# Structures and interactions of syndecans

Frank Gondelaud, Sylvie Ricard-Blum

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Frank Gondelaud, Sylvie Ricard-Blum. Structures and interactions of syndecans. FEBS Journal, 2019, 286 (15), pp.2994-3007. 10.1111/febs.14828. hal-04998471

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REVIEW ARTICLE

# Structures and interactions of syndecans

Frank Gondelaud and Sylvie Ricard-Blum

ICBMS, UMR 5246 CNRS – University Lyon 1, Univ Lyon, Villeurbanne, France

**Keywords**

chondroitin sulfate; extracellular matrix; heparan sulfate; interaction networks; intrinsic disorder; proteoglycans; structure; syndecans

**Correspondence**

S. Ricard-Blum, Institute of Molecular and
Supramolecular of Chemistry and
Biochemistry, ICBMS, UMR 5246,
University Lyon 1, 43 Boulevard du 11
novembre 1918, F-69622 Villeurbanne
Cedex, France
Tel: +33(0)4 72 44 82 32
E-mail: sylvie.ricard-blum@univ-lyon1.fr

(Received 24 November 2018, revised 4
February 2019, accepted 29 March 2019)

doi:10.1111/febs.14828

The four syndecans identified in mammals are membrane proteoglycans that play major roles in regulating cell behavior, cell signaling, and cell-matrix interactions. The membrane forms of these syndecans function as receptors and co-receptors. Their ectodomains, which are proteolytically released in the extracellular matrix by shedding, also regulate various biological processes. Apart from the cytoplasmic domain of syndecan-4, the 3D structures of syndecans are poorly characterized, which hinders our understanding of the molecular mechanisms underlying syndecan functions that are mediated by numerous interactions. This mini-review summarizes the structural data that are available for syndecans and provides a comprehensive syndecan interactome, which comprises three hundred and fifty-one partners, including those identified by the high-throughput method affinity purification-mass spectrometry. It also gives a perspective on future studies of syndecan structures and interactions, which are required to further elucidate the molecular recognition processes that mediate the biological roles of the membrane and shed forms of syndecans.

Introduction

Proteoglycans are comprised of a core protein and of at least one glycosaminoglycan (GAG) chain covalently attached to the protein part. They exhibit a high structural diversity [1]. Forty-three proteoglycan-encoding genes have been identified and in mammals four of them encode the core protein of syndecans 1–4 (SDCs 1–4), which are single-pass, type I transmembrane proteoglycans [2,3]. In addition, syndecan-4 exists as a soluble protein isoform lacking the trans-membrane and cytoplasmic domains and generated by alternative splicing [4]. The ectodomains of syndecan protein cores bear heparan sulfate chains and are often classified as heparan sulfate proteoglycans although syndecans 1 and 3 also bear two chondroitin sulfate chains [5]. Syndecans 1 and 3 are the largest members

of the syndecan family (33 and 41 kDa respectively), whereas syndecans 2 and 4 are smaller (23 and 22 kDa respectively) [6]. Syndecan-1 is found in leucocytes and is enriched in many epithelia, syndecan-2 is present in endothelia and fibroblasts, syndecan-3 in neural tissues, and syndecan-4 in most tissues [3].

Syndecans play numerous biological roles, which have been previously reviewed [3,7–14]. Their major roles are thus only briefly mentioned in this paragraph, which highlights the most recent findings. They act as receptors and co-receptors [15] and regulate cell behavior and cell signaling [3,7,16] such as Wnt signaling [17]. Syndecan-4 for example mediates cell-extracellular matrix interaction and cell-cell adhesion [18]. Syndecans regulate transient receptor potential canonical

**Abbreviations**

AP-MS, affinity purification-mass spectrometry; C1 and C2, conserved regions 1 and 2; CD, cytoplasmic domain; CS, chondroitin sulfate; ED, ectodomain; ESI-MS, electrospray ionization mass spectrometry; GAG, glycosaminoglycan; HS, heparan sulfate; MMP, matrix metalloproteinase; NMR, nuclear magnetic resonance; SAXS, small-angle X-ray scattering; SDC, syndecan; TMD, transmembrane domain; TRPC, transient receptor potential canonical; V, variable region.
(TRPC) channels to control cytoplasmic calcium equi-
libria and cell behavior [19,20]. They play a role in
lipid metabolism [21], tissue regeneration [8], angiogen-
esis [9] and are involved in the pathogenesis of numer-
ous diseases, including fibrosis and heart disease
[10,22], host-pathogen interactions [23], chronic inflam-
matory and autoimmune diseases [11], cartilage break-
down and synovial inflammation [24]. Syndecan-1 has
been recently shown to regulate homeostasis of Tγδ17
cells and to modulate psoriasis-like skin inflammation
[25], whereas syndecan-4 modulates epithelial gut bar-
rier function and epithelial regeneration in intestinal
inflammation [26]. Syndecans also play key roles in
cancer [12,27–31]. Syndecan-1, induced in the lung
microenvironment after mammary carcinoma cell dis-
semination, promotes outgrowth of metastases [32],
and controls lung tumorigenesis by regulating miR-
NAs packaged in exosomes [33]. Although syndecans
are membrane (co)receptors they may have other func-
tions in the nucleus. Nuclear translocation of synde-
can-1 inhibits the proliferation of fibrosarcoma cells,
activates the TGF-β pathway, and alters the activity of
several transcription factors [34].

