Identification, Purification, and Subcellular Localization of Prostate-Specific Membrane Antigen PSM' Protein in the Lncap Prostatic Carcinoma Cell Line

[CANCER RESEARCH 58. 4787-4789. November I. 1998] Advances in Brief

Identification, Purification, and Subcellular Localization of Prostate-specific Membrane Antigen PSM' Protein in the LNCaP Prostatic Carcinoma Cell Line

Lana S. Grauer,1 Kellie D. Lawler, JoÃ«lleL. Marignac, Abhay Kumar, Amila S. Goel, and Robert L. Wolfert

H\hrilt'i-h Incorporait-il, it Subsidiary of Bei-kman Coulter. Inc., San Diego. California 92196-9006

variant of PSMA RNA called PSM' was described (15). PSMA and Abstract PSM' cDNAs are identical except for a 266-nucleotide region near the An alternatively spliced variant of prostate-specific membrane antigen 5' end of PSMA cDNA (nucleotides 114-380) that is absent from (PSMA) designated PSM' was originally described following identification PSM'. This region codes for the transmembrane domain of PSMA, so of its mRNA in normal prostate. We have purified the PSM' protein from it was postulated that PSM' would be localized to the cytoplasm. To LNCaP cells using two immunoaffinity columns in tandem. The first determine whether the PSM' mRNA is translated and expressed in column contained a monoclonal antibody (7E11) that was reactive with the NH2 terminus of PSMA, which specifically depleted the LNCaP lysate prostate cancer cells and to identify its subcellular location, we used of full-length PSMA. The nonbinding fraction was then passed over a two mAbs that were specific to two different regions of the protein to affinity-purify PSM' and then sequence and characterize the protein. second column composed of a monoclonal antibody (PEQ226.5), the epitope of which was located within the 134-437 domain of PSMA and shared with PSM'. The protein eluted from the second immunoaffinity Materials and Methods column produced a .I/,. 95,000 band on SDS-PAGE, which was slightly lower than the full-length PSMA at MT 100,000. The band was NH2- Monoclonal Antibodies. The 7E11 antibody was obtained from Dr. Gerald terminally sequenced through 15 residues, and the assigned sequence Murphy (Pacific Northwest Cancer Foundation. Seattle. WA). Its development coincided with the predicted sequence for PSM' protein minus the first and clinical use with '"Indium has been described previously (4). 7E1I two Nil. terminus amino acids. The PSM' protein, therefore, began with recognizes the NH2-terminal portion of PSMA (residues 1-7; Ref. 16). residue 60 of PSMA (alanine). LNCaP cells were fractionated, and PSM' PEQ226.5 was developed from a BALB/c mouse inoculated i.p with plasma was localized to the cytoplasm. membranes prepared from prostate carcinoma tissue. Purification and Sequencing of PSM'. LNCaP cells obtained from Amer Introduction ican Type Culture Collection (Rockville. MD) were grown in RPMI medium with 10% horse serum (Life Technologies, Inc.. Grand Island. NY) and Prostate cancer is the second leading cause of cancer death in men (1). maintained in 5% CO, at 37Â°C.Cells were grown in a T160 flask until 80% Biomarkers of prostate cancer have been used in screening, diagnosis, cell confluency was obtained. Cells were lysed following the addition of 2 ml and predicting disease progression. Currently, the most accepted of of 1% Triton X-100, 10% glycerol, 15 mM MgCl2. 1 HIMphenylmethylsulfonyl these markers is prostate-specific antigen (2, 3). A newer marker of fluoride, 1 mM sodium vanadate. 10 Â¿IMaprotinin. and 50 mM HEPES (pH 7.5). prostate cancer, PSMA2 (2), was originally identified in LNCaP cells The crude lysate was incubated with 1.0 ml of 7E11-AminoLink agarose beads for 4 h at 4Â°C.Antibodies were covalently linked to the agarose according to by its immunoprecipitation with the mAb, 7E11-C5 (4). PSMA is manufacturer's instructions (Pierce. Rockford. IL). The unbound fraction was expressed in normal prostate (4-7) as well as a high proportion of then incubated with 1 ml of PEQ226.5-AminoLink beads for 4 h at 4Â°C.Beads prostate carcinomas (8). Expression of PSMA is enhanced in higher- were washed five times with 10 ml of 1% Triton X-100. 10% glycerol. 15 mM grade cancers, metastatic disease (8), and hormone-refractory prostate MgCl2. and 50 mM HEPES (pH 7.5) and then eluted with 1 ml of 2% SDS in carcinoma (7-9). Serum studies have suggested that PSMA levels 10 mM sodium phosphate-150 mM NaCl (pH 7.5). Aliquots of the eluents and may be linked to a more aggressive clinical phenotype (10). Low unbound fractions were reduced and denatured and subjected to SDS-PAGE on levels of the protein have also been reported in small intestine, colon 4-20% polyacrylamide gels (17). Following blotting to nitrocellulose paper, (11), and the capillary endothelium of a variety of tumors (11, 12). A the proteins were probed with both the 7E11 and PEQ226.5 mAbs. The radioimmunoconjugate of the 7E11-C5 antibody designated CYT356 remaining eluent from the PEQ226.5-AminoLink beads was concentrated to a minimal volume and loaded onto a single lane of a 4-20% gel. blotted to a is currently being used as an imaging agent for prostate cancer (13). The gene for human PSMA has been cloned and sequenced (5) and polyvinylidene difluoride membrane, and stained with Coomassie Blue. The band was cut out of the blot and sequenced on a Proci.se Protein Sequencer mapped to chromosome 1Iql4 (14). The DNA sequence revealed that (Perkin-Elmer Corp.. Foster City. CA). a portion of the coding region has a 54% homology to human Western Blot. Blots were incubated with primary mAbs followed by transferrin receptor. The sequence also indicated that the gene coded incubation with goat antimouse IgG conjugated to horseradish peroxidase for a putative type II transmembrane protein of M, â€”¿100,000con (Jackson Immunoresearch. West Grove, PA). Antibody reactivity was detected sisting of a short intracellular segment (amino acids 1-18), a trans- using the enhanced chemiluminescence reagent (Amersham. Arlington membrane domain (amino acids 19-43), and an extensive extracel Heights, IL). lular domain (amino acids 44-750). Recently, an alternatively spliced Cell Fractionation. Plasma membrane and cytosol fractions from LNCaP cells were prepared according to a previously published procedure (18). with minor modifications. Approximately 5X10* cells were disrupted by nitrogen Received 8/12/98; accepted 9/15/98. The costs of publication of this article were defrayed in part by the payment of page cavitation (50 pounds per square inch for 20 min), and plasma membranes charges. This article must therefore be hereby marked advertisement in accordance with were purified by differential centrifugation. Protein was quantitated using the 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Hybritech Incorporated. P.O. bicinchoninic acid assay (Pierce). Box 269006. San Diego. CA 92196-9006. Phone: (619)621-3259; Fax: (619)621-4610. Cloning and Purification of Fusion Proteins. Three DNA fragments, 2 The abbreviations used are: PSMA. prostate-specific membrane antigen: mAb. including PSMA 1-173, PSMA 134-437, and PSMA 438-750 were amplified monoclonal antibody: GST. glutathione 5-transferase. by PCR. Plasmids PACGHISNTA-PSMA1.9 and PDR2 were obtained from 4787

