Antigenic Prenylated Peptide Conjugates and Polyclonal Antibodies

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Bioconjugate Chem. 2004, 15, 270−277

Antigenic Prenylated Peptide Conjugates and Polyclonal Antibodies To Detect Protein Prenylation Xiao-hui Liu,† Dae-Yeon Suh,† Jeffrey Call,‡ and Glenn D. Prestwich*,† Department of Medicinal Chemistry and the Center for Cell Signaling, The University of Utah, 419 Wakara Way, Suite 205, Salt Lake City, Utah 84108-1257, and Biotechnology & Genomic Research Center, Utah State University, 4700 Old Main Hill, Logan, Utah 84322. Received October 30, 2003

Posttranslational modification of proteins with farnesyl and geranylgeranyl groups is a required modification for signaling proteins that includes the small GTPases in the Ras, Rho, and Rab families, heterotrimeric G proteins, and nuclear lamin proteins. To develop antibodies capable of detecting and distinguishing prenylated proteins, we synthesized two antigens, succinylglycine-(geranylgeranyl)cysteine methyl ester (SuccG-(gg)CMe, 1) and succinylglycine-(farnesyl)cysteine methyl ester (SuccG(f)CMe, 2). These prenylated peptides were covalently coupled to bovine serum albumin (BSA) and to keyhole limpet hemocyanin (KLH) to produce polyvalent, immunogenic bioconjugates. Immunization of rabbits with these immunogens generated polyclonal antisera that contained significant titers of anti-geranylgeranyl and anti-farnesyl antibodies. The selectivity of the polyclonal antisera was examined using ELISA and dot blotting methods. The anti-farnesyl and anti-geranylgeranyl antisera crossreacted with both antigens. Attempts to purify the polyclonal antisera by either positive or negative immunoaffinity selection protocols failed to produce selective anti-geranylgeranyl and antifarnesyl antibodies. Moreover, both crude antisera and purified antibodies also crossreacted with myristoylated and palmitoylated BSA conjugates. Immunofluorescence staining of EYFP-CVLL or EYFP-CVIM transfected CHO-K1 cells with rabbit polyclonal antisera showed colocalized membrane fluorescence. Thus, an important caveat for the use of antibodies raised against aliphatic antigens is that extensive controls must be performed to determine selectivity.

INTRODUCTION

Antibodies are synthesized predominantly by plasma cells, which are terminally differentiated cells of the B-lymphocyte lineage and constitute a crucial line of defense against foreign antigens (1). The unique specificities of the antibodies have made them indispensable tools in cell and developmental biology, therapeutic medicine, and pathology (2). While the majority of antigens used for immunization are proteins, peptides, nucleic acid, carbohydrates, or cell-surface antigens, small molecules can be immunogenic if delivered in a polyvalent form with an appropriate adjuvant. This often requires covalent linkage to a carrier protein such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or thyroglobulin. Recent studies revealed that some antigenic nonpeptide molecules can stimulate a strong T-cell response in humans and in mouse models. These molecules include aliphatic glycolipids, phosphoantigens, aromatic glycolipids, alkylamines, and lipids (3-11). Some anti-lipid antibodies can occur naturally. For example, anti-phospholipid antibodies (RPL) are an important clinical problem (12), occurring in autoimmune diseases (e.g., lupus erythematosus) and in infectious diseases such as tuberculosis, hepatitis, Lyme disease, malaria, and bacterial septicemia (13). Stereospecific antibodies against phosphatidylserine have been generated using liposomes (14) and acyl-linked antigens (15); * To whom correspondence should be addressed. Phone: 801-585-9051; fax: 801-585-9053; E-mail: gprestwich@deans. pharm.utah.edu. † The University of Utah. ‡ Utah State University.

these antigens have been employed for immunolocalization in human placenta (16) and other tissues (17). In recent studies of lipid signaling, selective antibodies against the phosphoinositides PI(4,5)P2 and PI(3,4,5)P3 have been developed (18, 19) and used to visualize changes in subcellular phosphoinositides in stimulated cells and in tissue sections (20). Prenylation is a required posttranslational modification of most small GTPases and a variety of other proteins (21-26). In particular, Ras, Rho, and Rab proteins play important roles in cell proliferation and signal transduction. Aberrant signaling is responsible for the growth and proliferation of cancer cells. Each of these mature small GTPases have isoprenyl moieties (farnesyl or geranylgeranyl) linked to a common cysteine methyl ester via a thioether bond. Proteins are prenylated by the transfer of a farnesyl (C-15) or geranylgeranyl (C-20) unit from farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP) to the cysteine of a C-terminal motif. Three carboxyl terminal motifs specify the type of prenylation: CA1A2X, CC, and CAC, where A is an aliphatic amino acid and X is any amino acid. Generally, the CA1A2X (X ) Ser, Met, Ala or Gln) sequence is a substrate for farnesylation unless X ) Leu or Phe, in which case geranylgeranylation occurs. The CC and CAC sequences are substrates for bisgeranylgeranylation and are found exclusively in the Rab family of small GTPases. Two groups have reported a polyclonal anti-farnesyl antibody to identify farnesylated proteins in cells (27, 28), developed using N-acetyl-S-farnesyl-L-cysteine, which was coupled to KLH using carbodiimide chemistry. However, the reported polyclonal antibodies crossreacted

