Identification of a critical site in Wlds:: Essential for Nmnat enzyme activity and axon-protective function

The chimeric Wlds protein consisting of the N-terminal 70 amino acids of Ufd2 and the complete sequence of nicotinamide mononucleotide adenylyltransferasel (Nmnat1), delays Wallerian degeneration in Wld(s) mice. Although Nmnatl enzyme activity was showed to be critical for the function of Wld(s) protein, the expected phenotype was not observed in Nmnatl transgenic mice. To further check whether Nmnatl enzyme activity is involved, we aligned sequences of eukaryotic Nmnats, and found that Phe in helix A is highly conserved not only in various species, but also in different homologues. The Phe is a residue located near to the highly conserved GXFXPX(T/H)XXH motif and resides in the same helix as the last His of this conserved motif. To investigate the role of the conserved Phe in Nmnat activity, we made the point mutation of Phe. The Phe28 mutation of mouse Nmnatl in Wlds completely abolished its Nmnat enzyme activity. To study the role of mutant Wlds in axon degeneration, herpes viruses were packaged to infect cultured SCGs. We found that the mutant Wld' failed to protect axon degeneration from morphological changes, microtubule integration and neurofilament degradation. Therefore, we have identified a Phe residue that critical for both enzyme activity of Nmnat and the axon-protective function of Wld', and further confirmed that Nmnat I enzyme activity is required in Wlds function. (c) 2006 Elsevier Ireland Ltd. All rights reserved.