Purification and characterization of the extracellular region of human receptor tyrosine kinase like orphan receptor 2 (ROR2)

Accepted Manuscript Purification and characterization of the extracellular region of human receptor tyrosine kinase like orphan receptor 2 (ROR2) Yuan Li, Xu Han, Wenqing Xu, Zihe Rao, Xin Li PII:

S1046-5928(19)30039-7

DOI:

https://doi.org/10.1016/j.pep.2019.02.015

Reference:

YPREP 5396

To appear in:

Protein Expression and Purification

Received Date: 17 January 2019 Revised Date:

25 February 2019

Accepted Date: 25 February 2019

Please cite this article as: Y. Li, X. Han, W. Xu, Z. Rao, X. Li, Purification and characterization of the extracellular region of human receptor tyrosine kinase like orphan receptor 2 (ROR2), Protein Expression and Purification (2019), doi: https://doi.org/10.1016/j.pep.2019.02.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Purification and characterization of the extracellular region of human receptor tyrosine kinase like orphan receptor 2 (ROR2)

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Yuan Li a, Xu Han a, Wenqing Xu b*, Zihe Rao a*, Xin Li a*

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a. Collage of Life Sciences, Nankai University, Tianjin 300071, China

* Correspondence: Xin Li: [email protected] Wenqing Xu: [email protected]

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Zihe Rao: [email protected]

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b. Department of Biological Structure, University of Washington, Seattle, WA 98195, USA

ACCEPTED MANUSCRIPT Abstract Receptor tyrosine kinase like orphan receptor 2 (ROR2) is a co-receptor for some Wnt proteins including Wnt5a that activate the noncanonical Wnt/planar cell polarity (PCP) signaling pathway. Upregulation of ROR2 is associated with several cancer forms. The extracellular region of ROR2,

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which contains an immunoglobulin (Ig)-like domain, a Frizzled like cysteine-rich domain (CRD) and a Kringle domain, is a potential anticancer drug target. The structural and biochemical properties of the ROR2 extracellular region remain largely unexplored. Here we describe the mapping and purification, using a baculovirus - insect cell system, of a near-full-length ROR2 extracellular

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fragment (residues 53-402), which is well-behaved and suitable for future structural and biochemical analysis. We show that the extracellular region of ROR2 per se is monomeric in solution. Different

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monoclonal antibodies raised against the purified ROR2 protein can specifically recognize the protein and can either inhibit or activate the PCP activity in a cell-based assay, and are thus potentially useful for future mechanistic and therapeutic/diagnostic studies. The biological relevance of these antibodies further demonstrates that the purified recombinant ROR2 protein is properly

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folded and biochemically active.

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Key words: ROR2; Deglycosylation; Monoclonal antibody; MMP-13

ACCEPTED MANUSCRIPT 1. Introduction Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is an essential co-receptor for Wnt5a and Wnt11 involved in the non-canonical Wnt/planar cell polarity (PCP) signaling pathway [1-3]. As an important regulatory protein, ROR2 is expressed in a variety of tissues during early embryonic

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development [4, 5], and plays a key role in skeletal, neurological and midgut development [6]. In adult tissues the expression of ROR2 is repressed and limited to parathyroid, testicular and uterine tissues [4, 7]. In contrast, ROR2 was reported to be up-regulated in numerous types of malignant tumor, including breast cancer [8], liver cancer [9], Renal cancer[10], gastric cancer [11], rectal

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carcinoma [12], oral cancer [13] and pancreatic cancer [14]. Furthermore, the increased ROR2 expression is associated with advanced clinical stage and poor prognosis in patients [15, 16]. As a

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result, ROR2 may be a tumor cell biomarker and a therapeutic target for cancer treatment.

As a member of the receptor tyrosine kinases (RTKs) superfamily, ROR2 is a single-pass transmembrane protein. The cytoplasmic region of ROR2 contains a putative tyrosine kinase (TK) domain and a proline-rich domain (PR) flanked by two serine/threonine (ST) rich domains [17]. The

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extracellular region of ROR2 contains an immunoglobulin (Ig)-like domain, a Frizzled like cysteine-rich domain (CRD) and a Kringle domain [17]. Among them, the CRD domain was demonstrated to be an essential binding site of Wnt5a [1], while the Ig domain and Kringle domain

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were both considered to mediate some protein-protein interaction and may modulate the function of the CRD domain (Fig 1A). Detailed structure-function relationships of the three extracellular

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domains await further investigations.

