The action of leucyl-leucine methyl ester on cytotoxic lymphocytes requires uptake by a novel dipeptide-specific facilitated transport system and dipeptidyl peptidase I-mediated conversion to membranolytic products.
The mechanism of toxicity for cytolytic lymphocytes of Leu-Leu-OMe and related dipeptide derivatives was examined. Selective inhibition of dipeptidyl peptidase I (DPPI), a lysosomal thiol protease highly enriched in cytotoxic lymphocytes, prevented all natural killer (NK) toxic effects of such agents. However, many DPPI substrates were found to possess no NK toxic properties. For some such agents, this lack of NK toxicity appeared to be related to the lack of uptake by lymphocytes. In this regard, Leu-Leu-OMe was found to be incorporated by lymphocytes and monocytes via a saturable facilitated transport mechanism with characteristics distinct from previously characterized mammalian dipeptide transport processes. This novel transport process was found to be specific for dipeptides composed of selective L-stereoisomer amino acids and enhanced by hydrophobic ester or amide additions to the COOH terminus of dipeptides. Maximal rates of Leu-Leu-OMe uptake by T8 and NK cell-enriched peripheral blood lymphocytes (PBL) were four- to sixfold higher than for T4-enriched PBL or PBL depleted of Leu-Leu-OMe-sensitive cytotoxic lymphocytes. All dipeptide amides or esters with NK toxic properties were found to act as competitive inhibitors of [3H]Leu-Leu-OMe uptake by PBL. However, some NK nontoxic DPPI substrates were found to be comparable with Leu-Leu-OMe in avidity for this transport process. Such agents were noted to possess one or more hydrophilic amino acid side chains and were found not to mediate red blood cell lysis when subjected to the acyl transferase activity of DPPI. Thus, uptake by a dipeptide-specific facilitated transport mechanism and conversion by DPPI to hydrophobic polymerization products with membranolytic properties were found to be common features of NK toxic dipeptide derivatives. The presence of a previously unreported dipeptide transport mechanism within blood leukocytes and the selective enrichment of the granule enzyme, DPPI, within cytotoxic effector cells of lymphoid or myeloid lineage appear to afford a unique mechanism for the targeting of immunotherapeutic reagents composed of simple dipeptide esters or amides.
Thiele
D L
DL
Liver Unit, University of Texas Southwestern Medical Center, Dallas 75235.
Lipsky
P E
PE
D000217:Acyltransferases / Q000378:metabolism; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D001693:Biological Transport, Active; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D020789:Cathepsin C; D004151:Dipeptides / Q000378:metabolism* / Q000494:pharmacology / Q000633:toxicity*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007694:Killer Cells, Natural / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects* / Q000378:metabolism; D013696:Temperature
D004151:Dipeptides; C045139:leucyl-leucine-methyl ester; C041865:glycylphenylalanine 2-naphthylamide; D000217:Acyltransferases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C