Biologically active interleukin 2-ricin A chain fusion proteins may require intracellular proteolytic cleavage to exhibit a cytotoxic effect.
DNA fusions encoding chimeric proteins in which human interleukin 2 (IL2) was fused to the A subunit of the plant cytotoxin ricin (RA) have been expressed in Xenopus oocytes. The constructs contained N-terminal IL2 and C-terminal RA, or N-terminal RA and C-terminal IL2. In the expressed chimeric proteins, the IL2 and RA moieties were joined by a peptide sequence containing a proteolytic cleavage site. Two proteolytically-sensitive peptide sequences were utilized; a peptide that forms the trypsin-sensitive disulfide-bonded loop in diphtheria toxin (DT) or a synthetic peptide containing the factor Xa recognition site in a sequence flanked by two cysteine residues. In an in vitro cell free system the RA component was biologically active in all chimeric proteins produced since it specifically depurinated 28S ribosomal RNA. Proteolytic cleavage of the chimeras with either trypsin or factor Xa as appropriate separated the IL2 and RA moieties, but they did not remain covalently linked by a disulfide bond. Because of this, the cytotoxicity of protease-treated chimeras could not be assessed. Chimeras not pretreated with factor Xa but which contained the factor Xa target sequence were not cytotoxic to CTLL-2 cells. Rather, these molecules had a stimulatory effect that was ascribed to the IL2 moiety. In contrast, recombinant chimeric toxins containing the DT loop sequence were cytotoxic to CTLL-2 cells. Taken together the data suggest that RA-containing chimeras require intracellular proteolytic cleavage to release the RA moiety to render them cytotoxic to target cells.
Cook
J P
JP
Department of Biological Sciences, University of Warwick, Coventry, UK.
Savage
P M
PM
Lord
J M
JM
Roberts
L M
LM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001711:Biotransformation; D004247:DNA / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007376:Interleukin-2 / Q000235:genetics / Q000493:pharmacokinetics* / Q000633:toxicity*; D051379:Mice; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000235:genetics; D012339:RNA, Ribosomal, 28S / Q000187:drug effects; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000493:pharmacokinetics* / Q000494:pharmacology / Q000633:toxicity*; D012276:Ricin / Q000235:genetics / Q000493:pharmacokinetics* / Q000633:toxicity*; D014158:Transcription, Genetic / Q000235:genetics; D014981:Xenopus
D007376:Interleukin-2; D012339:RNA, Ribosomal, 28S; D011993:Recombinant Fusion Proteins; D004247:DNA; D012276:Ricin; D010447:Peptide Hydrolases