The oligomerization mediated by the alanine 397 residue in the transmembrane domain is crucial to sydecan-3 functions

Journal Pre-proof The oligomerization mediated by the alanine 397 residue in the transmembrane domain is crucial to sydecan-3 functions

Hyejung Junimg, Minji Han, Bohee Jang, Eunhye Park, Eok-Soo Oh PII:

S0898-6568(20)30021-8

DOI:

https://doi.org/10.1016/j.cellsig.2020.109544

Reference:

CLS 109544

To appear in:

Cellular Signalling

Received date:

4 November 2019

Revised date:

17 January 2020

Accepted date:

17 January 2020

Please cite this article as: H. Junimg, M. Han, B. Jang, et al., The oligomerization mediated by the alanine 397 residue in the transmembrane domain is crucial to sydecan-3 functions, Cellular Signalling(2019), https://doi.org/10.1016/j.cellsig.2020.109544

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© 2019 Published by Elsevier.

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The oligomerization mediated by the alanine 397 residue in the transmembrane domain is crucial to sydecan-3 functions Hyejung Jung1,# , Minji Han2,#, Bohee Jang2, Eunhye Park3, Eok-Soo Oh1,2,* From the 1Skin QC Institute of Dermatological Sciences, Seoul 03759, Korea; 2Department of Life Sciences, Ewha Womans University, Seoul 03760, Korea #

Both authors contributed equally to this work.

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To whom correspondence may be addressed: Eok-Soo Oh, Department of Life Sciences, Ewha Womans University, 52, Ewhayeodae-gil, Seodaemoon-Gu, Seoul 03760, Korea, Tel.: (82)-2-3277-3761; Fax: (82)-2-3277-3760; E-mail: [email protected] Abstract

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Syndecans are single-pass transmembrane proteins on the cell surface that are involved in various cellular functions. Previously, we reported that both homo- and hetero-form of syndecan dimers affected their functionality. However, little is known about the structural role of the transmembrane domain of syndecan-3. A series of glutathione-S-transferase syndecan-3 proteins showed that syndecan-3 formed SDS-resistant dimers and oligomers. SDS-resistant oligomer formation was barely observed in the syndecan deletion mutants lacking the transmembrane domain. Interestingly, the presence of an alanine 397 residue in the transmembrane domain correlated with SDS-resistant oligomer, and its replacement by phenylalanine (AF mutant) significantly reduced SDS-resistant oligomer formation. Beside the AF mutant significantly reduced syndecan-3 mediated cellular processes such as cell adhesion, migration and neurite outgrowth of SH-SY5Y neuroblastoma. Furthermore, the alanine residue regulated hetero-oligomer formation of syndecan-3, and hetero-oligomer formation significantly reduced syndecan-3-mediated neurite outgrowth of SH-SY5Y cells. Taken together, all these data suggest that syndecan-3 has a specific feature of oligomerization by the transmembrane domain and this oligomerization tendency is crucial for the function of syndecan-3. Keywords: Syndecan-3, Oligomerization, Transmembrane domain, Cell adhesion Abbreviations

The abbreviations used are: SDC3, syndecan-3; TMD, transmembrane domain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;

1. Introduction Syndecan family is a class of transmembrane heparan sulfate proteoglycan that plays a key role in the processes of cell adhesion, migration, proliferation and differentiation [1]. It consists four members: syndecan-1, -2, -3, and -4. Syndecans have a large extracellular domain, a transmembrane domain 1

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(TMD) and a small cytoplasmic domain. The extracellular domain of syndecan interacts with a number of extracellular matrix (ECM) molecules and these interactions induce dimerization/ oligomerization of the TMD, which is an important process for activating intracellular signaling such as cytoskeletal reorganization and others [2, 3]. The TMD of syndecans is highly conserved and forms sodium dodecyl sulfate (SDS)-resistant dimer through a GXXXG motif, which induces a stable noncovalent interaction, even in the absence of ligand binding to their extracellular domains [4]. Since the GXXXG motif of TMD forms dimerization in syndecans, substitution of leucine for the glycine fails to form SDSresistant dimerization of all four syndecans [4, 5].

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For receptor activation, the TMD-induced dimerization is critical in cellular signaling. In case of the fibroblast growth factor receptor (FGFR), ligand binding to extracellular domain of the FGFR promotes conformational changes resulting TMD dimerization and activation of kinase domains [6]. The dimerization of the ErbB receptor by TMD leads to the oncogenic signaling, whereas TMD-derived peptides completely arrest proliferation of pancreatic cancer cells [7]. Like other receptors, TMDinduced dimerization seems to be crucial for the various functions of the syndecan family. Syndecan-2 TMD dimerization is important for recruitment of neurofibromin and CASK, which induces filopodia and dendritic spines [8]. Oligomerization of syndecan-4 TMD results in interaction of -actinin with cytoplasmic domain of syndecan-4 for the formation of focal adhesion and actin stress fiber, whereas its oligomerization-deficient mutant decreases focal adhesion [9]. Furthermore, syndecan-3 dimerization co-localizes with actin in the filopodia and several amino acid residues (K383, G392 and G396) in the TMD is important for the ligand-induced dimerization [10].