The molecular recognition processes underlying the numerous biological roles of the membrane and shed forms of syndecans rely on their ability to interact with ECM, membrane and intracellular partners but they have not been fully deciphered at the molecular level so far. The partners of syndecans listed in previous reviews [3,35,36] were mostly identified by low-throughput methods. We have added in this mini-review the partners of syndecans found in the adhésome dataset (http://www.adhesome.org/) and those recently identified by a high-throughput method (affinity purification-mass spectrometry, AP-MS) [37,38] to generate the first comprehensive syndecan interactome. We also review here the syndecan binding sites and the few 3D structures available in the Protein Data Bank [39] for syndecan domains or peptides, either alone or complexed to a partner. The ectodomains of syndecans are enriched in intrinsic disorder, and the study of their conformational flexibility will benefit from recent advances in the characterization of intrinsically disordered proteins by mass spectrometry [40–43] and computational modeling [44,45].

Domains and structures of syndecans

Full-length syndecans

The core proteins of syndecans are composed of three
domains, an extracellular domain (ectodomain, ED), a
transmembrane domain (TMD) and a cytoplasmic

domain (CD) (Fig. 1). Few experimental structural
data are available for syndecans 1, 2 and 4 and none
for syndecan 3 (Table 1 and Fig. 2). This might be
due to the fact that syndecans are enriched in intrinsic
disorder [13,46]. Indeed, human syndecans are pre-
dicted to contain 86% (SDC1), 62% (SDC2), 80%
(SDC3) and 55% (SDC4) of disordered amino acid
residues [46].

Although no experimental 3D structure of full-
length syndecans exists, models have been built for
syndecans 3 and 4. The molecular model of chick syn-
decan-3 without glycosaminoglycan chains was gener-
ated using secondary structure predictions and the
three-dimensional models of each domain built by
homology [47]. Then, these models were joined and
the entire structure was minimized to generate the
model of full-length syndecan-3. The N-terminal, the
juxtamembrane and the cytoplasmic domains are glob-
ular, whereas an extended stalk containing β-strands
connect the juxtamembrane and the N-terminal
regions [47]. Syndecan-4 was modeled as part of the
endothelial cell glycocalyx by splitting the proteogly-
can into three parts [48] and including GAG chains.
The ectodomain of syndecan-4 with three HS chains
made of various combinations of N-sulfated and non-
sulfated sequences, the transmembrane dimer modeled
from the dimer of glycophorin A embedded into a
lipid bilayer, and the 3D structure of the cytoplasmic
domain were connected to build the model of full-
length syndecan-4. There is thus a single model of a
full-length syndecan encompassing GAG chains but
models of the syndecans 1, 2 and 3 with GAG chains
could be generated using the computational modeling
approaches successfully used for syndecan-4 [48].

Ectodomains of syndecans

The ectodomains of syndecans are released in the
extracellular milieu by limited proteolysis. They con-
tain cleavage sites for metalloproteinases of the metz-
incin family, namely matrix metalloproteinases
(MMPs) [49], disintegrin and metalloproteinase
domain-containing proteins (ADAMs) [50], ADAMs
with thrombospondin domains (ADAMTSs) [51], and
also for plasmin and thrombin in syndecans 1 and 4
(Fig. 1) [6,52]. Further cleavage sites of syndecans
have been predicted by Bertrand and Bollman [53] but
no information of the prediction method(s) used is
available, and the existence of these sites has to be
experimentally confirmed. Several enzymes act as shed-
dases, which release the ectodomains of syndecans 1–4
under a soluble form [6,52] (Fig. 1). Although there
are evidences that syndecan-3 is shed by a member of
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Domain organization of human syndecans with proteolytic cleavage sites, GAG attachment sites, partners binding to the intracellular part of SDCs and receptor binding sites. Core protein: dark blue bars, transmembrane domain: yellow bars, conserved cytoplasmic regions C1 and C2: green bars, variable cytoplasmic region: blue bars, binding regions of receptors: red bars. Heparan sulfate (HS) and chondroitin sulfate (CS): curvy lines in black and grey respectively. Proteolytic cleavage sites: colored triangles for metzincins, white and black arrowheads for plasmin and thrombin respectively. Binding partners of the cytoplasmic domain are indicated and those common to the four syndecans are in red. (GAG: glycosaminoglycan).</figcaption></figure>

