Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins

Abstract

Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with 14 N-or 15 N-iodoacetamide. Absolute quantitation was achieved using 15 Niodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue-and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptornegative cell lines than in estrogen-receptor-positive cells. These findings demonstrate that individual human MT isoforms can be accurately quantified in cells and tissues at the protein level, complementing and expanding mRNA measurement as a means for evaluating MTs as potential biomarkers for cancers or heavy metal toxicity. Molecular & Cellular

The effect of metallothionein 2A core promoter region single-nucleotide polymorphism on accumulation of toxic metals in sinonasal inverted papilloma tissues

“…The literature provides numerous studies investigating the toxic and tumorigenic potential of metallic agents, heavy metals and their compounds in cancerous diseases, which confirm their association with progression of neoplastic processes (Ahamed and Alhadlaq, 2014; Krześlak Unfortunately, despite much research on the contribution of MT2A isoforms to tumorigenesis and behavior of various human cancers as well as patient prognosis, a literature review often demonstrates contradictory results and ambiguous conclusions (Forma et al, 2012;Kita et al, 2006;Krześlak et al, 2014Krześlak et al, , 2013Mehus et al, 2014;Pedersen et al, 2009). Most studies introduce some evidence for an association of increased MT1/MT2 isoform expression in neoplastic lesions with such clinicopathological parameters as higher stage and local tumor extension, lower grade and increased patient mortality.…”

“…It has been proven that the presence of singlenucleotide polymorphisms in MT genes near the TATA box, the carriage of different genotypes and the expression of various MT isoforms may affect the risk and prognosis of neoplasms of various origin (Forma et al, 2012;Kayaalti et al, 2010Kayaalti et al, , 2011Krześlak et al, 2013Krześlak et al, , 2014Mehus et al, 2014;Pedersen et al, 2009;Starska et al, 2014b). MT2A is the most widely expressed isoform (Krześlak et al, 2013(Krześlak et al, , 2014Mehus et al, 2014;Pedersen et al, 2009). The polymorphic forms of MT2A may determine the cellular activity of MTs and alter signaling pathways during tumor development and growth, contributes to deregulated cell proliferation, apoptosis, and oxidative stress (Kayaalti et al, 2010(Kayaalti et al, , 2011Raudenska et al, 2014).…”

“…They are required for cellular metal homeostasis by protecting cells against heavy metal toxicity, regulating metal distribution and donation to various enzymes and transcription factors as well as redox control. The metallothionein gene activity is dramatically induced in response to a wide range of stimuli, including metals, drugs, chemical agents, oxidative stress, hormones and cytokines (Mehus et al, 2014;Raudenska et al, 2014).…”

“…The conversion of nucleotide A to G in the core promoter region of the MT2A gene may reduce the effectiveness of MT gene transcription induced by metallic agents or toxic metals, which may consequently lead to an increase in sensitivity to metal toxicity, and may enable the use of MT issue in evaluating the risk of tumors of various origin(Forma et al, 2012;Kita et al, 2006;Krześlak et al, 2014Krześlak et al, , 2013Mehus et al, 2014;Pedersen et al, 2009).…”

“…They are required for cellular metal homeostasis by protecting cells against heavy metal toxicity, regulating metal distribution and donation to various enzymes and transcription factors as well as redox control. The metallothionein gene activity is dramatically induced in response to a wide range of stimuli, including metals, drugs, chemical agents, oxidative stress, hormones and cytokines (Mehus et al, 2014;Raudenska et al, 2014).A literature survey suggests that the expression and regulation of MT activity, and thus the different sensitivity among individuals to heavy metal escapement and their metabolic effect, can be determined by Toxicology and Applied Pharmacology 285 (2015) 187-197 Abbreviations: MT2A, metallothionein 2A; SNP, single-nucleotide polymorphism; IP, sinonasal inverted papilloma; NCM, non-cancerous sinonasal mucosa; RFLP, restriction fragment length polymorphism technique; FASS, flame atomic absorption spectrometer …”