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Although the TMD of syndecans is highly conserved, that of syndecans has a different tendency to dimerization. That of syndecan-2 dimerizes more strongly than those of syndecan-3 and syndecan-4, whereas syndecan-1 TMD dimerizes weakly [4]. These differences are explained by the different numbers of phenylalanines (Phe) in theirs TMD. Syndecan-2 TMD has three Phe and syndecan-4 only has two Phe.

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We previously reported that the syndecan-2 TMD has an additional Phe167 that causes additional intramolecular interaction, and that the loss of Phe167 reduces formation of SDS-resistant homo-dimer of syndecan-2 [11]. Conserved Phe on TMD of syndecan-2 also regulates heterodimerization between syndecan-2 and syndecan-4 [12]. Therefore, it is highly possible that the specific amino acid residue in the TMDs could be involved in the regulation of TMD-mediated dimerization/oligomerization of syndecan-3. Here, we analyzed the role of the alanine 397 residue (Ala397) in TMDs in the dimerization/oligomerization of syndecan-3, and its functional role.

2. Material and methods

2.1. Antibodies and materials 2

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Polyclonal anti-His and MAP2, monoclonal anti--actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibodies against site-specific phosphorylated FAK (Tyr397) and FAK were obtained from Cell Signaling (Danvers, MA, USA). Human recombinant laminins (laminin-111 and laminin-511) were from BioLamina (Sundbyberg, Sweden). Human plasma fibronectin was purchased from Millipore (Billerica, MA, USA). Collagen type I was purchased from Nitta Gelatin Inc. (Osaka, Japan) 2.2. Construction and transfection of expression vectors

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The extracellular and cytoplasmic domain of rat syndecan-2 (amino acids 1-154 and 180-211) were linked with the transmembrane domain of rat syndecan-3 (amino acids 385-409) to create a chimeric protein designed 2E3T2C, whereas the extracellular and cytoplasmic domain of rat syndecan-4 (amino acids 1-149 and 175-202) were linked with the TMD of syndecan-3 (amino acids 385-409) to generate chimeric protein 4E3T4C. The extracellular and cytoplasmic domain of rat syndecan-3 (amino acids 1384 and 410-442) were linked with the TMD of syndecan-2 (amino acids 155-179) to create a chimeric protein designed 3E2T3C. Single point mutant in the TMD of syndecan-3 (i.e S3T405L) was constructed by commercial gene synthesis (Bioneer, Daejeon, Korea), whereas single point mutant in the TMD of syndecan-3 (i.e S3A397F) was constructed with QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) following the manufacturer’s protocols. The chimera or point mutant were inserted into the N-terminal HA-tagging pcDNA3 expression vector (Invitrogen, Carlsbad, CA, USA).

 Expression and purification of recombinant His-syndecan core protein 

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The cDNAs encoding the full-length rat syndecan-2, 3, 4 core proteins (SDC3, SDC4) and substituted TMD mutants (3E2T3C, 2E3T2C, 4E3T4C) and the single point mutants [S3(T405L), S3(A397F)] were synthesized by PCR and subcloned into the His-tagging expression vector, pET32a+. which contained Trx (MW; 20.4 kDa) (Novagen, Madison, WI). The expression of fusion proteins in E.coli BL21 was induced by incubation with 1mM isopropyl-β-D-thiogalactopyranoside at 30℃ for 16 h. The E.coli cells were lysed with lysis buffer (20 mM Na2HPO4,(pH 8.0), 150 mM NaCl, 5 mM β-mercaptoethanol, and 0.5% Triton X-100 and 1% Sodium Dodecyl Sulfate (SDS)) containing a protease inhibitor mixture, with sonication on ice for 1min. The insoluble material was removed by centrifugation at 13,000×g for 30 min at 4℃, and the supernatants containing His-syndecan fusion proteins were applied to Ni-NTA-agarose columns (Qiagen, Hilden, Germany). Each column was washed three times with lysis buffer containing 50mM imidazole, and the bound proteins were eluted with lysis buffer containing 500 mM imidazole. 2.4. Analytical size exclusion gel chromatography The structural conformation of both 3eTC and 3eTC(A397F) proteins were confirmed using ACQUITY UPLC System (Waters, MA, USA) and BEH 200 Column (Waters, MA, USA). The Column were equilibrated in 50 mM Tris base (pH 8.0), 150 mM NaCl and 0.1% SDS/1% Triton X-100 and the proteins (3mg/ml) were load into the column at a flow rate of 0.5 ml/m. The oligomeric states of proteins were detected and analyzed by absorption of ultraviolet light at a wavelength of 280 nm. Molecular 3

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weight was calculated using the equations y = -0.162ln(x)+2.2157; y = (Elution - 45.15)/(120-45.15); x= Mw for Superdex 200 respectively. Molecular mass was calculated against the standard proteins aldolas(158kDa), conalbumin (75k Da), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa), and aprotinin (6.5 kDa). 2.5. Cell culture and transfection

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The SH-SY5Y human neuroblastoma cell line was purchased form ATCC (Manassas, VA, USA). SHSY5Y cells were maintained in 1:1 mix of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (DMEM/F12; Welgene, Daegu, Korea supplemented with 10% (v/v) fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and gentamycin (50 g/ml; Sigma-Aldrich, St Louis, MO, USA) at 37°C in a 5% CO2-containing humidified atmosphere. Transfections were performed using the Viva Magic transfection reagent (Vivagen, Gyeonggi-Do, Korea) according to the manufacturer's instructions. SHSY5Y cells (2.0x105 cells/well) were plated on 6-well plates, incubated at 37°C for 24 h, and then transfected with the generated expression vectors.

