Hagfish biopolymer: a type I/type II homologue of epidermal keratin intermediate filaments

Abstract

In contrast to most intermediate filaments (IF) which function intracellularly or constitute epidermal appendages, the single massive (≈60 cm length, ≈3 μm width) IF-rich ‘thread’ biopolymer synthesized by the specialized hagfish gland thread cell is released extracellularly via holocrine secretion to interact with mucins and seawater, thereby modifying the viscoelastic properties of the copious mucous exudate. Recently, using the Pacific hagfish ( Eptatretus stouti, class Agnatha), a jawless scaleless marine vertebrate of ancient lineage, we determined that the deduced amino acid sequence of one thread IF chain (α, 66.6 kDa, native pl 7.5) contained an atypical, threonine-rich central rod domain of low identity (< 30%) with other vertebrate IF types, but that the N- and C-terminal domains exhibited several keratin-like features. From these and other unexpected characteristics, it was concluded that hagfish α is best categorized as a type II homologue of an epidermal keratin. We now report the deduced sequence of a second thread IF subunit (γ, 62.7 kDa, native pl 5.3) which is co-expressed and co-assembles in vitro with α in a 1:1 ratio. As was found for α, the N- and C-terminal domains of γ have keratin-like parameters, but the central rod has low identity to IFs of types I-V (< 31%), a cephalochordate I F (< 29%) and invertebrate IFs (<20%) and no particular homology to type I or type II keratins. Central rod identity between γ and α is also low (≈23%), as is typical of comparisons between different rod types but atypical of similar rod types (>50%). The central rods of both γ and α lack the 42-residue insert of helix 1B present in lamins and invertebrate IFs, have unusually high threonine contents (γ, 10%; α, 13%) compared to other IF types (2–5%), contain a number of unexpected residues in consensus conserved sites, and employ a L12 segment of 21 residues rather than the 16 or 17 residues found in keratins. Theoretical analyses indicate that the hagfish molecules exist as coiled coil heterodimers (α/γ) in which the chains are parallel, in axial register, and stabilized by significant numbers of ionic interactions. Fast Fourier-transform analyses revealed that the linear distribution period of ≈9.55 for basic and acidic residues in other IF chains is not completely maintained, partly due to the high threonine content. The threonine residues occupy mainly outer sites b, c, f in the heptad substructure, possibly abetting parallel alignment of thousands of IFs within the thread, interactions with mucins at the thread periphery, and hierarchical IF chain assembly. It is suggested that the γ and α chains from this most primitive extant vertebrate are type I and type II homologues of epidermal keratin chains, possibly related to early specialized keratins.