Generation of an Antibody against the Protein Phosphatase 1 Inhibitor ...

ANTICANCER RESEARCH 30: 1573-1578 (2010)

Generation of an Antibody against the Protein Phosphatase 1 Inhibitor KEPI and Characterization of the Epitope KATJANA DASKALOW1,2*, PRISCA BOISGUERIN3, BURKHARD JANDRIG2, FRANK K.H. VAN LANDEGHEM5, RUDOLF VOLKMER3, BURKHARD MICHEEL1 and JÖRG A. SCHENK1,4 1Institute

of Biochemistry and Biology, University of Potsdam, Golm, Germany; for Molecular Medicine (MDC), Berlin, Germany; 3Institute of Medical Immunology, Charité - Universitätsmedizin, Berlin, Germany; 4UP Transfer GmbH, Hybrotec, Potsdam, Germany; 5Institute of Neuropathology, University of Bonn Medical Center, Bonn, Germany 2Max-Delbrück-Center

Abstract. A monoclonal antibody against the potential tumor suppressor kinase-enhanced protein phosphatase 1 (PP1) inhibitor KEPI (PPP1R14C) was generated and characterized. Human KEPI was expressed in Escherichia coli and used to immunize Balb/c mice. Using hybridoma technology, one clone, G18AF8, was isolated producing antibodies which bound specifically to the KEPI protein in ELISA, immunoblotting and flow cytometry. The antibody was also successfully applied to stain KEPI protein in paraffin sections of human brain. The epitope was mapped using peptide array technology and confirmed as GARVFFQSPR. This corresponds to the N-terminal region of KEPI. Amino acid substitution analysis revealed that two residues, F and Q, are essential for binding. Affinity of binding was determined by competitive ELISA as 1 μM. In Western blot assays testing G18AF8 antibody on brain samples of several species, reactivity with hamster, rat and chicken samples was found, suggesting a broad homology of this KEPI epitope in vertebrates. This antibody could be used in expression studies at the protein level e.g. in tumor tissues. The human kinase-enhanced protein phosphatase 1 (PP1) inhibitor KEPI, also known as protein phosphatase 1 regulatory subunit 14C (PPP1R14C) is expressed in brain,

heart and skeletal muscle (1). The gene of human KEPI consists of four exons and is located on chromosome 6q24q25. KEPI expression is regulated by morphine, and phosphorylated KEPI inhibits PP1, which modulates the physiological activities of several other proteins (1). Recently, a deletion in this region in a patient was found to cause growth failure, cardiac septal defect, thin upper lip and asymmetric dysmorphic ears (2). A frequent loss of heterozygosity (LOH) in 6q23-q25 in breast and other types of cancer suggested the presence of genes involved in tumor development. Microcell-mediated chromosome transfer studies showed that the introduction of a normal human chromosome 6 fragment encompassing the region 6q23.3q25 can suppress the neoplastic phenotype of breast cancer cell lines (3). In microarray studies on melanoma cell lines, the KEPI gene was found to be highly methylated and therefore down-regulated compared to normal cells (4). Expression studies of KEPI in breast cancer cell lines, breast tumors, and metastases by reverse transcriptase-polymerase chain reaction (RT-PCR) showed a reduced or complete loss of KEPI expression (5). Therefore, expression studies on the protein level would help to identify KEPI as a tumor suppressor protein. We describe here the generation of a KEPI-specific monoclonal antibody, the characterization of the epitope bound by this antibody and the use of the antibody to identify the KEPI protein in tissue samples.

*Present address: AJ Innuscreen GmbH, Berlin, Germany.

Materials and Methods

Correspondence to: Jörg A. Schenk, UP Transfer GmbH, Hybrotec, c/o Fraunhofer Institute for Biomedical Engineering, Am Mühlenberg 13, D-14476 Potsdam, OT Golm, Germany. Tel: +49 33158187231, Fax: +49 3319771143, e-mail: [email protected]

Cloning of KEPI cDNA, protein production and purification. The cDNA of kinase C-enhanced PP1 inhibitor (KEPI) (GenBank AL096708) was cloned in pET-30 Xa/LIC and pET-32 Xa/LIC vectors (Novagen, Schwalbach, Germany) according to the manufacturer’s protocol. Escherichia coli BL21-Gold(DE3)pLysS (Stratagene, Amsterdam, the Netherlands) were transformed with the generated plasmids by heat shock. For induction of KEPI fusion protein, an overnight culture was grown (37˚C, 250 rpm) in LB

Key Words: Hybridoma, monoclonal, KEPI, peptide array, substitutional analysis.

