The International Journal of Biochemistry & Cell Biology 68 (2015) 8791

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The International Journal of Biochemistry

& Cell Biology
journal homepage: www.elsevier.com/locate/biocel

Molecules in focus

Glycosaminoglycans: Sorting determinants in intracellular protein

trafc
Deyan Mihov , Martin Spiess
Biozentrum, University of Basel, Basel, Switzerland

a r t i c l e

i n f o

Article history:
Received 26 June 2015
Received in revised form 29 July 2015
Accepted 27 August 2015
Keywords:
Proteoglycans
Glycosaminoglycans
Protein transport
TGN export
Endocytosis

a b s t r a c t
Intracellular transport of proteins to their appropriate destinations is crucial for the maintenance of cellular integrity and function. Sorting information is contained either directly in the amino acid sequence
or in a proteins post-translational modications. Glycosaminoglycans (GAGs) are characteristic modications of proteoglycans. GAGs are long unbranched polysaccharide chains with unique structural and
functional properties also contributing to protein sorting in various ways. By deletion or insertion of
GAG attachment sites it has been shown that GAGs affect polarized sorting in epithelial cells, targeting to and storage in secretory granules, and endocytosis. Most recently, the role of GAGs as signals for
rapid trans-Golgi-to-cell surface transport, dominant over the cytosolic sorting motifs in the core protein,
was demonstrated. Here, we provide an overview on existing data on the roles of GAGs on protein and
proteoglycan trafcking.
2015 Elsevier Ltd. All rights reserved.

1. Introduction

2. Structure

Many physiological functions depend on the proper localization of proteins to specic cellular compartments. The sorting
information is contained directly within the amino acid sequence
or alternatively in the proteins posttranslational modications
(Bonifacino and Traub, 2003; Seaman, 2008). Even glycosylation,
i.e. modications in the lumen of the endoplasmic reticulum (ER) or
the Golgi compartments and thus without direct access to cytoplasmic machineries, has been shown to act as sorting determinants.
Best understood is late endosomal/lysosomal transport of proteins carrying the mannose-6-phosphate modication of N-linked
glycans by interaction with the mannose-6-phosphate receptors
(Ghosh et al., 2003). In addition, N- and O-linked glycans have been
found to mediate apical sorting in polarized cells by incompletely
understood mechanisms most-probably involving lectins as cargo
receptors (Potter et al., 2006; Vagin et al., 2009). Since N- and Oglycosylation are also found on basolateral proteins, only a subset
of carbohydrate chains seems to be responsible for polarized sorting (Kitagawa et al., 1994). Finally, glycosaminoglycans (GAGs),
the dening modication of proteoglycans, are emerging as sorting
determinants for various transport steps.

Unlike N- and O-linked glycans, GAGs are long unbranched

polysaccharide chains made of two alternating monosaccharides
(Fig. 1A), an uronic acid or galactose, and an amino sugar (Mikami
and Kitagawa, 2013; Sarrazin et al., 2011). Structurally, GAGs are
extended semi-rigid polymers adopting helical conformation with
an axial rise of about 1 nm per disaccharide unit (Almond and
Sheehan, 2000; Rodriguez-Carvajal et al., 2003). The length of
GAG chains is variable, typically from 20 to 60 kDa (40120 disaccharides) in proteoglycans and 6 to 34 kDa for xyloside-primed
protein-free chains (Victor et al., 2009).

Corresponding author.
E-mail address: deyan.mihov@unibas.ch (D. Mihov).
http://dx.doi.org/10.1016/j.biocel.2015.08.019
1357-2725/ 2015 Elsevier Ltd. All rights reserved.

3. Synthesis and degradation

GAG chains synthesis is initiated in the early secretory pathway by formation of a tetra-saccharide linker on serine residues
(Fig. 1B), except for keratan sulfate chains which are initiated on Nor O-linked glycans. Chain extension, deacetylation and epimerization continue during the transport through the Golgi complex. In
the trans-Golgi and the trans-Golgi network (TGN), GAGs are sulfated at specic positions (Mikami and Kitagawa, 2013; Sarrazin
et al., 2011). Degradation of GAG chains takes place in the lysosomes by the help of endo-type hydrolases, exolytic glycosidases
and sulfatases to liberate monosaccharide moieties.