Unlike the canonical RTKs that form dimers or oligomers with other RTK molecules during signaling, ROR2 acts as a co-receptor that forms a receptor complex with the Frizzled (Fzd) family receptors, which belongs to the seven-transmembrane G protein-coupled receptor (GPCR) superfamily to facilitate the Wnt5a signaling [18]. Matrix metalloproteinases (MMPs) form a family of enzymes that remodel tissues in both normal tissue development and cancer progression by degrading the extracellular matrix. The Wnt5a-ROR2 signaling was reported to regulate the expression of various MMPs through a Wnt5a/JNK signaling pathway (Fig 1A) [1, 2, 10, 19]. Specifically, the expression of MMP-13 was identified as a downstream readout of Wnt5a-ROR2

ACCEPTED MANUSCRIPT signaling in human osteosarcoma cell lines which constitutively express both ROR2 and Wnt5a [19, 20]. Repressing the expression of either ROR2 or Wnt5a also down-regulates the expression of MMP-13 and reduces the invasiveness of the osteosarcoma cells [19, 20].

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In this study, we expressed and purified four extracellular regions of ROR2 using the baculovirus insect cell expression system, which led us to a well-behaved ROR2 extracellular fragment

containing all three Ig, CRD and Kringle domains. Using this highly purified ROR2 extracellular fragment, we generated and isolated eleven variant mouse monoclonal antibodies. Out of these

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eleven antibodies, two were found to be more capable of inhibiting or irritating the Wnt5a-ROR2 signaling pathway in the osteosarcoma cell line SaOS-2. The monoclonal antibodies and methods for

and potentially therapeutic studies.

2. Materials and methods 2.1 Materials and cell culture

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expression and purification of ROR2 may further contribute to ROR2-related structural, functional

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The cDNA encoding the full-length ror2 gene was a generous gift from Dr. Jiahuai Han’s laboratory at the Xiamen University. Commercial mouse monoclonal antibody against human ROR2 was purchased from Abcam (catalogue No. ab90120). Spodoptera frugiperda 9 (Sf9) cells from

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Invitrogen were cultured at 28°C using the SFX-Insect medium (Hyclone) without serum. A human osteosarcoma cell line, SaOS-2 (BNCC catalogue #338486), was cultured in McCoy’s 5A medium (BI) supplemented with 10% Fetal Bovine Serum (FBS, Hyclone) at 37°C with 5% CO2. For Sf9

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cells, cellfectin® II reagent (Life Technologies) was used for transfection experiments.

2.2 Plasmid construction

The DNA fragments for ROR2 overexpression (encoding ROR2 protein fragments 34-402, 53-402, 77-402 and 173-402, respectively) were PCR amplified from the full-length ROR2 cDNA using the forward primers ROR2-34F (ATAGAATTCGAGAACCTGTACTTCCAGGGAGGCTCTGGAGAAGTGGAGGTTCTGGATC C), ROR2-53F (ATAGAATTCGAGAACCTGTACTTCCAGGGAGGCTCTGGACCGATTCCAACTCTGAAAG

ACCEPTED MANUSCRIPT G), ROR2-77F (ATAGAATTCGAGAACCTGTACTTCCAGGGAGGCTCTGGACAGACGGCAATTCTGCACT G) and ROR2-173F (ATAGAATTCGAGAACCTGTACTTCCAGGGAGGCTCTGGATTCTGCCAGCCTTACCGGGG

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AATTG), respectively, and the reverse primer ROR2-402R (CTACTCGAGTTACATCTTGCTGCTGTCTCGGGGACTAC). A Tobacco etch virus (TEV) protease cleavage site encoding sequence (GAGAACCTGTACTTCCAGGGAGGCTCTGGA) is included in all the four forward primers between the His6 tag and ROR2. The TEV-ROR2 DNA

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fragments were then double digested with the EcoRI and XhoI restriction enzymes (ThermoFisher Scientific), and cloned into the secretory expression transfer vector pFastBacTM-2 (Life Technologies)

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which contains an HB2K signal peptide and a His6 tag before the MCS. Recombinant bacmids were generated following the Bac-to-Bac Baculovirus expression system manual (Life Technologies). In brief, the pFastBacTM-2-His-TEV-ROR2 plasmids were transformed into the DH10BacTM Escherichia coli (Life Technologies). Recombinant bacmids with transposition were identified using the blue/white colony screening method, and the resulting ROR2-containing bacmids were verified

2.3 Baculovirus production

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by PCR.

