Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of
thiopurine drugs such as
6-mercaptopurine. Levels of TPMT activity in human tissue are controlled by a common genetic polymorphism that is an important factor responsible for individual variation in
thiopurine drug toxicity and therapeutic efficacy. Our goal was to purify, to obtain a partial amino acid sequence for, and to clone and express
cDNA for human TPMT as a first step in determining the molecular basis for this genetic polymorphism. Human kidney TPMT was purified, the
protein was subjected to limited proteolysis, and amino acid sequence information was obtained from the resultant
peptide fragments. Primers based on the amino acid sequence information were used to amplify a unique sequence from human liver
cDNA by use of the polymerase chain reaction. Because TPMT has been reported to be present in the colon, T84 human colon
carcinoma cells were studied and were found to express TPMT activity with biochemical properties similar to those of the human kidney and liver
enzymes.
Oligonucleotide probes based on the human kidney TPMT amino acid sequence were then used to screen a T84 human colon
carcinoma cell cDNA library. A 2.7-kilobase
cDNA clone was isolated that contained an open reading frame of 735
nucleotides, which encoded a
protein of 245
amino acids. The deduced amino acid sequence of the encoded
protein included one 24- and two separate 12-amino
acid sequences identical to those obtained by sequencing proteolytic fragments of purified human kidney TPMT. Transcripts were made in vitro from the open reading frame of the
cDNA clone. These transcripts were translated in a rabbit reticulocyte lysate system, and the resulting translation product comigrated with human kidney TPMT in
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis. The T84 cell
cDNA clone, truncated within the
3' untranslated region at an Sstl restriction site, was then used to create an expression construct with the eukaryotic expression vector P91023(B), and this construct was used to transfect COS-1 cells. The transfected cells expressed a high level of TPMT enzymatic activity, and this activity displayed a pattern of inhibition by TPMT inhibitors identical to that of human kidney and T84 human colon
carcinoma cell TPMT. Cloning of
cDNA for this important
drug-metabolizing
enzyme may make it possible to define the molecular basis of the TPMT genetic polymorphism in humans.