10.1021/bc0342027 CCC: $27.50 © 2004 American Chemical Society Published on Web 02/18/2004

Polyclonal Anti-Isoprenyl Antibodies

with geranylgeranyl moieties. Since selective anti-geranylgeranyl and anti-farnesyl antibodies could be very useful tools for the study of prenylated proteins in cells, we designed and synthesized two new antigens: SuccG(gg)CMe 1 and SuccG-(f)CMe 2. (To avoid confusion with single letter amino acid abbreviations, geranylgeranyl is abbreviated as “gg” and farnesyl as “f”.) The introduction of a succinylglycine spacer was selected to enhance immunogenicity and to increase the likelihood of obtaining prenyl selective antibodies. Antisera that contained polyclonal antibodies were obtained from immunized rabbits. Antibodies were purified by affinity and size exclusion chromatography and were characterized using ELISA, dot blots, and immunofluorescence. EXPERIMENTAL PROCEDURES

General. All reactions were conducted using ovendried glassware. Column chromatography was performed on silica gel (60 Å, 240-400 mesh ASTM) from Whatman Co. BSA and KLH were from Sigma. Affigel-10 was from Bio-Rad. Dot blotting and western blotting reagents were from Amersham. Myristoyl-Gly was from BACHEM, and all other organic reagents were from Sigma-Aldrich Chemical Co. CHO-K1 cells were from ATCC. Lipofectamine and cell culture medium were from Invitrogen Life Technologies. G418 (Geneticin) was from Gibco. L-(Trityl)-Cys 3 was prepared from L-cysteine as described (29). The synthetic compounds were visualized on TLC with anisaldehyde-H2SO4-AcOH solution. Vector pcDNA3.1 encoding YFP-KMSKDGKKKKKKSKTKCVIM was the generous gift of Dr. Tamas Balla (Endocrinology and Reproduction Research Branch, National Institutes of Health). EYFP-CVLL was constructed by site-directed mutagenesis from the EYFP-CVIM, using QuickChange mutagenesis kit (Invitrogen). The mutagenesis primers were sense, 5′-ACAAAGTGTGTACTTCTTTAAAGATCTCGG-3′; antisense, 5′-CCGAGATCTTTAAAGAAGTACACACTTTGT-3′. The PCR conditions were 95 °C for 45 s, followed by 18 cycles at 95 °C for 45 s, 55 °C for 1 min, and 68 °C for 13 min. 2-Amino-3-(tritylsulfanyl)propionic Acid Methyl Ester (L-(trityl)-CysMe, 4). A mixture containing concentrated HCl (1.25 mL) and L-(trityl)-Cys 3 (1 g, 2.75 mmol) in 20 mL of methanol was heated to reflux for 15 h. Then the solution was diluted with water, neutralized with Na2CO3, and extracted with EtOAc. The organic layer was concentrated and then purified on SiO2 (EtOAc) to afford a thick colorless oil, 560 mg (54%). 1H NMR (CDCl3, 400 MHz) δ 7.41-7.44 (m, 6H, ArH), 7.19-7.31 (m, 9H, ArH), 3.66 (s, 3H, OCH3), 3.21 (br s, CH), 2.62 (m, 1H, CHS), 2.52 (m, 1H, CHS), 1.71 (br s, NH2). 2-(2-(tert-Butoxycarbonylamino)acetylamino)-3(tritylsulfanyl)propionic Acid Methyl Ester (BocGly-(trityl)-CysMe, 5). A solution of L-(trityl)-CysMe 4 (377 mg, 1 mmol), N-(tert-butoxycarbonyl)glycine (210 mg, 1.2 mmol), and DCC (247 mg, 1.2 mmol) in CH2Cl2 (50 mL) was stirred at room temperature for 3 h. The mixture was filtered and concentrated. Chromatography on SiO2 (EtOAc/hexanes 3:2) afforded 480 mg (90%) of a white solid. 1H NMR (CDCl3, 400 MHz) δ 7.39-7.36 (m, 5H, ArH), 7.31-7.20 (m, 10H, ArH), 5.01 (br s, 1H, NH), 6.38 (d, J ) 7.6 Hz, 1H, NH), 4.54 (m, 1H, CH), 3.76 (m, 2H, CH2), 3.71 (s, 3H, OCH3), 2.66 (m, 2H, CH2), 2.04 (s, 1H, CH), 1.48 (s, 9H, 3CH3). 13C NMR (CDCl3, 400 MHz) δ 170.8, 169.2, 144.4, 129.7, 128.2, 127.1, 67.2, 60.6, 52.9, 51.3, 33.8, 28.5. EI-MS m/z 478, 434.1, 316.1, 243, 239, 165, 101, 57. HRMS (EI) (m/z) calcd for C30H34SN2O5 (M+) 534.66744; found 534.2176.