To produce recombinant baculoviruses for expression of the four different ROR2 extracellular

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fragments, logarithmic phase Sf9 cells with density of about 1.5 × 106 ml-1 in 6-wells tissue culture plate were transfected with each recombinant bacmid DNA, using the cellfectin II reagent following

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manufacturer’s instructions. After 72 hours incubation at 28°C, cell culture supernatants containing recombinant baculoviruses (P1) were collected and transferred into 15 cm plates with 1.5 × 106 ml-1 Sf9 cells and then cultured for another 72 hours at 28°C to generate the P2 viruses. P3 viruses with higher titers were obtained by amplifying the P2 viruses in 50ml shaking flasks, using the same cell density and culture time as for the P2 virus generation. After removing cell debris by centrifuging at 120 g for 5 min, 50 mL P3 baculoviruses for expressing each of the four ROR2 protein fragments were harvested and stored at 4°C.

2.4 Protein expression and purification

ACCEPTED MANUSCRIPT For recombinant ROR2 protein production, each P3 virus was mixed with 1 × 106 ml-1 mid-logarithmic phase Sf9 cells at 1:20 (v/v). The infected cells were then cultured at 28°C in 1 liter shaking flasks with orbital rotation at 121 rpm for 72 hours. The cell culture supernatants containing secreted His6-tagged ROR2 proteins were harvested by centrifugation at 2,600 g for 20 min. To

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concentrate ROR2 proteins and remove undesired cell culture ingredients, ROR2-containing supernatants were filtered through a 0.45µm membrane (Millipore), and buffer exchanged against a MES buffer (150 mM NaCl, 50 mM MES pH6.0) using a Vivaflow 200 crossflow cassette (Sartorius,

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10,000 MWCO Hydrosart).

The concentrated His6-tagged ROR2 proteins were applied to 10 ml of Ni-NTA sepharose beads (GE

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healthcare) pre-equilibrated with MES buffer. The beads were then washed with 100 ml washing buffer (150 mM NaCl, 30 mM imidazole, 50 mM MES pH6.0). His6-tagged ROR2 proteins were eluted with an elution buffer (150 mM NaCl, 300 mM imidazole, 50 mM MES pH6.0). His6-tagged TEV protease and His6-tagged endoglycosidases (expressed and purified in our laboratory) were then added to remove the His6-tag and polysaccharide chains of ROR2. The mixtures were dialyzed

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against the MES buffer (150 mM NaCl, 50 mM MES pH6.0) overnight at 4°C. To remove the His6-tagged enzymes and the His6-tag cleaved from ROR2, the dialyzed samples were centrifuged at 14,000 g for 10 min at 4°C and applied to a 5ml His-Trap HP column (GE, Healthcare). After

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washing the column with the MES buffer for 5 column volumes (CVs), bound proteins were eluted with a linear gradient of 0-1000 mM imidazole in the MES buffer and the ROR2 protein fractions

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were collected.

The collected ROR2 proteins were buffer changed to 25 mM NaCl, 25 mM Tris pH8.5 using a 10 kDa Amicon Ultra (Millipore) centrifugal filter and applied to a 1ml Hi-trap Resource Q anion exchange column (GE, Healthcare). The ROR2 proteins were then eluted with a 25-1000 mM NaCl gradient. The ROR2 peak frations were concentrated and further purified by size-exclusion chromatography (SEC) using a Superdex 200 10/300 GL column (GE, Healthcare), in a Tris buffer (150 mM NaCl, 25 mM Tris-HCl, pH8.5). The purified ROR2 proteins were concentrated to ~6 mg/ml and frozen with liquid nitrogen for further characterization. Protein samples in the purification procedure were analyzed by SDS-PAGE and western blot.

