ANNIVERSARY
COMM
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anel Discussion

B

iological functions ofglycosaminoglycans

S hoko Nishihara

Department of Bioinformatics,
Graduate School of Engineering,Soka University, Japan

Professor Shoko Nishihara received her Ph.D. in 1982 from Dept. of Chemistry,
Graduate School of Science, the University of Tokyo. She continued her research asa research associate at Dept. of Microbiology, the Jikei University School of Medicine. Then she worked on Drosophila oocyte maturation as a postdoctoral fellow
at Dept. of Biochemistry, University of North Carolina (Prof. Laura Kalfayan) andcontinued her Drosophila work as a postdoctoral fellow at Dept. of Molecular Biology, Mitsubishi Kasei Institute of Life Sciences. In 1991 she moved as an assistantprofessor to Institute of Life Science, Soka University (Prof. Hisashi Narimatsu) and
was introduced to the field of glycobiology. In this period she contributed to themolecular cloning and characterization of the fucosyltransferase gene family. In 2001
she moved to her current position, Professor, at Dept. of Bioinformatics, Graduate
School of Engineering, Soka University. She is a member of the Editorial Board of theGlycoconjugate Journal. Research projects within her group are focused on analysisof glycan functions in embryonic stem cells, analysis of glycan functions by Drosophila model system and the functional analysis of PAPS transporters.

Introduction
Proteoglycans are composed of negatively charged glycosaminoglycans (GAGs) and core proteins. GAGs include heparan sulfate(HS), chondroitin (CS)/dermatan sulfate (DS), keratan sulfate (KS)and hyaluronan (HA). Various growth factors, morphogens and cytokines interact with GAGs, which regulate the signaling pathwaysof these factors. Therefore, GAGs have important roles in variousdevelopmental processes such as embryogenesis, morphogenesis,neurogenesis and immunogenesis, in addition to progressions ofdiseases including cancer and hereditary disorder.

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the 25th Anniversary of Mizutani Foundation for Glycoscience

Heparan sulfate and chondroitin sulfate are conserved
across a wide range of species
The glycosylation of proteins and lipids is performed in the Golgi
apparatus by various glycosyltransferases. Molecular evolutionary
study about glycosyltransferases showed that the divergence ofgene families had finished before the branching of the deuterostome and protostome such as C. elegans and Drosophila melanogaster 1). After that, early in the history of the vertebrate lineage,
intrafamilial genes increased through gene duplication to obtaina variety of substrate specificities for synthesizing various glycanstructures. A prototype set of glycosyltransferases including HSand CS-synthesizing enzymes existed before the protostome-deuterostome split, suggesting conservation of these GAG structuresMEMORATIVE SYMPOSIUM
and functions from C. elegans and Drosophila to mammals. However, DS, KS and HA could not be found in C. elegans and Drosophila.
Whole body or tissue-specific knockdown (KD) of 125 Drosophila
glycosylation-related genes were performed 2). Whole body gene
KD of HS- and CS-related genes resulted in lethality before eclosion. However, sulfotransferases showed lower ratio of lethality
than glycosyltransferases synthesizing HS and CS backbone structure. The tissue-specific KD of HS- and CS-related genes showed thehighest ratio of abnormalities in the corresponding tissue among
all glycosylation-related genes, indicating that HS and CS were essential for development. The similar malformed phenotypes wereobserved between HS-related and CS-related genes, suggestingthat they regulate similar signaling pathways.
Biological functions of heparan sulfates
HS proteoglycans (HSPGs) or CS proteoglycans (CSPGs) are composed of negatively charged HS or CS and core proteins. There havebeen many studies of the biological functions of HSPGs in Drosophila 3),4). They play important roles in the regulation of fundamental
signaling pathways; for example, Hedgehog (Hh), Decapentaplegic(Dpp; a bone morphogenetic protein (BMP)-type ligand that belongs to the TGFβ family), Wingless (Wg), fibroblast growth factor(FGF) and Slit-Roundabout (Slit-Robo) signaling. Hh, Dpp and Wg,
which are secreted signaling molecules, function as morphogens

