariant chain, thrombomodulin and the
transferrin receptor are ‘part-time’ PGs (Fransson, 1987),
alternatively spliced variants having GAG-initiation sites. PGs
such as versican and CD44 also occur as alternatively spliced
forms (Greenfield et al., 1999; Dours-Zimmermann and
Zimmermann, 1994; Naso et al., 1994) whose sugar
modifications vary. A variant of versican without CSattachment sites has been discovered, and thus the PG versican
could also be regarded as a part-time PG (Iozzo, 1998).
GAG (except for in keratan sulphate (KS); Baker et al.,
1975; Stein et al., 1982) synthesis is initiated by sequential
addition of four monosaccharides (xylose (Xyl), galactose
(Gal), galactose and glucuronic acid (GlcA); see Fig. 1). From
this linker tetrasaccharide, the sugar chains are extended by
addition of two alternating monosaccharides, an aminosugar
and GlcA. In heparin and HS, the aminosugar is N-acetylglucosamine (GlcNAc) and in CS/DS it is N-acetylgalactosamine (GalNAc; see Fig. 2). The extent of
Key words: Proteoglycan, Glycosaminoglycan, Sorting, Polarised
cell, Epithelial cell, Glycan
K. Prydz and K. T. Dalen
are initiated as N-linked or O-linked oligosaccharides and
extended by addition of GlcNAc and Gal.
There is also regional variability to the epimerisation and
COO -
CH2 OH
H
H
O
H
H
H
O
UDP-Xyl
transferase
O
H
H
OH
O
H
NH
H
O
OH
CH
C
OH
H
OH
H
CH 2
H
H
H
H
OH
H
O
HO
H
HO
H
CH2 OH
O
HO
O
H
UDP-Gal
transferase I
UDP-GlcA
transferase I
Diverging point
epimerisation of GlcA to iduronic acid (IdoA) and the
sulphation pattern of the disaccharide units distinguish heparin
from HS, and DS from CS (see Figs 1 and 2). In KS, the GAGs
UDP-Gal
transferase II
194
O
GlcA β(1→3) Gal β(1→3) Gal β(1→4) Xyl β O−Serine
EXTL2
GalNActransferase I
GlcNAc- transferase I
GalNAc α(1→4)
GlcNAc α(1→4)
EXT1 and EXT 2
GalNAc β(1→4)
C-5 GlcA
epimerase
GlcAtransferase II
GlcA → IdoA
epimerisation
GlcA β(1→3)
[GlcA β(1→4) GlcNAc α(1→4)]n
GalNActransferase II
Alterating activity of GalNAc β(1→4)
GlcA-transferase II and
GalNAc-transferase II
(Until now, three different
NDSTs have been identified).
[GlcA β(1→3) GalNAc β(1→4)]n
followed by GlcA →IdoA
epimerisation
C-5 GlcA
epimerase
4-0-GalNAc sulphotransferase
COO
CH2 OH
O
H
H
OH
OH
O
H
H
H
H
H
H
Chondroitin
sulphate
NHCOCH 3
C5 GlcA epimerisation
O
COO OH
H
6-0-GlcN
sulphotransferase
O
HO
H
O
O
H
H
H
2-0-IdoA- 2-0-GlcAsulphation sulphation
CH2 OH
H
H
2-0-GlcA/IdoA
sulphotransferase
C5 GlcA epimerase
4-0-GalNAc sulphotransferase
2-0-GlcA sulphotransferase
H
O
HO
O
H
GlcA β(1→4) GlcNSO42- α(1→4)
N-Sulphation
6-0-GalNAc sulphotransferase
6-0-GalNAc sulphotransferase
GlcNAc N-deacetylase/
N-sulfotransferase
(NDST)
GlcA β(1→4) GlcN α(1→4)
N-Deacetylation
GalNAc β(1→4)
[GlcA β(1→3) GalNAc β(1→4)]n
(a hetero-oligmeric complex
that contains both GlcNActransferase and GlcAtransferase activity)
GlcA β(1→4)
?
OH
H
H
H
Dermatan
sulphate
6-0-GlcN- 6-0-GlcNsulphation sulphation
6-0-GlcN- 6-0-GlcNsulphation sulphation
NHCOCH 3
3-0-GlcN sulphotransferases
(Several are known, with different substrate specificities).
3-0-GlcNsulphation
COO
_
CH 2OH
O
H
H
OH
H
H
OH
O
H
OH
H
H
H
CH 2OH
H
O H
H
H
O
O
NHCOCH
COCH 33
COO
OH
_
O
O H
H
O
H
H
OH
H
H
NHCOCH 3
H
H
OH
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.