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2.5 Anti-ROR2 antibodies production The 11 Anti-ROR2 mouse monoclonal antibody variants (#1-#11) were produced at the Sino

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Biological Inc., using our purified deglycosylated and tag-free ROR2(53-402) protein as the antigen.

2.6 Immunoprecipitation assay

Purified ROR2(53-402) protein was mixed for 1h at 4°C with the 11 Anti-ROR2 antibody variants, respectively, at a 1:1 molar ratio, in a Tris buffer (150 mM NaCl, 25 mM Tris-HCl, pH8.5). The

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mixtures were then applied to the Protein A Sepharose beads (GE, Healthcare) and incubated for 2 hours at 4°C with gentle shaking. Unbound proteins were removed by washing with the buffer (150

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mM NaCl, 25 mM Tris-HCl, pH8.5, 0.1% CA630) for 5 times, and bound proteins were eluted using the SDS loading buffer and subjected to SDS-PAGE and stained with Coomassie Brilliant Blue.

2.7 Anti-ROR2 antibodies related MMP-13 activity measurement

About 1.0 × 105 ml-1 SaOS-2 cells in McCoy’s 5A medium (BI) without FBS were seeded into each

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well of a 48-wells tissue culture plate. Anti-ROR2 monoclonal antibody variants (#1-#11, except #6) or PBS (control) were immediately added to the wells, respectively. Eight hours later, the supernatants of each well were collected by centrifugation at 120 g for 10 min and transferred to

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wells of a 96-well black flat bottom microplate (Corning) for MMP-13 activity assay. The MMP-13 fluorescence substrate (MCA-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2, from Millipore) was added to each well (at a final concentration of 5 µM). With excitation at 325nM, fluorescence emission at

BioTek).

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393nM was measured at 120 minutes at 25°C, using a fluorescent microplate reader (CYTATION 5,

3. Results and discussion

3.1 Construction of ROR2 expression vectors As a potential therapeutic target, we focused on the extracellular N-terminal region of the single transmembrane protein ROR2. This region is composed of an Ig, a CRD and a kringle domain, and contains multiple disulfide bonds and three predicted glycosylation sites located in each of the three domains, respectively. We used the Bac-to-Bac baculovirus - insect cell expression system and a

ACCEPTED MANUSCRIPT secretory expression vector pFastBacTM-2 to generate properly folded ROR2 extracellular fragments. ROR2 possesses a signal peptide at its N-terminal region (residue 1-33) as predicted by the online Signal 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/). Because signal peptides vary in different organisms, for secretory expression of the ROR2 extracellular region, we replaced the

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ROR2 native signal peptide with the HB2K signal peptide included in the pFastBacTM-2 vector (Fig 1B). The ROR2(34-402) fragment corresponds to the full-length extracellular region. The slightly shorter ROR2 (53-402) fragment leaves out the more variable N-terminal segment (19 residues), and starts at a conserved proline residue. ROR2(77-402) starts at the first residue of the predicted Ig

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domain, and ROR2(173-402) consists of the CRD and kringle domains only (Fig 1B). The

pFastBacTM-2 plasmids integrated DNA sequences of these ROR2 fragments in frame were

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transferred into DH10BacTM E. coli cells, where the ROR2 coding sequences were transposed to the bacmids (Fig 1B). The resultant recombinant bacmids were used to produce baculoviruses for ROR2 protein production as described in Materials and methods.

3.2 Purification and deglycosylation of ROR2 protein fragments

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All of the four ROR2 extracellular fragments were expressed in Sf9 insect cells as described in Materials and methods. After Ni-NTA affinity chromatography purification, to confirm the identity of His6-TEV-ROR2 fragments, these ROR2 fragments were subjected to TEV protease treatment.

