The structure and organization of the human heavy neurofilament subunit (NF-H) and the gene encoding it.

Abstract

Genomic clones for the largest human neurofilament protein (NF‐H) were isolated, the intron/exon boundaries mapped and the entire protein‐coding regions (exons) sequenced. The predicted protein contains a central region that obeys the structural criteria identified for alpha‐helical ‘rod’ domains typically present in all IF protein components: it is approximately 310 amino acids long, shares amino acid sequence homology with other IF protein rod domains and displays the characteristic heptad repeats of apolar amino acids which facilitate coiled‐coil interaction. Nevertheless, anomalies are noted in the structure of the NF‐H rod which could explain observations of its poor homopolymeric assembly in vitro. The protein segment on the carboxy‐terminal side of the human NF‐H rod is uniquely long (greater than 600 amino acids) compared to other IF proteins and is highly charged (greater than 24% Glu, greater than 25% Lys), rich in proline (greater than 12%) and impoverished in cysteine, methionine and aromatic amino acids. Its most remarkable feature is a repetitive sequence that covers more than half its length and includes the sequence motif, Lys‐Ser‐Pro (KSP) greater than 40 times. Together with the recent identification of the serine in KSP as the main target for NF‐directed protein kinases in vivo, this repetitive character explains the massive phosphorylation of the NF‐H subunit that can occur in axons. The human NF‐H gene has three introns, two of which interrupt the protein‐coding sequence at identical points to introns in the genes for the two smaller NF proteins, NF‐M and NF‐L.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…The amino acid sequence of coil 2 was found to be very highly conserved with respect to both rat NF-L and NF-M and also mouse and human NF-H, demonstrating a homology of approx. 80°7o and 90o70, respectively [6,[11][12][13]. This is in good agreement with previous results which have shown a high degree of conservation between the coiled regions of intermediate filaments from a number of different species [4][5][6].…”

“…There is, however, a greater divergence in the first and sixth amino acids of the sextet, but this difference is unlikely to influence greatly the secondary structure of the region, this probably being determined by the triplet repeat sequence. The Lys-Ser-Pro sequence has previously been identified as the phosphorylation site within NF-H [18] and has also been recognized in other neurofilament proteins as well as in neurofilament and microtubule-associated proteins including [12] and human [13].…”

“…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].…”

Characterization of the post‐translational modifications in tubulin from the marginal band of avian erythrocytes

“…In the case of the p6 site a member of the serine -proline-directed protein kinases may be responsible for phosphorylation. These kinases are also responsible for phosphorylation of tau proteins (Drewes et al, 1992;Ishiguro et al, 1991) and neurofilaments (Geisler et al, 1987;Lees et al, 1988). In this context we note that the sequence motif ASPEK at positions 440-444 of /36 tubulin occurs several times in chicken neurofilament protein M (Zopf et al, 1991).…”

Regional modulation of neurofilament organization by myelination in normal axons

“…All three ofthese proteins share a central rod domain, but they differ in their C-terminal regions. In NF-H, this region contains over 40 lysine-serine-proline repeats (Julien et al, 1986(Julien et al, , 1988Lees et al, 1988), which provide potential phosphorylation sites (Julien et al, 1983;Geisler et al, 1983;Lee et al, 1988). The extent of phosphorylation of this region, which contributes to the sidearms of the neurofilament (Hirokawa et al, 1984;Carden et al, 1985;Hisanaga and Hirokawa, 1988) appears to differ within different regions of the neuron, with a higher phosphorylation state in the axon than in the cell body and dendrite (Stemberger and Sternberger, 1983;Lee et al, 1986Lee et al, , 1987.…”