0250-7005/2010 $2.00+.40

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ANTICANCER RESEARCH 30: 1573-1578 (2010) media containing appropriate antibiotic to an optical density OD680nm of 0.4-0.6 before induction with isopropyl-thio-beta-Dgalactopyranoside (IPTG) to a final concentration of 100 μM. Maximum concentration of pET30 and pET32 fusion protein was obtained after 16 h and 4 h, respectively. The osmotic shock fraction of whole cell extract (OS) was isolated from pelleted bacteria by adding 5 mM MgSO4 (1/5 volume of the culture) and extracting the clarified supernatant by centrifugation for at least 30 min. OS of non-transformed E. coli were used as control. The KEPI protein was purified using TALONspin columns (BD Biosciences, Heidelberg, Germany) according to the manufacturer’s instructions. For mammalian expression of KEPI and KEPI-GFP fusion proteins, plasmids pEGFP-C2 (Clontech, Heidelberg, Germany) or pcDNA3.1 (Invitrogen, Heidelberg, Germany) were used. Generation and characterization of monoclonal antibodies. Hybridoma technology was applied for the generation of murine monoclonal KEPI-specific antibodies. Female Balb/c mice were immunized three times with unpurified heat-denatured KEPI pET30 fusion protein. The booster immunization was performed with purified KEPI pET32 fusion protein. Five days later, electrofusion of spleen cells with myeloma cells (Sp2/0, ATCC CRL-1581) in the presence of polyethylene glycol 8000 was performed as described elsewhere (6). Selected hybrids were cultivated in RPMI-1640 medium (containing 10% (v/v) fetal calf serum (FCS), 2 mM glutamine, 50 μM β-mercaptoethanol) and subcloned by limiting dilution on mouse peritoneal feeder cells. Culture supernatants of clones and subclones were tested in an enzyme immunoassay (ELISA) for antigen binding with KEPI pET32 fusion protein bound to the solid phase. Class and subclass of monoclonal antibodies were determined as described elsewhere (7). The antibodies were purified from cell culture supernatant by affinity chromatography using Protein A columns (GE Healthcare, Munich, Germany). SDS-PAGE and Western blot. Samples were separated in a gradient (12.5 to 20%) SDS-polyacrylamide gel according to Laemmli (8). Proteins were stained in 0.25% Coomassie Brilliant Blue R-250 (Serva, Heidelberg, Germany) and destained. For Western blot, the separated proteins were blotted on nitrocellulose membranes (PROTRAN NC BA3; Schleicher & Schuell, Dassel, Germany) using a semi-dry blotting apparatus. After staining with Ponceau S, the membrane was blocked in Western blocking reagent (Roche, Mannheim, Germany) (diluted in PBS, pH 7.4, 0.1% Tween 20), incubated for 1 h in 0.5 μg/ml purified antibody G18AF8 and incubated with secondary peroxidase conjugated goat anti-mouse IgG(H+L) antibody (Southern Biotech, Birmingham, AL, USA) (1:20,000 in blocking reagent). Detection was performed with the ECL™-substrate (Lumigen, Southfield, MI, USA) according to the manufacturer’s instructions. To detect the reactivity with KEPI protein of other species, the Mega Western Protein Array: Multiple Species (normal) system (BioChain, Hayward, CA, USA) was used according to the manufacturer’s instructions with the same reagents as described above. Transfection of HEK293, MCF-10 cells and intracellular FACS staining. Human embryonal kidney cell line HEK293 (ATCC: CRL1573) and human breast cancer cell line MCF-7 (ATCC: HTB-22) were cultivated as described elsewhere (9, 10) and transfected with 10 μg of plasmids pEGFP-C2-KEPI, pEGFP-C2, pcDNA3.1-KEPI or pcDNA3.1, respectively, using calcium phosphate co-precipitation