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ROR2 protein samples with or without His6-tag cleavage were analyzed by SDS-PAGE. Although a recent phage display study has reported the expression of full-length ROR2 extracellular domain with HEK293F cells [21], in our case, the complete ROR2 extracellular fragment (residues 34-402;

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theoretically about 44 kDa without His6-tag and glycosylation), is hardly detectable (Fig. 2A). Because the previous report did not include any protein purification data, such as ROR2 purity and behavior, we were not able to further compare our data with this previous study. Excluding His6-tag and glycosylation, the other three ROR2 fragments (residues 53-402, 77-402 and 173-402) have nominal molecular weights of about 42 kDa, 39 kDa and 28 kDa, respectively. Taking glycosylation into consideration, bands with proper molecular weights for ROR2 fragments 53-402 and 173-402 were observed (Fig. 2A). Western blot using a commercial anti-ROR2 antibody confirmed that ROR2 fragments 53-402 and 173-402 can be overexpressed in decent yield and behave well in solution (Fig. 2A). The protein bands with sizes comparable with ROR2(77-402) on the Coomassie

ACCEPTED MANUSCRIPT Brilliant Blue-stained SDS-PAGE gel were not detected by the antibody, indicating that these are contaminating proteins. Indeed, unlike ROR2 (53-402) and ROR2 (173-402), these bands could not be digested by TEV protease (Fig. 2A). Since ROR2(53-402) contains all three extracellular domains and only leaves out a short N-terminal variable region, we focused on ROR2(53-402) for further

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characterization and follow-up experiments.

As indicated in Uniprot (https://www.uniprot.org/uniprot/Q01974), the extracellular region of human ROR2 can be glycosylated at residues Asn70, Asn188 and Asn318, respectively (Fig. 1B). Since

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insect cell-derived glycosylation is different from that in human cells, we intended to remove the glycosyl chains from the purified ROR2 fragment. Endoglycosidase F1 (Endo F1), endoglycosidase

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F3 (Endo F3) and endoglycosidase H (Endo H) or their combinations were employed to digest the glycosyl chains. As shown in Fig. 2B, treatment of Endo F3 or endoglycosidase combinations including Endo F3, but not Endo F1, Endo H or their combination, can reduce the size of the ROR2 fragment, indicating that only Endo F3 is capable of trimming the glycosyl chains of the ROR2 fragment. Endo F3 treatment also compressed the ROR2 fragment double bands in SDS-PAGE gel

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into a single lower band (Fig. 2A and B), suggesting a chemically more uniform ROR2(53-402) protein. Thus we treated the Ni-NTA-purified ROR2(53-402) fragment with both TEV protease and

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Endo F3 to remove the His6-tag and glycosyl chains (Fig. 2C) for further purification and analysis.

3.3 Further purification and characterization of the ROR2(53-402) fragment Both the TEV protease and Endo F3 we used are His6-tagged and supposedly removable by an

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additional Ni-NTA purification step. However, the His6-tag free ROR2 fragment also bound to the Ni-NTA affinity beads, likely due to some consecutive histidine residues in the ROR2 fragment (such as His344, His346, His348 and His349). We thus carefully purified the ROR2(53-402) fragment using a His6-trap HP affinity column and a linear gradient of 0-1000mM imidazole. In this way, as shown in Fig 3A, the untagged ROR2 fragment can be purified in a peak apart from the His6-tagged TEV and Endo F3 peaks.

The pooled ROR2(53-402) fragment was buffer changed to the 25 mM NaCl buffer. Then a standard ion-exchange chromatography with a Hi-trap Resource Q column and a subsequent size-exclusion

ACCEPTED MANUSCRIPT chromatography (SEC) with a Superdex 200 10/300 GL column were performed. ROR2(53-402) showed as single and sharp peaks in both anion-exchange and SEC. Importantly, our SEC data show that ROR2(53-402) migrates predominantly as a monomer (Fig. 3B and C). The protein purity was validated by SDS-PAGE analyses (Fig. 3B and C). The total yield of the purified ROR2(53-402)

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protein was ~2 mg per liter supernatant of cultured cells.

We had intended to crystallize the purified ROR2 extracellular region for further structural studies. Although the highly purified ROR2(53-402) demonstrated a monodispersive peak with the

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monomeric size in SEC, the ROR2(53-402) fragment did not yield a usable crystal after extensive screening. It is possible that the junction regions between Ig, CRD and Kringle domains of ROR2 are

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flexible to some extent, and the entire extracellular region of ROR2 may lack a rigid conformation in the absence of its ligand.