O
Synthesis and sorting of proteoglycans
sulphation in each GAG chain.
Studies of these patterns have
defined the motifs required for
specific interactions with growth
factors,
cytokines,
matrix
components, enzymes and other
proteins (Salmivirta et al., 1996).
The minimal requirement for
binding to GAGs may differ from
protein to protein – for instance
fibroblast growth factors FGF-1 and
FGF-2 are recognised by different
HS structures expressed in discrete
domains of the HS polymers
(Kreuger et al., 1999). Chlorate
treatment (5-20 mM) of MDCK
cells reduces 6-O-sulphation; higher
concentrations (50 mM) also reduce
2-O-sulfation, but N-sulphation of
HSPG is not affected (Safaiyan et
al., 1999). In parallel with the dosedependent loss of 6-O-sulphation
there is a reduction in binding to
FGF-1, whereas binding to FGF-2 is
essentially unchanged (Kreuger et
al., 1999).
GAG
Hexuronic or
Iduronic acid
Galactose
Hexosamine
195
Disaccharide composition
COO
H
_
CH2OH
O
O H
H
H
OH
H
H
OH
O
H
OH
H
H
NHCOCH3
O
H
Heparan
sulphate/
Heparin
D-glucuronic
acid
(GlcA)
L-iduronic acid
(IdoA)
D-glucosamine
(GlcNAc)
GlcA β(1→4) GlcNAc α( (1→4)
-
CH2OH
H
H
O
_
O H
H
COO
OH
H
O
H
OH
H
H
NHCOCH3
O
H
H
OH
IdoA α(1→4) GlcNAc α(1→4)
CH2OH
CH2OH
Keratan
sulphate
-
Galactose
(Gal)
D-glucosamine
(GlcNAc)
H
H
OH
O
H
H
O
HO
O
HO
H
H
H
O
H
H
OH
NHCOCH 3
Gal β(1→4) GlcNAc β(1→3)
COO-
Chondroitin
sulphate
D-glucuronic
acid
(GlcA)
-
D-galactosamine
(GalNAc)
H
CH2OH
O
H
OH
H
O
H
O
H
H
H
O
HO
H
H
OH
H
NHCOCH3
GlcA β(1→3) GalNAc β(1→4)
WHERE ARE
PROTEOGLYCANS
SYNTHESISED?
H
Dermatan
sulphate
D-glucuronic
acid
(GlcA)
L-iduronic acid
(IdoA)
-
D-galactosamine
(GalNAc)
H
CH2OH
O
COOOH
H
H
O
O
H
H
H
O
HO
H
H
OH
H
NHCOCH
The cell takes up the building
IdoA β(1→3) GalNAc β(1→4)
blocks
for
GAG
synthesis,
monosaccharides and sulphate,
COOCH OH
through specialised transporter
O
H
O
H
complexes in the plasma membrane.
H
D-glucuronic
D-glucosamine
H
O
Hyaluronic
O
OH
H
H
acid
(GlcNAc)
Sugars (with a few exceptions) and
H
acid
H
HO
(GlcA)
sulphate are then activated by
OH
H
H
NHCOCH
nucleotide consumption in the
GlcA β (1→3) GlcNAc β(1→4)
cytosol to form UDP-sugars and
3′-phosphoadenosine 5′-phosphoFigure 2: Structure of the different glycosaminoglycan chains.
sulphate (PAPS), respectively (Fig.
The structure of the repeating disaccharides in the different types of glycosaminoglycan chains is
3). Specific transporters then
drawn without sulphation. The different sulphation positions in each GAG are marked by encircling
translocate UDP-sugars and PAPS
with a dashed red line (
).
(Mandon et al., 1994) into the
endoplasmic reticulum (ER) and
have also put forward kinetic arguments for Golgi localisation
Golgi lumens (Hirschberg and Snider, 1987; Hirschberg et
of xylosyl transferase (XT) in rat chondrosarcoma cells. CHO
al., 1998). Glycoproteins and glycolipids are also often
cells lacking either XT (Esko et al., 1985), galactosyl
sulphated. PAPS is the universal donor of sulphate to all
transferase I (GT I; Esko et al., 1987) or glucuronic acid
sulphotransferases, both in the Golgi and the cytosol.
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
the linker tetrasaccharides of these GAG types share enzymes.