the MMP family [54], and that numerous cleavage sites by MMPs have been predicted in its sequence [53], no specific protease has been experimentally

identified so far as a syndecan-3 sheddase. The shedding of syndecans is regulated by external stimuli (i.e. molecular and biological context) [6]. Cancer [55],
<table><caption>**Table 1.** Structural studies of syndecans. 3D structures deposited in the Protein Data Bank (PDB, https://www.rcsb.org/, UniprotKB release 2018.10) and in the Biological Magnetic Resonance Data Bank (BMRB, http://www.bmrb.wisc.edu/). The sequence of peptides used for structural studies and those found in the PDB file are indicated. CD, cytoplasmic domain; ED, ectodomain; NMR, nuclear magnetic resonance; SDCBP, syndecan-binding protein-1 (syntenin-1); TMD, transmembrane domain; X-ray, X-ray crystallography.</caption><thead><tr><th colspan="6">Syndecans (SDCs)</th></tr><tr><th>Peptide sequence used</th><th>Peptide sequence observed in the structure</th><th>Domain</th><th>Partners</th><th>Method & PDB/ BMRB entries</th><th>Referencs (PMID or DOI)</th></tr></thead><tbody><tr><td><strong>Syndecan-1 (P18827)</strong><br>303TKQEEFYA<sup>310</sup> (Tyr<sup>309</sup>: phosphorylated)</td><td>Identical</td><td>CD (part of C2 domain)</td><td>TIAM1 (Q13009)</td><td>X-ray 4GVC</td><td>23395182</td></tr><tr><td>303TKQEEFYA<sup>310</sup></td><td>Identical</td><td>CD (part of C2 domain)</td><td>TIAM1 (Q13009)</td><td>X-ray 4GVD</td><td>23395182</td></tr><tr><td>201PAAEGSGEQDFT<sup>212</sup></td><td>202AAEGSGEQ<sup>210</sup></td><td>ED</td><td>XYLT1 (Q86Y38)</td><td>X-ray 6EJE</td><td>29681470</td></tr><tr><td><strong>Syndecan-2 (P34741)</strong><br>GSRMRKKDEGSYDLGERKPSSAAYKAPTKEFYA<br>(the residues in bold are those found in the TMD of SDC2, 146–170)</td><td>–</td><td>CD (full-length)</td><td>–</td><td>NMR</td><td>10.5012/bkcs. 2008.29. 12.2449</td></tr><tr><td colspan="6"><strong>Syndecan-3 (O75056)</strong> No 3D structure available in the Protein Data Bank</td></tr><tr><td><strong>Syndecan-4 (P31431)</strong><br>176RMKKKDEGSYDLGKPIYKKAPTNEFYA<sup>202</sup> (rat)<br>(Identical to residues 171–198 of human SDC4)</td><td>Identical</td><td>CD (full-length)</td><td>–</td><td>NMR 1EJP/4591</td><td>11456484</td></tr><tr><td>170RMKKKDEGSYDLGKPIYKKAPTNEFYA<sup>197</sup><br>chicken, and rat SDC4 (residues 171–198 in human SDC4)</td><td>Identical</td><td>CD (full-length)</td><td>PIP<sub>2</sub> Not visible in the PDB file</td><td>NMR 1EJQ/4592</td><td>9582338</td></tr><tr><td>193TNEFYA<sup>198</sup></td><td>1st chain:<br>194NEFYA<sup>198</sup><br>2nd chain:<br>193TNEFYA<sup>198</sup></td><td>CD (part of C2 domain)</td><td>SDCBP (O00560)</td><td>X-ray 1OBY</td><td>12842047</td></tr><tr><td>193TNEFYA<sup>198</sup></td><td>1st chain:<br>193TNEFYA<br>1982nd chain:<br>194NEFYA<sup>198</sup></td><td>CD (part of C2 domain)</td><td>SDCBP (O00560)</td><td>X-ray 1YBO</td><td>16533050</td></tr><tr><td>195APTNEFYA<sup>202</sup> (rat) (Identical to residues 191–198 of human SDC4)</td><td>Identical</td><td>CD (part of C2 domain)</td><td>SDCBP (O00560)</td><td>X-ray 5G1D</td><td>27830760</td></tr><tr><td>195APTNEFYA<sup>202</sup> (rat) (Identical to residues 191–198 of human SDC4)</td><td>Identical</td><td>CD (part of C2 domain)</td><td>C-terminal truncated SDCBP (O00560)</td><td>X-ray 5A2P</td><td>27830760</td></tr><tr><td>Zebrafish syndecan-4<br>174RIKKKDEGSYDLGKTP1<br>YKKAPTTEIYA<sup>201</sup> of zebrafish SDC4</td><td>Identical</td><td>CD (full-length)</td><td>–</td><td>NMR</td><td>18753130</td></tr><tr><td>MVLAALIVGGVVGILFAVLILLVYGGKKKKM<br>(the residues in bold are those found in the TMD of human SDC4, 146–170)</td><td>–</td><td>TMD (with N- and C-terminal extra residues)</td><td>–</td><td>NMR</td><td>10.1016/j.procbio. 2011.02.006</td></tr><tr><td>Rat syndecan-4 (P34901) (exact sequence unknown)</td><td>–</td><td>CD</td><td>NUDT16L1 (Q9BRJ7)</td><td>NMR</td><td>26100207</td></tr></tbody></table>

