William W. Idler

Self-assembly of bovine epidermal keratin filaments in vitro.

The seven α-keratin polypeptides which comprise the subunits of the in situ epidermal keratin filaments of bovine epidermis polymerize in vitro in dilute salt solution into filaments that have the same structure as the in situ keratin filaments. Most combinations of two or three of the purified polypeptides also polymerize into filaments of the same general structure and these contain the polypeptides in the precise molar ratios of 1:2 or 1:1:1. On the basis of these polypeptide stoichiometries and the characteristic α-type X-ray diffraction pattern by all filaments, it is concluded that the keratin filaments polymerized in vitro and thus the in situ epidermal keratin filaments are comprised of a three-chained unit structure. Turbidometric measurements of the kinetics of filament assembly revealed a biphasic mechanism, involving the initial rate-limiting formation of a hexamer nuclear particle, followed by a more rapid rate of polymerization to filaments. The nuclear particle probably consists of a pair of triple-chained units.

The fate of trichohyalin. Sequential post-translational modifications by peptidyl-arginine deiminase and transglutaminases

Trichohyalin (THH) is a major structural protein of the inner root sheath cells and medulla layer of the hair follicle and, to a lesser extent, of other specialized epithelia. THH is a high molecular weight insoluble α-helix-rich protein that forms rigid structures as a result of postsynthetic modifications by two Ca2+-dependent enzymes, transglutaminases (TGases) (protein cross-linking) and peptidyl-arginine deiminase (conversion of arginines to citrullines with loss of organized structure). The modified THH is thought to serve as a keratin intermediate filament matrix protein and/or as a constituent of the cell envelope. In this paper, we have explored in vitro the order of processing of THH to fulfill these functions, using an expressed truncated, more soluble form THH-8. THH-8 is a complete substrate for three known TGases expressed in epithelia, but the kinetic efficiency with TGase 3 is by far the greatest. Following maximal conversion of its arginines to citrullines, THH-8 is cross-linked even more efficiently by TGase 3, using most glutamines partially and all lysines. In addition, we show that insoluble aggregates of THH-8 or native pig tongue THH can be solubilized following peptidyl-arginine deiminase modification. Together, these data suggest an in vivo model in which THH located in insoluble cytoplasmic droplets is first modified by peptidyl-arginine deiminase which denatures it and makes it more soluble. This renders it available for efficient cross-linking by TGase 3 to form highly cross-linked rigid structures in the cells. This temporal order of reaction is supported by the observation that THH is expressed in hair follicle cells before the TGase 3 enzyme.

Transglutaminase Cross-linking Properties of the Small Proline-rich 1 Family of Cornified Cell Envelope Proteins INTEGRATION WITH LORICRIN

Small proline-rich 1 (SPR1) proteins are important for barrier function in stratified squamous epithelia. To explore their properties, we expressed in bacteria a recombinant human SPR1 protein and isolated native SPR1 proteins from cultured mouse keratinocytes. By circular dichroism, they possess no α or β structure but have some organized structure associated with their central peptide repeat domain. The transglutaminase (TGase) 1 and 3 enzymes use the SPR1 proteins as complete substrates in vitro but in different ways: head domain A sequences at the amino terminus were used preferentially for cross-linking by TGase 3, whereas those in head domain B sequences were used for cross-linking by TGase 1. The TGase 2 enzyme cross-linked SPR1 proteins poorly. Together with our data base of 141 examples of in vivo cross-links between SPRs and loricrin, this means that both TGase 1 and 3 are required for cross-linking SPR1 proteins in epithelia in vivo. Double in vitro cross-linking experiments suggest that oligomerization of SPR1 into large polymers can occur only by further TGase 1 cross-linking of an initial TGase 3 reaction. Accordingly, we propose that TGase 3 first cross-links loricrin and SPRs together to form small interchain oligomers, which are then permanently affixed to the developing CE by further cross-linking by the TGase 1 enzyme. This is consistent with the known consequences of diminished barrier function in TGase 1 deficiency models.