Dr. Gerald Murphy and were used as vectors to amplify PSMA. These fragments were cloned into PGEX (Pharmacia Biotech, Uppsala, Sweden) or pETSa (Novagen, Madison. WI| vectors to generate GST and T7 PSMA fusion proteins, respectively (19). Following transformation into Escherichia coli and induction, the fusion proteins were purified by high-performance liquid chromatography. 200 kD Results and Discussion 116 kD The identification of an alternatively spliced form of PSMA mRNA PSMA 97 kD called PSM' (15) and the observed changes in ratios of PSMA:PSM' PSM1 mRNA between normal prostate and prostate cancer tissue suggested that the PSM' protein might be a useful diagnostic or prognostic 66 kD marker of prostate cancer. Although PSM' mRNA was identified in prostate tissues, the protein has not been identified to date. A two-step immunouffinity chromatography procedure was used to purify PSM' from a crude lysate of the human prostate carcinoma cell line LNCaP. Because PSM'-specific mAbs are not currently available, we used two 45 kD mAbs that recognize full-length PSMA. The first mAb (7E11) has previously been shown to bind to the NH2 terminus of PSMA (16). Because the PSM' cDNA is missing the 266-nucleotide region at the 5' end of PSMA (15), 7E11 presumably would not bind PSM'. We used a second PSMA mAb, PEQ226.5, which recognizes PSMA and could potentially bind PSM'. 34

