An unusual intermediate filament subunit from the cytoskeletal biopolymer released extracellularly into seawater by the primitive hagfish (Eptatretus stouti)

Abstract

Each slime gland thread cell from the primitive Pacific hagfish (Eptatretus stouti) contains a massive, conical, intermediate filament (IF)-rich biopolymer (‘thread,’ approximately 60 cm length, approximately 3 microns 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, alpha (pI 7.5), which is coexpressed with a second polypeptide, gamma (pI 5.3). These two polypeptides coassemble in vitro into approximately 10 nm filaments. The alpha-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 (< 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.(ABSTRACT TRUNCATED AT 400 WORDS)