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D. Mihov, M. Spiess / The International Journal of Biochemistry & Cell Biology 68 (2015) 8791

Fig. 1. Structure and synthesis of glycosaminoglycan chains. GAGs are long unbranched polysaccharide chains made of the following monosaccharides: xylose (Xyl), galactose (Gal), glucuronic acid (GlcA), iduronic acid (IdoA), N-acetyl-glucosamine (GlcNAc), N-acetyl-galactosamine (GalNAc). (A) The GAG chains consist of long repeats of
disaccharide units, whose composition is specic for the different GAG types: CS (4GlcA13GalNAc1), DS (4IdoA13GalNAc1), KS (3Gal14GlcNAc1), HS
(4GlcA14GlcNAc1) and heparin (4IdoA14GlcNAc1). GAGs can be sulfated at different sites (orange arrowheads) and the sulfation pattern further increases the
complexity of their structures. (B) The synthesis of GAGs is initiated by the generation of a tetra-saccharide linked to a serine residue: GlcA13Gal13Gal14Xyl1O-Ser.
This linker is common for CS/DS/HS/heparin proteoglycans with the exception of keratan sulfate, which can be generated on both N- and O-linked glycans. (For interpretation
of the references to color in this gure legend, the reader is referred to the web version of this article.)

4. Biological function: GAGs as sorting determinants

Due to their many carboxylate groups and extensive sulfation,
GAG chains attract water and cations forming hydrated matrices.
Distinct saccharide and sulfation patterns allow specic interactions with various ligands, thus regulating cell signaling, migration,
and differentiation (Mikami and Kitagawa, 2013; Sarrazin et al.,
2011). Proteoglycans are prominent components at the cell surface
and of the extracellular matrix (ECM), but are also found intracellularly in secretory granules and in the endosomallysosomal system.
Initially, the protein core was believed to be solely responsible
for the correct proteoglycan localization. However, several studies have demonstrated that GAGs contribute to protein trafc in a
variety of ways (Fig. 2).

medium upon modication with a sequence containing multiple

CS attachment sites (Hafte et al., 2011). In the contrary, however, fusion of a single CS attachment site to the asialoglycoprotein
receptor H1 (ASGPR-H1), which is targeted to the basolateral surface by a cytoplasmic tyrosine motif interacting with the clathrin
adaptor AP-1B (Sugimoto et al., 2002), did not alter its polarity
(Kobialka et al., 2009). Similarly, natural splice variants of amyloid precursor-like protein 2 (APLP2) with and without a single
CS chain were equally targeted to the basolateral surface, both as
membrane-bound and secretory forms (Lo et al., 1995). The basolateral sorting information in the cytoplasmic portions of these
proteins thus is dominant over the apical signal of the CS chain.
Alternatively, the afnity to the apical transport machinery might
depend on the number of GAG chains.

4.1. Polarized surface expression

4.2. Biosynthetic export

In polarized epithelial cells, proteoglycans are found to be differentially distributed to the apical and basolateral sides of the cell
monolayer, reecting their specic roles on either surface. Heparan sulfate (HS) modication was proposed to mediate basolateral
transport, since glypican, a glycosylphosphatidylinositol-anchored
and predominantly basolateral proteoglycan in CaCo-2 and MDCK
cells, was transported strictly to the apical surface, when its HS
attachment sites were mutated (Mertens et al., 1996). On the
contrary, chondroitin sulfate (CS) proteoglycans and free xylosideattached CS were mainly found to be secreted apically (Kolset et al.,
1999). Rat growth hormone, normally secreted in a non-polarized
manner from MDCK cells, was found predominantly in the apical

To analyze the mechanism of proteoglycan export from the Golgi

to the cell surface, HS proteoglycan-containing post-Golgi vesicles
were isolated from [35 S]sulfate-labeled rat hepatocytes (Barthel
et al., 1995; Nickel et al., 1994). Surprisingly, other secretory
proteins, such as serum albumin, apolipoprotein E and brinogen, were not detected in this vesicle fraction, indicating that
HS-proteoglycans are sorted into distinct types of constitutive
secretory carriers at the TGN.
Additional evidence for GAG-mediated sorting in biosynthetic
export was obtained by comparison of the transport kinetics of proteins with and without GAG modication (Kobialka et al., 2009).
The ASGPR-H1 and 1 -protease inhibitor (A1Pi) were used as