A series of PSMA fusion proteins were synthesized to map the 7E11 226.5 epitope of PEQ226.5. By Western blot analysis, PEQ226.5 reacted Fig. 2. Western blot of PSM' purified from LNCaP lysate by sequential immunoaf- with fusion protein PSMA-GST-134-437 but did not bind fusion finity chromatography. Equivalent volumes of eluent from both immunoaffinity resins proteins PSMA-GST-1-173 or PSMA-GST-438-750. On the basis of (7E11-E and PEQ226.5-E) were reduced and loaded onto 5-20% SDS-polyacrylamide these results, PEQ226.5 was mapped to the 173-437 domain of gels and blotted to nitrocellulose. The blot was probed with mAbs 7E11 (Lanes I and 2) PSMA (Fig. 1). Both antibodies were covalently bound to agarose and PEQ226.5 (Lanes 3 and 4) at 10 /ig/ml. resin and used sequentially to first remove full-length PSMA from the lysate (7E11-agarose beads) and then capture the unbound PSM' on the second antibody resin (PEQ226.5-agarose beads). Eluted protein and unbound fractions from both columns were evaluated with 7E11 and by Western blot (Fig. 2). Both 7E11 and PEQ226.5 detect PSMA, which is captured and eluted from the 7E11-agarose beads (Lanes 1 200 kD and 3). In contrast. 7E11 fails to detect any protein in the PEQ226.5 eluent (Lane 2), whereas PEQ226.5 detects a protein that is migrating slightly faster than PSMA (Lane 4). Because 7E11 cannot detect PSM' but PEQ226.5 can. we speculated that the band in Lane 4 was PSM'. 116KD To confirm the identity of the PEQ226.5-reactive protein, the entire 97 kD PEQ226.5 eluent was concentrated and loaded in a single lane of a reduced SDS-polyacrylamide gel and blotted to a polyvinylidene difluoride membrane. The blot was stained with Coomassie blue and the Mr 95,000 kDa band was cut out and NH-,-terminally sequenced 66 kD

PEQ226.5

7E11 (1-7) 173I 437TR]//////////AY//////////Ã€418 7E11 PEQ226.5 C PSMA ^'8- 3' Subcellular localization of PSM' by Western blot analysis. LNCaP cytosol (Lanes 1 and 3} was loaded at 1 /ig/lane and LNCaP plasma membranes (Limes 2 and 4) 19 43 567PEQ226.5750 were loaded at 0.05 Â¿tg/laneand then probed with the PSMA-specific mAb 7E11 at 10 Hg/ml or PEQ226.5, which recognizes both PSMA and PSM'. at 10 /ig/ml.

through 15 residues. The following sequence for PSM' was obtained: n TR Ala-Phe-Leu-Asp-Glu-Leu-Lys-Ala-Glu-Asn-Ile-Lys-Lys-Phe-Leu. -c PSM The sequence matched the predicted protein sequence for PSM' 80 deduced from the cDNA sequence (11). The only deviation from the Fig. I. Mapping of PSMA mAbs 7E11 and PEQ226.5 to PSMA and PSM'. Shown is a schematic of full-length PSMA and alternatively spliced PSM' showing the transmem predicted sequence was the absence of residues 58 (Met) and 59 (Lys) at the NH2 terminus. Although the putative translation initiation site brane domain (TM) and transferrin receptor homologous region (77?). Arrows, binding for PSM' was identified at residue 58 (Met), the actual NH2-terminal regions for 7E1I and PEQ226.5. 4788

