Characterization of the Methylthioadenosine Phosphorylase Polymorphism rs7023954 - Incidence and Effects on Enzymatic Function in Malignant Melanoma

Abstract

Deficiency of methylthioadenosine phosphorylase (MTAP) supports melanoma development and progression through accumulation of its substrate 5'-methylthioadenosine (MTA), which leads amongst others to a constitutive inhibition of protein arginine methyltransferases (PRMTs) and activation of the transcription factor AP-1 via the receptor ADORA2B. Genetic association studies have also suggested that genetic polymorphism in MTAP may modulate the risk of melanoma. Here, we investigated the only globally common non-synonymous single nucleotide polymorphism (SNP) reported to date for MTAP. The SNP rs7023954 is located in exon 3 (c.166G>A), and leads to the conservative substitution of one branched-chain amino acid residue (valine) for another (isoleucine) at position 56 (p.Val56Ile). Whereas genotype frequencies in normal and primary melanoma tissues or cell lines were in Hardy-Weinberg equilibrium based on cDNA amplicon sequencing, a marked (P = 0.00019) deviation was observed in metastatic melanoma tissues and cell lines due to a deficit of heterozygotes. Enzyme assays conducted on the co-dominantly expressed alleles revealed no difference in the conversion rate of MTA to adenine and 5-methylthioribose-1-phosphate, indicating that this known enzymatic activity does not modulate the tumor suppressive function of MTAP.

Fig 1. Determination of MTAP rs7023954 in cell lines and tissue samples.

The non-synonymous MTAP SNP c.G166A was genotyped by sequencing of cDNA that had been produced from total RNA extracted from both normal as well as primary (prim.) and metastatic (met.) melanoma cell lines and tissues. Distribution of the genotypes AA, AG, and GG is shown. A detailed description of the genotypes of the samples investigated is given in S1 Table.

Fig 2. Enzyme kinetics of both in vitro-translated MTAP variants.

(A) Western Blot for determination of MTAP expression by in vitro transcription/translation of the pCMX-PL1 (1.0) control plasmid and the expression constructs for MTAP-56I (2.56-fold increased) and MTAP-56V (2.28-fold increased). Densitometric quantification revealed increased MTAP amount. The original Western blot is shown in S1 Fig. (B) Analysis of the metabolic activity of the in vitro-translated products by liquid chromatography–tandem mass spectrometry. Catalytic rates of IVT-MTAP-56I and IVT-MTAP-56V were corrected for the rate of the control, which was set at 1.

Fig 3. Effect of both MTAP variants in transiently cells.

Quantification of MTAP expression at the mRNA (A) and protein level (B) in the melanoma cell line Mel Juso after transient transfection of expression constructs for MTAP-56I and MTAP-56V or control plasmid (pCMX-PL1). The original Western blot is shown in S2 Fig. (C) Analysis of intracellular MTA levels in the transiently transfected Mel Juso cells.

Fig 4. Enzyme kinetics of both MTAP variants in stably transfected cells.

Quantification of MTAP expression at the mRNA (A) and protein level (B) in the melanoma cell line Mel Juso after stable transfection of control plasmid (pCMX-PL1) or expression constructs for MTAP-56I and MTAP-56V. The original Western blot is shown in S3 Fig. (C) Analysis of intracellular MTA levels in the stably transfected Mel Juso cell clones.