Although the lumen of the Golgi apparatus is the main site for
Cells lacking GT I still synthesise GAGs on xylosides*,
GAG synthesis, the formation of the linker tetrasaccharide
however (Esko et al., 1987), which indicates that an alternative
might start earlier in the secretory pathway (Fig. 4). In chicken
GT activity can synthesise xyloside-based GAGs. Actually,
chondrocytes, xylosylation clearly takes place in a pre-Golgi
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.
3
2
3
196
K. Prydz and K. T. Dalen
decorin protein cores contain xylose and one or two Gal
residues in the presence of xylosides (Moses et al., 1999). The
competition between synthesis of xyloside-based GAGs and
endogenous CSPG might thus be mainly at the level of chain
polymerisation. Recently, a cDNA encoding a novel GT was
transfected into GT-I-deficient CHO cells, restoring the PG
synthesis (Okajima et al., 1999). Patients with Ehlers-Danlos
syndrome have previously been shown to exhibit reduced GT
I activity (Quentin et al., 1990), and two missense substitutions
have now been identified in the GT I gene (Almeida et al.,
1999).
A mutant MDCK cell line that exhibits reduced import of
UDP-Gal into the Golgi lumen, has dramatically reduced
synthesis of KS, but essentially normal CS and HS production
(Toma et al., 1996). Gal is incorporated into the polymerising
GAG chain in KS, whereas in CS/DS and HS/heparin Gal is
found only in the linker tetrasaccharide (see Fig. 1). GTs
involved in KS chain polymerisation (GT III and IV) and linker
tetrasaccharide synthesis (GT I and II) might have different
Kms for UDP-Gal, but another possibility is that linker
tetrasaccharide synthesis is localised to a compartment
excluded from the MDCK cell Golgi fraction initially
characterised by Brändli et al. (1988). A pre-Golgi UDP-Gal
transporter activity has not been demonstrated (Kawakita et al.,
1998), but a functional GT has been localised to the ER
(Sprong et al., 1998). UDP-Gal therefore must also be available
in the lumen of this compartment.
Enzyme studies and a study using xylosides to prime GAG
synthesis indicated that GT I and II localise to different subregions of rat liver Golgi (Sugumaran et al., 1992; Etchison et
al., 1995). Also, the last enzyme needed for synthesis of the
linker tetrasaccharide, GlcAT I, has a dual density distribution
after gradient fractionation. The distribution resembles that of
GlcAT II, which is involved in CS polymerisation, but is clearly
different from those of GT I and II (Sugumaran et al., 1998).
Dual localisation of enzymes involved in GAG synthesis could
be interpreted in the context of the recently revived Golgi
cisternal maturation model, in which a fraction of the
transferases is constantly being transported retrogradely in
vesicles from maturing Golgi cisternae (Bonfanti et al., 1998;
Mironov et al., 1998; Glick and Malhotra, 1999).
WHAT DETERMINES WHETHER THE
GLYCOSAMINOGLYCAN CHAIN BECOMES
CHONDROITIN SULPHATE/DERMATAN SULPHATE
OR HEPARAN SULPHATE/HEPARIN?
After completion of the linker tetrasaccharide, the addition of
the fifth saccharide determines whether the GAG chain
becomes CS/DS or HS/heparin. This sugar is GlcNAc in the
case of HS/heparin and GalNAc in the case of CS/DS.
GlcNAcT I (Fritz et al., 1997) and GalNAcT I mediate addition
of these sugars (Sugumaran et al., 1998) and are postulated to
be distinct from those enzymes used in elongation of the GAG
chains of both CS (Rohrmann et al., 1985) and HS (Fritz et al.,
1994). Nadanaka et al. (1999), however, provide evidence that
the same enzyme catalyses the addition of the fifth sugar
(GalNAc) to the linker tetrasaccharide and GalNAc addition
during CS poymerisation.
Kitagawa et al. (1995) have shown that an α-GalNAcT is
able to add GalNAc to the linker tetrasaccharide to produce
structures that have ‘unproductive’ pentasaccharides because a
β-linkage is required for further elongation of CS chains. The
same group has recently shown that this enzyme also catalyses
the addition of α-GlcNAc (Kitagawa et al., 1998, 1999), which
is the proper sugar in HS chain synthesis. This indicates that
the enzyme is the GlcNAcT I active in HS and heparin
synthesis. This enzyme could be important for regulation,
capping some linker tetrasaccharides with α-GalNAc and
preventing their elongation. Several other factors might also
regulate GAG-type synthesis (see below).
