Cloning and specific polymerised‐chain‐reaction amplification of a third charge‐separable human metallothionein isoform

Abstract

Metallothionein (MT) fractions isolated from human adult liver tissue are readily separated by anion-exchange chromatography in two isoforms, MT-1 and MT-2, which differ from each other in the nature of the amino acid residue at position 11. In fetal liver tissue, the presence of a third chargeseparable MT isoform has been previously reported. We determined its partial amino acid sequence and the sequence of a cDNA clone encoding this M T form. This confirmed the existence of another human MT isoform, hereafter named MT-0, which is characterized by the presence of a negatively charged amino acid at position 8, and by a Glu23 to Lys substitution in a strictly conserved region of the protein. Taking into account these substitutions, we are able to classify human MT isoforms into three instead of two charge-separable groups, based on the nature of three amino acid residues.The unique presence of Glu8 in MT-0 enabled us to develop an MT-0-specific amplification by the polymerase chain reaction, which revealed the presence of MT-0 mRNA in adult liver RNA samples, in spite ofthe total absence of this isoform at the protein levcl. This suggests the involvement of post-transcriptional regulatory mechanisms in the expression of this fetal MT form.Metallothioneins (MT) are ubiquitous low-molecularmass, sulphydryl-rich proteins with a high affinity for essential as well as toxic trace metals, and to which possible functions in metal metabolism and detoxification have been attributed [I). In humans, MT constitute a rather complex multiprotein family, of which the exact number of closely related isoforms is still unknown. MT fractions from human liver and kidney, as well as from different cell cultures, have been isolated and separated by anion-exchange chromatography in two isoforms, MT-1 and MT-2 (reviewed in [2]). Sequence analysis has demonstrated that MT-2 fractions consist of a single polypeptide, characterized by an Asp residue at position 11, while MT-1 fractions show microheterogeneity at several positions but contain only Glyll or Val11 residues [2]. Reverse-phase (RP) HPLC of MT fractions from human adult liver revealed at least six peaks, which where identified as one MT-2 and five distinct M r -1 forms [3].At the genomic level, Southern-blot analysis showed that these proteins are encoded by a multigene family of at least 12-15 members [4], which are distributed to five autosomes [5]. The nucleotide sequence of several human MT genes has been determined and their expression pattern characterized in

Nitric oxide induces Zn2+ release from metallothionein by destroying zinc–sulphur clusters without concomitant formation of S-nitrosothiol

“…MT1 and MT2 are generally found in the liver and kidney [13]. A third isoform, MT0, is also known to exist in human fetal liver (HFL) [14]. Other isoforms such as MT3 and MT4 are reported to be expressed in the brain [15,16] and in mouse tissues containing stratified squamous epithelial cells [17], respectively.…”

Human metallothionein gene MT1L mRNA is present in several human tissues but is unlikely to produce a metallothionein protein

“…2). In addition, MT1G, MT1H (also known as MTO [4]) and MT1X mRNAs were observed in at least one of the samples (Fig. 2) whilst MT1A, MT1B, MT1E and MT1F mRNA were not detected (data not shown).…”

“…The MT2A gene, and 13 MT1 isogenes have been linked to one cluster [2], whilst evidence suggests that the MT3 and MT4 genes are located nearby [3]. Much work has been devoted to establishing which of these genes are functional, and the proteins encoded by MT2A, MT3, and the MT1 isogenes MT1E, MT1H (also known as MTO, [4]) and MT1X have been isolated from human tissues [5,6], although additionally, specific mRNA transcripts have been identified in human tissues from MT1F and MT1G ( [7], unpublished data). Up to six isoforms of MT have been isolated from human liver by HPLC [5], and it is likely that additional separation techniques such as capillary electrophoresis [8] will allow a further estimate of how many isoforms are actually present at the protein level.…”

Cloning and sequencing a novel metallothionein I isoform expressed in human reticulocytes

“…MT IV has only been detected in stratified squamous epithelia [l l] and is thought to play a unique role in its differentiation. Previously thought to be a foetal liver isoform, MT 0 has been detected in stimulated adult monocytes [5,12]. Monocytes induced with zinc express among others MT Io and MT 0, which in adults may be specific to these cells and have a role in monocyte activation and/or differentiation.…”

“…Abbreviations: MT(s), metallothionein(s); PCR, polymerase chain reaction able isoform, MT 0 [5]. To date, sequence analysis has identified only one MT II isoform, with aspartic acid at position 11.…”