3.4 Characterization of anti-ROR2(53-402) monoclonal antibodies

The purified and deglycosylated ROR2(53-402) extracellular fragment was used for generating

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mouse antibodies at Sino Biological Inc. and eleven variants of anti-ROR2(53-402) monoclonal antibodies were finally isolated. To validate these antibody variants, each monoclonal antibody was used to immune-precipitate the purified ROR2(53-402) fragment. As shown in Fig. 4A (upper panel),

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nine of the eleven antibody variants specifically interacted with ROR2(53-402). For the two exceptions, the #5 variant showed no ROR2(53-402) binding activity, while the #6 variant had a poor solubility by itself although it also recognized ROR2(53-402). We then performed Western blot

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experiments with ten of these eleven antibody variants (except the #6 variant) to examine whether these antibodies recognize linear epitopes of purified ROR2(53-402). Comparing with the commercial antibody which was raised against an ROR2 peptide, the #3, #4, #8, #9, #10 and #11 variants showed almost identical binding affinity to ROR2(53-402), while the #1, #2 and #7 variants exhibited significant decreased ROR2(53-402) affinity, especially the #7 variant. Interestingly, the #5 variant, which did not bind with ROR2(53-402) in the immunoprecipitation experiment, most strongly interacted with denatured ROR2 (Fig. 4A, lower panel). These data indicated that some of our antibody variants prefer to bind with folded ROR2 protein (especially the #2 and #7 variants), while some more favor ROR2 linear epitope (the #5 variant).

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The previous study on ROR2 antibodies has mainly focused on ROR2 targeting [21], while we are more interested in whether our monoclonal antibody variants are biologically functional. Consequently, we examined the PCP/c-Jun activity of the ten anti-ROR2(53-402) variants (except #6)

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in the SaOS-2 cell line. The human osteosarcoma cell line constitutively expresses Wnt5a and ROR2 and induces high level secretory expression of MMP-13 [19, 20] (Fig. 1A). Therefore, whether a ROR2 monoclonal antibody variant can inhibit or enhance the Wnt5a activity can be monitored by measuring the MMP-13 activity in cell culture media. A quenched fluorescence substrate

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(MCA-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2) specifically recognized by MMP-13 was used for the assay and the fluorescence signal was continuously measured [22]. As shown in Fig. 4B, the effects

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of the monoclonal antibodies on Wnt5a-ROR2 signaling varied from inhibition to stimulation. Among them, the #11 variant exhibited the highest enhancing activity, while the #4 variant demonstrated the most significant inhibition. To verify the biological function of these two antibodies, we performed antibody dose-dependent MMP-13 activity assays. As shown in Fig. 4C and D, the #4 and #11 variants each showed inhibition or augmentation activity, respectively, in a

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dose-dependent manner, validating the biological relevance of these two monoclonal antibodies. Since ROR2 is a potential cancer therapeutic target [23, 24], these monoclonal antibodies may be useful for cancer immunotherapy after appropriate humanization [21, 25]. Humanized ROR2

4. Conclusion

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monoclonal antibodies can also be used for targeting drugs to cancer cells.

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The extracellular region of ROR2 is a potential target for cancer treatment. Here we provide an optimized protocol for expressing and purifying the extracellular region of ROR2. To obtain well-folded protein, we tried four different constructs using the Bac-to-Bac baculovirus - insect cell expression system. The ROR2(53-402) fragment behaved best among them, with excellent expression/purification yield and solubility. Since the very N-terminal 33 residues of ROR2 is the predicted signaling peptide, ROR2(53-402) covers all conserved domains and could be counted as a near-full-length ROR2 extracellular region. Monoclonal antibody variants generated against purified ROR2(53-402) extracellular fragment exhibited biological function in a cell-based assay, indicating that the ROR2 extracellular fragment we purified was properly folded and suitable for further

ACCEPTED MANUSCRIPT biomedical studies. The inhibitory monoclonal antibody might interact with ROR2 and block Wnt5a binding, while stimulatory antibodies might directly stimulate ROR2 or enhance the Wnt5a binding. Nonetheless, these antibodies provide a tool set that may be useful for both mechanistic and

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therapeutic studies.