 RT-PCR 

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Total RNA was extracted from cells and reverse transcribed. Aliquots of the resulting cDNAs were

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amplified using the following primers: rat syndecan-3 5′-GCTACCCACGACCGTTATCC-3′ (forward) and 5′-ATGCCGGTGGTCCTTATGTC -3′ (reverse); rat syndecan-4, 5′-

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ATGGCGCCTGTCTGCCTGTT-3′ (forward) and 5′-GCTGCCCTGGGAAGTGCTGG-3′ (reverse); human GAPDH 5´-CCACCCATGGCAAATTCCATGGCA-3´ (forward) and 5´TCTAGACGGCAGGTCAGGTCCACC-3´ (reverse); After an initial denaturation at 94°C for 5 minutes, the samples were subjected to 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 60 seconds, and extension at 72°C for 60 seconds. Human GAPDH were amplified as internal controls. The generated PCR products were separated by 1% agarose gel electrophoresis. 2.7. Monitoring Cell Spreading and Migration Cell spread and migration were monitored using the xCELLigence system (Roche Diagnostics GmbH, Basel, Switzerland). For cell spreading, E-plate 16 assemblies (Roche Diagnostics GmbH, Basel, Switzerland) were coated with ECM and seeded with cells (1.0x104 cells/well). Each plate was assembled on the RTCA DP Analyzer (Roche Diagnostics GmbH, Basel, Switzerland), and data were gathered at 5 m intervals for 20 h at 37°C in 5% CO2. The obtained data were analyzed using the provided RTCA software. For cell migration, the lower chambers of a CIM-plate 16 (8- µm pore size) were filled with fresh medium containing 10% FBS, the upper chambers were filled with serum-free medium (30 µl/well), and the plate was incubated at 37 °C in 5% CO2 for 1 h. The background was measured using an RTCA DP Analyzer (RTCA software version 1.2, ACEA Biosciences). Transfected SH-SY5Y cells (1 x104 cells/well) were added to each well, and the plate was incubated at 25 °C. After 30 min, the plate was assembled onto the RTCA DP Analyzer, and cell migration was assessed at 5-min intervals for 24 h under conditions of 37 °C and 5% CO2. The data obtained were analyzed using the provided RTCA software. 4

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2.8. Immunofluorescence analysis Cells cultured coverslips in 12-well plates were fixed with 4% paraformaldehyde for 10 min. The cells were then washed with PBS, permeabilized with 0.5% Triton X-100 in PBS for 10 min before the blocking step blocked with 0.5% BSA, and incubated overnight with FITC-conjugated phalloidin antibody (Sigma-Aldrich, St Louis, MO, USA), anti-MAP2 antibody at 4°C. After a further wash with PBS, the cells were incubated with fluorescent dye-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 1 h at 25°C. The coverslips were mounted on glass slides with mounting solution, and the results were imaged under a confocal fluorescence microscope (Carl Zeiss, Gottingen, Germany).

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2.9. Transwell migration assay

2.10. Centrifugal detachment assay

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Transwell plates (Costar; 8-µm pore size) were coated with various ECMs and then the membranes were allowed to dry at 25 °C for 1 h. The Transwell plates were assembled in a 24-well, and the lower chambers were filled with the culture medium containing 10% FBS. Cells (1 X 105) were added to each upper chamber, and the plates was incubated at 37 °C in 5 % CO2 for 4 h. The cells that had migrated to the lower surface of the filters were stained with 0.6% hematoxylin and 0.5% eosin and counted.

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2.11. Immunoblotting

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Cell substratum adhesiveness was quantified with an inverted centrifugal detachment assay. Cells were plated on each substrate and incubated for the indicated periods. After removing unattached cells, plates were filled with SFM, sealed with Parafilm and centrifuged inverted for 30 min at 2,000 g at 25 °C with a large-capacity table-top centrifuge (Hanil Science Industrial, Inchun, Korea). Before and after the centrifugation, cells on the plates were photographed with a phasecontrast microscope (Zeiss, Oberkochen, Germany) attached to a digital camera (Olympus, Tokyo, Japan) and the numbers of cells were counted. Detached cells were collected by centrifugation and counted with a haemocytometer.

