Sequence and expression patterns of two forms of the middle molecular weight neurofilament protein (NF-M) of Xenopus...

The middle molecular weight neurofilament protein (NF-M) is relevant to our understanding of vertebrate neurofilaments in growing axons, both because it exists in all vertebrates and because it undergoes characteristic changes in its phosphorylation state during axonal development. Indeed, all vertebrate neurofilament proteins are believed to have originated by gene duplication from an ancestral, NF-M-like protein. The role of NF-M in axonal development has been studied extensively in the frog, Xenopus laevis, through the use of monoclonal antibodies. To acquire a better understanding of the relationship of X. laevis NF-M to that of other vertebrates and to obtain additional reagents to study and perturb neurofilaments in developing axons, we isolated cDNA clones from the nervous system. These clones encoded two forms of NF-M, which exhibited 93% amino acid identity overall and 94%, 96% and 90% identity over their head, rod, and tail domains, respectively. Synonymous nucleotide substitution rates between the two forms tied their origin to an ancestral duplication of the Xenopus genome, which occurred approximately 30 million years ago. Non-synonymous substitution rates indicated that the tail domain is evolving more rapidly than the rod domain. Both forms shared structural features in common with other vertebrate NF-Ms but had only a single example of the KSP phosphorylation motif that is repeated multiple times in the NF-Ms of bird, goldfish and mammal. In post-metamorphic frogs, the NF-M(1) transcript was consistently expressed at higher levels than that of NF-M(2), although their anatomical patterns of expression were qualitatively similar. During development, however, only NF-M(2) was detectable in retinal ganglion cells through stage 42. We speculate that the differences observed between these two forms may represent early stages of protein diversification akin to what occurred after the gene duplications that gave rise to other vertebrate neurofilament proteins.