The amino acid sequence flanking the serine
residue
Some differences in the consensus amino acid sequences for
attachment of CS and HS have been found. Most HSPGs
contain repetitive (Ser-Gly)n segments (n >1) and a nearby
cluster of acidic residues (Zhang et al., 1995), whose position
and composition are quite flexible. Substitution of these acidic
residues in betaglycan gave more CS and less HS (Zhang and
Esko, 1994). Three Ser-Gly-Asp motifs accepted mainly HS if
an acidic motif was present N-terminally. Changing acidic
residues to amines increased the proportion of CS as did
deletion of one or two GAG sites (Dolan et al., 1997). Acidic
clusters also occur in some CSPGs (Bourdon et al., 1987;
Brinkmann et al., 1997) and are thus necessary, but not
sufficient, for HS modification. Tryptophan residues also
influence the choice of GAG for a particular site. In betaglycan,
a Trp ⇒ Ala mutation near the Ser-Gly pair causes a shift from
HS to CS production, whereas introduction of a tryptophan
residue near a CS site caused a shift to HS production (Zhang
and Esko, 1994). Removal of 110 residues from the basement
membrane PG perlecan reduced the HS content slightly (Dolan
et al., 1997).
Many isoforms of CD44 are modified by addition of GAGs.
Most sites accept CS only, but a Ser-Gly-Ser-Gly motif in exon
V3 also accepts HS. Substituting an acidic eight residue
sequence downstream of this motif with eight residues from
downstream of a CS site in exon E5 changes the modification
correspondingly (Greenfield et al., 1999). Incubation of
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
segregated from CSPG core proteins early during biosynthesis
(Moses et al., 1999) and that different CSPG core proteins
might even be segregated from each other (Vertel et al., 1989;
Wong-Palms and Plaas, 1995). It is thus not clear whether some
protein cores have more stringent requirements for
modification by CS-GAG than others.
Access of UDP-sugars and the presence of
glycosaminoglycan-synthesising enzymes in the
Golgi
Formation of CS/DS and HS/heparin requires UDP-Xyl, UDPGal, UDP-GlcA, UDP-GlcNAc and UDP-GalNAc in the Golgi
and/or ER lumens. Uptake of UDP-sugars, ATP and PAPS into
Golgi vesicles in vitro increases the concentration 50- to 100fold in comparison with the incubation medium (Hirschberg
and Snider, 1987). The UDP-Gal transporter (Ishida et al.,
1996; Miura et al., 1996) and the UDP-GlcNAc transporter
(Guillen et al., 1998) from mammalian species have
Synthesis and sorting of proteoglycans
been characterised and cloned, whereas the UDP-GalNAc
transporter from rat liver has been characterised but not yet
cloned (Puglielli et al., 1999a).
Reduced levels of UDP-Gal in the Golgi lumen in intact
MDCK cells give reduced levels of KS (Toma et al., 1996). In
an in vitro system, the chain length of heparin GAGs produced
is determined by the ratio of UDP-GlcNAc to UDP-GlcA
(Lidholt et al., 1988). In a similar way, the ratio of UDPGalNAc and UDP-GlcNAc could influence the extent of
CS/DS versus HS/heparin synthesis. Normally, UDP-GalNAc
is formed by epimerisation of UDP-GlcNAc, which is
catalysed in the cytosol by UDP-GlcNAc-4-epimerase, an
enzyme that also catalyses epimerisation of UDP-glucose to
UDP-Gal (Piller et al., 1983). A kidney cell line deficient in
this enzyme has defects in the synthesis of glycolipids and the
N-linked and O-linked carbohydrate chains of glycoproteins.
The defect can be corrected by exogenous Gal and GalNAc
(Kingsley et al., 1986). Interestingly, a UDP-GlcNAc
pyrophosphorylase isolated from kidney can use GalNAc-1-P
and UTP to catalyse the formation of UDP-GalNAc (Szumilo
et al., 1996). Thus, alternative pathways for formation of UDPGal and UDP-GlcNAc seem to exist. The enzymatic systems
producing the different UDP-sugars, and the translocators, are
thus likely to influence the concentration of UDP-sugars in the
Golgi lumen and therefore also GAG-chain synthesis.
Modifications of the linker region
Several modifications of the linker region have been
discovered: phosphorylation of C-2 of xylose, sulphation at
various positions and epimerisation of the last sugar of the
linker tetrasaccharide. The C-2 of xylose is a major
phosphorylation site in both CSPG (Oegma et al., 1984;
Sugahara et al., 1992a,b,c) and HSPG (Fransson et al., 1985)
in certain tissues (Fig. 5). However, Xyl is generally not
phosphorylated in PGs derived from tissues that have abundant
extracellular matrix (Sugahara et al., 1995a,b; Cheng et al.,
1996). Phosphorylation of C-2 in Xyl is most prominent after
addition of the two Gal residues (Moses et al., 1999), whereas
addition of the first GlcA residue is followed by rapid
dephosphorylation (Moses et al., 1997). In PGs produced by
melanomas, this dephosphorylation is complete (Spiro et
al., 1991), whereas chondrosarcoma aggrecan contains
phosphate even in secreted PGs (Oegma et al., 1984). This is
also the case when high concentrations of xylosides are added
to cells (Greve and Kresse, 1988; Moses et al., 1999).