traumatic brain injury and heparanase enhance syndecan-1 shedding [56,57], whereas chemicals, and increased oxidative stress enhance syndecan-4 shedding in dysfunctional endothelial cells [58]. In contrast, resveratrol suppresses the shedding of syndecan-4 [59]

and the inhibition of syndecan-1 shedding induced by ischemia/reperfusion may protect against ischemic acute kidney injury [60].

The ectodomains of syndecans have a number of biological functions [61]. They may act as competitive
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** 3D structures of syndecan domains and syndecans complexes. Upper panel from left to right: X-ray structures of the complex formed by the AAEGSGEQD sequence located in the ectodomain (ED) of syndecan-1 (SDC1) with xylosyltransferase 1 (6EJE), the complex formed by TKQEEFYA sequence located in the cytoplasmic domain (CD) of syndecan-1 with the PDZ domain of Tiam-1 (4GVC), and the complex of the C-terminal sequence TNEFYA of the cytoplasmic domain of syndecan-4 (SDC4) with the PDZ domain of syntenin. Middle panel: X-ray structures of the complex formed by the APTNEFYA sequence located in the cytoplasmic domain of syndecan-4 with syntenin-1 (5GID) or with the C-terminal truncated syntenin-1 (5A2P). Lower panel: NMR structures of the full-length cytoplasmic domain of syndecan-4 with (1EJP) or without (1EJQ) phosphatidylinositol-2 phosphate (PIP₂). C1 and C2 regions: orange, V region: blue.</figcaption></figure>

inhibitors and also as agonists of the membrane forms of syndecans [53]. The ectodomains of syndecans 1–4 bearing GAG chains suppress osteoclast differentiation [62], and the ectodomain of syndecan-4 promotes tubule-interstitial fibrosis [58]. Shed syndecan-1 can be translocated to the nucleus of tumor cells, where it inhibits the activity of histone acetyltransferase and delivers growth factors such as hepatocyte growth factor [63]. In addition, soluble ectodomains of syndecans serve as biomarkers for several diseases such as inflammatory diseases, sepsis, leukemia, celiac disease or systemic sclerosis (ectodomain of syndecan-1), colon cancer (shed syndecan-2), and atopic dermatitis (shed

syndecan-4) as reviewed in [53]. The soluble form of syndecan-4 is a predictor of heart failure [64–66] and the increase in soluble syndecan-1 level in cerebrospinal fluid is correlated with the severity of neuromyelitis optica, an inflammatory demyelinating disease of the central nervous system [67].

The ectodomains of syndecans regulate various biological processes both outside and inside the cells, and this might be due, at least partly, to their structural plasticity. Indeed, the comparison of the sequences of the ectodomains of syndecans 1–4 in 25 animals has shown that they are enriched in glycine, alanine, glutamic acid and proline residues and depleted in
order-promoting amino acid residues [13]. The high
amount of intrinsic disorder might explain why no
experimental 3D structure is available for the ectodo-
mains of syndecans in absence of binding partners.
The only X-ray structure available for a syndecan
ectodomain comprises a dodecapeptide of syndecan-1
in complex with human xylosyltransferase 1 [68]
(Table 1 and Fig. 2). A 3D model of the syndecan-4
ectodomain has been built and shown to partially fold
into α-helices at its N- and C-termini, whereas the dis-
ordered region containing the attachment sites of HS
chains remains flexible [48].