Aduring various stages of development. HSPGs bind to them, stabilize them, determine their distribution, probably work as co-receptors, and then regulate the expression of downstream target genesof their signals. HSPGs also bind to FGFs and work as co-receptors.
In addition, HSPGs bind to major axon guidance cues, such as Slitand Netrin, and determine their distribution. Thus, HS, rather thancore protein, binds to various ligands and regulate their signals inmany aspects of development in Drosophila.
The fundamental functions of HS are conserved in mammals.
Again, HS binds to Hh, BMP, Wnt, FGF, and so on to regulate theirsignals 5). In early mouse embryogenesis, HS on the surface of extraembryonic endoderm (ExE) cells are essential for the stable and local retention of FGF ligands to the FGF receptor and subsequent activation of FGF signaling required for ExE development 6). In mouseembryonic stem (ES) ells, HS also regulates the important extrinsic
signals for their maintenance and differentiation (Figure 1A) 7). HS
is present at higher levels than other GAGs such as CS/DS and HA
on mouse ES cells, and the levels of each GAG increases during differentiation. HS contributes to the maintenance of the undifferentiated naïve state of mouse ES cells through BMP/Smad signaling andprobably through Wnt/β-catenin signaling. BMP4 and Wnt3a bindto heparin (KD = 69.4 nM and 26.0 nM, respectively). However, thesituation of Wnt signaling is bit complex, and not yet resolved 7). TheB

Figure 1

Overview of heparan sulfate (HS) functions in undifferentiated naïve mouse ES cells and during differentiation from ES cells.
A.	HS contribution to the signaling events triggered by extrinsic factors.	The undifferentiated naïve state of mouse ES cells is regulated by many extrinsic factors. HS contributes to the maintenance of this state throughWnt/β-catenin and BMP/Smad signaling, and to the exit from this state through FGF4/ERK signaling. HS proteoglycan Glypican 4 specificallyregulates Wnt/β-catenin signaling. 3-O sulfated HS (3-O-HS) promotes to the differentiation from the undifferentiated naïve state through Fassignaling.
B.	Mechanism of Fas signaling via 3-O-HS in promotion of differentiation. During the differentiation from the undifferentiated naïve state, 3-O-HS
are upregulated, and then bind to Fas. They translocate intracellular Fas into lipid rafts on mouse ES cell surfaces. The accumulation of Fas in thelipid rafts activates the Fas/caspase-8/caspase-3 signaling cascade, and the subsequently activated caspase-3 degrades the Nanog protein, which
is a key transcription factor for maintaining self-renewal and pluripotency of ES cells.

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contributions of HS to Wnt signaling may depend on core proteins.
For example, HS on proteoglycans other than Glypican 4 may act asa reservoir, inhibiting binding of Wnt to cognate receptors, whereas
HS on Glypican 4 may act as a co-receptor or a stabilizer. In addition,
HS contributes to exit from the naïve state via FGF4 signaling, andthen 3-O sulfated HS promotes the differentiation through Fas signaling from naïve mouse ES cells into primitive endoderm or primitive ectoderm and the subsequent three germ layers (Figure 1B).
Biological functions of chondroitin sulfates
Some studies have been done for the function of CSPGs in C. elegans. There is only one chondroitin synthase sqv-5 in C. elegans,of which mutant showed squashed vulva phenotype. Whole body
KD by using synthesized double-stranded RNA of sqv-5 proved thatChn, non-sulfated forms of CS, is involved in embryonic cell division, namely cytokinesis and chromosome partition 8). Cytokinetic
regression caused by loss of CS is also observed in the embryo ofGlcAT-I knockout (KO) mouse, indicating that the Chn/CS functionin embryonic cell division is conserved from C. elegans to mammals.
In addition, GlcAT-I KO ES cells demonstrated that CS, probably
CS-A and CS-E, promotes initial ES cell commitment to differentiation via binding to E-cadherin. Furthermore, CS-E shows strong
binding affinity to Wnt3a (KD = 27.3 nM), comparable to heparin, as
well as various growth factors, neurotrophic factors and cytokines
in vitro 9). During differentiation from ES cells to cardiomyocytes,
CS-E inhibits Wnt signaling, leading to a reduction in early cardiac
progenitors and an enhancement of later differentiation steps into
mature cardiomyocytes.
CSPGs and CS-E are also involved in neuronal plasticity 9),10),11). Perineuronal nets (PNNs) are a specialized extracellular matrix surrounding parvalbumin-positive interneurons, a subclass of GABAergic inhibitory neurons. PNNs in adult consist of CSPGs, HA,
tenascin R, Otx2 and Semaphorin 3A, which strongly interacts withCS containing CS-E and inhibits neurite outgrowth. Transgenic
mice overexpressing C6ST-1 inhibited the formation of adult 4-Osulfation-enriched PNNs and showed ocular dominance plasticity

