Nickel-induced proteins in human HaCaT keratinocytes: annexin II and phosphoglycerate kinase.
It has been established in previous in vitro experiments with human HaCaT keratinocytes that nickel becomes cytotoxic at concentrations higher than 100 microM and that it is accumulated mainly in the cytosolic fraction (Ermolli et al., 2000). The aim of this work was to search possible biomarkers of metal insult, i.e. nickel-binding proteins or proteins differentially expressed in the cytosolic fraction of nickel-exposed cells (up to 1 mM nickel) as compared to controls. Cytosolic proteins were studied by isoelectric focusing (IEF) and two-dimensional gel electrophoresis (2-DE). Separation by IEF revealed nickel-induced changes in the abundance of cytosolic proteins as visualised with nickel-nitrilo-triacetic-alkaline phosphatase (Ni-NTA-AP) in blots. The cytosolic fraction of cells incubated with nickel, at concentrations over 100 microM, showed nickel binding components which were absent or present in significantly lower amounts in control cells. These proteins had isoelectric points (pIs) 6.9, 7.7 and 8.5. After 2-DE silver- and protein staining significantly increased abundance of four proteins was observed. Their pI values corresponded to those of the nickel binding ones seen after IEF. A protein with pI 6.9 had a molecular weight estimated to 38 kDa, two proteins with pI around 7.7 showed molecular weights of 57 and 22 kDa, respectively and another protein with pI of 8.5 had a molecular weight of 33 kDa. The increased abundance of these components, both in IEF experiments and in 2-DE, correlated with the nickel concentration in the culture media. N-terminal amino acid sequencing and database search allowed identification of one a protein as phosphoglycerate kinase and another one as annexin II. The involvement of these proteins in cellular functions and their possible implications in the mechanism of nickel toxicity in keratinocytes are discussed. Some of these proteins may be biomarker candidates for effects of nickel exposure in human keratinocytes.
Acevedo
F
F
National Institute for Working Life, S-17184, Solna, Sweden. fernado.acevedo@niwl.se
Serra
M A
MA
Ermolli
M
M
Clerici
L
L
Vesterberg
O
O
D017306:Annexin A2 / Q000096:biosynthesis*; D015415:Biomarkers; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D003877:Dermatitis, Contact / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007525:Isoelectric Focusing; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism / Q000633:toxicity*; D010735:Phosphoglycerate Kinase / Q000096:biosynthesis*
D017306:Annexin A2; D015415:Biomarkers; D009532:Nickel; D010735:Phosphoglycerate Kinase