Cells were lysed with RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, and 2 mM Na3VO4, pH 8.0) containing several protease inhibitors (1 g/ml aprotinin, 1 g/ml antipain, 1 mM dithiothreitol, 5 g/ml leupeptin, 1 g/ml pepstatin A, and 20 g/ml phenylmethylsulfonyl fluoride). Cell lysates were clarified by centrifugation at 13,000 rpm for 15 min at 4°C, denatured with sample buffer, boiled, and analyzed by SDS-PAGE. Proteins were transferred to nitrocellulose blotting membranes (Amersham Biosciences, Piscataway, NJ, USA) and probed with the appropriate primary antibodies and fluorescently labelled secondary anti-mouse (IRDye 800) or anti-rabit (IRDye 680) antibodies (LI-COR Biosciences). Signals were detected with an Odyssey CLx imager (LI-COR Biosciences, Lincoln, NE, USA) and analyzed using the Image Studio Lite software (LI-COR Biosciences). 2.12. Statistical analysis

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Data are presented as the means from at least three independent experiments. Statistical analysis was performed using an unpaired Student’s t test. A p-value less than 0.05 or 0.01 was considered statistically significant.

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3. Results

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3.1. Syndecan-3 forms SDS-resistant oligomerization through the transmembrane domain Previously, we reported that syndecan-2 and -4 formed SDS-resistant homo- and hetero-dimer through their TMDs [11, 12]. Accordingly, we expressed all four syndecans as His-tagged proteins and compared their SDS-resistant dimer formation ability (Fig. 1A). Wild-type syndecan-2 and -4 (SDC2 and SDC4, respectively) showed strong SDS-resistant dimer formation, whereas syndecan-1 (SDC1) showed weaker SDS-resistant dimer formation. Interestingly, in addition to SDS-resistant dimer, syndecan-3 (SDC3) showed an additional larger band than the dimer (eg, SDS-resistant oligomer), suggesting that syndecan-3 might have a unique ability to form higher order oligomer unlike the other syndecans. To exclude the effects of ectodomain, we generated deletion mutants lacking the extracellular domain, but containing TMD and cytoplasmic domain. Compared to the other syndecan mutants (1eTC, 2eTC and 4eTC), only syndecan-3 mutant (3eTC) showed SDSresistant oligomers (Fig. 1B). Furthermore, mass spectrometry confirmed that all three bands (e.g. monomer, SDS-resistant dimer and additional larger band than the dimer) were syndecan-3 (Fig. 1C), implying that syndecan-3 forms the different levels of SDS-resistant oligomer formation. Collectively, these data suggest that syndecan-3 core protein forms strong SDS-resistant oligomers in vitro. 3.2. The TMD is essential for the SDS-resistant oligomer formation of syndecan-3

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To investigate whether the cytoplasmic domain of syndecan-3 had an influence on SDS-resistant dimer/oligomer formation, we constructed and expressed cytoplasmic domain deletion mutant of syndecan-3 (3ET). Like wild-type syndecan-3, the His-tagged 3ET showed in SDS-resistant oligomer formation (Fig. 2A), suggesting that the cytoplasmic domain of syndecan-3 is not directly involved in the regulation of SDS-resistant oligomer formation.

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Because the TMDs of syndecan-2 and -4 had shown a distinct ability to form SDS-resistant dimers [11], we further investigated the role of TMD on the SDS-resistant oligomerization of syndecan-3. We constructed TMD mutants of syndecan-2 and -4 whose TMDs were substituted to syndecan-3 TMD (2E3T2C, 4E3T4C, respectively) (Fig. 2B) and/or TMD of syndecan-3 was substituted to syndecan-2 TMD (3E2T3C) (Fig. 2C). As expected, both 2E3T2C and 4E3T4C showed enhanced formation of SDS-resistant oligomer, comparable to the size of tetramer (Fig. 2B). Consistent with above results, 3E2T3C showed monomer and SDS-resistant dimer, but SDS-resistant oligomer of syndecan-3 in stacking gel was disappeared in the 3E2T3C mutant (Fig. 2C). Collectively, these data suggest that the TMD of syndecan-3 are capable of forming SDS-resistant oligomer. 3.3 The alanine 397 residue in the transmembrane domain enhances the SDS-resistant oligomer formation of syndecan-3 It has been known that the conserved GxxxG motif is crucial for SDS-resistant dimer formation of all syndecans [4, 13]. In addition, amino acid residues located nearby GxxxG motif further regulated SDS-resistant dimer/oligomer formation of each syndecans. For instance, the unique phenylalanine in the syndecan-2 TMD enhances SDS-resistant dimer formation of syndecan-2 TMD [11]. Therefore, we presumed the unique amino acid residue in the syndecan-3 TMD contributed the SDS-resistant oligomer formation of syndecan-3. There are two interesting unique amino acid residues, one is Ala397 and Thr405. Therefore, we replaced Ala397 of syndecan-3 TMD with Phe167 at the same position of 7

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syndecan-2 TMD (S3(A397F)) and Thr405 with Leu177 at the same position of syndecan-2 TMD (S3(T405L)) (Fig. 3A). Interestingly, S3(A397F) mutant decreased SDS-resistant oligomer and increased SDS-resistant dimer ratio compared to wild-type syndecan-3, but S3(T405L) showed SDSresistant oligomer formation as comparable to the wild type syndecan-3 (Fig. 3A), suggesting that Ala397 might be involved the SDS-resistant oligomer formation. On the other hand, the replacement of Phe167 in syndecan-2 TMD with Ala, which is found at the corresponding position of the syndecan-3 TMD, S2(F167A) showed SDS-resistant oligomer (Fig. 3A). Since the wild type syndecan is difficult to observe oligomer formation due to its large size, we repeated the experiment using deletion mutants lacking the extracellular domain (Fig. 3B). Consistently, 2eTC(F167A) containing alanine residue of syndecan-2 showed increased SDS-resistant oligomer formation, but 3eTC(A397F) lacking Ala397 in syndecan-3 showed lower oligomer formation compared to 3eTC (Fig. 3B). In addition, our gel filtration data revealed that syndecan-3 formed higher oligomer than S3(A397F) (Fig. 3C). Taken together, these data indicate that Ala397 of syndecan-3 regulates SDS-resistant oligomer formation via the molecular interactions of TMD.

