Expression and purification of a histidinetagged Clostridium perfringens enterotoxin COOH-terminal fragment (H₁₀PER). H₁₀PER was expressed in E. coli BL21 (DE3) and purified as described in Materials and Methods. A total cell lysate (lane 1), flow through fraction (lane 2), and purified H₁₀PER (lane 3) were separated on SDS-15% polyacrylamide gel and stained with Coomassie brilliant blue. Purified H₁₀PER was transferred to a PVDF membrane, and Western blot analysis was carried out using rabbit polyclonal anti-CPE antibody, followed by alkaline phosphatase–conjugated anti–rabbit IgG (lane 4). The positions of molecular weight standards are indicated on the left in kD.

Detection of the CPE receptor by a flow cytometric analysis. Vero (A), Hep3B (B), Henle Intestine 407 (C), L929 (D), K562 (E), and JY (F) cells were treated with biotinylated H₁₀PER (thick lines) or buffer alone (dotted lines) followed by PE-conjugated streptavidin. For (A), cells were preincubated with unlabeled H₁₀PER as a competitor (thin line). The plots show cell numbers on the ordinate and relative fluorescences on a log scale on the abscissa.

Cloning and sequence analysis of the CPEreceptor cDNA. (A) Cloning of the CPE receptor. L929pyT18 cells were transfected with 10 μg of pME18Sf(−)pyori vector (thick line) or 10 μg of the Vero cDNA library rescued after the third round of screening (thin line). Flow cytometric analysis was done as described in Materials and Methods. The plot displays cell number on the ordinate and relative fluorescence on a log scale on the abscissa. (B) Nucleotide and deduced amino acid sequence of CPE-receptor cDNA. The deduced amino acid sequence (single letter code) is shown under the nucleotide sequence. The underlines indicate possible phosphorylation sites. A putative polyadenylation signal is indicated by bold letters. These sequence data are available from GenBank/EMBL/DDBJ under accession number D88492. (C) Hydrophobicity profile of CPE receptor. The hydrophobicity index was determined by the Kyte and Doolittle algorithm (Kyte and Doolittle, 1982). The four putative transmembrane segments are indicated by numbers above the line. (D) Alignment of the amino acid sequence of CPE receptor with those of the rat androgen withdrawal apoptosis protein RVP1 (Genbank/EMBL/DDBJ accession number M74067) and the mouse oligodendrocyte specific protein (GenBank/EMBL/DDBJ accession number U19582). Identical amino acids are shown by white letters in black boxes, and conservative replacements are shadowed. Triple alignment was done by the CLUSTAL method (Higgins and Sharp, 1989).

Northern blot analysis of CPE-R. Total RNA samples (15 μg/lane) from Vero (lane 1), K562 (lane 2), Hep3B (lane 3), JY (lane 4), and Intestine 407 (lane 5) cell lines were subjected to Northern blot analysis. The blots were hybridized with ³²Plabeled DNA probes from the entire sequence of clone 706 containing CPE-R cDNA (top). The blots were rehybridized with ³²P-labeled human EF-1α probe (bottom) to confirm the amounts and integrities of the samples. The positions of 28 and 18S ribosomal RNA are indicated on the right.

Functional analysis of stable cell lines expressing the CPE receptor. (A) Clonal L929 cell lines expressing the CPE receptor were isolated as described in Materials and Methods. Expression of the CPE-R gene product was examined by flow cytometric analysis in a typical cell line expressing CPE-R (thick line, indicated as Neo706) and parental L929 cell line (thin line, indicated as control), as shown in Fig. 2. Cell numbers are shown on the ordinate and relative fluorescence on a log scale on the abscissa. (B) Comparison of sensitivities of typical cell line expressing the CPE receptor (top) and the parental L929 cell line (bottom) to CPE. The cells were not treated with CPE (top and bottom, denoted as none) or with CPE at 100 ng/ml (top, denoted as CPE), or 5 μg/ml (lower right, denoted as CPE) in DME supplemented with 10% FCS for 30 min at 37°C. Bar, 30 μm. (C) Scatchard plot analysis of the binding of ¹²⁵I-CPE to the L929 cell line expressing CPE-R. Scatchard plot analysis giving a K_a value of 1.49 × 10⁸ M⁻¹ for ¹²⁵I-CPE binding. (Inset) Various concentrations of ¹²⁵I-CPE were incubated with the L929 cell line expressing CPE-R (0.4 × 10⁶ cells each concentration), and specific binding was determined as described before (Horiguchi et al., 1985). Symbols represent means ± SD (n = 6).

Specific and direct binding of CPE to the CPEreceptor. L929 cell lines expressing the CPE receptor with FLAG tag peptide at the COOH-terminal end were established as described in Materials and Methods, and a typical cell line (706FLAG) was selected. Cell lysates were prepared from the parental L929 (lanes 1, 3, and 5) and 706FLAG (lanes 2, 4, and 6) cell lines and subjected to Western blot analysis with anti-FLAG antibody (lanes 1 and 2) and ligand overlay assay (lanes 3 to 6). Ligand overlay assay was carried out using ¹²⁵I-CPE with (lanes 5 and 6) or without (lanes 3 and 4) cold CPE. The positions of molecular weight standards are indicated on the right in kD.

Presence of both the CPE and CPE receptor in a CPE-induced high molecular weight complex. (A) 706 FLAG cells were labeled with [³⁵S]methionine and treated with buffer alone (lanes 1 and 2) or buffer containing either 140 nmol of H₁₀PER (lanes 3 and 4) or CPE (lanes 5 and 6) for 30 min at 4°C (odd-numbered lanes) or 37°C (even-numbered lanes). The cells were solubilized with PBS-0.5% NP-40, and the lysates were immunoprecipitated with anti-FLAG antibody. Precipitated proteins were separated by SDS-15% PAGE, and protein bands were located with Bioimaging Analyzer. The positions of molecular weight standards are indicated on the left in kD. (B) 706FLAG cells (lanes 1–3 and 5–7), and parental L929 cells (lanes 4 and 8) were treated with buffer alone (lanes 1 and 5) or buffer containing either 140 nmol of H₁₀PER (lanes 2 and 6) or CPE (lanes 3, 4, 7, and 8). The cells were solubilized with PBS-0.5% NP-40 and immunoprecipitated with anti-FLAG antibodies. The precipitates were separated by SDS-15% PAGE (top) or SDS-2–15% gradient PAGE (bottom; note that only the relevant area is shown), transferred to a PVDF membrane, and then treated with biotinylated anti-CPE (lanes 1–4) or anti-FLAG (lanes 5–8) antibodies followed by alkaline phosphatase–conjugated streptavidin. As a control, 7 nmol of purified CPE was applied to SDS-2–15% polyacrylamide gradient gel, and then subjected to Western blotting with biotinylated anti-CPE antibody (bottom, lane 9). The positions of molecular weight standards are indicated on the left in kD.