In conclusion, no structural data are available for
the entire ectodomains of syndecans 1–4. Since they
contain a significant amount of disorder, which would
likely prevent their crystallization, low-resolution
structural techniques such as small-angle X-ray scatter-
ing (SAXS) should be favored. Intrinsically disordered
proteins can fold upon binding to their partners, and
crystallogenesis studies of the syndecan ectodomains in
presence of partners interacting with their protein part
would give new insights on their ability to fold upon
binding, differently or not depending on their partners,
and on the extent of their folding. Mass spectrometry
(MS) approaches such as native electrospray ionization
(ESI)-MS and native ion mobility MS would be useful
to evaluate the conformational space of the syndecan
ectodomains, dissect their conformational ensembles
and capture compact conformations from their charge-
state distributions [40,42,43,69]. In addition, 3D mod-
els of the ectodomains of syndecans could be gener-
ated using computational modeling approaches used
for intrinsically disordered proteins by coarse-grained
molecular dynamics simulations restrained by SAXS
data [45] or by approaches based on the dock-and-coalesce
mechanism of intrinsically disordered protein bind-
ing (IDP-LZerD [70]). The 3D structures and/or
models of syndecans would be used for docking
experiments with their partners to study the mole-
cular recognition processes associated with their bio-
logical roles.

Last, to get a global structure of syndecans it will be
necessary to investigate the influence, if any, of GAGs
chains on the conformations of the syndecan ectodo-
mains. A single model has been built for a syndecan
ectodomain with three HS chains [48] and no experi-
mental data are available. Structural analyses by
SAXS of the ectodomains of syndecans 1–4, expressed
in eukaryotic cells allowing the synthesis of GAG
chains, would provide new insights on the role of
GAGs on the flexibility, folding and conformation of
the syndecan ectodomains when bound to partners.
Another challenge would be to determine the effect on

the syndecan core proteins of GAG-modifying
enzymes such as sulfatases and heparanase.

Transmembrane domains of syndecans

The transmembrane domains of syndecans modulate
intracellular signaling by regulating the oligomeric sta-
tus of the cytoplasmic domain [71]. Their self-association,
which is crucial for their functions is driven by the
consensus sequence GXXXG [72] A conserved pheny-
lalanine residue of the transmembrane domain regu-
lates heteromeric interactions of syndecans [73], which
form homodimers and heterotrimers [72,73].

No data are available either for transmembrane
domains of syndecans -1, -2 and -3, whereas nuclear
magnetic resonance (NMR) and circular dichroism
experiments have been carried out on the transmem-
brane domain of syndecan-4. It forms an asymmetrical
α-helical dimer in micelles [74] but the corresponding
structure is not available in the Protein Data Bank
(Table 1). The 3D structure or models of syndecan
transmembrane homodimers and heterodimers are
required to decipher the molecular mechanisms of the
dimerization. This process, which is of major impor-
tance for the regulation of syndecan functions [75,76],
has not been fully characterized so far although several
amino acids critical for the dimerization have been iden-
tified [73].

Cytoplasmic domains of syndecans

The cytoplasmic domains of syndecans are comprised
of two constant, conserved, regions, C1 and C2, flank-
ing a variable region V containing two conserved
amino acid residues (Tyr and Lys), which promotes
syndecan-specific signaling [3,7].

