Fig. 5

Grx2 acts via CRMP2. Knockdown of zfCRMP2 by morpholino injection impaired formation of axon scaffold (A). Impaired formation of axon scaffold by knockdown of zfGrx2 was rescued by simultaneous injection of 20 pg/embryo capped zfCRMP2 mRNA (B). Densitometric analyses of the resulting pattern of CRMP2-specific spots after separation by 2D redox blots demonstrated Grx2-dependent changes in the thiol redox state of CRMP2 (C-E). At 24 hpf zfCRMP2 was more oxidized in embryos lacking zfGrx2 compared to wild-type (C), whereas in SH-SY5Y cells overexpressing hGrx2c the redox state of CRMP2 was more reduced compared to control cells (D). The redox state of recombinant zfCRMP2 was more reduced after incubation with recombinant zfGrx2 for 1 h under anaerobic conditions in a GSH/GSSG buffer adjusted to -300 mV compared to a buffer with a potential of -180 mV (E). Thiol redox modifications changed secondary structure of zfCRMP2 (F). (F, I) Ellipticity of reduced recombinant zfCRMP2 (solid line) and after incubation with H₂O₂ for 1 min (dashed/dotted line), 15 min (dotted line), and 30 min (dashed line). (F, ii) Spectra of oxidized zfCRMP2 before (dashed line) and after incubation with GSH/zfGrx2 (solid line). (F, iii) Spectra of oxidized zfCRMP2 before (dashed line) and after incubation with reduced zfGrx2 at a ratio of 161.2 (solid line). (G) A Coomassie blue-stained PAGE comparing quarternary structure of oxidized zfCRMP (60 kDa) before and after incubation with reduced zfGrx2 (16 kDa) at a ratio of 161.2.