The human mid-size neurofilament subunit: a repeated protein sequence and the relationship of its gene to the intermediate filament gene family.

Abstract

We report the isolation and sequence of cDNA and genomic clones for one of the two large subunits of human neurofilament, NF-M. Analysis of the sequence has allowed us to investigate the structure of the carboxy-terminal tail of this protein, and to compare it to that of the small neurofilament as well as to other intermediate filaments. The carboxyterminal region of the protein contains a 13 amino acid prolineand serine-rich sequence repeated six times in succession. Within each repeat unit are two smaller repeats of the sequence Lys-Ser-Pro-Val. The four amino acid repeat may represent a kinase recognition site in a region of the protein that is known to be highly phosphorylated. We also note the presence of an additional heptad repeat at the extreme carboxy terminus of the protein. This region of 60 amino acids may be involved in coiled-coil interactions similar to those that facilitate the filament formation in the rod region. The human gene contains only two introns. Their positions correspond to two of the three introns found in the small neurofilament of the mouse. Thus, two of the three neurorflament genes of mammals have similar structures which are quite different from those of the other intermediate ifiaments. This finding suggests a common origin of the neurofilament subunits, whose evolutionary relationship to other intermediate filament genes is uncertain.

Partial sequence of the rat heavy neurofilament polypeptide (NF‐H) Identification of putative phosphorylation sites

“…The mouse and human NF-H messages are fairly similar with 43 and 40 repeat sequences, respectively, whereas the rat NF-H has 51 repeats ( fig.3). A similar interspecies difference has also previously been ...... noted in the corresponding region of NF-M [4,[9][10][11]. In the rat NF-H cDNA sequence, 41 of the triplet Lys-Ser-Pro repeats occur in a regular pattern of six amino acids ( fig.3).…”

“…A small number of triplet repeats are also present in NF-M [9][10][11]18], but are absent from NF-L [6][7][8]. Previous studies have shown that the number of alkaline phosphatase-sensitive phosphate groups on NF-H and NF-M are approximately proportional to the number of triplet repeats, these phosphate groups being absent from NF-L [19].…”

“…The NF-L and NF-M genes and cDNAs have Haute de Recul6e, 49045 Angers, Cedex, France been cloned and sequenced [6][7][8][9][10][11] and also those of the mouse and human NF-H [12,13]. More recently, a partial cDNA clone for rat NF-H has been sequenced [14].…”

Location and sequence characterization of the major phosphorylation sites of the high molecular mass neurofilament proteins M and H

“…Although all peptides must have contained phosphoserine residues after the first dimension, a filled circle above a serine is only used for those peptides where the particular phosphorylated serine is clearly identified (see text). Sequences Ml-M3 are tentatively arranged along the corresponding regions of the human and rat proteins [14,15]. Numbers on the right reflect position number of the last residue from these regions.…”

“…The less abundant species (5%) started 4 residues prior to the methionine. From the isolation of a tryptic peptide corresponding to residues 861-872 in the human sequence [14] we assume that the M tail isolated after chymotryptic cleavage lacks the carboxy-terminal 43 residues of porcine NF-M.…”