Brain-specific metallothionein-3 has higher metal-binding capacity than ubiquitous metallothioneins and binds metals noncooperatively

摘要

Zinc metabolism in the cells is largely regulated by ubiquitous small proteins,metallothioneins (MT).Metallothionein-3 is specifically expressed in the brain and is down regualted in Alzheimer's disease.We demonstrate by mass spectrometry that MT-3,in contrast to comon MTs,binds Zn~(2+) and Cd~(2+) in a noncooperative manner and can also bind higher stoichiometries of metals than seven.MT-3 reconstituted with seven metals exists in a dynamic equilibrium of different metalloforms,where the prevalent metalloform is Me_7MT-3,but metalloforms with 6,8,and even 9 metals are also present.The results from pH and stability studies demosntrae that the heterogeneity of metalloforms origninates from the N-terminal #betA#-cluster,whereas the C-terminal #alpha#-cluster of MT-3 binds four metal ions such as that of common MTs.Experiments with EDTA demonstrate that the #beta#-cluster of ZnMT-3 loses metals during ultrafiltration.MT-3,reconstitted with an excess of Zn~(2+) or Cd~(2+),exists as a dynamic mixture of metalloforms with higher than 7 metal stoichiometries (8-11).Such forms of ZnMT-3 are unstable and decompose partly already during a rapid gel filtration,whereas CdMT-3 forms are more stable.Extra metal ions may bind to the #beta3-cluster region as well as to the carboxylates of MT-3.The specific metal-binding properties of MT-3 could be functionally implemented for buffering of fluctuating concentrations of zinc in zincergic neurons and for transfer of ainc to synaptic vesicles.