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89

Fig. 2. Intracellular protein trafcking pathways regulated by the glycosaminoglycan chains. (1) Polarized TGN-to-cell surface transport; (2) non-polarized biosynthetic
export; (3) sorting to secretory granules; (4) recruitment to retraction bers; (5) protein internalization.

model transmembrane and secretory proteins, respectively. Both

proteins were either tagged with a short tyrosine sulfation motif
of procholecystokinin or a CS attachment sequence of APLP2,
expressed in HeLa cells, and analyzed by pulse-chase experiments
with [35 S]sulfate-labeling in the trans-Golgi/TGN. CS attachment
accelerated TGN-to-cell surface transport kinetics of both the transmembrane and the soluble protein by eliminating a 10-min lag
phase observed for both GAG-free proteins. This supported distinct
sorting mechanisms, requiring a GAG-recognition machinery at the
TGN (Kobialka et al., 2009).
These results were conrmed in a more physiologically relevant context where the transport of the amyloid precursor protein
(APP), a part-time proteoglycan, was studied in HeLa cells (Mihov
et al., 2015). APP is naturally tyrosine-sulfated and undergoes alternative splicing, leading to formation of CS-proteoglycan variants.
Again, CS attachment resulted in fast, lag-free TGN exit. Deletion of
the entire cytosolic domain of APP slowed down surface transport
of the GAG-free form, but did not affect the transport kinetics of
the proteoglycan forms. Fast CS-mediated transport is thus independent of and dominant over cytosolic sorting signals of APP.
Consistent with this notion, xyloside-attached CS chains displayed
the same fast transport kinetics. Interestingly, both GAG-free and
proteoglycan isoforms of APP were routed via transferrin-positive
endosomes to the plasma membrane (Mihov et al., 2015).
Another part-time proteoglycan, MHCII invariant chain, was
shown to have fast TGN-to-PM transport kinetics, acquiring 40%
sensitivity to external chondroitinase after 15 min sulfate labeling
(Arneson and Miller, 2007). Since the proteoglycans form trimers
with GAG-free invariant chains, one CS chain appeared to be dominant over cytoplasmic di-leucine signals for endosomal targeting.
GAG chains allow proteoglycans to simultaneously connect
multiple ligands and binding partners, thus creating supramolecular complexes at the cell surface (Mikami and Kitagawa, 2013;
Sarrazin et al., 2011). Rapid segregation into distinct post-TGN vesicles and fast surface transport might serve to reduce the risk of

premature intracellular assembly and formation of dysfunctional

protein aggregates. Currently, the mechanisms of GAG-mediated
TGN export are unknown. The extended, semi-rigid conformation
and large size of GAGs might hinder proteoglycan entry into small
transport vesicles and divert them into distinct carriers for large
cargo, as has been observed for TGN exit of large collagen assemblies (Polishchuk et al., 2003). Alternatively, proteoglycans might be
actively sorted at the TGN via interaction with yet unknown cargo
receptors, linking the lumenal GAGs with the cytosolic machinery. To distinguish these possibilities, it would be interesting to
test whether GAG chains other than CS similarly affect TGN-to-cell
surface transport.

4.3. Regulated secretion

Serglycin is a proteoglycan found in hematopoietic and endothelial cells. It has important functions related to secretory granule
homeostasis and constitutive secretion of various cargoes (Kolset
and Pejler, 2011). By interaction with its GAG chains (mainly CS),
granule cargo such as chemokines, cytokines, proteases, as well as
histamine are concentrated before regulated secretion. In pancreatic acinar cells, serglycin is present in zymogen granules and is
involved in packing secretory cargo (Biederbick et al., 2003). Two
approaches were used to generate GAG-free proteins and study
the role of GAGs for serglycin sorting: deletion of the GAG attachment domain and inhibition of GAG attachment by addition of
exogenous xyloside as a competing substrate for GAG synthesis.
In both cases, secretory granule sorting was disrupted and serglycin was detected predominantly in perinuclear Golgi structures.
Additionally, granule sorting of secretory enzymes like amylase and
procarboxypeptidase A was impaired in cells expressing GAG-free
serglycin (Biederbick et al., 2003). The data suggest that the sorting of serglycin into secretory granules at the TGN and its function
depends on the attached GAGs.

