Distinct roles for the N- and C-terminal regions in the cytotoxicity of pierisin-1, a putative ADP-ribosylating toxin from cabbage butterfly, against mammalian cells

Abstract

Pierisin-1 is an 850-aa cytotoxic protein found in the cabbage butterfly, Pieris rapae, and has been suggested to consist of an N-terminal region with ADP-ribosyltransferase domain and of a C-terminal region that might have a receptor-binding domain. To elucidate the role of each region, we investigated the functions of various fragments of pierisin-1. In vitro expressed polypeptide consisting of amino acid residues 1-233 or 234-850 of pierisin-1 alone did not show cytotoxicity against human cervical carcinoma HeLa cells. However, the presence of both polypeptides in the culture medium showed some of the original cytotoxic activity. Introduction of the N-terminal polypeptide alone by electroporation also induced cell death in HeLa cells, and even in the mouse melanoma MEB4 cells insensitive to pierisin-1. Thus, the N-terminal region has a principal role in the cytotoxicity of pierisin-1 inside mammalian cells. Analyses of incorporated pierisin-1 indicated that the entire protein, regardless of whether it consisted of a single polypeptide or two separate N- and C-terminal polypeptides, was incorporated into HeLa cells. However, neither of the terminal polypeptides was incorporated when each polypeptide was present separately. These findings indicate that the C-terminal region is important for the incorporation of pierisin-1. Moreover, presence of receptor for pierisin-1 in the lipid fraction of cell membrane was suggested. The cytotoxic effects of pierisin-1 were enhanced by previous treatment with trypsin, producing "nicked" pierisin-1. Generation of the N-terminal fragment in HeLa cells was detected after application of intact entire molecule of pierisin-1. From the above observations, it is suggested that after incorporation of pierisin-1 into the cell by interaction of its C-terminal region with the receptor in the cell membrane, the entire protein is cleaved into the N- and C-terminal fragments with intracellular protease, and the N-terminal fragment then exhibits cytotoxicity.

Figure 1

Fluorescence micrographs of Cy2-pierisin-1-treated HeLa and MEB4 cells. (A) HeLa cells were incubated with 2 μg/ml Cy2-pierisin-1 for 1 h at 37°C. (B) HeLa cells were incubated with the same concentration of Cy2-pierisin-1 for 1 h at 4°C. (C) The medium containing Cy2-pierisin-1 was then removed, and the cells were further incubated in a medium free of Cy2-pierisin-1 for 1 h at 37°C. (D) MEB4 cells were incubated with 2 μg/ml Cy2-pierisin-1 for 1 h at 37°C.

Figure 2

Cytotoxicity of the N- and C-terminal polypeptides of pierisin-1 against HeLa cells. Each translated protein in lysate was added to a culture medium containing HeLa cells and incubated for 72 h. Horizontal bars represent polypeptides of pierisin-1. Dashed vertical line is the trypsin cleavage site. The 165th glutamic acid residue is considered to be the putative NAD binding site and indicated as “E.” The mutation site by replacement of the 165th glutamic acid with glutamine is indicated as “Q” (4). (I) The reticulocyte lysate containing 1–850 full-length pierisin-1; the concentration of lysate in culture medium was 0.04%. (II) 1–233 N-terminal polypeptide, >6.4%. (III) 234–850 C-terminal polypeptide, >6.4%. (IV) 1–233 N-terminal plus 234–850 C-terminal polypeptides, 0.02% N + 0.06% C. (V) E165Q 1–233 N-terminal polypeptide, >6.4%. Under the above conditions, about 50% of cell death was detected. The translation efficacy of each polypeptide in rabbit reticulocyte lysate used in this study generally depended on its length. The amounts of the 1–233 N-terminal and 234–850 C-terminal polypeptides in lysate were around 10- and 3-fold greater than that of 1–850 full-length pierisin-1, respectively. Consequently, the activity of the mixture of N- plus C-terminal polypeptides was estimated to be about one-tenth of that of the full-length protein.

Figure 3

Dose-dependent cytotoxic effects of the N- and C-terminal polypeptides of pierisin-1. Cells were electroporated with various concentrations of lysates, incubated for 72 h, and subjected to a cell proliferation assay. ■, control lysate without any translated proteins; ●, lysate containing 1–233 N-terminal polypeptide; □, 234–850 C-terminal polypeptide; ○, N-terminal E165Q polypeptide. (A) HeLa cells. (B) MEB4 cells. Each experiment was carried out twice, and an average value of proliferation activity at each concentration of lysate in electroporation cuvette is given.

Figure 4

Morphological changes in HeLa and MEB4 cells that incorporated the N- and C-terminal polypeptides of pierisin-1 by electroporation. HeLa cells were electroporated with 1.6% control lysate alone (A), with 1.6% lysate containing 1–233 N-terminal polypeptide (B and E), with 1.6% lysate containing 234–850 C-terminal polypeptide (C), or without lysate (D). (F) MEB4 cells were electroporated with 1.6% lysate containing 1–233 N-terminal polypeptide. Electroporated cells were incubated for 18 h at 37°C. (A–D, F) Phase-contrast micrographs. (E) Fluorescence micrograph of Hoechst 33342-stained cells.

Figure 5

Detection of various incorporated pierisin-1 polypeptides. (A) Western blot analysis of HeLa cells treated with native pierisin-1. Cells were incubated with 1 μg/ml pierisin-1 for 0 min (lane 1) and 15 min (lane 2) at 37°C. Medium containing pierisin-1 was then removed from the culture, and the cells were further incubated without pierisin-1 for another 45 min (lane 3) and 3 h (lane 4). Anti-purified pierisin-1 (full-length) and anti-peptide corresponding to amino acids 101–123 of pierisin-1 (N-specific) antibodies were used to detect pierisin-1. (B) SDS/PAGE of radioisotope-labeled pierisin-1 fragment incorporated into HeLa cells. Cells were incubated with 2% lysate containing the [³⁵S]methionine-labeled C-terminal polypeptide plus 2% control lysate (*C) or plus 2% lysate containing the nonradioisotopic N-terminal polypeptide (*C + N) for 0, 30, and 180 min. Cells were also incubated with 2% lysate containing [³⁵S]methionine-labeled 1–233 N-terminal polypeptide plus 2% control lysate (*N) or plus 2% lysate containing nonradioisotopic 234–850 C-terminal polypeptide (*N + C) for 0, 30, and 180 min.

Figure 6

Effect of membrane fraction from HeLa or MEB4 cells on cytotoxicity of pierisin-1 to HeLa cells. A sample of 0.5 ng of native pierisin-1 was preincubated in a 50-μl aliquot solution containing membrane fraction from HeLa and MEB4 cells for 1 h at 4°C. The appropriate volume of preincubated mixture was combined with the cultured medium of HeLa cells and incubated for 15 min at 37°C. Then, cells were washed and further incubated for 72 h. ■, pierisin-1 only. ●, pierisin-1 plus membrane fraction from HeLa cells. □, pierisin-1 plus membrane fraction from MEB4 cells. Each experiment was carried out twice, and an average value of proliferation activity is given.