Dephosphorylation might be inefficient in some tumour cells
and at high production rates and, in some way, cause the altered
PG synthesis seen in these cells. The role of C-2
phosphorylation is thus not clear, but it might provide a signal
for secretory transport of PGs or for further modifications of
the growing glycan chain (Moses et al., 1997, 1999). The
phosphorylation of the linker residues might occur in the ER,
in the Golgi or in both locations, since functional ATP
transporters are present in both membrane systems (Guillen
and Hirschberg, 1995; Puglielli et al., 1999b).
Sulphation of the linker region has been observed only in
DS/CS and not in heparin/HS. Both CS and DS may have C4 sulphation of the second Gal (Sugahara et al., 1988) and C4 or C-6 sulfation in the first GalNAc after the linker region
(Sugahara et al., 1991). Modification by sulphate might
influence the degree of synthesis of CS/DS versus HS/heparin.
197
Distinguishing a CS site from a DS site, and an HS site from
a heparin site, probably involves regulation of enzymes
involved in GAG elongation in the Golgi complex. The
regulation of HS synthesis versus heparin synthesis is complex
and probably determined by the proportions of the different
sulphotransferases and epimerases (Aikawa and Esko, 1999).
CS and DS differ in the critical C5 epimerisation of GlcA to
IdoA in DS. Once the first IdoA is formed, the C5 epimerase
seems to make more IdoA (Malmstroem et al., 1993). In some
PGs isolated from bovine aorta, this epimerisation starts with
the GlcA of the linker region (Sugahara et al., 1995a,b). Thus,
the first epimerisation reaction might be a trigger for DS
synthesis and at the same time prevent the synthesis of CS and
HS.
Synthesis of heparan sulphate/heparin
Lidholt et al. suggested that addition of the sixth sugar and of
all the following sugars, to HS and heparin precursors is
catalysed by a bifunctional 70-kDa enzyme (i.e. it catalyses
addition of alternating GlcA and GlcNAc units; Lidholt et al.,
1992; Lind et al., 1993). More recently, it has become evident
that two different proteins are involved in the catalysis of HS
and heparin polymerisation (McCormick et al., 1998; Lind et
al., 1998). Both of these are products of tumor suppressor
genes (EXT1 and EXT2) of the hereditary multiple exostoses
gene family – as is GlcNAc transferase I (EXTL2). The EXT1
and EXT2 gene products are both needed for normal HSPG
synthesis in CHO cells, but the exact role of each protein is not
clear. EXT1 and EXT2 must form larger hetero-oligomeric
complexes to aquire proper Golgi localisation. Such complexes
have previously been described for other Golgi enzymes
(Nilsson et al., 1993, 1994). Overexpression of only one of
EXT1 or EXT2 resulted in ER localisation of the monomeric
protein (McCormick et al., 2000). Overexpressed EXTL2 also
localised to the ER and might also require a ‘partner’ protein
to localise to the Golgi. If the transferases involved in linker
tetrasaccharide synthesis also exist in ER and Golgi forms, this
would explain some of the difficulties investigators have had
in unequivocally localising these enzymes to a particular region
of the Golgi.
The growing GAG chains are modified at various positions.
The modifications include the following: (1) deacetylation/Nsulphation of GlcNAc units in HS and heparin; (2) epimerisation of GlcA to IdoA in HS and heparin (and also DS); (3)
O-sulphation in various positions of the disaccharides of HS,
heparin (and CS/DS).
As already mentioned, heparin is modified more extensively
than HS but, whereas HS is expressed in virtually all cell types
of the body, heparin is synthesised mainly by connective tissue
mast cells. However, heparin-like structures have been found
in glial cells (Stringer et al., 1999), and highly sulphated HS
regions with heparin-like segments have also been found
in thymic epithelial cells (Werneck et al., 1999). Three
bifunctional N-deacetylase/N-sulphotransferases (DASTs or
NDSTs; Wei et al., 1993) have been cloned (Hashimoto et al.,
1992; Eriksson et al., 1993; Orellana et al., 1994; Humphries
et al., 1997, 1998; Aikawa and Esko, 1999), but the specificity
and function of these enzymes are not established. A putative
heparin-specific NDST is also expressed in cells that do not
produce heparin (Toma et al., 1998).