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2-(2-Aminoacetylamino)-3-(tritylsulfanyl)propionic Acid Methyl Ester (Gly-(trityl)-Cys-Me, 6). A solution of Boc-Gly-(trityl)-CysMe 5 (400 mg, 0.748 mmol) in 4 mL of TFA/CH2Cl2 (1:1) was stirred at room temperature for 1 h. The mixture was concentrated and evaporated 3× with ether. Filtration with ether afforded 494 mg of a white solid. 1H NMR (CD3OD, 400 MHz) δ 7.38-7.20 (m, 15H, 3Ph), 3.86 (s, 2H, CH2), 3.61 (s, 3H, OCH3), 2.69-2.51 (m, 3H), 2.00 (4H, 2CH2). 13C NMR (CD3OD, 400 MHz) δ 170.8, 166, 144, 129.5, 127.9, 126.9, 110, 52.2, 51.8, 40.2, 33.2. EI-MS m/z 434, 357, 309.8, 243, 239, 165, 117.9, 68.9, 44.9. HRMS (EI) (m/z) calcd for C25H26SN2O3 (M+) 434.55162; found 434.1663. N-[(1-(Methoxycarbonyl)-2-(tritylsulfanyl)ethylcarbamoyl)methyl]succinamic Acid (Succ-Gly-(trityl)-CysMe, 7). A mixture containing Gly-(trityl)-CysMe 6 (420 mg, 0.966 mmol) and succinic anhydride (146 mg, 1.45 mmol) in 25 mL of HOAc was stored at room temperature for 2 days. The mixture was concentrated under vacuum and purified on SiO2 (EtOAc/HOAc 100: 3). Pale yellow oil (414 mg, 80%) was obtained. 1H NMR (CD3OD, 400 MHz) δ 7.38-7.20 (m, 15H, 3Ph), 3.86 (s, 2H, CH2), 3.61 (s, 3H, OCH3), 2.69-2.51 (m, 3H), 2.00 (4H, 2CH2). 13C NMR (CD3OD, 400 MHz) δ 175.2, 174.0, 173.9, 171.8, 170.8, 170.4, 144.6, 129.5, 127.8, 126.8, 67.0, 60.4, 52.0, 51.7, 42.1, 33.2, 30.3, 29.0, 19.7, 19.6, 13.3. FAB-MS m/z 533, 470, 437, 356.9, 311.1, 291, 275, 239, 205, 183, 167, 113. HRMS (FAB) (m/z) calcd for C29H30SN2O6 (M-1)+ 533.17463; found 533.17565 N-[(2-Mercapto-1-(methoxycarbonyl)ethylcarbamoyl)methyl]succinamic Acid (Succ-Gly-CysMe, 8). To a solution of Succ-Gly-(trityl)-CysMe 7 (55 mg, 102.8 µmol) in 4 mL of 50% TFA/CH2Cl2 was added triethylsilane (66 µL, 411 µmol). The mixture was stored at room temperature overnight. After evaporation to remove solvent, the residue was purified on SiO2 (EtOAc/MeOH 4:1) to afford 57.7 mg (71%) of Succ-Gly-CysMe 8. 1H NMR (CD3OD, 400 MHz) δ 3.90 (s, 2H, CH2), 3.73 (s, 3H, OCH3), 2.67-2.50 (m, 3H), 2.00 (4H, 2CH2). 13C NMR (CD3OD, 400 MHz) δ 174.3, 171.9, 170.6, 60.4, 55.0, 51.8, 42.4, 30.2, 29.0, 19.7, 19.6, 13.2. EI-MS m/z 244, 207, 167, 165, 152, 115, 91, 71, 57. N-{[2-(3,15-Dimethylhexadeca-2E,6E,10E,14-tetraenylsulfanyl)-1-(methoxycarbonyl)ethylcarbamoyl]methyl}succinamic Acid (SuccG-(gg)CMe, 1). A solution that contained geranylgeranyl chloride (35 mg, 113 µmol), Succ-Gly-CysMe 8 (33 mg, 113 µmol), and Zn(OAc)2‚2H2O (37 mg, 169 µmol) in 4 mL of DMF/ CH3CN/0.025% TFA (2:1:1) was stirred at 0 °C and then brought to room-temperature overnight. The mixture was concentrated and purified on SiO2 (EtOAc/HOAc 100:3) to afford 36.7 mg (57.5%) of pale yellow oil. 1H NMR (CD3OD, 400 MHz) δ 5.20 (m, 1H), 5.12 (m, 2H), 4.64 (m, 1H), 3.90 (d, J ) 2 Hz, 2H), 3.7 (s, 3H, OCH3), 3.24 (m, 1H), 3.13 (m, 1H), 2.95 (m, 1H), 2.74 (m, 1H), 2.63 (t, J ) 6.4 Hz, 2H), 2.50 (t, J ) 6.4 Hz, 2H), 2.14-1.98 (m, 18H), 1.69, 1.66, 1.60, 1.59 (5s, 15H). 13C NMR (CD3OD, 400 MHz) δ 174.2, 139.5, 124.3, 120.2, 52.4, 51.7, 42.2, 39.7, 32.0, 30.3, 29.1, 26.6, 26.2, 19.6, 15.0. N-{[1-Methoxycarbonyl-2-(3,7,11-trimethyl-dodeca2E,6E,10-trienylsulfanyl)ethylcarbamoyl]methyl}succinamic Acid (SuccG-(f)CMe, 2). A solution containing farnesyl bromide (29 mg, 97.5 µmol), Succ-GlyCysMe 8 (57 mg, 195 µmol), and Zn(OAc)22H2O (66 mg, 292.5 µmol) in 4 mL of DMF/CH3CN/0.025% TFA (2:1:1) was stirred at 0 °C overnight. The mixture was condensed and purified on SiO2 (EtOAc) to afford 56 mg (∼100%) of pale yellow oil. 1H NMR (CD3OD, 400 MHz) δ 5.21 (m,

