involucrin and Ichthyosis

Harlequin ichthyosis (HI) is a life-threatening disorder characterized clinically by massive generalized hyperkeratosis and ultrastructurally by an absence of lamellar bodies. However, infants who survive the perinatal period develop a phenotype resembling the nonbullous ichthyosiform erythrodermic (CIE) form of autosomal recessive ichthyosis. We studied a child with a severe hyperkeratotic skin disorder present at birth that developed into a CIE-like phenotype. Electron microscopy demonstrated an absence of lamellar bodies consistent with HI. Abnormalities of filaggrin and involucrin expression by immunostaining were evident. However, transglutaminase 1 (TGase1) was expressed in the epidermis in a pattern consistent with other diseases that involve epidermal acanthosis. Analysis of patient keratinocytes grown in vitro demonstrated expression of normal amounts of TGase1 mRNA and full length TGase1 protein, as well as normal levels of transglutaminase enzymatic activity.

Topics: Biomarkers; Child, Preschool; Epidermis; Filaggrin Proteins; Gene Expression Regulation, Enzymologic; Humans; Ichthyosis; Immunohistochemistry; Intermediate Filament Proteins; Microscopy, Electron; Protein Precursors; Transglutaminases

The cornified cell envelope is a highly insoluble and extremely tough structure formed beneath the cell membrane during terminal differentiation of keratinocytes. Its main function is to provide human skin with a protective barrier against the environment. Sequential cross-linking of several integral components catalyzed by transglutaminases leads to a gradual increase in the thickness of the envelope and underscores its rigidity. Key structural players in this cross-linking process include involucrin, loricrin, SPRRs, elafin, cystatin A, S100 family proteins, and some desmosomal proteins. The recent identification of genetic skin diseases with mutations in the genes encoding some of these proteins, including transglutaminase 1 and loricrin, has disclosed that abnormal cornified cell envelope synthesis is significantly involved in the pathophysiology of certain inherited keratodermas and reflects perturbations in the complex, yet highly orderly process of cornified cell envelope formation in normal skin biology.

Topics: Amino Acid Sequence; Animals; Cell Membrane; Cornified Envelope Proline-Rich Proteins; Cystatins; Filaggrin Proteins; Humans; Ichthyosis; Intermediate Filament Proteins; Keratinocytes; Keratoderma, Palmoplantar, Diffuse; Membrane Proteins; Protein Precursors; Proteinase Inhibitory Proteins, Secretory; Proteins; Skin; Skin Diseases, Genetic; Transglutaminases

Transglutaminases (TGases) are defined as enzymes capable of forming isopeptide bonds by transfer of an amine onto glutaminyl residues of a protein. Here we show that the membrane-bound form of the TGase 1 enzyme can also form ester bonds between specific glutaminyl residues of human involucrin and a synthetic analog of epidermal specific omega-hydroxyceramides. The formation of a approximately 5-nm-thick lipid envelope on the surface of epidermal keratinocytes is an important component of normal barrier function. The lipid envelope consists of omega-hydroxyceramides covalently linked by ester bonds to cornified envelope proteins, most abundantly to involucrin. We synthesized an analog of natural omega-hydroxyceramides N-[16-(16-hydroxyhexadecyl)oxypalmitoyl]sphingosine (lipid Z). When recombinant human TGase 1 and involucrin were reacted on the surface of synthetic lipid vesicles containing lipid Z, lipid Z was attached to involucrin and formed saponifiable protein-lipid adducts. By mass spectroscopy and sequencing of tryptic lipopeptides, the ester linkage formation used involucrin glutamine residues 107, 118, 122, 133, and 496 by converting the gamma-carboxamido groups to lipid esters. Several of these residues have been found previously to be attached to ceramides in vivo. Mass spectrometric analysis after acetonide derivatization also revealed that ester formation involved primarily the omega-hydroxyl group of lipid Z. Our data reveal a dual role for TGase 1 in epidermal barrier formation and provide insights into the pathophysiology of lamellar ichthyosis resulting from defects of TGase 1 enzyme.

Topics: Amino Acid Sequence; Ceramides; Enzyme Inhibitors; Epidermis; Esterification; Glutamine; Humans; Ichthyosis; Mass Spectrometry; Molecular Sequence Data; Molecular Structure; Peptide Fragments; Protein Precursors; Putrescine; Recombinant Proteins; Sequence Analysis; Substrate Specificity; Transglutaminases; Trypsin

Loricrin is a glycine-, serine-, and cysteine-rich protein expressed very late in epidermal differentiation in the granular layers of normal human epidermis. Subsequently, loricrin becomes cross-linked by the activity of transglutaminases TGK/E as a major component of the cornified cell envelope by N epsilon-(gamma-glutamyl)lysine isopeptide bonds. In this study, 115 biopsy specimens from patients with various cutaneous diseases with a morphologically altered epidermal differentiation were analyzed with use of immunohistology with antibodies to loricrin and to involucrin. In addition, antibodies to filaggrin were used for ichthyotic lesions. In contrast to involucrin, loricrin expression was consistently down-regulated in agranulotic, parakeratotic keratinization as observed in psoriasis, dermatitis, pityriasis lichenoides, porokeratosis, or precancerous and malignant squamous lesions. High levels of loricrin were found in hypergranulotic and hyperorthokeratotic epidermis as observed in lichen planus, benign papillomas, and pseudocarcinomatous hyperplasia. Eleven biopsy specimens from patients with ichthyosis vulgaris showed a normal staining in the granular layers. Our results demonstrate that loricrin expression is closely linked to an orthokeratotic phenotype of human epidermal keratinization. The different expression patterns of loricrin and involucrin provide further evidence that these proteins are regulated by different mechanisms and serve different functions during terminal epidermal differentiation.

Topics: Filaggrin Proteins; Humans; Ichthyosis; Immunohistochemistry; Intermediate Filament Proteins; Membrane Proteins; Protein Precursors; Skin; Skin Diseases; Skin Neoplasms