The legless lizard Anguis fragilis (slow worm) has a potent metal-responsive transcription factor 1 (MTF-1)

Abstract

Abstract:The metal-responsive transcription factor-1 (MTF-1) is a key regulator of heavy metal homeostasis and detoxification. Here we characterize the first MTF-1 from a reptile, the slow worm Anguis fragilis. The slow worm, or blind worm, is a legless lizard also known for its long lifespan of up to several decades. Anguis MTF-1 performs well and matches the strong zinc and cadmium response of its human ortholog, clearly surpassing the activity of rodent MTF-1s. Some amino acid positions critical for metal response are the same in humans and slow worm but not in rodent MTF-1. This points to a divergent evolution of rodent MTF-1, and we speculate that rodents can afford a less sophisticated metal handling than humans and (some) reptiles.

Short-lived mammals (shrew, mouse) have a less robust metal-responsive transcription factor than humans and bats

“…Transfection efficiency was normalized with a co-transfected reference gene. Besides the standard 0.5 μg of mouse effector plasmid, a lower amount (0.1 μg) was also tested, showing that the concentration of MTF-1 hardly affected metal response (see also Georgiev et al 2014 ). The transcript level with the zinc-induced human MTF-1 is set to 100. b Experimental setting as in a but with MTF-1s of human, mouse, shrew and bat.…”

“…Note that in the NES region, typical mammals are more closely related to birds and reptiles than to rodents (see also (Georgiev et al 2014 )). The MTF-1s of the animals have not been functionally tested, except those of slow worm (Georgiev et al 2014 ), and fugu (Auf der Maur et al 1999 ), both of which functioned very well in our standard reporter assay (for zebrafish, see also Cheuk ( 2008 )). The extensive alterations in the rabbit (Oryctolagus) and the two lysines replacing leucine and threonine in opossum (Monodelphis) might be worth a further investigation; both of them are relatively short-lived, rapidly multiplying species.…”

“…To determine the metal responsiveness of shrew and bat MTF-1s, their cDNAs were cloned into an expression vector driven by the widely used HCMV enhancer–promoter region (Boshart et al 1985 ), and tested by transfection into mouse Dko7 cells which lack MTF-1 due to targeted gene disruption (Heuchel et al 1994 ; Radtke et al 1995 ). Dko7 cells have been used to produce active MTF-1 of various species, from mammals to reptiles to fish (Georgiev et al 2014 ; Günther et al 2012 ). It should be mentioned that the Dko7 cell clone was established more than 20 years ago; several generations of cells were frozen and used over the years and we note two tendencies: (i) originally transfected mouse MTF-1 induced a 2-3 fold higher transcript level of a 4xMRE reporter when cells were exposed to zinc or cadmium load, while induction by human MTF-1 was 5-12 fold.…”

“…Also included were two control MTF-1s, one from the South American rodent capybara ( H. hydrochaeris ) and one from a long-lived European reptile, the slow worm Anguis fragilis . With regard to metal induction, the former responds poorly and the latter well (Lindert et al 2008 ; Georgiev et al 2014 ). Mouse and shrew MTF-1s were particularly well expressed, which in this context is however not important; the overall transcript level of the 4xMRE reporter might be a bit higher but the fold induction was shown to remain constant for a given MTF-1 over an almost 100-fold expression range (Georgiev et al 2014 ).…”

“…With regard to metal induction, the former responds poorly and the latter well (Lindert et al 2008 ; Georgiev et al 2014 ). Mouse and shrew MTF-1s were particularly well expressed, which in this context is however not important; the overall transcript level of the 4xMRE reporter might be a bit higher but the fold induction was shown to remain constant for a given MTF-1 over an almost 100-fold expression range (Georgiev et al 2014 ). Skipping of the entire exon 7 in shrew MTF-1 thus seems to have the same adverse effect as the amino acid substitutions in the same region of the mouse (Fig.…”