Acknowledgement

This work was supported by grants from the National Key Research and Development Program of China (Grant No. 15 2017YFC0840300), the Strategic Priority Research Program of the Chinese

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Academy of Sciences (Grant No. XDB08020200), the State Key Development Program for Basic Research of the Ministry of Science and Technology of China (973 Project Grant Nos.

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2014CB542800, 2014CBA02003 to ZR and 2014CB910700 to FS), and the National Natural

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Science Foundation of China (Grant Nos. 813300237 and 81520108019).

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Wnt-mediated, pro-tumourigenic role in colon cancer. Molecular Cancer 9 (2010) 170. [13] M. Kobayashi, Y. Shibuya, J. Takeuchi, M. Murata, H. Suzuki, S. Yokoo, M. Umeda, Y. Minami, T. Komori, Ror2 expression in squamous cell carcinoma and epithelial dysplasia of the oral cavity. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics 107 (2009) 398-406. [14] D. Zhu, Z. Yang, Z. Liu, Q. Zou, Y. Yuan, C. Hu, Association between Wnt inhibitory factor 1 and receptor tyrosine kinase-like orphan receptor 2 protein expression and the clinical pathological significance in benign and malignant pancreatic lesions. Oncology Letters 13 (2017) 2244-2252. [15] B.J. Lu, Y.Q. Wang, X.J. Wei, L.Q. Rong, D. Wei, C.M. Yan, D.J. Wang, J.Y. Sun, Expression of WNT-5a and ROR2 correlates with disease severity in osteosarcoma. Molecular Medicine Reports 5 (2012) 1033-1036. [16] M.P. O'Connell, J.L. Fiori, M. Xu, A.D. Carter, B.P. Frank, T.C. Camilli, A.D. French, S.K. Dissanayake, F.E. Indig, M. Bernier, D.D. Taub, S.M. Hewitt, A.T. Weeraratna, The orphan tyrosine kinase receptor, ROR2, mediates Wnt5A signaling in metastatic melanoma. Oncogene 29 (2010) 34-44. [17] P. Masiakowski, R.D. Carroll, A novel family of cell surface receptors with tyrosine kinase-like domain. The Journal

ACCEPTED MANUSCRIPT of Biological Chemistry 267 (1992) 26181-26190. [18] M. Nishita, S. Itsukushima, A. Nomachi, M. Endo, Z. Wang, D. Inaba, S. Qiao, S. Takada, A. Kikuchi, Y. Minami, Ror2/Frizzled complex mediates Wnt5a-induced AP-1 activation by regulating Dishevelled polymerization. Molecular and Cellular Biology 30 (2010) 3610-3619. [19] M. Enomoto, S. Hayakawa, S. Itsukushima, D.Y. Ren, M. Matsuo, K. Tamada, C. Oneyama, M. Okada, T. Takumi, M. Nishita, Y. Minami, Autonomous regulation of osteosarcoma cell invasiveness by Wnt5a/Ror2 signaling. Oncogene 28 (2009) 3197-3208.

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[20] K. Yamagata, X. Li, S. Ikegaki, C. Oneyama, M. Okada, M. Nishita, Y. Minami, Dissection of Wnt5a-Ror2 signaling leading to matrix metalloproteinase (MMP-13) expression. The Journal of Biological Chemistry 287 (2012) 1588-1599. [21] H. Peng, T. Nerreter, J. Chang, J. Qi, X. Li, P. Karunadharma, G.J. Martinez, M. Fallahi, J. Soden, J. Freeth, R.R. Beerli, U. Grawunder, M. Hudecek, C. Rader, Mining naive rabbit antibody repertoires by phage display for monoclonal antibodies of therapeutic utility. Journal of Molecular Biology 429 (2017) 2954-2973. The Journal of Biological Chemistry 271 (1996) 1544-1550.

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[22] V. Knauper, C. Lopez-Otin, B. Smith, G. Knight, G. Murphy, Biochemical characterization of human collagenase-3. [23] Z. Debebe, W.K. Rathmell, Ror2 as a therapeutic target in cancer. Pharmacology & Therapeutics 150 (2015) 143-148.

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Cytotherapy 18 (2016) 1393-1409.