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according to standard protocols. Stable cell lines were generated by selection in 600 μg/ml G418 containing medium for approximately 2 weeks. For protein detection in Western blot, the cells were washed twice in phosphate-buffered saline (PBS) and lysed in 300 mM NaCl, 50 mM Tris-HCl pH 7.6, 0.5% Triton X-100 and 1 mM phenylmethylsulfonylfluoride (PMSF). FACS analysis was performed on a FACScalibur (Becton-Dickinson, Heidelberg, Germany). Cells were incubated with 4% formaldehyde for 10 min and intracellular staining was performed with 10 μg/ml G18AF8 or murine anti-GFP (1 μg/ml, Roche Diagnostics, Penzberg, Germany), followed by biotinylated goat anti-mouse-Ig (Dianova, Hamburg, Germany) and streptavidin-phycoerythrin (Pharmingen Biosciences, San Diego, CA, USA). All reagents were diluted in 5% (w/v) saponin. Immunohistochemical and immunofluorescence examinations. The study was performed with approval of the Institutional Review Board of the Charité, in accordance with Berlin law. Tissue samples were obtained from ten female patients with an intracranial metastasis of a known ductal mamma carcinoma. They were fixed in buffered 4% formaldehyde overnight and paraffin embedded. Four-μm paraffin sections were used for hematoxylin and eosin staining, alcian bluePAS reaction and immunohistochemical staining with antibodies against cytokeratins (Lu5; DAKO, Hamburg, Germany) and Ki-67 (MIB-1; DAKO) to identify the tumor region. According to a standard protocol for immunohistochemistry, sections were dewaxed and subjected to microwave pretreatment (3×4 min in 10 mM citrate buffer, pH 6.5, 600 W; Bosch, Berlin, Germany). Endogenous peroxidase activity was blocked by incubating sections with 0.6% hydrogen peroxide for 15 min at room temperature. Subsequently, sections were incubated with normal goat serum diluted 1 to 20 in PBS for 20 min and then incubated with the antibody against KEPI overnight (diluted 1 to 100, in 10% FCS in PBS). Goat anti-mouse IgG labelled with horseradish peroxidase (diluted 1 to 100, 90 min; DAKO) was applied and after detection with Vectastain ABC Elite kit (Vector Laboratories, Wertheim, Germany) immunopositive cells were visualized with 3,3’-diaminobenzidine (Sigma, Germany). Nuclei were counterstained with hematoxylin. For negative control, the primary antibody was omitted. Peptide array synthesis and binding studies. Cellulose membranebound peptides were automatically prepared according to standard SPOT synthesis protocols using a MultiPep synthesizer (Intavis AG, Cologne, Germany) as described in detail previously (11). For generation of the sequence files, the software LISA 1.78 (in-house software) was used. The generated peptide arrays were synthesized on an N-modified cellulose-amino-hydroxypropyl ether membrane (N-CAPE) (12). The cellulose-bound peptides were pre-washed once with ethanol (1×10 min), with Tris-buffered saline (TBS), pH 8.0, (3×10 min) and then blocked for 3 h with blocking buffer (Sigma-Genosys, Cambridge, MA, USA) in TBS pH 8.0, containing 5% sucrose. The membranes were incubated with antibody G18AF8 (1 μg/ml in blocking buffer) overnight at 4˚C and thereafter washed with TBS pH 8.0 (3×10 min). All antibodies were protein Apurified. The interaction of the antibodies with the membrane-bound peptides were detected by a peroxidase labelled goat anti-mouse IgG (Sigma-Genosys) (1:500 in blocking buffer) for 2.5 h at room temperature. To remove excess of antibody, the membrane was washed with TBS, pH 8.0, (3×10 min). An Uptilight HRP blot chemiluminescent substrate (Uptima-Interchim, Montluçon, France) was applied for detection using a Lumi-Imager (Boehringer

Daskalow et al: Monoclonal Antibody against KEPI

Figure 1. Flow cytometric evaluation of GFP (A) and G18AF8 staining (B) of HEK293 cells transfected with GFP-KEPI (open curve) or untransfected HEK293 cells (shaded curve). An unrelated protein GFP-profilin (C, D) was used. Binding of primary antibody was detected after incubation with biotinylated secondary antibody and enhancement with streptavidin-phycoerythrin.

