An unusual intermediate filament subunit from the cytoskeletal biopolymer released extracellularly into seawater by the primitive hagfish (Eptatretus stouti)
Authors: Koch Elizabeth A. , Spitzer Robert H. , Pithawalla Ron B. , Parry David A. D. 
Published: April 2021

ABSTRACT
Each slime gland thread cell from the primitive Pacific hagfish (Eptatretus stouti) contains a massive, conical, intermediate filament (IF)-rich biopolymer (‘thread,’ ~60 cm length, ~3 µm width). In view of the unusual ultrastructure of the thread, its extracellular role in modulation of the viscoelastic properties of mucus, and the ancient lineage of this primitive vertebrate, we report the nucleotide and deduced amino acid sequences of one major thread IF subunit, α (pI 7.5), which is coexpressed with a second polypeptide, γ (pI 5.3). These two polypeptides coassemble in vitro into ~10 nm filaments. The α-thread chain, a 66.6 kDa polypeptide, has an unusual central rod domain containing 318 residues flanked by N- and C-terminal domains of 192 and 133 residues, respectively. Each peripheral region exhibits some epidermal keratin-like features including peptide repeats and a high total content of glycine and serine residues. The terminal domains, however, lack the H1 and H2 subdomains characteristic of known keratins. Moreover, when the central rod is aligned either in relation to established homology profiles (J. F. Conway and D. A. D. Parry (1988) Int. J. Biol. Macromol. 10, 79-98) of other IF subunits (types I-V, nestin, non-neuronal invertebrate), or by computer-based homology searches of the GenBank™/EMBL Data Bank, a low identity (&lt;30%) is evident, with no preferred identity to keratins or other known IF types. Although the central rod of 318 residues consists of the canonical apolar heptad repeats interspersed with three linker regions, a discontinuity in phasing of the heptad substructure in rod 2B, and conserved sequences at either end of the rod domain, other collective characteristics are atypical: overall high threonine content (13.2% vs 2.3-5.4% for other IFs), high threonine content in rod 1B (18.8% vs 1-6%), five Thr-Thr repeats in coiled coil segments, L12 of length greater than in keratins, substitution of phenylalanine for a highly conserved glutamate in the sixth position of L2, and a glycine-proline sequence in segment 2B. Possibly as a result of the high threonine content, the percentage of both acidic and basic residues in most helical subdomains is reduced relative to type I and II chains. Fast Fourier transform analyses show that only the acidic residues in segment 1B and basic residues in segment 2 have near typical IF periods. The results are concordant with the low immunocrossreactivity of the hagfish α polypeptide with intracellular keratins from higher vertebrates and may reflect structural requirements for macromolecular packaging of IFs in parallel alignment prior to extracellular export by holocrine secretion for organization of water and mucins. This function is in contrast to that of other IFs sequenced to date, which function intracellularly or correspond to hard keratins. Although the hagfish α-component has a unique central rod that bears no preferred identity to any other currently sequenced IF subunit, it also has several characteristics of a conventional type II keratin chain. For these reasons α has been classified as a type II homologue of an epidermal keratin rather than a new type of IF. It is recognized, nonetheless, that if sequence data from other species are ultimately shown to display a high degree of identity to α then this would accord α the designation of a new chain type rather than that of a simple homologue.