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1998 American Association for Cancer Research. IDENTIFICATION OF PSM' PROTEIN amino acid by protein sequencing was identified as alanine at residue of patients with prostate cancer: the Johns Hopkins experience. In: W. J. Catalona. 60 of PSMA. NH2-terminal methionine is usually not important for D. S. Coffey. and J. P. Karr (eds.). Clinical Aspects of Prostate Cancer, pp. 247-251. New York: Elsevier Scientific, 1989. protein function and is often removed shortly after its incorporation by 4. Horoszewicz. J. S., Kawinski, E.. and Murphy. G. P. Monoclonal antibodies to a new the limited proteolysis of a few residues that occurs at the amino antigenic marker in epithelial prostatic cells and serum of prostalic cancer patients. terminus of many proteins in eukaryotes (20). Unlike full-length Anticancer Res., 7: 927-936. 1987. PSMA from LNCaP cells (5), PSM' was not NH2-terminally blocked 5. Israeli. R. S., Powell. C. T.. Fair. W. R.. and Heston. W. D. W. Molecular cloning of a complementary DNA encoding a prostate-specific membrane antigen. Cancer Res.. and, therefore, was sequenced directly without digestion. This result 53: 227-230, 1993. indicates that the two-column procedure is quite effective in first 6. Lopes. A. D., Davis, W. L.. Rosenstraus. M. J.. Uveges, A. J., and Oilman. S. C. Immunohistochemical and pharmacokinetic characterization of the site-specific im- depleting the lysate of the majority of full length PSMA and then munoconjugale CYT-356 derived from antiprostate monoclonal antibody 7EI1-C5. capturing the PSM' on the second column. Cancer Res.. 50: 6423-6429, 1990. Because PSM' mRNA lacks the transmembrane domain expressed 7. Israeli, R. S.. Powell. C. T.. Corr. J. G., Fair, W. R., and Heston. W. D. W. Expression by PSMA, it was speculated that PSM' would be a cytosolic protein of the prostate-specific membrane antigen. Cancer Res., 54: 1807-1811. 1994. 8. Wright. G. L., Jr.. Haley. C.. Beckett. M. L.. and Schellhammer. P. F. Expression of (15). To test this hypothesis, we prepared plasma membrane and prostate-specific membrane antigen in normal, benign and malignant prostate tissues. cytosol fractions of LNCaP cells and examined each fraction by Urol. Oncol.. /.- 18-28, 1995. 9. Diamond. S. M.. Fair. W. R.. and Heston, W. D. W. Modulation of prostate specific Western blot with the PSMA antibodies. The 7E11 antibody detected membrane antigen (PSM) expression in vitro by cytokines and growth factors. Proc. PSMA in the plasma membrane fraction (Fig. 3, Lane 2). In contrast, Am. Assoc. Cancer Res., 36: 643. 1995. 7E11 detected only a minor band in the cytosol fraction. This sub- 10. Murphy. G.. Ragde. H.. Kenny. G.. Barren. R.. Erickson, S.. Tjoa. B.. Boynton, A., Holmes. E.. Gilbaugh. J.. and Douglas. T. Comparison of prostate specific membrane cellular location is consistent with PSMA being a transmembrane antigen, and prostate specific antigen levels in prostatic cancer patients. Anticancer protein. When the cytosol was probed with PEQ226.5, the faster- Res.. 15: 1473-1480. 1995. migrating form (PSM') was detected (Lane 3). PEQ226.5 detected 11. Silver, D. A.. Pellicer. I., Fair. W. R.. Heston. W. D. W.. and Cordon-Cardo. C. full-length PSMA in the plasma membrane fraction (Lane 4). A Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin. Cancer Res.. 3: 81-85. 