Hessler J L, Kreitman R J
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Biochemistry. 1997 Nov 25;36(47):14577-82. doi: 10.1021/bi971447w.
During the intoxication process, Pseudomonas exotoxin (PE) and immunotoxins containing PE internalize into the target cell and become processed into two fragments, and the carboxyl fragment translocates into the cytosol where it inactivates elongation factor 2. We have proposed that after internalization into cells the carboxyl-terminal fragment of PE (amino acids 280-613), which ends in REDLK, binds to the KDEL receptor (ERD2) which carries it to the endoplasmic reticulum, from which the PE fragment translocates to the cytosol. Earlier experiments showing that REDL but not REDLK binds to the KDEL receptor suggested that the terminal lysine is removed sometime during the intoxication process. To determine if and where this occurs, we exposed a peptide ending in REDLK to malignant cells in culture and found that binding to the KDEL receptor was restored. Restoration of receptor binding also occurred if a peptide or toxin ending in REDLK at its carboxyl terminus was incubated with plasma, indicating that the terminal lysine is removed prior to entry of the toxin into the cell. We conclude that plasma carboxypeptidase(s) cleave(s) the lysine residue from the carboxyl terminus of PE and PE-containing immunotoxins as an early and essential step in their cellular intoxication pathway.
在中毒过程中，铜绿假单胞菌外毒素（PE）和含PE的免疫毒素内化进入靶细胞并被加工成两个片段，羧基片段转移到细胞质中，在那里它使延伸因子2失活。我们提出，PE的羧基末端片段（氨基酸280 - 613）在细胞内化后，以REDLK结尾，与KDEL受体（ERD2）结合，该受体将其携带到内质网，PE片段从内质网转移到细胞质中。早期实验表明，REDL而非REDLK与KDEL受体结合，这表明末端赖氨酸在中毒过程中的某个时候被去除。为了确定这种情况是否发生以及在哪里发生，我们将以REDLK结尾的肽暴露于培养中的恶性细胞，发现其与KDEL受体的结合得以恢复。如果将羧基末端以REDLK结尾的肽或毒素与血浆一起孵育，受体结合也会恢复，这表明末端赖氨酸在毒素进入细胞之前就被去除了。我们得出结论，血浆羧肽酶从PE和含PE的免疫毒素的羧基末端切割赖氨酸残基，这是它们细胞中毒途径中的一个早期且关键的步骤。