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 GAGHirschberg, 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
Synthesis of chondroitin sulfate/dermatan sulfate
(Li et al., 1997) and one each of the STs that catalyse 2-O- (Bai
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
ATP
different levels in different human
tissues (Shworak et al., 1999).
Gln
Glu
As soon as modification
ADP
UTP
reactions are initiated by an NDST,
Acetyl-CoA
the other enzymes are able to
GlcN-6-P
Glc-1-P
Glc-6-P
PPi
CoA-SH
act. Most of the disaccharides
in heparin undergo ‘default’
modification by addition of three
UDP-Gal
sulphate groups per disaccharide,
UDP-Glc
GlcNAc-6-P
2 NAD+
whereas only a minor fraction of
H 2O
HS disaccharides acquire this
Gal-1-P
structure (Salmivirta et al., 1996).
ADP
Successful GAG elongation has
2 NADH
been postulated to depend on
UDP-GlcA
GlcNAc-1-P
efficient sulphation of the polymer
ATP
UTP
(Lidholt et al., 1989; Lidholt and
Gal
Golgiapparatus
apparatus
Golgi
Lindahl, 1992). In this context,
sulphation is a measure of
PPi
completion of all the modification
steps, but the modification
UDP-GlcNAc
generating the possible message
UDP-Xyl
to the bifunctional polymerase
could also be the epimerase,
which introduces a more flexible
ATP
ADP
conformation to the GAG chain.
UDP-GalNAc
The concept of a coupling
PAPS
APS
between the polymerisation and
ERGIC
PPi
modification steps has arisen from
experiments with microsomal
?
fractions, in which the organisation
+
H
of the synthesis machinery may be
suboptimal. Alternative studies of
ATP + SO42PG synthesis have also been
Endoplasmic reticulum
reticulum
carried out in intact cells in
the presence of inhibitors of
Abbreviations:
Gl
cA:
Glucuronic acid
sulphation. One such inhibitor, Abbreviations:
GlcA:
acid
APS:
APS: Adenosine-5′-phosphosulphate
Adenisone-5’-phosphosulphate
Gl
cN: Glucuronic
N-Glucosamine
selenate, blocks synthesis of GAG
GlcN:
N-Glucosamine
ERGIC:
Intermediate
ERGIC:ER-Golgi
ER-Golgi
intermediate
GlcNAc: N-acetyl-Glucosamine
GlcNAc: N-acetyl-Glucosamine
Compartment
chains, both short and long
Compartment
IdoA:
Iduronic acid
IdoA:
Iduronic acid
Gal:
Galactose
Gal:
Galactose
P
APS: 3′-Phosphoadenosine-5′-phosphosulphate
3’-Phosphoadenosine-5’-phosphosulphate
PAPS:
(Dietrich et al., 1988). Incubations
GalNAc:
N-acetyl-Galactosamine
GalNAc:Glucose
N-acetyl-Galactosamine
X
yl:
Xylose
Xyl:
Xylose
Glc:
in the presence of other inhibitors,
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
Brefeldin A treatment
Brefeldin
trans-Golgi
network
STs
GlcAT
trans-Golgi
medial-Golgi HS/heparinPolymerising
enzymes
cis-Golgi
GT I
GT II
XT
CS/DSPolymerising
enzymes
Cis-Golgi
network
Some enzymes are
retained in TGN, which
in some cells results in
reduced synthesis of HS
polymers.
All or some enzymes
necessary for CS/DS
elongation are retained in
the TGN, and CS/DS
synthesis does not take place.
Enzymes necessary for HS/
heparin elongation are transported to the ER, where they
catalyse the elongation of HS
polymers.
ERGIC
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
identified
in
Sulphate is is
notnot
yet yet
identified
in
HS or
linker
regions.
6-O-ST (Uchimura et al., 1998) and the 2-O-ST
HS
orheparin
heparin
linker
regions.
Phosphate has
never
beenbeen
foundfound
SO42(Kobayashi et al., 1999), whereas a 4-O-ST has
has
never
SO42- Phosphate
together with sulphate groups in
2together
with sulphate
been purified from the culture medium of a rat
SO4
the linker region
of CS/DS. groups in
the linker region of CS/DS.
chondrosarchoma cell line (Yamauchi et al.,
1999).