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3.4. Oligomerization of syndecan-3 is crucial for cell adhesion and migration of SH-SY5Y cells

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Since oligomerization is crucial for syndecan functions, we then investigated whether syndecan-3 oligomerization mediated by Ala397 was involved in regulation of syndecan-3 function. Syndecan-3 predominantly expressed in the brain plays important role in neuronal development and differentiation [15], extracellular matrix (ECM) regulates differentiation of neuronal cells [16], and syndecans provide essential links to ECM for its function [17, 18]. Thus, we investigated the effect of Ala397mediated oligomerization on adhesion of SH-SY5Y neuroblastoma cells on various ECMs (Fig. 4). SH-SY5Y cells were transfected with wild-type syndecan-3 (SDC3) or its mutant S3(A397F) (Fig. 4A) and cells were placed on variety of ECM-coated E-plates to measure cell spreading using xCELLigence system. Our data revealed that overexpression of syndecan-3 enhanced cell spreading of SH-SY5Y cells on all ECMs tested, but this effect was diminished in cells overexpressing S3(A397F) (Fig. 4A). Especially, the spreading effect of syndecan-3 was most prominent on laminin 511, consistent with the previous report [19] and it was also significantly increased on collagen type I (Fig. 4A). The activity of focal adhesion kinase (FAK), a key regulator of cell spreading and migration, is known to be essential for neuronal cell adhesion and migration [20]. Consistently, overexpression syndecan-3 enhanced tyrosine phosphorylation of FAK, but S3(A397F) showed much reduced phosphorylation of FAK (Fig. 4B). Cell-detachment assay revealed that syndecan-3, but not S3(A397F), enhanced cell attachment onto collagen type I (col) and laminin511(LN511) (Fig. 4C). In addition, syndecan-3, but not S3(A397F), enhanced cell migration on collagen type I and laminin511 (Fig. 4D). Taken together, all these data suggest that the oligomerization mediated by Ala397 plays an important role in regulating cell adhesion to ECM and migration of SH-SY5Y cells. 3.5. Syndecan-3 oligomerization regulates laminin-mediated neurite outgrowth in SH-SY5Y neuroblastoma Because and cell adhesion and migration is required for neuronal cell differentiation (e.g. neurite outgrowth, [21, 22] and syndecan-3 activates Src family kinases (SFKs) leading to hippocampal neurite outgrowth and neuronal migration [23, 24], we further analyzed whether oligomerization of syndecan-3 affects dendrite formation SH-SY5Y cells (Fig. 5). SH-SY5Y cells grown on the indicated ECM were immunostained with anti-microtubule-associated protein 2 (MAP2) antibody. Similar to 8

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the results shown in Fig. 4, SH-SY5Y cells spread most on laminin 511. Interestingly, syndecan-3 transfected cells on collagen or LN511 increased dendrite length compared to S3(A397F) transfected cells (Fig. 5). Interestingly, however, syndecan-3 did not affect the number of dendrites on SH-SY5Y cells. Taken together, Ala397 of syndecan-3 TMD is a key amino acid residue that gives syndecan-3 some of its unique functions. 3.6. Alanine 397 residue enhances SDS-resistant hetero-oligomer formation of syndecan-3.

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We previously reported that syndecan-2 and -4 formed SDS resistant hetero-dimers through the GXXXG motif in their TMDs in addition to homo-dimer [12, 14]. We next invested the effect of Ala397 of syndecan-3 TMD on SDS-resistant hetero-oligomer formation (Fig. 6). Firstly, 3eTC or 3eTC(A397F) mutant was mixed with the wild type syndecan-2 (SDS2) or syndecan-4 (SDC4) and compared a degree of SDS-resistant hetero-oligomer formation (Fig. 6A). Expectedly, 3eTC clearly increased SDS-resistant hetero-oligomer formation with syndecan-2 (Fig. 6A, left) or syndecan-4 (Fig. 6A, right). Interestingly, the hetero-oligomer was predominant form of interaction between syndecan-2 and 3eTC, but not syndecan-4 and 3eTC (Fig. 6A), supporting the stronger intermolecular interaction tendency of syndecan-2 TMD than that of syndecan-4. On the other hand, 3eTC(A397F) failed to show hetero-oligomer formation with syndecan-2, or much reduced heterooligomer with syndecan-3 than 3eTC (Fig. 6A). Consistently, in contrast 2eTC, which show strong SDS-resistant dimer formation with syndecan-2, 2eTC(F167A) mediated SDS-resistant heterooligomer formation with syndecan-2 or syndecan-4 (Fig. 6B). Collectively, these data suggested that Ala397 appears to regulate hetero-oligomer formation of syndecan-3. 3.7. Hetero-oligomerization inhibits syndecan-3-mediated nerite outgrowth that depends on homo-oligomerization

