Role of tyrosine residues in modulation of claudin-4 by the C-terminal fragment of Clostridium perfringens enterotoxin.

The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) modulates the barrier function of claudin-4 via its C-terminal 16 amino acids. In the current study, we investigated the roles of tyrosine residues (Y306, Y310 and Y312) in this region in the modulation of TJs by C-CPE. Single mutations of Y306, Y310 and Y312 to alanine resulted in partial reduction of claudin-4 binding. We also prepared double mutants of C-CPE to further evaluate the roles of these tyrosine residues. Replacement of Y310 and Y312 with alanine (Y310A/Y312A) partly reduced the ability of C-CPE to bind to claudin-4. Double mutants Y306A/Y310A and Y306A/Y312A, however, lost the ability to bind to claudin-4 and to modulate the TJ barrier. We also found that a triple mutant (Y306A/Y310A/Y312A) lost the ability to bind claudin-4, modulate the TJ barrier, and enhance jejunal absorption in rats. These results indicate that tyrosines 306, 310, and 312 are critical for the interaction of C-CPE with claudin-4 and for the modulation of TJ barrier function by C-CPE. This study provides information that should help in the development of claudin modulators based on C-CPE.

Role of Tyr306 in the C-terminal fragment of Clostridium perfringens enterotoxin for modulation of tight junction.

We previously reported that the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is a novel type of absorption enhancer that interacts with claudin-4 and that Tyr306 of C-CPE plays a role in ability of C-CPE to modulate barrier of tight junctions. In the current study, to investigate effects of Tyr306 on the C-CPE activity, we prepared some C-CPE mutants substituted Tyr306 with Trp (Y306W), Phe (Y306F) and Lys (Y306K). We found that Y306W and Y306F mutants of C-CPE had claudin-4 binding affinities and effects on the barrier function of tight junctions, whereas both of these properties were greatly reduced with the Y306K mutant. Finally, the Y306K but not the Y306F and Y306W mutants had reduced abilities to enhance absorption in rat jejunum. These results indicate that aromatic and hydrophobic properties, not hydrogen bonding potential, of Tyr306 are involved in the interaction of C-CPE with claudin-4 and in the modulation of the tight junction barrier function by C-CPE.

Induction of zinc transporters by forskolin in human trophoblast BeWo cells.

During pregnancy, the zinc level in fetal serum is up to two-fold higher than that in the maternal serum at the end of pregnancy, but the mechanism of zinc release from the placenta into fetal circulation is not well understood. In this study, we determined the expression profiles of zinc transporters in human trophoblast BeWo cells, a representative human trophoblast cell line. Zn transporter 1-8 (ZnT1-8), Zrt/IRT-like protein 1 and Zn transporter-like transporter 1 were detected in BeWo cells by reverse transcription-polymerase chain reaction. Forskolin (FK) is a representative inducer of differentiation of BeWo cells cytotrophoblast into syncytiotrophoblast. Treatment of BeWo cells with FK resulted in morphological changes of BeWo cells into syncytiotrophoblast cells and secretion of human chorionic gonadotropin, which is a characteristic of syncytiotrophoblast cells. Treatment of BeWo cells with FK elevated ZnT1, 2 and 4 mRNA levels. These data about expression profiles of ZnTs may be useful for further investigation of placental biology.

Preparation of a claudin-targeting molecule using a C-terminal fragment of Clostridium perfringens enterotoxin.

Although most malignant tumors are epithelia-derived carcinomas, methods for specific and effective delivery of antitumor agents to carcinomas have not been developed. Recent reports indicate that epithelia overexpress claudin-3 and -4, which are integral membrane proteins of epithelial tight junctions. This suggests that claudins can be targeted for tumor therapy, but there is not currently a method for delivering drugs to claudin-expressing cells. In the present study, we evaluated whether a potent claudin-4-binding C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) would allow targeting to claudin-4-expressing cells. We fused C-CPE to the protein synthesis inhibitory factor (PSIF), which lacks the cell binding domain of Pseudomonas exotoxin. This fusion protein, C-CPE-PSIF, was cytotoxic to MCF-7 human breast cancer cells, which express endogenous claudin-4, but it was not toxic to mouse fibroblast L cells, which lack endogenous claudin-4. The cytotoxicity of C-CPE-PSIF was attenuated by pretreating the MCF-7 cells with C-CPE but not bovine serum albumin. Also, deletion of the claudin-4-binding region of C-CPE reduced the cytotoxicity of C-CPE-PSIF. Finally, we found that C-CPE-PSIF is toxic to L cells expressing claudin-4 but not to normal L cells or cells expressing claudin-1, -2, or -5. These results indicate that use of the C-CPE peptide may provide a novel way to target drugs to claudin-expressing cells.

Expression profiles of zinc transporters in rodent placental models.

Zinc is a vital metal that is a structural and functional component of many proteins. The precise mechanism of zinc transport in the placenta remains unclear. In this study, we investigated the expression of zinc transporters (ZnTs) in the mouse placenta and in two rat trophoblast cell lines, TR-TBT cells, which are syncytiotrophoblast cells of the labyrinth zone, and Rcho-1 cells, which retain trophoblast cell features and differentiate into trophoblast giant cells of the junctional zone. All of the ZnTs that have been identified in mice (ZnT1-7) were detected in the mouse placenta by RT-PCR. The expression profiles of ZnTs in the placenta during pregnancy were different. The mRNA levels of ZnTs, with the exception of ZnT7, did not change during pregnancy. The ZnT7 mRNA level in placenta was elevated during pregnancy. In TR-TBT cells, ZnT1, ZnT3 and ZnT4 were detected by RT-PCR analysis. In Rcho-1 cells, all of the ZnTs that have been identified in rats (ZnT1-4) were detected by RT-PCR analysis. There were no differences between the mRNA expression levels of ZnT family members in undifferentiated Rcho-1 cells and differentiated Rcho-1 cells. This is the first report of expression profiles of ZnTs during differentiation of the placenta in the mouse placenta and rat placental cell models.