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4.4. Sorting at the plasma membrane

In some cases, GAGs are important for proteoglycan recruitment
to specic membrane domains at the cell surface. Neural/glial antigen 2 (NG2 or CSPG4) CS proteoglycan is a membrane-spanning
protein involved in the formation of retraction bers and expressed
by tumors and immature progenitor cells including oligodendrocyte progenitors, chondroblasts, and pericytes/smooth muscle
cells (Stallcup and Dahlin-Huppe, 2001). The amount of GAG-free
mutant NG2 at cellular retraction bers was signicantly lower
compared to wild-type NG2. Similarly, chondroitinase treatment
of cells expressing wild-type NG2 resulted in similar reduction of
NG2 localization to retraction bers (Stallcup and Dahlin-Huppe,
2001).
In addition, GAGs are determinants for protein endocytosis.
Early studies showed that GAGs are involved in receptor binding
and cellular uptake of secreted HS proteoglycans in arterial and
human skin broblasts (Kresse et al., 1975; Kruger and Kresse,
1986). The same phenomenon was later demonstrated for the
small DS proteoglycan decorin: its uptake in the mouse skeletal
muscle cell line C2C12 is mediated by simultaneous and cumulative interaction of the protein core and the GAGs with lipoprotein
receptor-related protein (Brandan et al., 2006).
Transmembrane HS proteoglycans have also been observed to
act themselves as uptake receptors for a variety of ligands including
lipoproteins, FGF2, anti-HS antibodies, cell-penetrating peptides,
cationic polymers and polyplexes (Payne et al., 2007; Ram et al.,
2008; Tkachenko et al., 2004; Williams and Fuki, 1997; Wittrup
et al., 2010). The internalization of proteoglycanligand complexes
occurs through relatively slow clathrin-independent, but lipid raftdependent pathways as well as macropinocytosis. Clustering of the
HS proteoglycans is required for internalization, but the mechanism and the precise role of HS chains in the process remain to be
claried.
Interestingly, CS chains were found to negatively inuence
the rapid clathrin-mediated endocytosis of transmembrane proteins. The ASGPR-H1 is a typical recycling receptor, mediating
hepatic uptake of galactose-terminal glycoproteins. Insertion of
a GAG attachment site and resulting CS addition caused strong
inhibition of receptor internalization, which was reversed upon
chondroitinase digestion of the cell surface (Kobialka et al., 2009).
The inhibitory effect of CS on endocytosis was further demonstrated
for APP, which in its GAG-free forms is also actively internalized via
clathrin-coated vesicles at a rate of 8%/min (Mihov et al., 2015;
Schneider et al., 2008). Endocytosis of the CS-containing isoform
was three-fold reduced. GAG chains thus act dominantly over existing cytosolic internalization signals in transmembrane proteins
(Mihov et al., 2015).
How CS chains inhibit endocytosis is not known. They might
sterically hinder entry of the proteoglycan into clathrin-coated
pits. Alternatively, the CS chain might interact with components of
the extracellular matrix, thus restricting its lateral diffusion in the
plasma membrane and its capture at endocytic sites. Experiments
employing uorescence recovery after photobleaching (FRAP),
however, did not detect signicant immobilization of CS-ASGPR-H1
(Kobialka et al., 2009).

5. Signicance and future development

Proteoglycans participate in a plethora of cellular functions,
both at the cell surface and intracellularly, and are thus indispensable for many essential processes including morphogenesis,
neuronal development, tumor formation, and pathogen uptake
(Mikami and Kitagawa, 2013; Sarrazin et al., 2011). Correct localization is therefore a prerequisite for their proper cellular functions.

Good evidence has been accumulated that GAGs act as sorting

determinants at different transport steps. However, the mechanisms underlying GAG-mediated sorting are still largely unknown.
By analogy to established sorting mechanisms, specic cargo receptors might specically recognize GAGs and distinguish between
different types. They could be either transmembrane proteins linking the lumenally exposed GAG chains with cytosolic transport
machinery, or lumenal linker proteins sequestering the proteoglycans into specic lipid domains. Alternatively, GAGs might have
a passive effect on protein sorting via their physical and sterical
properties. Unraveling the role of GAGs in protein trafcking is an
important key for a better understanding of proteoglycan biology
in health and disease.
Acknowledgments
Our work was supported by the Swiss National Science Foundation (grant 31003A-125423).
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