H
CH2OH
CH2OH
COOAlthough linker tetrasaccharide synthesis
O
O
O
NH
H
HO
O
HO
H
seems to be a common pathway for both HS
H
H
H
H
O
O
O
synthesis and CS synthesis, not only are
OH
H
H
H
O CH2 CH
H
H
H
H
H
elongation and modification of CS/DS and
HO
C O
OH
H
OH
OH
H
H
OH
H
HS/heparin GAG chains catalysed by different
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
the
attachmentof of
each
whereas
the attachment
each
Gal,Gal,
whereas
Localisation of enzymatic activities to
addition ofofthe
first
GlcA
is followed
by
addition
the
first
GlcA
is followed
by
different regions of the Golgi apparatus has been
PO42rapid dephosphorylation.
rapid
dephosphorylation.
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
modifications are not known.
(Lippincott-Schwartz et al., 1990; Sandvig et al.,
200
K. Prydz and K. T. Dalen
substrates, which consequently do not undergo TGN
modifications. In several cell types, synthesis of HSPG is still
detectable in the presence of BFA, whereas CSPG synthesis is
blocked, which indicates that separate enzymes in different
subcompartments of the Golgi complex are involved in HSPG
and CSPG synthesis (Spiro et al., 1991; Sugumaran et al.,
1992; Fransson et al., 1992; Uhlin-Hansen and Yanagishita,
1993; Calabro and Hascall, 1994). The enzymes necessary for
the completion of the synthesis of HSPGs are located in the
cis-, medial- and trans-Golgi cisternae, whereas those needed
for CSPG synthesis are localised to the trans-Golgi network.
SORTING OF PROTEOGLYCANS
PGs are expressed by virtually all vertebrate cells and are also
expressed in Drosophila melanogaster (Campbell et al., 1987;
Cambiazo and Inestrosa, 1990; Spring et al., 1994; Graner et
al., 1994) and C. elegans (Rogalski et al., 1993; Schimpf et al.,
1999). How PGs are sorted and transported to their proper
destinations can therefore be studied in many different
organisms. After reaching the plasma membrane, PGs can take
part in binding and uptake of signalling molecules, such as
growth factors and γ-interferon. Cell surface GAGs are
internalised and have been detected in endocytic organelles and
in the nucleus (Fedarko and Conrad, 1986; Ishihara et al., 1986;
Tumova et al., 1999), co-localised with growth factors, but the
pathway taken from the cell surface to the nucleus is largely
unknown. In rat hepatocytes, HSPG is transported from the
TGN to the cell surface in a population of vesicles different
from that which transports serum albumin, apolipoprotein E
and fibrinogen (Nickel et al., 1994; Barthel et al., 1995).
Hepatocytes are polarised cells in vivo, but it is now evident
that in non-polarized cells there are also at least two alternative
carriers from the TGN to the cell surface (Keller and Simons,
1997). Many neural and endocrine cells possess two secretory
pathways from the trans-side of the Golgi apparatus: a
regulated pathway and a constitutive pathway. Frequently PGs
are sorted to the regulated pathway, being released together
with the other contents of the storage granules. The negatively
charged PGs are engaged in binding of small positively
charged molecules, such as histamine (Grimes and Kelly, 1992;
Brion et al., 1992; Castle and Castle, 1998) and proteases
(Huang et al., 1998; Lützelschwab et al., 1997). Mast cells
from mice that lack the enzyme NDST 2, which is involved in
heparin synthesis, fail to store several proteins that are
normally bound to heparin in secretory granules. Mast cells
from NDST 2 knockout mice contained smaller granules and
large empty vacuoles, which indicates that heparin GAGs are
needed for normal granule formation (Humphries et al., 1999;
Forsberg et al., 1999).
The role of glycans in apical sorting in epithelial
cells
Similarly to hepatocytes and neurons, epithelial cells have their
plasma membranes divided into two domains, the apical and
basolateral surfaces. To maintain the polarised organisation,
efficient sorting mechanisms target newly synthesised and
recycling molecules to the proper surface domain or
intracellular location. Epithelial cells must ensure that PG
components of the extracellular matrix (ECM) and those that
attach the basolateral side of the epithelium to the ECM are
transported to the correct side of the monolayer. Basolateral
distribution of syndecan-1 in MDCK cells requires the terminal
12 amino acids of the cytoplasmic tail (Miettinen et al., 1994),
while basolateral secretion of the major basement membrane
PG occurs by a pH-dependent mechanism (Caplan et al.,
1987). The fact that different sets of PGs are secreted apically
and basolaterally indicates that these molecules are actively
sorted. The recent discovery that endogenously synthesised
CSPG, and hexyl-β-D-xyloside that has CS chains, are mainly
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
glypican was detected predominantly at the basolateral surface
of CaCo-2 and MDCK cells (Mertens et al., 1996). A form of
glypican that lacks sites for HS attachment was transported
strictly to the apical surface in MDCK cells, presumably
because of its glycosylphosphatidylinositol (GPI) membrane
anchor or N-linked sugars, although the latter question was not
addressed. HS chains thus either promote basolateral sorting of
glypican or interfere with the recognition of the apical sorting
information in the molecule.