272 Bioconjugate Chem., Vol. 15, No. 2, 2004

1H), 5.11 (m, 2H), 4.62 (m, 1H), 3.90 (d, J ) 2 Hz, 2H), 3.71 (s, 3H, OCH3), 3.22 (m, 1H), 3.12 (m, 1H), 2.78 (m, 1H), 2.65 (t, J ) 6.8 Hz, 2H), 2.53 (t, J ) 6.8 Hz, 2H), 2.13-1.96 (m, 8H), 1.69, 1.66, 1.60, 1.59 (4s, 12H). 13C NMR (CD3OD, 400 MHz) δ 222.0, 163.9, 124.3, 123.9, 120.2, 72.6, 52.4, 51.8, 42.2, 39.7, 39.6, 36.0, 32.0, 30.6, 30.3, 29.0, 26.6, 26.2, 24.8, 19.6, 16.6, 15.1, 15.0. FABMS m/z 497.1, 416.8, 354.9, 336.9, 304.9, 69. HRMS (FAB) (m/z) calcd for C25H40SN2O6 (M - 1)+ 495.24288; found 495.25174. 2-[2-(2,5-Dioxopyrrolidin-1-yl)acetylamino]-3-(3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenylsulfanyl)propionic Acid Methyl Ester (Succinimide-G(gg)CMe, 11). SuccG-(gg)-CMe 1 (36.7 mg, 65 µmol), SDPP (33 mg, 97.4 µmol), and triethylamine (37 µL, 260 µmol) were dissolved in 10 mL of CH3CN. The mixture was stored at room temperature for 16 h and then concentrated and purified on SiO2 (EtOAc) to afford 14.7 mg (41%) of oil. 1H NMR (CDCl3, 400 MHz) δ 6.52 (s, 1H), 5.19 (t, J ) 8 Hz, 1H), 5.09 (s, J ) 6 Hz, 2H), 4.76 (m, 1H), 4.23 (m, 2H), 3.76 (s, 3H, OCH3), 3.2-2.97 (m, 2H), 2.97-2.78 (m, 6H), 2.09 (m, 8H), 1.98(m, 4H), 1.67, 1.65 (2s, 6H, 2CH3), 1.59 (s, 9H, 3CH3). 13C NMR (CDCl3, 400 MHz) δ 176.4, 170.9, 169.1, 168.2, 165.5, 165.2, 140.2, 135.4, 134.9, 131.2, 126.1, 124.4, 124.2, 123.7, 120.3, 119.4, 52.7, 52.0, 41.1, 39.7, 39.6, 33.2, 30.0, 28.2, 26.7, 26.6, 26.4, 25.7, 25.6, 25.3, 17.7, 17.5, 16.1, 16.0. 2-[2-(2,5-Dioxopyrrolidin-1-yl)acetylamino]-3-(3,7,11-trimethyldodeca-2,6,10-trienylsulfanyl)propionic Acid Methyl Ester (Succinimide-G-(f)CMe, 12). SuccG-(f)-CMe 2 (47 mg, 94.6 µmol), SDPP (47.7 mg, 142 µmol), and triethylamine (53 µL, 378 µmol) were dissolved in 10 mL of CH3CN. The mixture was stored at room temperature for 4 h and then concentrated and purified on SiO2 to afford 10 mg (22%) of a colorless oil. 1 H NMR (CDCl3, 400 MHz) δ 5.19 (t, J ) 8.4 Hz, 1H), 5.09 (t, J ) 6 Hz, 2H), 4.77 (m, 1H), 4.23 (m, 2H), 3.77 (s, 3H, OCH3), 3.2-3.0 (m, 2H), 2.98-2.86 (m, 2H), 2.80 (s, 4H, 2CH2), 2.10-1.95 (m, 8H), 1.68, 1.66, 1.60, 1.54 (4s, 12H, 4CH3). 13C NMR (CDCl3, 400 MHz) δ 184.4, 176.7, 171.1, 165.4, 140.4, 135.6, 131.5, 130.0, 126.3, 124.5, 123.9, 120.5, 120.4, 119.6, 60.6, 52.9, 52.3, 41.3, 39.9, 39.8, 33.5, 30.3, 28.4, 26.9, 26.4, 25.9, 25.5, 17.9, 16.3, 16.2. N-(2-Methoxyethyl)succinamic Acid (13). A solution containing 2-methoxyethylamine (100 mg, 1.33 mmol) and succinic anhydride (134 mg, 1.33 mmol) in HOAc (2 mL) was stored at room temperature overnight. The solvent was evaporated, and the residue was recrystallized in EtOAc to afford needles, 185 mg (79%). 1H NMR (CDCl3, 400 MHz) δ 6.18 (br s, NH), 3.46 (m, 4H), 3.36 (s, 3H), 2.70 (m, 2H), 2.53 (m, 2H). 1-(2-Methoxyethyl)pyrrolidine-2,5-dione (14). To a solution of acid 13 (5 mg, 28.5 µmol) in 0.5 mL of CH3CN were added SDPP (14.2 mg, 28.5 µmol) and triethylamine (16 µL). The reaction was allowed to proceed overnight, and the residue was purified on SiO2 (EtOAc) to give 2 mg of succinimide. 1H NMR (CDCl3, 400 MHz) δ 3.74 (m, 2H), 3.56 (m, 2H), 3.32 (s, 3H), 2.72 (s, 4H). 2-Amino-3-(3,7,11-trimethyldodeca-2,6,10-trienylsulfanyl)propionic Acid Methyl Ester (FarnesylCysMe, 20). A solution containing farnesyl bromide (30 mg, 105 µmol), L-cysteine methyl ester hydrochloride (36 mg, 210 µmol), and Zn(OAc)22H2O (71.4 mg, 315 µmol) in 6 mL of DMF/CH3CN/ 0.025%TFA (2:1:1) was stirred at 0 °C for 2 h. The mixture was concentrated and purified on SiO2 (EtOAc) to afford 57.8 mg (91.6%) of pale yellow oil. 1H NMR (CD3OD, 400 MHz) δ 5.24 (t, J ) 7.2 Hz, 1H), 5.13 (m, 2H), 3.78 (s, 3H, OCH3), 3.16 (m, 1H),