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Since homo-oligomerization is crucial for syndecan-3 functions, hetero-oligomer formation might result in inhibition of syndecan-3 function. The fact that syndecan-4 reduced homo-oligomer of syndecan-3 by hetero-oligomer formation (Fig. 6A) led us to investigate whether the formation of hetero-oligomers inhibited syndecan-3-mediated neurite outgrowth in SH-SY5Y cells. Expectedly, whereas neurite outgrowth was increased in cells expressing syndecan-3, co-expression of syndecan-3 and -4 inhibited syndecan-3-mediated increase of dendrite length (Fig. 7). In particular, most cells overexpressing syndecan-3 displayed one or two long dendrites per cell, while cells overexpressing either syndecan-4 or co-expressing syndecan-3 and -4 developed several short dendrites (Fig. 7), confirming that the specific feature of oligomerization is crucial to the function of syndecan-3.

4. Discussion Although the TMDs of the other syndecans including syndecan-1, -2 and -4 are known to specifically regulates the unique function of each syndecan through dimerization/oligomerization, the importance of syndecan-3 TMD has not been directly assessed. In this study, we investigated the unique characteristic of syndecan oligomerization through the TMD domain. Our data revealed that recombinant syndecan-3 core protein formed SDS-resistant dimer and oligomer, and SDS-resistant oligomer was only observed in syndecan-3 but not other syndecans (Fig 1) and SDS-resistant oligomer formation was barely observed in the syndecan-3 mutant lacking the transmembrane 9

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domain, suggesting that syndecan-3 may have different inter-molecular interaction of TMDs for oligomerization. Since SDS-resistant oligomer of syndecan-3 was too big to enter the running gels and thus SDS-resistant oligomer was observed in the stacking gels (Fig. 1), we generated the deletion mutants of all syndecans lacking the extracellular domain. Among 4 syndecan mutants, only 3eTC showed clear SDS-resistant oligomer on the running gels, supporting the unique characteristic of syndecan-3 oligomerization. Interestingly, the presence of the Ala397 in the TMD correlated with SDSresistant oligomer formation (Fig. 2).

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Syndecans have a transmembrane domain consisted of 25 hydrophobic amino acids which are conserved within four family members and have a tendency to gather each other in a way of generating homo- or hetero dimerization through the interactions of α-helices which hold van der Waals force and weak hydrogen bond [25]. Particularly, the GxxxG motif is important for initially inducing syndecan dimerization. In addition, the polarity and the size of side chains influence helixhelix interfaces to regulate stability of their interaction. Therefore, the oligomerization of syndecan is decided by amino acids of TMD and these associations of TMD further influence cytoplasmic domain-induced signal cascades related to maintain unique cellular functions of each syndecan. Interestingly, among the unique amino acid in the syndecan-3 TMD, Ala397 is crucial for the oligomerization of syndecan-3. Indeed, the replacement of Ala397 of syndecan-3 TMD with Phe167 at the same position of syndecan-2 TMD (S3(A397F)) decreased SDS-resistant oligomer formation and the replacement of Phe167 in syndecan-2 TMD with Ala, which is found at the corresponding position of the syndecan-3 TMD, S2(F167A) induced SDS-resistant oligomer of syndecan-2 (Fig. 3), indicating that Ala397 of syndecan-3 regulates SDS-resistant oligomer formation via the molecular interactions of TMD, supporting the unique role of syndecan TMDs for strong inter-molecular interaction tendency. All these data support the critical role of Ala397 for SDS-resistant oligomer formation.

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TMD-mediated oligomerization is crucial for syndecan functions and the unique TMD endow their unique functions. For example, although the TMDs of syndecan-2 and -4 are highly homologous, the syndecan-4 TMD cannot mimic the functions of the syndecan-2 TMD, and vice versa [11, 12, 14]. Thus, proper dimerization of the TMD of a syndecan contributes to regulating cell function. Since syndecan-3 predominantly expressed in the brain, and ECM regulates differentiation of neuronal cells, we investigated the effect of syndecan-3 oligomerization on the effect of Ala397-mediated oligomerization on adhesion, migration and neurite outgrowth of SH-SY5Y cells (Figs. 4,5). Indeed, S3(A397F) significantly reduced syndecan-3 mediated cellular processes such as adhesion on ECM, migration and neurite outgrowth on laminin 511 of SH-SY5Y cells (Figs. 4,5), supporting the importance of TMD of syndecan-3 as a regulatory molecule. As we reported previously, syndecans formed SDS-resistant heterodimers in addition to the homodimer [12, 14], Ala397 of syndecan-3 TMD was involved in SDS-resistant hetero-oligomer formation (Fig. 6). Ala397-containing 3eTC clearly increased SDS-resistant hetero-oligomer formation with syndecan-2 or -4 (Fig. 6A), and 2eTC(F167A), but not 2eTC, mediated SDS-resistant heterooligomer formation with syndecan-2 or syndecan-4 (Fig. 6B), supporting that Ala397 also regulates hetero-oligomer formation of syndecans. The hetero-oligomer formation reduced homo-oligomer of syndecan-3 (Fig. 6A) and inhibited syndecan-3-mediated neurite outgrowth in SH-SY5Y cells (Fig. 7), further supporting the importance of TMD of syndecan-3 as a regulatory molecule.