No structural data are available for the cytoplasmic
domains of syndecans 1 and 3. The cytoplasmic
domain of syndecan-2 has been analyzed by NMR [77]
but no model of the domain was generated and the
data are available neither in the PDB nor in the
Biological Magnetic Resonance Data Bank (BRMB).
Several structures of the cytoplasmic domain of synde-
can-4, either alone or in complex with a partner, have
been determined by NMR (Table 1). The cytoplasmic
domain of syndecan-4 is a compact intertwined dimer
with a symmetric clamp shape in the variable region
and two parallel β-strands forming a cavity, whereas
the C1 region appears to be disordered within the
dimeric cytoplasmic domain [78,79]. This structure is
conserved in the cytoplasmic domain of zebrafish syn-
decan-4 although two of its amino acid residues are
different [80,81]. The X-ray structure of the syntenin-
1/syndecan-4 complex has shown that syntenin-1 forms a symmetrical pair of dimers anchored by a syndecan-4 dimer. The cytoplasmic domain of syndecan-4 wraps the syntenin-1 dimers [82]. The binding of syndesmos to rat syndecan-4 induces changes in the C1 and V regions, and the amino acid residues of rat syndecan-4 directly involved in the interaction are Lys177, Lys178, Lys179, Gly182, and Ser183 in the C1 region, and Lys193 and Lys194 in the variable region [83]. The cytoplasmic domain of syndecan-4 also undergoes a shape change upon binding to phosphatidylinositol 4,5 bisphosphate [78]. Two 3D structures of the complexes formed by regions of the cytoplasmic domains of syndecans 1 and 4 encompassing the EFYA tetrapeptide and PDZ protein domains are also available (Table 1 and Fig. 2). In contrast, the TKEFYA sequence of syndecan-2 does not bind to the PDZ-like domain of synbindin although NMR titration experiments showed that the cytoplasmic domain, comprised of 32-amino acid residues, interacts with it [84]. No 3D structure of the complex or of the cytoplasmic domain of syndecan-2 is reported in the article or is available in the Protein Data Bank. Only the solution structure of the PDZ domain of synbindin (2JSN) is deposited in the PDB [84]. Whether the cytoplasmic domains of syndecans 1, 2 and 3 are able to form twisted clamp dimers remains to be determined. However, their 3D structures are needed to decipher the molecular mechanisms of signaling mediated by the interactions between the syndecans and their partners as previously done for syndecan-4-syntenin-1 and syndecan-4-phosphatidylinositol 4,5 bisphosphate interactions [78,82].

## The syndecan interaction networks

### The global interaction network of syndecans

We report here the first comprehensive interaction network of syndecans 1–4 by combining interaction data from previous reviews [35,36], manual curation of the literature, queries of interaction databases, namely MatrixDB [85,86] and IntAct [87], the large scale BioPlex 2.0 interaction dataset [38] obtained by high-throughput affinity purification mass spectrometry (AP-MS), and from the adhosome (http://www.adhe some.org/) (Figs 3 and 4). The entire dataset of syndecan interactions, including the names of their binding partners, the location of their binding sites on syndecans (i.e. core protein and/or glycosaminoglycan chains) when available, and the references reporting experimental evidences of these interactions, is provided in Table S1.

The four syndecans have 351 partners in total. Syndecans 1, 2 and 4 have a high number of binding partners

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Partners of syndecans 1–4. A: total number of partners of syndecans 1–4, B: number of specific partners of syndecans 1–4 and number of partners shared by two, three or four syndecans. The names of the partners common to four syndecans are listed below the Venn diagram built with InteractiVenn (http://www.inte ractivenn.net/).</figcaption></figure>

(131, 103 and 84 respectively), whereas syndecan-3 has only 33 partners (Fig. 3A and Table S1). Syndecan-3 has a more restricted distribution than the other syndecans, which might partly explain the lower number of its partners. There might be also a research bias. Indeed, syndecans 1 and 4 are the most studied syndecans with 670 and 304 publications in PubMed respectively (queries made with syndecan-1 and syndecan-4 in the title of the publication), whereas there are 123 and 56 publications on syndecans 2 and 3 respectively.

### The consensus interaction network of syndecans

The partners specific to each syndecan and those shared by two, three and four syndecans are listed in Table S2. The four syndecans share 18 binding proteins corresponding to the syndecan consensus interactome, including the four syndecans, which are able to form homo- and hetero-dimers (Fig. 3B and Table S2). In addition to the four syndecans, the consensus interactome comprises 14 proteins, namely
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** The interaction network of syndecans 1–4. SDCs are represented as green diamonds. Black lines: direct interactions. Red lines: interactions detected by affinity purification-mass spectrometry (Bioplex 2.0 dataset) and listed in the adhésome (http://www.adhésome.org/), which may be either direct or indirect. Partners binding to syndecan GAG chains: yellow, partners binding to syndecan protein core: blue, lacritin binding to a hybrid site made of GAG saccharides and protein core: purple, partners without information on binding site: white, partners of the BioPlex 2.0 dataset and adhésome: grey.</figcaption></figure>