Liu et al.

3.03 (m, 1H) 2.82 (m, 1H), 2.16-2.04 (m, 8H), 1.96 (m, 2H), 1.70 (m, 3H), 1.66 (s, 3H), 1.61 (s, 3H), 1.59 (s, 3H). 13C NMR (CD OD, 400 MHz) δ 172.8, 141.1, 135.6, 131.5, 3 124.5, 123.9, 119.1, 53.6, 53.4, 39.9, 34.3, 29.6, 26.9, 26.7, 25.9, 17.9, 16.6, 16.2, 14.3. 2-(Acetylamino)-3-(hexadecanoylsulfanyl)propionic Acid (Palm-AcCysOH, 21). A mixture containing N-acetylcysteine (16.3 mg, 0.1 mmol), palmitoyl chloride (36.4 µL, 0.12 mmol), and DIEA (52 µL, 0.3 mmol) in 1 mL of DMF was stirred at room temperature overnight. The product was purified with silica gel (EtOAc/HOAc 100:2) to afforded 8.88 mg (21.4%) of Palm-AcCysOH. 1H NMR (CDCl3, 400 MHz) δ 7.20 (s, 1H), 6.72 (m, 1H), 4.62 (m, 1H), 4.14 (m, 2H), 3.37 (m, 1H), 2.85 (s, 2H), 2.05 (s, 3H), 1.90-1.30 (m, 30H). Coupling of SuccG-(gg)-CMe 1, SuccG-(f)-CMe 2, (gg)-CysMe 19, (f)-CysMe 20, Palm-AcCysOH 21, and Myr-GlyOH to BSA with EDCI. To a solution of haptens (1.2 µmol, 33 equiv to BSA) and BSA (0.36 µmol) in 30% DMSO buffer (10 mM NaH2PO4, 200 mM NaCl, pH 7.2) was added EDCI (4.5 mg, 23.4 µmol). The mixture was stirred at room temperature for 2 h and then purified (Sephadex G-25) by elution with 10 mM NaH2PO4, 200 mM NaCl, pH 7.2. Sample concentrations were measured using the Bradford reagent. Coupling of SuccG-(gg)-CMe 1, SuccG-(f)-CMe 2 to KLH with EDCI. To a solution containing prenylated peptides 1 and 2 (0.8 µmol, 833 eq. to KLH) and KLH (0.96 µmol) in 30% DMSO buffer (31 mM NaH2PO4, 0.96 M NaCl, pH 7.4) was added EDCI (2 mg, 10.4 µmol). The mixture was stirred at room temperature for 2 h and then purified (Sephadex G-25) by elution with with 10 mM NaH2PO4, 200 mM NaCl, pH 7.2. Sample concentrations were measured using the Bradford reagent. ELISA. A Maxisorp plate (Nunc) was washed twice with PBS 200 µL/well and then coated with BSA-SuccG(gg/f)CMe 1 µg/well in the amount of 50 µL and incubated at room temperature for 1 h. The plate was rinsed 3× with 100 µL/well of PBS. Blocking solution (100 µL/well, 5% BSA/TBST) was added, and the plate was shaken for 1 h at room temperature. Blocking solution was removed, and the plate was washed 3× with 100 µL/well of TBST. Next, 50 µL/well of primary antibody (1:300) was added to the plate and shaken at room temperature for 1 h. The plate was washed 4× with 100 µL/well of TBST. Then 50 µL/well of anti-rabbit IgG-HRP at 1:2000 in TBST was applied and shaken for 1 h at room temperature. Antibodies were removed and washed 4× with 100 µL/well of TBST; TMB (Sigma) was added (100 µL/well). The plate was placed in a dark drawer for 15-20 min as the blue color developed. The reaction was stopped with 50 µL/well of 0.5 M H2SO4 to yield a yellow color. Absorbance at 430 nm was measured by a UV spectrometer. CHO-K1 Cell Culture and Transfection. CHO-K1 epithelial cells (1 × 105 ) were seeded in a 6-well plate and incubated at 37 °C for 19 h to reach 30% confluence. Vector EYFP-CVLL (6 µg) was dissolved in 60 µL of serum-free medium. Lipofectamine (30 µL) was dissolved into 180 µL of serum-free medium, mixed at room temperature for 30 min, and then diluted with 1.6 mL of serum-free medium. Cells were treated with 1 mL of this mixture after aspiration of the original medium. The transfection continued for 6 h at 37 °C and was stopped by addition of medium containing G418 (0.5 mg/mL). The transfected cells were visualized by fluorescence microscopy. In the blank cell cultures, most cells were killed with G 418 antibiotic by day 9. Cell colonies could be seen in the Petri dishes.

