We have isolated cDNA and genomic clones for the small, mid- size, and heavy neurofilament subunits of humans. These cDNA clones were sequenced to determine the amino acid sequence of the protein and the genomic clones were sequenced to define the organization of the genes. The predicted protein of the mid-size neurofilament contains a serine rich 13 amino acid sequence repeated tandemly six times in the carboxyl terminus tail region. The sequence is missing from the small neurofilament, but a related sequence has been identified in the carboxyl terminal region of the heavy neurofilament subunit. In the latter case, there may be as many as 50 to 100 tandem repeats of this sequence. The fact that these repeats occur in regions of the proteins known to be phosphorylated, prompted us to speculate that they were the site of phosphorylation. The repeated region appears to allow the otherwise, helical protein to bend, indicating that phosphorylation at the site might alter the protein's conformation. Our speculations that this was the site of phosphorylation have been confirmed. We have synthesized peptides corresponding to the repeat subunit and a triplication of the repeat subunit. Samples of these peptides were chemically phosphorylated. A large panel of monoclonal antibodies recognizing neurofilaments was assembled and tested for their ability to recognize the various forms of the synthetic peptides. Of the 500 monoclonal antibodies tested, 96 recognized one or another form of the synthetic peptides. Those monoclonal antibodies which were completely specific for the phosphorylated form of neurofilaments recognized only the phosphorylated form of the synthetic peptides, proving that this site is, in fact, the major site of phosphorylation of the neurofilaments. Circular dichroism measurements substantiated our prediction that phosphorylation at these sites dramatically changes the conformation of the protein.