protein kinases (the peripheral plasma membrane protein CASK, the tyrosine-protein kinase Fyn, and the proto-oncogene tyrosine-protein kinase Src), proteins involved in actin cytoskeletal organization (cortactin, syntenin-1 and neurofibromin) and in microtubule formation (tubulins α and β), a protein playing a role in vesicular transport from endoplasmic reticulum to Golgi (synbindin or TRAPPC4), and proteins involved in signaling (synectin or PDZ domain-containing protein GIPC1 involved in G protein-coupled receptor signaling pathway and TIAM1 or T-lymphoma invasion and metastasis-inducing protein 1). TIAM1 connects extracellular signals to cytoskeletal activities like integrin α6β4, another partner common to the four syndecans, which mediates bidirectional signaling between the cytoskeleton and the extracellular matrix. In addition, all syndecans bind to fibroblast growth

factor-2 (FGF2) and to gp120, a glycoprotein from the human immunodeficiency virus. Most partners interacting with the four syndecans are intracellular or membrane proteins involved in signaling and the organization of actin cytoskeleton.

### The specific networks of syndecans 1–4

The cellular location and the molecular functions of the partners specific to each syndecan together with the biological processes and the biological pathways they are involved in have been analyzed using the Functional Enrichment analysis tool FunRich [88] we have used to analyze the interaction network of the extracellular matrix with the amyloid β-42 peptide [89]. The annotations used by the FunRich tool, discussed below, are put into quotation marks.
The 74 proteins interacting only with syndecan-1 (Fig. 4 and Table S2) are mostly membrane and extracellular proteins involved in ‘cell communication’ (43%) and ‘cell transduction’ (43%). 77% of them are involved in ‘integrin family cell surface interactions’ and 68% in ‘glypican pathway’. Syndecan-1 is the only syndecan to interact with fibrillar collagens I, III and V, and it binds to more pathogens and pathogen proteins than the other syndecans (Fig. 4 and Table S1). It is shed by the metalloproteinase virulence factor ZmpC of *Streptococcus pneumonia* and plays a role in bacterial infections [90].

No significant feature is observed for the 56 specific partners of syndecan-2 (Fig. 4 and Table S2), except that 25% of them are associated with lysosomes. 75% of the 11 specific partners of syndecan-3 (Fig. 4 and Table S2) are extracellular, 25% of them are ‘extracellular matrix constituents’ and 25% are found in basement membranes. Furthermore, 67% are involved in ‘cell communication’ and 67% in ‘cell transduction’, and 58% of them are associated with axon guidance. Syndecan-3 interacts with the extracellular sulfatase Sulf-1 and Sulf2, which have endoglucosamine-6-sulfatase activity, and modulate heparan sulfate sulfation. The 33 specific partners of syndecan-4 (Fig. 4 and Table S2) are extracellular (42%) or associated with exosomes (39%). 39% are involved in ‘cell communication’, 45% in ‘cell transduction’, and 71% in ‘integrin family cell surface interactions’.

In conclusion, a number of the partners of syndecans 1, 2, and 4 are associated with signal transduction and cell communication, and interact with integrins for the partners of syndecans 1 and 4. A significant percentage of the partners of syndecans 1 and 4 (43% and 39% respectively) are associated with exosomes. The exosomes are involved in cell–cell communication [91], and play crucial roles in cancer development [92]. They may thus be associated with syndecans 1 and 4 in these processes.

## Syndecan binding sites

According to the location of their binding sites on syndecans (Table S1), the vast majority of syndecan partners interacts with their GAG chains (71% for SDC1, 67% for SDC2, 91% for SDC3 and 68% for SDC4). Lacritin, a partner of syndecan-1, requires the combination of HS and CS saccharides and of the ⁴⁸GAGAL⁵² sequence of the syndecan ectodomain to bind to syndecan-1 [93]. Structural differences of HS and CS chains dictate different unbinding pathways for syndecan complexes as shown by single-molecule force spectroscopy used to rupture SDC1 and SDC4

complexes with monoclonal antibodies. The unbinding pathways comprises two energy barriers for syndecan-1, whereas syndecan-4 unbinds by crossing over one energy barrier [94]. Furthermore, the same protein may bind either to the protein core or to the GAG chains depending on the syndecan such as ADAMTS-4, which interacts with heparan sulfate chains of SDC1 but binds to the core protein of SDC4 [95,96]. Integrins, metalloproteinases and few other proteins (VEGFR2, HER2, and IGF1R for syndecan-1, TGF-β and PTRJ receptor for syndecan-2, and TFPI for syndecan-3) directly interact with syndecan core proteins (Fig. 4 and Table S1). In conclusion, most interactions established by syndecans in the extracellular matrix or at the cell surface are mediated by their GAG chains. Only few interactions occurring in the extracellular matrix or at the cell surface are mediated by the syndecan core proteins, most of them being receptors and proteinases.