Polyclonal Anti-Isoprenyl Antibodies

Bioconjugate Chem., Vol. 15, No. 2, 2004 273

Scheme 1. Synthesis of Farnesylated and Geranylgeranylated Cysteine Haptensa

a Reagents and conditions: (a) (Ph) COH, TFA, 75%; (b) concentrated HCl, MeOH, 54%; (c) N-Boc-glycine, DCC, CH Cl , 90%; (d) 3 2 2 TFA/CH2Cl2; 100%; (e) succinic anhydride, HOAc, 80%; (f) (Et)3SiH, TFA/CH2Cl2; 71%; (g) farnesyl bromide or geranylgeranyl chloride, Zn(OAc)2‚2H2O, DMF/CH3CN/TFA.

Scheme 2. Side Product Succinimides 11 and 12 in the Attempted Synthesis of NHS Esters 9 and 10

Purification of Anti-gg/f from Rabbit Sera with Positive Immunoaffinity Chromatography. Rabbit antisera (1 mL, 26.7 mg/mL, serum-15, immunized with KLH-SuccG-(gg)CMe 15) was diluted in 4 mL of NaOAc (60 mM, pH 4.0). Caprilic acid (125 µL) was added dropwise, and the mixture was stirred gently at room temperature for 30 min. The mixture was centrifuged to remove precipitate at 10 000 × g at room temperature for 30 min. The supernatant was dialyzed against 20 mM Tris-Cl (pH 7.5) overnight (protein concentration was 1.01 mg/mL, total 6.5 mg). Affigel-10 (Bio-Rad, 0.3 mL) was mixed with 0.5 mL of BSA-(gg)CMe 22 (0.54 mg/mL) or BSA-(f)CMe 23 (0.79 mg/mL) and incubated at 4 °C overnight. The Affigel beads were centrifuged at 5000 × g. The beads were washed twice with 0.4 mL of 10 mM Na3PO4/200 mM NaCl (pH 7.0) and then incubated with 0.2 mL of 7 M urea/1 M NaCl at room temperature for 1 h. The beads were further washed 3× with 0.4 mL of 10 mM Na3PO4/200 mM NaCl (pH 7.0) and then once with 0.4 mL of 0.2 M glycine, pH 7.2. Next, the beads were washed 3× with 0.4 mL of 10 mM Na3PO4/200 mM NaCl (pH 7.0) and then incubated with 0.4 mL of pretreated antibody solution at room temperature for 1 h. The beads were washed 3× with 0.4 mL of 10 mM Na3PO4/200 mM NaCl (pH 7.0). The beads were transferred to a column and eluted with 0.2 M glycine buffer (pH 2.0), and 150 µL of each fraction was collected into 150 µL of 2 M TrisCl, pH 8.8. The diluted antibodies were dialyzed against PBS and concentrated to about 1 mg/mL by ultrafiltration. Fluorescence Imaging of CHO-K1 and EYFPCVLL CHO-K1 Cells with Rabbit Antiserum. Cells were seeded on cover slips for 10 h to reach ∼30%

confluence. The localization of EYFP was visualized by fluorescence microscopy. The medium was aspirated, and then cells were washed with 2 × 1 mL of PBS. The cells were fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were washed with 2 × 1 mL of PBS and permeabilized by addition of 0.8 mL of 0.2% Triton X-100 in PBS for 5 min at room temperature. The cells were washed with 3 × 1 mL of 0.2% Triton X-100 in PBS. The cover slips were blocked with 3% BSA/0.2% Triton X-100 in PBS 25 µL/each for 0.5 h. After adding serum-15 (1:150, 25 µL, 3% BSA/0.2% Triton X-100 in PBS), incubation continued for 1 h. The cells were washed with 3 × 1 mL of 0.2% Triton X-100 in PBS, 5 min/each and then incubated with 25 µL of anti-rabbit IgG-Texas Red (1:500, 3% BSA/0.2% Triton X-100 in PBS) for 1 h. Next, the cells were washed with 3 × 1 mL of 0.2% Triton X-100 in PBS, 5 min/each, followed by dehydration with 2 × 1 mL of ethanol, and 10 mL of xylene. Cover slips were mounted on glass slides. RESULTS AND DISCUSSION