Mannheim GmbH, Mannheim, Germany). The signal intensities were recorded as Boehringer light units (BLU) using the LumiAnalyst™ software. The binding affinity (Kd) was measured by the BIACORE × system (Uppsala, Sweden) according to standard protocols (12).

Results The cDNA of kinase C-enhanced PP1 inhibitor (KEPI) was successfully cloned into pET30 and pET32 vectors, which was proven by sequencing the cloning sites. After transformation of E. coli, both KEPI pET30 and KEPI pET32 fusion proteins were expressed after induction with IPTG. Analysis of the recombinant proteins in SDS-PAGE and Coomassie staining showed an estimated size of about 31 kDa (for KEPI pET30) and 35 kDa (for KEPI pET32). For antibody generation mice were immunized with KEPI-pET30 and KEPI–pET32 fusion proteins. Out of several hybridoma cell lines, one clone was established whose antibody showed a specific binding to both KEPI-fusion proteins. This antibody, G18AF8 belonged to the IgG2b subclass. In Western blot analysis with lysates from KEPI-transfected MCF-7 breast cancer cell line, monoclonal antibody G18AF8 recognized a specific band of about 18 kDa which corresponds to the molecular weight of KEPI. Mocktransfected MCF-7 cell lysates did not react (5). In previous experiments it was shown that most breast cancer cell lines do not express KEPI mRNA (5). Cell lysates from HEK293 cells transfected with GFP-KEPI fusion plasmid (pEGFP-C1-

Figure 2. Immunochemistry of a human brain metastasis showing groups of carcinoma cells expressing KEPI (brown) within the cytoplasm, some of them also within the nucleus (blue; arrow). The staining intensity was weak to moderate. Other parts of this metastasis of ductal mamma carcinoma show no expression of KEPI. KEPI immunohistochemistry (brown) with hematoxylin counterstaining (blue), magnification ×400.

KEPI) were also stained by G18AF8. The bands correspond to about 48 kDa (GFP-KEPI) and several degradation products (32 kDa, 20 kDa) (data not shown). In flow cytometric experiments, it was shown that antibody G18AF8 recognized HEK293 cells transfected with GFP-KEPI, but not

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ANTICANCER RESEARCH 30: 1573-1578 (2010) GFP-profilin (as a control fusion gene) transfected or nontransfected HEK293 cells. Transfection was validated by flow cytometry using GFP fluorescence (Figure 1). An unrelated murine monoclonal antibody was not able to stain these cells in FACS analysis (data not shown). To determine the applicability of antibody G18AF8 to paraffin-embedded tissue, we stained intracranial metastases of ductal mamma carcinomas. The immunohistochemical examination using paraffin sections revealed a moderate to faint nuclear and cytoplasmic staining of solitary tumor cells and smaller groups of cells in six patients. Four metastases did not show expression, neither nuclear nor cytoplasmic (Figure 2). Our results clearly indicate the capacity of the KEPI antibody to work on formaldehyde-fixed, paraffin-embedded tumor tissue. As the antibody G18AF8 was able to detect KEPI in Western blot and flow cytometry, the detected epitope should be linear. Therefore, it was further characterized using a pepscan approach for antibody epitope determination (data not shown). In a first approach, 6mer peptides from the KEPI sequence with an overlap of 4 amino acids were used. The antibody gave the strongest signal with the peptide sequence, RVFFQS, and weaker signals with overlapping peptides GARVFF and FFQSPR. The main epitope was therefore concluded to be GARVFFQSPR, corresponding to N-terminal amino acids 18-27 in the human KEPI protein. Kd measurements of the 10mer epitope revealed an affinity in the low micromolar range (1 μM ± 0.5 μM). By downsizing the epitope to a 6mer (RVFFQS), a clearly loss of affinity (22 μM ± 0.1 μM) was found. To further analyze the key positions of the KEPI epitope, a substitutional analysis was performed. Every amino acid from the epitope GARVFFQSPR was substituted with 20 of the naturally occuring L-amino acids (Figure 3A). Variance analysis revealed that two amino acids (F and Q) are essential for antibody binding and three others have a variance from 40-50% (Figure 3B). The exchange of a single amino acid at any other position did not influence the antibody binding. Therefore, a minimal antibody epitope with the sequence GAxxxFQSxx could be assumed. Knowing the bound epitope, we searched for homologies of human KEPI in different species. As KEPI should be expressed in brain (1, 13), we stained a commercially available brain tissue blot from different animal species with our monoclonal antibody. We were able to detect the protein in human brain tissue, as well as in hamster, rabbit, chicken and rat. There were very low signals in dog, pig and monkey (Figure 4). According to the Swiss Prot database, the only known KEPI sequences are human, murine and rat, where the epitope GARVFFQSPR is identical.