even in the adult mice, indicating that 6-O-sulfated CS is essential
for neuronal plasticity. After CNS injury including spinal cord injury, functional recovery is difficult because of glial scar formation,
which inhibits axon regeneration 10),11). Reactive astrocytes secrete
extracellular matrix proteins including CSPGs and KS proteoglycans (KSPGs), and then they form a glial scar. In this case, CSPGs andKSPCs inhibit axon regeneration, while HSPGs promotes it, becauseof the different type of binding to receptor protein tyrosine phosphatase sigma.
Conclusion
As mentioned above, GAGs bind to morphogens and growth factors to regulate their signals as co-receptors. In addition, GAGs
directly bind to GAG receptors as ligands to regulate downstream
signals. These signals work as key regulators for many biologicalprocesses including development, although I covered just several
examples of their functions here. Therefore, the defects of GAGrelated genes induce many types of hereditary diseases such as
Ehlers-Danlos syndrome, bipolar disorder and hereditary multiple
exostoses. When we are faced with the challenges of GAG functions, we should consider the following two points: 1) sulfation pattern of GAG and 2) core-protein, though it may not be identified
easily. The former determines the binding specificity to each signal
molecule and the latter controls GAG localization, which may determine whether GAG acts as a co-receptor or as a reservoir, namelyinhibiting binding of ligands to cognate receptors. GAGs also bindto cytokines and chemokines and regulate their signals, indicating
their functions in immunogenesis, inflammation and also cancer
progression. HA in the tumor microenvironment modulates thefunction of tumor-associated macrophages to support self-renewalof cancer stem cells. It is worth mentioning that many viruses andother pathogens bind to GAGs on cell surface and use them as entry sites of infection. Thus, GAGs have huge variety of functions in awide range of biological and medical fields, and therefore, the applications of GAGs based on their functions will be spread into various arenas including regenerative medicine and cancer therapy.

References1)	Kaneko M, Nishihara S, Narimatsu H, Saitou N. The evolutionary history of glycosyltransferase genes, TIGG (13): 147-155, 2001.
2)	Yamamoto-Hino M, Yoshida H, Ichimiya T, Sakamura S, Maeda M, Kimura Y, Sasaki N, Aoki-Kinoshita KF, Kinoshita-Toyoda A, Toyoda H, Ueda R, Nishihara S,Goto S. Phenotype-based clustering of glycosylation-related genes by RNAi mediated gene silencing. Genes to Cells (20): 521-542, 2015.
3)	Nishihara S. Glycosyltransferases and transporters that contribute to proteoglycan synthesis in Drosophila: Identification and functional analyses using the heritable
and inducible RNAi system. Method in Enzymnology (480): 323-351, 2010.
4)	Nakato H, Li JP. Functions of heparan sulfate proteoglycans in development: insights from Drosophila models. Int Rev Cell Mol Biol. (325): 275-293, 2016.
5)	Poulain FE, Yost HJ. Heparan sulfate proteoglycans: a sugar code for vertebrate development? Development (142): 3456-3467, 2015.
6)	Shimokawa K, Kimura-Yoshida C, Nagai N, Mukai K, Matsubara K, Watanabe H, Matsuda Y, Mochida K, Matsuo I. Cell surface heparan sulfate chains regulate local
reception of FGF signaling in the mouse embryo. Dev Cell. (21): 257-272, 2011.
7)	Nishihara S. Glycans define the stemness of naïve and primed pluripotent stem cells.Glycoconj J. 2016 Oct 28. [Epub ahead of print]8)	Mikami T, Kitagawa H. Biosynthesis and function of chondroitin sulfate. Biochim Biophys Acta, (1830): 4719-4733, 2013.
9)	Mizumoto S, Yamada S, Sugahara K. Molecular interactions between chondroitin-dermatan sulfate and growth factors/receptors/matrix proteins. Curr Opin Struct
Biol. (34): 35-42, 2015.
10)	Kadomatsu K, Sakamoto K. Mechanisms of axon regeneration and its inhibition: roles of sulfated glycans. Arch Biochem Biophys. (558): 36-41, 2014.
11)	Miyata S, Kitagawa H. Mechanisms for modulation of neural plasticity and axon regeneration by chondroitin sulphate. J Biochem. (157): 13-22, 2015.

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