The haptens for coupling to BSA or KLH were synthesized from L-cysteine (Scheme 1). The free thiol of L-cysteine was protected with triphenylmethanol, and the S-protected cysteine was then converted to the methyl ester and coupled to N-Boc glycine (29, 30). Subsequent removal of the Boc group afforded dipepetide 6, which was converted to compound 8 by reaction with succinic anhydride, followed by deprotection of the trityl group (31, 32). Compound 8 is a common intermediate used to synthesize the desired farnesylated and geranylgeranylated haptens SuccG-(gg)CMe 1 and SuccG-(f)CMe 2 (33).

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Liu et al.

Scheme 3. Model Reaction To Form Succinimidea

a Reagents and conditions: (a) Succinic anhydride, HOAc, 79%; (b) SDPP, CH3CN.

Table 1. Haptens, Carrier Proteins, and Immunogens for Preparation and Characterization of Anti-Isoprenyl Antibodies haptens

carrier

conjugates

SuccG-(gg)CMe 1 SuccG-(f)CMe 2 SuccG-(gg)CMe 1 SuccG-(f)CMe 2 (gg)-CMe 19 (f)-CMe 20 Palm-AcCys 21 Myr-Gly

KLH KLH BSA BSA BSA BSA BSA BSA

KLH-SuccG-(gg)CMe 15 KLH-SuccG-(f)CMe 16 BSA-SuccG-(gg)CMe 17 BSA-SuccG-(f)CMe 18 BSA-(gg)CMe 22 BSA-(f)CMe 23 BSA-Palm-AcCys 24 BSA-Myr-Gly 25

Originally, we planned on using the N-hydroxysuccimidyl (NHS) esters 9 and 10 of compounds 1 and 2 as active haptens ready for coupling to BSA or KLH (Scheme 2). Pure products were obtained when 1 and 2 were treated with SDPP in CH3CN (34). However, immunization of chickens with their BSA or KLH conjugates did not produce any anti-isoprenyl reactivity in the IgY fractions. After careful examination of the chemistry of the immunogen preparation, we discovered an unexpected side reaction. When SDPP is added to SuccG-(gg)CMe 1 or SuccG-(f)CMe 2, the NHS esters 9 and 10, respectively, appear to form as unstable intermediates that rapidly undergo intramolecular cyclization to the more stable succinimide side products 11 and 12. To confirm this hypothesis, a model reaction using methoxyethylamine was conducted, and 1-(2-methoxyethyl)-pyrrolidine-2,5-dione 14 was isolated as the main product (Scheme 3). Successful preparation of immunogens was achieved by conjugation of SuccG-(gg)CMe 1 or SuccG-(f)CMe 2 to BSA and KLH using EDCI (Table 1) (35). The reactions were complete in 2 h as determined by TLC. Longer reaction times resulted in the formation of the succinimide byproducts. Four immunogens, KLH-SuccG-(gg)CMe 15, KLH-SuccG-(f)CMe 16, BSA-SuccG-(gg)CMe

Figure 1. Dot blotting of antisera from rabbits; dilution 1:500.

17, and BSA-SuccG-(f)CMe 18 were obtained and then used to immunize both rabbits and mice. Six weeks later the animals were bled and sacrificed to collect antisera. Rabbit and mouse antisera containing polyclonal antibodies were checked with dot blotting. Figure 1 shows that the rabbit antisera contained antibodies against all four immunogens (15, 16, 17, 18). None of the mousederived antisera contained sufficiently high titers of either anti-farnesyl or antigeranylgeranyl antibodies to allow us to proceed further with fusion and cloning (data not shown). Antisera from rabbits immunized with KLH-SuccG-(gg)CMe 15 (designated serum-15) or KLHSuccG-(f)CMe 16 (serum-16) had the same patterns and did not recognize unmodified BSA. In contrast, antisera from rabbits immunized with BSA-SuccG-(gg)CMe 17 (serum-17) and BSA-SuccG-(f)CMe 18 (serum-18) were similar and showed a cross reaction with KLH. We thus selected serum-15 as the starting material for subsequent studies using BSA conjugates. To determine whether serum-15 could recognize (gg)CMe and (f)CMe epitopes, two additional immunogens, BSA-(gg)-CMe 22 and BSA-(f)-CMe 23, were synthesized from geranygeranylated or farnesylated cysteine methyl esters 19 and 20 (Scheme 4A) (36). ELISA on Maxisorp plate (Figure 2) further showed that serum-15 recognized both farnesylated and geranylgeranylated immunogens (17, 18, 22, 23) and that the selectivity was low. We also asked whether, given the lack of selectivity between the farnesyl and geranylgeranyl groups, the serum-15 antibodies could distinguish prenylation from simple fatty acid modifications such as S-palmitoylation and N-terminal myristoylation. To test this hypothesis, first, we synthesized BSA-Palm-AcCys 24 and BSA-

Scheme 4. Synthesis of Immunogens for Characterization of Polyclonal Antibodiesa

a Reagents and conditions: (a) farnesyl bromide or geranylgeranyl chloride, Zn(OAc) ‚2H O, DMF/CH CN/TFA; (b) EDCI; (c) 2 2 3 palmitoyl chloride, Et3N, 21%.

