Enzyme kinetics of both <i>in vitro</i>-translated MTAP variants.

<p>(A) Western Blot for determination of MTAP expression by <i>in vitro</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160348#pone.0160348.s001" target="_blank">S1 Fig</a>. (B) Analysis of the metabolic activity of the <i>in vitro</i>-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.</p

Effect of both MTAP variants in transiently cells.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160348#pone.0160348.s002" target="_blank">S2 Fig</a>. (C) Analysis of intracellular MTA levels in the transiently transfected Mel Juso cells.</p

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

<div><p>Deficiency of methylthioadenosine phosphorylase (<i>MTAP</i>) 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 <i>MTAP</i> may modulate the risk of melanoma. Here, we investigated the only globally common non-synonymous single nucleotide polymorphism (SNP) reported to date for <i>MTAP</i>. 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.</p></div

Determination of <i>MTAP</i> rs7023954 in cell lines and tissue samples.

<p>The non-synonymous <i>MTAP</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160348#pone.0160348.s004" target="_blank">S1 Table</a>.</p

Enzyme kinetics of both MTAP variants in stably transfected cells.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160348#pone.0160348.s003" target="_blank">S3 Fig</a>. (C) Analysis of intracellular MTA levels in the stably transfected Mel Juso cell clones.</p

Confirmation of cDNA genotype by liquid chromatography-tandem mass spectrometry (LC-MS/MS) based protein expression analysis.

<p>Sole expression of the 56I-allele was observed in melanoma cell lines Mel Im, Mel Ho, and A375, whereas the 56V-allele was detected exclusively in WM793, WM1366, and WM293A. In the fibroblast cell line 3F0379, both alleles were co-expressed equally.</p