Discussion We were able to generate a monoclonal antibody specifically binding to the human PP1 inhibitor KEPI. This antibody binds its antigen in ELISA, immunoblotting, flow cytometry, and immunohistochemistry on paraffin sections. The epitope

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Figure 3. Substitutional analysis of the epitope. A: Each residue of the KEPI epitope GARVFFQSPR was replaced with 20 naturally occuring L-amino acids. Black spots denote interactions of the peptide analogs with antibody G18AF8. All spots in the left hand columns are identical and represent the wild-type (wt) peptide. All other spots are single substitution analogs, the rows defining the sequence position replaced and the columns defining the amino acid used as a replacement. B: To determine objectively the number of countable spots for substitutional analysis, the signal intensities for each spot in the substitutional analysis were measured as BLU values for antibody G18AF8. The percentage of replacement variability (V) of each sequence position was calculated (V=BLU/20X100) and plotted against the sequence position. As a result, we observed three classes: a class of low variability (VV>20%), and a class of high variability (V>50%).

detected by the antibody was mapped using cellulose bound peptide array (pepscan) and the binding motif is further depicted using amino acid substitution analysis and Kdmeasurements. The affinity of G18AF8 to its epitope peptide was in a micromolar range, which should be sufficient for diagnostic purposes. A minimal antibody epitope with the sequence GAxxxFQSxx was discovered. Only a few other publications have studied KEPI expression so far, mostly at the mRNA level or using polyclonal antisera (1, 13, 14). Recently, KEPI knock out mice were generated to investigate the influence of KEPI on the morphine receptors in the brain

Daskalow et al: Monoclonal Antibody against KEPI

Figure 4. Detection of KEPI protein in brain tissue of different species. Western blot was performed with purified antibody G18AF8 on a MegaWestern protein array (Biochain Institute, Inc.) representing brain tissue of 15 different species (see Materials and Methods).

(15). The authors suggest a role for KEPI action in adaptive responses to repeated administration of morphine that include analgesic tolerance and drug reward (15). As the biological role of KEPI, especially in cancer and metastasis, is not yet absolutely clear, antibody G18AF8 should be a powerful reagent for examining KEPI expression in different cell types and tissues, and might also be a diagnostic tool. Possibly, the expression of KEPI might correlate with cancer stage or prognosis. As the epitope is identical in mouse, rat and hamster, this antibody could also be valuable for studies in these and in other species. The antibody was also binding chicken and rabbit KEPI. Surprisingly, the antibody is not able to detect murine KEPI, suggesting that either KEPI was not expressed in the murine brain or that post-translational modifications hide the peptide epitope. Although the KEPI sequence of other species is not known, we can state that hamster KEPI should be very homologous or identical in amino acids 18-27. Even the avian KEPI homolog should be similar in this region. In summary, a murine monoclonal antibody specific for the PP1 inhibitor KEPI was generated and characterized. It is the first monoclonal antibody binding this antigen described so far and might help understanding the role of KEPI in cardiovascular and brain function (16) as well as its role as a potential tumor suppressor (5).

Acknowledgements We are grateful to B. Vogt and A. Menning for help with FACS analysis and K. Messerschmidt for help in the cell fusion. This work was supported by: Deutsche Krebshilfe (grant number: 10-1249). P.B. is supported by the Deutsche Forschungsgemeinschaft (DFG Grant VO885/3-2).

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Received October 12, 2009 Revised March 16, 2010 Accepted March 19, 2010