Subunit structure of the mouse epidermal keratin filament

The two proteins which are the subunits of mouse epidermal keratin filaments have been isolated from fully differentiated epidermis (stratum corneum), viable differentiating cells and cells grown in culture. The proteins have molecular weights of 68 000 and 60 000, consist of families of very similar species, have common N-terminal (N-acetylserine) and C-terminal (glycine) residues, contain 35--40% alpha-helix and are immunologically cross-reacting. In mixtures, the two proteins polymerize in vitro into native-type keratin filaments that are 70--80 angstrom in diameter, up to 30 micrograms long, possess a characteristic alpha-type X-ray diffraction pattern and contain the subunits in the precise molar ratio of 1 : 2 or 2 : 1.

STRUCTURAL AND TRANSGLUTAMINASE SUBSTRATE PROPERTIES OF THE SMALL PROLINE-RICH 2 FAMILY OF CORNIFIED CELL ENVELOPE PROTEINS

The small proline-rich (SPR) proteins are components of the cornified cell envelope of stratified squamous epithelia and become cross-linked to other proteins by transglutaminases (TGases). The SPR2 family is the most complex, as it consists of several differentially expressed members of the same size. To explore their physical and cross-linking properties, we have expressed in bacteria a human SPR2 family member, and purified it to homogeneity. By circular dichroism, it possesses no α or β structure but has some organized structure associated with the central peptide repeat domain. The TGase 1, 2, and 3 enzymes expressed in epithelia use the recombinant SPR2 protein as a complete substratein vitro, but with widely differing kinetic efficiencies, and in different ways. With TGase 1, only one glutamine on the head domain and one lysine on the tail domain were used for limited interchain cross-linking. With TGase 3, multiple head and tail domain residues were used for extensive interchain cross-linking. The total usage of glutamine and lysine residues in vitro by TGase 3 was similar to that seen in earlier in vivo studies. We conclude that SPR2 proteins are cross-linked in epithelia primarily by the TGase 3 enzyme, a minor extent by TGase 1, and probably not by TGase 2.

Co-assembly of Envoplakin and Periplakin into Oligomers and Ca2+-dependent Vesicle Binding IMPLICATIONS FOR CORNIFIED CELL ENVELOPE FORMATION IN STRATIFIED SQUAMOUS EPITHELIA

Plakin family members envoplakin and periplakin have been shown to be part of the cornified cell envelope in terminally differentiating stratified squamous epithelia. In the present study, purified recombinant human envoplakin and periplakin were used to investigate their properties and interactions. We found that envoplakin was insoluble at physiological conditions in vitro, and co-assembly with periplakin was required for its solubility. Envoplakin and periplakin formed soluble complexes with equimolar stoichiometry. Chemical cross-linking revealed that the major soluble form of all periplakin constructs and of envoplakin/periplakin rod domains was a dimer, although co-assembly of the full-length proteins resulted in formation of higher order oligomers. Electron microscopy of rotary-shadowed periplakin demonstrated thin flexible molecules with an average contour length of 88 nm for the rod-plus-tail fragment, and immunolabeling EM confirmed the molecule as a parallel, in-register, dimer. Both periplakin and envoplakin/periplakin oligomers were able to bind synthetic lipid vesicles whose composition mimicked the cytoplasmic side of the plasma membrane of eukaryotic cells. This binding was dependent on anionic phospholipids and Ca2+. These findings raise the possibility that envoplakin and periplakin bind to the plasma membrane upon elevation of intracellular [Ca2+] in differentiating keratinocytes, where they serve as a scaffold for cornified cell envelope assembly.

Structural Changes of Human Epidermal α-Keratin in Disorders of Keratinization

AbstractThe chemistry and structure of the epidermal α-keratin extracted from the skin of patients with a variety of disorders of keratinization have been investigated using biochemical, biophysical, and electron microscopic techniques developed for the characterization of normal mammalian epidermal keratin. Generally, the α-keratin polypeptides of the diseased epidermis differed from those of uninvolved epidermis or of normal volunteers in having varying numbers of polypeptide components of lower molecular weights, numerous free amino-terminal and increased numbers of carboxyl-terminal amino acids, higher contents of α-helix, and only limited facility for polymerization in vitro into native-type epidermal keratin filaments. As the α-helix-enriched fragments, which represent up to two-thirds of the polypeptide chains, isolated after limited tryptic digestion ofthe keratin filaments of normal, uninvolved, and involved epidermis, were physicochemically identical, it seems that the end-terminal non-α-helical regions of the polypeptides of diseased epidermis are abnormal. These differences may be a result of degradation or of altered protein synthesis.