Related Experiment Video
Updated: Sep 23, 2026
Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods
Published on: July 17, 2019
Molecular characterization of 5'-deoxy-5'-methylthioadenosine phosphorylase-deficient mutant clones of murine
Yuwaraj Kadariya1, Junji Nishioka, Akiko Nakamura
1Department of Laboratory Medicine, Mie University School of Medicine and Central Clinical Laboratories, Mie University Hospital, Tsu, Mie 514-8507, Japan. nobori@clin.medic.mie-u.ac.jp
Abstract:
5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAP), the polyamine and purine metabolic enzyme, is ubiquitously expressed in normal human tissues and cells, but is deficient in a variety of human and murine malignant cell lines. MTAP-deficient mutants were previously selected from murine lymphoma cell line R1.1 that contains abundant MTAP activity, to analyze the metabolic consequences of MTAP deficiency. Two mutants, one with partial deficiency (F clone) and the other with complete deficiency (H5 clone), were found to have the MTAP protein by immunoblotting. However, the molecular mechanism of enzyme deficiency in these mutants has not been established. In this study, we cloned the mouse cDNA and analyzed MTAP mRNA in the two mutants by reverse transcription-polymerase chain reaction (RT-PCR) followed by direct sequencing. Both mutants have a single nucleotide substitution at the third base of codon 223, which results in a change of cysteine to tyrosine (C223Y). The MTAP mRNA level determined by RT-PCR was significantly lower in the mutant with complete deficiency than in wild-type (WT) R1.1 cells, whereas the mRNA level in the mutant with partial deficiency was comparable to that in WT R1.1 cells. C223Y mutation may cause conformational alteration of the methylthioribose-binding site and decrease the substrate binding. Thus, C223Y may account for the partial deficiency in the F clone, but the complete deficiency with a remarkably low MTAP mRNA level in the H5 clone may be a result of transcriptional abnormality in addition to C223Y mutation.
Insights
5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAP) deficiency in cancer cells was investigated. A specific mutation (C223Y) was identified, impacting enzyme function and mRNA levels, potentially explaining varying deficiency levels in mutants.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- 5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAP) is a key enzyme in purine and polyamine metabolism.
- MTAP is widespread in normal tissues but often deficient in malignant cells.
- Understanding MTAP deficiency mechanisms is crucial for cancer research.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying MTAP deficiency in selected murine lymphoma cell mutants.
- To characterize the genetic and molecular basis of partial and complete MTAP deficiency.
Main Methods:
- Selection of MTAP-deficient mutants (F and H5 clones) from murine lymphoma cell line R1.1.
- Cloning of mouse MTAP cDNA.
- Analysis of MTAP mRNA levels using reverse transcription-polymerase chain reaction (RT-PCR).
- Direct sequencing of MTAP cDNA to identify mutations.
Main Results:
- Both F and H5 mutants exhibited a C223Y mutation in the MTAP gene, altering cysteine to tyrosine at codon 223.
- The H5 clone (complete deficiency) showed significantly lower MTAP mRNA levels compared to wild-type.
- The F clone (partial deficiency) had MTAP mRNA levels comparable to wild-type.
- The C223Y mutation likely affects the enzyme's substrate-binding site.
Conclusions:
- The C223Y mutation is a key factor in MTAP deficiency, potentially causing altered enzyme conformation and reduced substrate binding.
- Complete MTAP deficiency in the H5 clone may result from both the C223Y mutation and additional transcriptional abnormalities.
- Further investigation into MTAP's role in cancer metabolism is warranted.