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Since SDS-resistant dimer/oligomer formation is an experimental model, it is not known whether syndecan-3 actually forms higher order oligomers than other syndecans inside of cells. However, unlike other syndecans, we can predict that syndecan-3 which form SDS-resistant oligomer can easily form higher order of clustering, because it can form oligomers more easily in cells that are not affected by SDS. This could be important for the regulatory role of syndecan-3. Unlike common growth factor receptors, which primarily induce dimerization, clustering is commonly among cellECM adhesion receptors such as syndecans. We believe that oligomerization has an advantage over dimerization for syndecan-3 to act as an adhesion receptor. Since most ECM molecules are insoluble and huge in size, it is difficult to bring the molecule together in a particular location. Therefore, clustering is essential and good means for clustering a sufficient concentration of cell surface ECMs, particularly in the regulation of cell-ECM-associated cellular progresses. For this reason, we believe that oligomerization-defective mutant were less potent than wild type syndecan-3 in cell adhesion, migration and differentiation processes of SH-SY5Y cells.

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In sum, the results of this study suggest that syndecan-3 has a unique feature of higher order oligomerization in members of syndecan family, and that syndecan-3 TMDs can regulate the oligomeric status through the Ala 397 residue and its function on neuronal cells. This unique oligomerization feature of syndecan-3 TMD provides addition signaling capabilities of cell surface receptors and insights into the underlying signaling mechanisms of high-order oligomers of cell surface receptors.

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5. Conclusion

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We demonstrate that syndecan-3 has a specific feature of oligomerization by the Ala397 in the transmembrane domain, and this tendency toward oligomerization is crucial for the functions of syndecan-3. This unique oligomerization feature provides additional cell surface receptor signaling and insights into the underlying signaling mechanisms of high-order oligomers of cell surface receptors.

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2017R1A2B4008680, 2019R1A2C2009011).

Declaration of competing interest The authors declare that they have no competing interests. References [1]

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FIGURE LEGENDS Fig. 1. Syndecan-3 forms SDS-resistant oligomerization through the transmembrane. (A) Purified His-tagged syndecan core proteins were separated by SDS-PAGE on 8% gels followed by Coomassie blue staining (CB, left) or Western blotting using anti-His-tag antibody (-His). Molecular mass 13

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markers, in kilo-daltons, are shown. Migration positions of SDS-resistant syndecan oligomer (O), dimer (D) and monomer (M) of each syndecan are indicated. S.L; stacking line. (B) Schematic representation of syndecan mutants. The number indicates each syndecan family member. The transmembrane domain (T), the cytoplasmic domain (C), and four amino acid residues in the membrane flanking region (ERTE, KRTE) are shown (left). Syndecan proteins were separated by SDS-PAGE on 8% gels followed by Coomassie blue staining (CB) or Western blotting using antiHis-tag antibody (-His). (C) Mass spectrometry analysis of purified His-tagged syndecan -3 proteins from silver staining. The table reports the spectral data for the indicated proteins. Fig. 2. Transmembrane domain is essential for the SDS-resistant oligomer formation of syndecan-3. (A) Schematic representation of the syndecan-3 deletion mutants (top). Purified His-tagged syndecan proteins were separated by SDS-PAGE on 8% gels followed by Coomassie blue staining (CB) or

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Western blotting using anti-His-tag antibody (-His) (middle). The oligomer ratios were quantitated using Image Studio software (bottom). (B) Schematic representation of the syndecan-2 and -4 mutants of which transmembrane domain was replaced with syndecan-3 transmembrane domain (2E3T2C and 4E3T4C) (top). Purified His-tagged syndecan proteins were separated by SDS-PAGE on 8% gels

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followed by Coomassie blue staining (CB) or Western blotting using anti-His-tag antibody (-His) (middle). Western blotting using anti-His antibody was analyzed using Image Studio software to quantify the oligomer ratio (bottom). (C) Schematic representation of the syndecan-3 mutants of which transmembrane domain was replaced with syndecan-2 transmembrane domain (3E2T3C) (top). Purified His-tagged syndecan proteins were separated by SDS-PAGE on 8% gels followed by Coomassie blue staining (CB) or Western blotting using anti-His-tag antibody (-His) (middle). The oligomer ratios were quantitated using Image Studio software (bottom).