Polyclonal Anti-Isoprenyl Antibodies

Figure 2. ELISA of rabbit serum-15 on Maxisorp plate. Immunogens (1 µg/well) were added to 96-well plate. In the negative control, no antiserum was added. See text for experiment details.

Figure 3. ELISA of BSA conjugates (17, 18, 22, 23, 24, and 25) using immunoaffinity purified anti-gg antisera in a series of concentration (anti-f antisera gave similar results). Aliquots (1 µg) of BSA and BSA conjugates were added into each well on 96-well plate.

Myr-Gly 25 (Scheme 4B and Table 1). Second, we attempted to obtain selective polyclonal anti-gg or anti-f antibodies from serum-15 by positive immunoaffinity chromatography. Thus, BSA-SuccG-(gg)CMe (17) or BSA-SuccG-(f)CMe (18) was coupled to Affigel-10, and these resins were then incubated with pretreated serum15 to bind the desired specific antibodies. After washing the resin beads with sodium phosphate buffer, followed by elution with glycine buffer, pH 2.0, putative anti-gg-

Bioconjugate Chem., Vol. 15, No. 2, 2004 275

or anti-f-containing fractions were collected in Tris-Cl buffer (2 M, pH 8.8). To our surprise, both the anti-gg and the anti-f fractions showed strong cross-reactivity with these aliphatic modifications as well (Figure 3). It is apparent that rabbit polyclonal antibodies poorly distinguish aliphatic-modified peptides. One future opportunity will be exploring the use of these antibodies as “universal” detectors of lipidated proteins in cells for a proteomic approach to identify differentially expressed lipidated proteins in cells (37-40). To address whether serum-15 could be used to stain isoprenylated proteins in cells, we transfected CHO-K1 cells with EYFP-CVLL (41-43), a fluorescent protein with a C-terminal CAAX sequence for geranylgeranylation. In the G1 phase of the cell cycle, tranasfected cells overexpressed and geranylgeranylated EYFP-CVLL, thus facilitiating movement of the EYFP transport to cell plasma membrane (Figure 4A). When the cells were fixed and treated with serum-15 followed by anti-rabbit IgGTexas Red, red fluorescence was observed, which indicated colocalization with EYFP in the cell plasma membrane (Figure 4B). As a control, staining of native CHOK1 cells with the same protocols gave dim red fluorescence in cytosol (Figure 4C). When the cells were treated with lovastatin, which blocks HMG CoA reductase and thus stops the production of FPP and GGPP, EYFP-CVLL could not be geranylgeranylated and thus remained localized in cytosol (Figure 4D) (44-48). Only dim red fluorescence could be observed in transfected and native CHO-K1 cells (Figure 4E, 4F). Taken together, these results demonstrate that serum-15 could be used to visualize overexpressed isoprenylated proteins in cells. The background fluorescence can be attributed either to nonspecific binding or to low-abundance of naturally occurring prenylated proteins. In summary, the synthetic immunogens KLH-SuccG(gg)CMe (15), KLH-SuccG-(f)CMe (16), BSA-SuccG(gg)CMe (17), and BSA-SuccG-(f)CMe (18) stimulated strong immune responses in rabbits, affording antisera containing high titers of polyclonal anti-lipid antibodies. The lack of selectivity of these antisera suggest that distinguishing among specific lipidated proteins in cells would not be feasible. However, the antisera can visualize overexpressed isoprenylated cell proteins localized to the cell membrane, suggesting that perhaps a more universal

Figure 4. Immunofluorescence of EYFP-CVLL CHO-K1 cells and native CHO-K1 cells. Panel A: EYFP-CVLL cell, 10 h after seeding; Panel B: EYFP-CVLL cells stained with serum-15 (1:150), 10 h after seeding; Panel C: Native CHO-K1 cells stained with serum-15 (1:150), 10 h after seeding; Panel D: EYFP-CVLL cells was treated with 20 µM lovastatin for 24 h; Panel E: EYFP-CVLL cells were treated with 20 µM lovastatin for 24 h and then stained with serum-15 1:150; Panel F: Native CHO-K1 cells were treated with 20 µM lovastatin for 24 h and then stained with serum-15 (1:150).

276 Bioconjugate Chem., Vol. 15, No. 2, 2004

tool for identification of electrophoretically separated lipidated proteins could be developed (37-40). ACKNOWLEDGMENT

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