Since many GPI-linked proteins are delivered to the apical
surface in epithelial MDCK cells (Lisanti et al., 1988), it has
been suggested that GPI anchors function as apical sorting
signals. However, both secreted and GPI-linked rat growth
hormone required N-glycans to be transported apically
(Scheiffele et. al., 1995; Benting et al., 1999). O-linked sugars
might also direct apical sorting in MDCK cells (Yeaman et al.,
1997), and both N-linked and O-linked sugars play a role in
apical targeting of bovine enteropeptidase (Zheng et al., 1999).
For transmembrane proteins, both N-glycans (Gut et al., 1998)
and transmembrane domains (Scheiffele et al., 1997) have been
proposed to play a role.
The mechanisms underlying carbohydrate-mediated
transport and sorting are not known. Simons and co-workers
have proposed that sorting is mediated by lectin molecules that
accumulate in sphingolipid-cholesterol rafts within the
exoplasmic leaflet of the TGN membrane (Simons and Ikonen,
1997; Keller and Simons, 1997). VIP36 has been proposed as
a candidate raft-associated lectin (Fiedler et al., 1994), but
more recently this protein has been localised to the cis region
of the Golgi (Füllekrug et al., 1999) and, thus, to date no
sorting lectin has been identified.
Proteins may also be transported apically in a glycanindependent manner (Rodriguez-Boulan and Gonzalez, 1999),
and proteins both with (Zheng et al., 1999) and without
(Alonso et al., 1997) N-linked sugars are transported to the
apical surface without being integrated into rafts, according to
the detergent extraction criteria applied for raft association.
Thus, different kinds of determinants might mediate apical
sorting. A particular protein may contain more than one sorting
signal, and glycan signals are often recessive to other sorting
signals contained in the protein portion of a molecule
(Mellman et al., 1993). This could explain why attachment
of a CS chain to a variant of the amyloid-precursor-like
protein 2 influences the basolateral sorting of neither the
transmembrane-anchored form nor the secretory form of this
protein (Lo et al., 1995).
In an alternative model, the sorting lectin is not a
transmembrane protein that recycles but a secreted PG.
Synthesis and sorting of proteoglycans
Versican (Zimmermann and Ruoslahti, 1989) might be the
CSPG secreted apically by MDCK cells (Svennevig et al.,
1995). This PG is a member of a group called lecticans that,
in addition to CS chains, contain several interesting protein
core domains (Iozzo, 1998). Among these are two hyaluronic
acid (HA)-binding modules near the N terminus and a C-lectin
domain at the C terminus (Halberg et al., 1988). Versican has
been proposed to bind tenascin-R (Aspberg et al., 1995), Lselectin (Kawashima et al., 1999) and sulphated glycolipids
through the C-type lectin domain (Miura et al., 1999). Lselectin has been shown to bind to, in addition to versican,
many of the same kinds of ligands as versican and some
additional ones (Needham and Schnaar, 1993; Kimura et al.,
1999; Li et al., 1999; Watanabe et al., 1999). In the Golgi
apparatus, the C-lectin domain of versican could bind to
sulphated glycolipids. Since HA has been proposed to be
synthesised at the cell surface, the two HA-binding domains
may be free to bind other sugars in the Golgi apparatus. The
HA-binding motifs are structurally similar to C-type lectins,
apart from a possible calcium-binding loop (Kohda et al.,
1996). Lectin domains at both ends of the versican molecule
could thus allow binding of one end to the membrane and
binding of glycanated molecules, such as glycoproteins, to the
other end. Cross-linking into larger complexes may be
necessary for apical sorting of soluble molecules.
More studies are needed if we are to determine the extent to
which glycans contain sorting information and to identify the
structural determinants involved. The indications that
glycosaminoglycans influence the subcellular localisation of
proteoglycans raises the possibility that the tissue-specific
variability seen in GAG modification directs PGs to different
destinations in different tissues. This possibility will link future
studies of the regulation of PG synthesis to studies of PG
sorting.
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