## Ternary complexes formed by syndecans

Besides the binary interactions included in the global interaction network, the syndecans are engaged in ternary complexes such as those formed by the ectodomain of syndecan-1 with α3β1 integrin and HER2 (receptor tyrosine-protein kinase erbB-2), and with either αvβ3 integrin or αvβ5 integrin and IGF1R [97]. The shed ectodomain of syndecan-1 also forms a ternary complex with VEGFR2 and α4β1 integrin [98]. In addition, the ectodomain of syndecan-4 forms a complex with the integrin α3β1 and EGFR [99], and the formation of a ternary complex comprising syndecan-4, integrin α5β1 and Thy-1 (CD90) has been reported [100]. The structural characterization of these ternary complexes would provide new clues of the regulation at the molecular level of cell signaling by syndecans.

## Conclusion and perspectives

Syndecans -1 and -4 are the most frequently studied syndecans, both at the structural and interaction levels. In addition, they share the highest number of partners among the syndecans. Less data are available for syndecans 2 and 3, although fifty-two new partners of syndecan-2 have been recently identified by affinity purification-mass spectrometry [37,38]. Syndecan-3 has been even less characterized, and the enzyme(s) releasing its shed ectodomain remain(s) to be identified. Regarding the structures of full-length syndecans, the major challenge is to collect experimental structural data and/or to generate reliable 3D models for the individual domains of syndecans, and then to connect
them by integrating sequence-to-conformation relationships of disordered regions tethered to folded domains [101]. Native ESI-MS will be also useful to characterize the conformational transitions of the membrane forms of syndecans [102]. In parallel, coarse-grain models of heparan and chondroitin sulfate chains of syndecans should be generated by previously developed methods [103,104]. GAG models could also be generated from conformational maps using the bioinformatics pipeline we have recently developed [105]. It is freely available on the web site of the extracellular matrix interaction database (MatrixDB, http://matrixdb.univ-lyon1.fr/), which has been recently updated with a focus on GAGs and GAG interactions [86].

The comprehensive interaction network of syndecans 1–4 combines low- and high-throughput interaction data, direct and potentially indirect interactions, and warrants more in-depth analyses. Few data are available on GAG sequences of syndecans interacting with their protein partners. The identification of GAG chemical groups involved in the binding of syndecan partners, their spatial distribution along the GAG sequences and a 3D model of these sequences would help to decipher the molecular mechanism(s) of actions of syndecans and to understand how their biological effects are triggered by GAG-protein interactions in the extracellular matrix and at the cell surface. The global interactome of syndecans will provide new clues on how the syndecans are connected, and how they regulate biological processes in a concerted and/or in a specific manner. It will be used for identifying protein-protein interactions, which are crucial for a specific biological process, and could be targeted for a therapeutic purpose. Furthermore, it provides a scaffold to be contextualized with *i*) transcriptomics and quantitative proteomics data to build tissue- or cell-specific syndecan networks, *ii*) binding sites to determine the interactions occurring simultaneously and those that compete with each other, and *iii*) the amount of predicted intrinsic disorder to identify the zones of structural flexibility in the network, which are prone to rewiring of interactions depending on the biological context (e.g. changes in growth factor concentration, and upregulation or downregulation of gene expression). Kinetics parameters (association and dissociation rates) will be added when available to identify transient and stable interactions, whereas the integration of equilibrium dissociation constants will discriminate weak from strong interactions. The integration of all these data will generate a dynamic syndecan interaction network mimicking the *in vivo* situation. The ultimate goal is to build a 3D syndecan interactome.

## Acknowledgements

We acknowledge Sylvain D. Vallet (ICBMS, UMR 5246, Villeurbanne, France) for his valuable help in handling the BioPlex 2.0 and the adhandsome datasets, in drafting Fig. 4 and for his critical reading of the revised manuscript. MatrixDB, the database used among other sources, to build the syndecan interactome, is supported by the Fondation pour la Recherche Médicale (grant DBI20141231336 to SRB), and by the Institut Français de Bioinformatique (ANR-11-INS-0013, grant GlycomatrixDB to SRB).

## Conflicts of interest

The authors declare no conflict of interest.

## Author contributions

FG and SRB performed literature searches and database queries and designed the tables. FG drafted the manuscript and the figures, and revised the Tables and the figures. SRB wrote the final version of the manuscript, revised the manuscript and contributed to the figure design.

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## Supporting information

Additional supporting information may be found online in the Supporting Information section at the end of the article.

**Table S1.** List of the partners of syndecans.

**Table S2.** List of partners specific to each syndecans and of their shared partners.