1997. Western blot of LNCaP lysate probed with PEQ226.5 (data not 12. Liu, H., Moy. P., Kim, S., Xia. Y.. Rajasekaran, A., Navarro, V., Knudsen. B.. and shown) exhibited a strong PSMA band and a weak PSM' band, Bander. N. H. Monoclonal antibodies to the extracellular domain of prostate-specific membrane antigen also react with tumor vascular endothelium. Cancer Res.. 57: suggesting that PSMA is the predominant form in LNCaP cells. This 3629-3643, 1997. result is in agreement with previous reports that have shown that, in 13. Babaian. R. J.. Sayer, J.. Podoloff. D. A.. Stcclhammcr. L. C.. Bhadkamkar. V. A., LNCaP cells, the ratio of PSMA:PSM' RNA is -10:1 (15). PSM' was and Gulfo. J. V. Radioimmunoscintigraphy of pelvic lymph nodes with '"indium- labeled monoclonal antibody CYT-356. J. Urol.. 152: 1952-1955. 1994. not detected in the plasma membranes of LNCaP cells. 14. Rinker-Schaeffer. C. W.. Hawkins. A. L.. Su. S. L., Israeli. R. S.. Griffin. C. A.. This report is the first study outlining methods to identify and Isaacs. J. T., and Heston, W. D. W. Localization and physical mapping of the purify PSM' protein from LNCaP cell lysates. NH->-terminal sequence prostate-specific membrane antigen (PSM) gene to chromosome 11. Genomics, 30: analysis has confirmed the identity of PSM' and shown that the 105-108, 1995. 15. Su, S. L.. Huang. !.. Fair. W. R., Powell. C. T.. and Heston, W. D. W. Alternatively protein begins at Ala-60 in the PSMA sequence. The protein has been spliced variants of prostate-specific membrane antigen RNA: ratio of expression as a shown to reside in the cytoplasm, as speculated previously. PSM' potential measurement of progression. Cancer Res.. 55: 1441-1443. 1995. protein is a minor component in comparison to full-length PSMA in 16. Troyer, J. K.. Qi Feng. B. S.. Beckett. M. L., and Wright. G. L. Biochemical characterization and mapping of the 7EI 1-C5.3 epitope of the prostate-specific these cells. Studies are ongoing in our laboratory to determine whether membrane antigen. Urol. Oncol.. /: 29-37. 1995. ratios of PSMA:PSM' are of value in the detection and prognosis of 17. Towbin, H.. Staehelin. T.. and Gordon. J. Electrophorctic transfer of proteins from prostate cancer. polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Nati. Acad. Sci. USA. 76: 4350-4354, 1979. 18. Howard. F. D.. Ledbetter. J. A.. Mehdi. S. Q.. and Herzenberge. L. A. A rapid method References for the detection of antibodies to cell surface antigens: a solid phase radioimmuno- assay using cell membranes. J. Immunol. Methods. Ã•8:75-84. 1980. 1. Murphy. G. P. Report on the American Urology Association/American Caneer Society Scientific Seminar on the detection and treatment of early-stage prostate 19. Maniatis, T.. Fritsch, E., and Sambook. J. Extraction, purification, and analysis of cancer. CA Cancer J. Clin.. 44: 91-95. 1994. mRNA from eukaryotic cell. In: T. Maniatis. E. Fritsch. and J. Sambook (eds.). 2. Catalona. W. J.. Smith. D. S., Ratliff, T. L.. Dodds. K. M, Copien. D. E.. Yuan, J. J., Molecular Cloning: A Laboratory Manual, pp. 188-209. Cold Spring Harbor. NY: Petros. J. A., and Andriole. G. L. Measurement of prostate-specific antigen in serum Cold Spring Harbor Laboratory. 1982. as a screening test for prostate cancer. N. Engl. J. Med.. 324: 1156-1161. 1991. 20. Alberts. B.. Bray. D.. Lewis. J.. Raff, M.. Roberts. K.. and Watson. J. D. Basic genetic 3. Carter. H. B., Partin, A. W.. Oesterling. J. E.. Weber. J. P., Epstein, J. I.. Chan. D. W., mechanisms. In: Molecular Biology of the Cell. pp. 199-253. New York: Garland Rock. R. C. and Walsh. P. C. The use of prostate specific antigen in the management Publishing. Inc., 1983.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1998 American Association for Cancer Research. Identification, Purification, and Subcellular Localization of Prostate-specific Membrane Antigen PSM' Protein in the LNCaP Prostatic Carcinoma Cell Line

Lana S. Grauer, Kellie D. Lawler, Joelle L. Marignac, et al.

Cancer Res 1998;58:4787-4789.

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