ACCEPTED MANUSCRIPT Figure legends

Fig 1. Domain structure of ROR2 and the scheme for ROR2 extracellular domain overexpression. (A) Among several downstream events, the interaction between Wnt5a and ROR2 induces expression of

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the MMP-13 which is secreted to the extracellular matrix through signal transmission by c-Src, Dvl, Rac, JNK and c-Jun/ATF-2. IG: Immunoglobulin-like domain; CRD: Cysteine-rich domain; KD: kringle domain; TKD: tyrosine kinase domain; PRD: Proline-rich domain; MMP-13: matrix

metalloproteinase-13. (B) Schematic representation for construction of expression vectors of the

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ROR2 extracellular domain fragments. SP: signal peptide; TM: transmembrane; Transposition region of recombinant bacmid occurs between the mini-Tn7 element on the pFastBacTM-2 secretory

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expression vector and mini-attTn7 target site on the bacmid. All constructions were confirmed by PCR.

Fig 2. Secretary overexpression and affinity-purification of four different ROR2 extracellular fragments. (A) SDS-PAGE and Wester blotting analysis of four ROR2 extracellular fragments, and

ROR2(53-402) protein. Endo F1: endoglycosidase H.

endoglycosidase F1; Endo F3: endoglycosidase F3; Endo H:

(C) Digestion of His6-tagged ROR2(53-402) protein by TEV protease and

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Endo F3.

Deglycosylation of purified His6-tagged

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their confirmative digestion by TEV protease. (B)

Fig 3. Further purification of ROR2(53-402). (A) Removal of His6-tagged TEV protease and

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His6-tagged Endo F3 by His-trap HP affinity column, using an imidazole gradient. (Left) Chromatogram; (Right) SDS-PAGE analysis of peak a (ROR2) and peak b (contaminants). Lane M: Page RulerTM Prestained Protein Ladder; Lane R’: digested ROR2(53-402) with TEV protease and Endo F3; Lane 1-25: fraction samples. (B) Anion exchange purification of the ROR2(53-402) protein. (Left) Chromatogram; (Right) SDS-PAGE analysis of peak c. Lane M: protein MW marker; Lane R: pooled ROR2(53-402) protein from peak a; Lane pre: protein precipitation from procedure of lower salt density; Lane 1-12: fraction samples. (C) Final purification of the ROR2(53-402) protein using size-exclusion chromatograph (SEC). Migration positions of standard proteins are marked.

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Fig 4. Characterization of anti-ROR2(53-402) monoclonal antibodies. (A) Immunoprecipitation (upper panel) and Western blot (lower panel) of ROR2(53-402) with our anti-ROR2(53-402) antibodies. For the immunoprecipitation the result was analyzed using reducing SDS-PAGE gels

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stained with Coomassie Brilliant Blue, in which the antibodies were denatured to heavy chain and light chain (upper panel). For the Western blot (lower panel), the commercial antibody (Ctl) and our antibody variants were all diluted to a same concentration (0.5 µg/ml) to detect the purified

ROR2(53-402). (B) Histogram illustration of analysis of antibody-dependent MMP-13 activities

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after SAOS-2 cells were incubated with antibodies #1-#11 (except #6, 50 µg/ml) for 8 hours. The antibody-modulated MMP-13 activity was detected by fluorescence reader at 120 min. The

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fluorescence signals in the well without adding any antibody was used as the base line. (C, D) Histogram illustration of analysis of antibody-dependent MMP-13 activities after incubating with #4 or #11 antibody with different doses (from 0 µg/ml to 100 µg/ml) for 8 hours, detected by

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fluorescence reader at the 120 min time point.

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ACCEPTED MANUSCRIPT Highlights A well-behaved, near-full-length ROR2 extracellular fragment (residues 53-402) was overexpressed using an insect cell secretory expression system.

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The purification and deglycosylation procedures for ROR2(53-402) were optimized. Our SEC analysis demonstrates that ROR2(53-402) is monomeric in the absence of ligand.

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Monoclonal antibodies raised against the purified ROR2(53-402) fragment can specifically recognize the ROR2 protein, and can either inhibit or activate the PCP activity in a cell-based assay. These antibodies can be useful for future mechanistic and biomedical studies.