Identification of a critical site in Wld(s): essential for Nmnat enzyme activity and axon-protective function.
The chimeric Wld(s) protein consisting of the N-terminal 70 amino acids of Ufd2 and the complete sequence of nicotinamide mononucleotide adenylyltransferase1 (Nmnat1), delays Wallerian degeneration in Wld(s) mice. Although Nmnat1 enzyme activity was showed to be critical for the function of Wld(s) protein, the expected phenotype was not observed in Nmnat1 transgenic mice. To further check whether Nmnat1 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 Nmnat1 in Wld(s) completely abolished its Nmnat enzyme activity. To study the role of mutant Wld(s) in axon degeneration, herpes viruses were packaged to infect cultured SCGs. We found that the mutant Wld(s) 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(s), and further confirmed that Nmnat1 enzyme activity is required in Wld(s) function.
Jia
Haiqun
H
Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China.
Yan
Tingting
T
Feng
Yan
Y
Zeng
Chengming
C
Shi
Xianglin
X
Zhai
Qiwei
Q
D000818:Animals; D001369:Axons / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics; D051381:Rats; D014855:Wallerian Degeneration / Q000235:genetics / Q000473:pathology / Q000503:physiopathology
D009419:Nerve Tissue Proteins; D009612:Nicotinamide-Nucleotide Adenylyltransferase; D010649:Phenylalanine; C442580:Wld protein, mouse; C510972:Nmnat1 protein, mouse