Brain-specific metallothionein-3 has higher metal-binding capacity than ubiquitous metallothioneins and binds metals noncooperatively.
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 regulated in Alzheimer's disease. We demonstrate by mass spectrometry that MT-3, in contrast to common 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(7)MT-3, but metalloforms with 6, 8, and even 9 metals are also present. The results from pH and stability studies demonstrate that the heterogeneity of metalloforms originates 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 has a higher metal transfer potential than the beta-cluster of Zn(7)MT-2. Moreover, ZnMT-3 loses metals during ultrafiltration. MT-3, reconstituted 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 beta-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 zinc to synaptic vesicles.
Palumaa
Peep
P
Centre for Gene Technology, Tallinn Technical University, Ehitajate tee 5, EE-19086 Tallinn, Estonia. peepp@staff.ttu.ee
Eriste
Elo
E
Njunkova
Olga
O
Pokras
Lesja
L
Jörnvall
Hans
H
Sillard
Rannar
R
D000544:Alzheimer Disease / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002104:Cadmium / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D000090023:Metallothionein 3; D008670:Metals / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D011817:Rabbits; D021241:Spectrometry, Mass, Electrospray Ionization; D015032:Zinc / Q000378:metabolism
D007527:Isoenzymes; D000090023:Metallothionein 3; D008670:Metals; D009419:Nerve Tissue Proteins; D002104:Cadmium; D015032:Zinc