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Fig. 3. The Ala397 in the transmembrane domain enhances the oligomer formation of syndecan-3. (A) Amino acid sequences of the transmembrane (TM) of syndecan-2 and -3 are shown. The Ala residue at 397 and Thr reside at 405 of syndecan-3 was replaced with Phe (S3A397F) or Leu (S3T405L), respectively. The Phe residue at 167 of syndecan-2 was replaced with Ala (S2F167A) (top). Purified His-tagged syndecan proteins were separated by SDS-PAGE followed by Coomassie blue staining (CB) or Western blotting using anti-His-tag antibody (-His) (bottom). (B) Purified His-tagged syndecan core proteins (2eTC, 2eTC(F167A), 3eTC and 3eTC(A397F) were separated by SDS-PAGE on 8% gels followed by Coomassie blue staining(CB) or Western blotting using anti-His-tag antibody (-His). (C) Sephadex gel filtration chromatography of recombinant protein 3eTC, 3eTCA397F mutant are shown. Fig. 4. Oligomerization of syndecan-3 is crucial for cell adhesion and migration of SH-SY5Y neuroblastoma. (A) SH-SY5Y cells were transfected with syndecan-3 or syndecan-3 mutant constructs. After 48 h, total RNA levels were analyzed by RT-PCR. GAPDH was used as a loading control (top panel). Cells were plated on E-plate coated with the indicated ECM. Cell spreading was monitored using the xCELLigence system and analyzed using the RTCA software. The data shown are representative of three independent experiments; *, p<0.05 versus syndecan-3. (B) Cells plated on the indicated ECM for 1 h were lysed with RIPA buffer and cell lysates were analyzed by Western blotting with phospho-FAK, FAK antibody. GAPDH was used as a loading control (top). The band intensity was quantitated using Image Studio software (bottom). The data shown are representative of 14

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four independent experiments; *, p<0.05, **, 0.01. (C) Cells plated on the indicated ECM for 1h were centrifuged the inverted plates for 3 min at 2,000 g. The number of detached cells was counted with a hemocytometer and percentage of cells detached is shown. Results are means S.E.M. for three independent experiments. (D) Transwell migration assays were performed with SH-SY5Y cells transfected with the indicated cDNA using 10% FBS in the lower chamber. Cells (1 X 105) were allowed to migrate on various ECM (10 μg/ml) transwell plates for 18 hours (top). SH-SY5Y cells were transfected with 1μg of vectors encoding syndecan-3(SDC3) or the mutants syndecan-3 A397F (S3(A397F)) and plated on RTCA CIM-plates. After the indicated times, cell migration was monitored using an xCELLigence system (bottom). Representative results from three independent experiments are shown as means ± SEM (n=3); *, p<0.05, **, 0.01. versus SDC3. Fig. 5. Oligomerization of syndecan-3 is crucial for migration of SH-SY5Y neuroblastoma. SH-SY5Y

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cells (1.0 × 105 cells/well) plated on indicated ECM were incubated at 37°C for 24 h. Cells were fixed

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and immunostained with the MAP2 antibody. Scale bars represent 20μm. The dendrite length was given as the means ± SEM (n=3); *, p<0.05, **, 0.01.

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Fig. 6. Ala397 enhances SDS-resistant hetero-oligomer formation of syndecan-3. (A, B) The indicated syndecan wild type and mutants were purified and mixed for 10 min on ice, separately by 10% SDSPAGE, and followed by Coomassie blue staining (CB, left) or Western blotting using anti-His-tag antibody (-His) (H.D, hetero dimer; H.O, hetero oligomer). Molecular mass markers, in kilo-daltons, are shown (left). The hetero-oligomer ratios were quantitated using Image Studio software (right).

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Fig. 7. Hetero-oligomerization inhibits syndecan-3-mediated nerite outgrowth that depends on homooligomerization. SH-SY5Y cells (1.0 × 105 cells/well) plated on with SH-SY5Y cells transfected with

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the indicated cDNA were incubated at 37°C for 24 h. Total RNA levels were analyzed by RT-PCR.

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GAPDH was used as a loading control (top panel). Cells were fixed and immunostained with the MAP2 antibody. Scale bars represent 20 μm (middle panel). The dendrite length was given as the means ± SEM (n=15, bottom panel); *, p<0.05, **, 0.01.

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Credit Author Statement Hyejung Jung, Minji Han, Bohee Jang, Eok-Soo Oh: Conceived and coordinated the study and wrote the paper Hyejung Jung, Minji Han, Bohee Jang, , Eunhye Park, Eok-Soo Oh: Performed the experiments and analyzed the data

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All authors reviewed the results and approved the final version of the manuscript

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The oligomerization mediated by the alanine 397 residue in the transmembrane domain is crucial to sydecan-3 functions Hyejung Jung1,# , Minji Han2,#, Bohee Jang2, Eunhye Park3, Eok-Soo Oh1,2,* From the 1Skin QC Institute of Dermatological Sciences, Seoul 03759, Korea; 2Department of Life Sciences, Ewha Womans University, Seoul 03760, Korea #

Both authors contributed equally to this work.

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To whom correspondence may be addressed: Eok-Soo Oh, Department of Life Sciences, Ewha Womans University, 52, Ewhayeodae-gil, Seodaemoon-Gu, Seoul 03760, Korea, Tel.: (82)-2-32773761; Fax: (82)-2-3277-3760; E-mail: [email protected]

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Highlights

Recombinant syndecan-3 protein forms SDS-resistant dimers and oligomers

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Alanine 397 residue in the transmembrane domain mediates the oligomer formation of syndecan-3.

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The oligomerization tendency is crucial for the function of syndecan-3

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