A novel function for transglutaminase 1: Attachment of long-chain ω-hydroxyceramides to involucrin by ester bond formation

Abstract

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 -hydroxyceramides. The formation of a Ϸ5-nm-thick lipid envelope on the surface of epidermal keratinocytes is an important component of normal barrier function. The lipid envelope consists of -hydroxyceramides covalently linked by ester bonds to cornified envelope proteins, most abundantly to involucrin. We synthesized an analog of natural -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 ␥-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 -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.Terrestrial vertebrates protect themselves from chemical and physical damage and uncontrolled water loss by maintaining a water-impermeable barrier function of their epidermis. In mammals, this function is essentially accomplished by forming a highly insoluble protein structure on the surface of the corneocytes termed the cornified envelope (CE) and by impeding water diffusion across the stratum corneum by mortaring the corneocytes together by layers of skin-specific lipids (1, 2). These lipids differ in composition from other bilayerforming lipids found in living cells. Notably, their phospholipid content is lost, and instead they contain increased amounts of free fatty acids, cholesterol and its acyl and sulfate esters, and several classes of ceramides, including epidermal-specific longchain -hydroxy-and -hydroxyacylceramides (3). Synthesis of these lipids is initiated in the spinous layer, and they are temporarily stored in lamellar bodies of stratum granulosum, wherein they are arranged as stacks of tetralaminar sheets. Preceding or paralleling the formation of the protein envelope, the contents of the lamellar bodies are extruded into the intercellular space. One component of these lipids is epidermal-specific long-chain -hydroxyceramides that become covalently attached onto the outer surface of the CE as a Ϸ5-nm monomolecular layer. These protein-linked ceramides interdigitate with the intercellular lipid in a comb-like fashion, presuma...

Harlequin ichthyosis and other autosomal recessive congenital ichthyoses: The underlying genetic defects and pathomechanisms

“…Similarly, transglutaminase 1 is an enzyme that was reported to crosslink hydroxyceramide to the cornified cell envelope [23,24]. Cornified cell envelope formation is essential as the scaffold upon which normal intercellular lipid layer formation in the stratum corneum can then take place [25].…”

Cholesterol 3-Sulfate Interferes with Cornified Envelope Assembly by Diverting Transglutaminase 1 Activity from the Formation of Cross-links and Esters to the Hydrolysis of Glutamine

“…Occasionally, involucrin expression was induced in a LB broth containing 100 nmol (0. Preparation of SLV-The following mixtures were made in chloroform/methanol (2:1): 55 mol % dimyristoyl phosphatidylcholine, 15 mol % dipalmitoyl phosphatidylserine, 0 -10 mol % CSO 4 , cholesterol up to 99 mol % (all from Sigma), and 1 mol % of the synthetic ceramide analog N- [16-(16-hydroxyhexadecyl)oxypalmitoyl]sphingosine (lipid Z) (27). The solvent was evacuated, and the lipids were taken up in aqueous buffer and dispersed by sonication as before (44).…”

“…However, the thioester intermediate can also be transferred to primary alcohols (25,26). We have shown that in the epidermis, the terminal () hydroxyl group of -hydroxyceramides is an effective substrate for membrane-bound TGase 1, and this route links these lipids to protein-bound glutamines by an ester bond (27). Lastly, water can also enter the active site of TGases to attack the acyl-enzyme intermediate, which leads to a net deamidation of a reactive glutamine to a glutamic acid residue (28,29).…”

“…TGase 1 is expressed as a 106-kDa monomeric protein, which is constitutively N-myristoylated and S-palmitoylated on its amino-terminal 10-kDa domain, thereby directing the enzyme to plasma membranes (34 -36). Membrane-bound TGase 1 enzyme is essential for both the assembly of the protein envelope by cross-linking CE structural proteins located in the intimate vicinity of the cellular membrane, and the esterification of the -hydroxyceramides to proteins, primarily involucrin (27). Genetic defects of TGase 1 cause the often devastating disease lamellar ichthyosis (37,38).…”

Loricrin and involucrin expression is down-regulated by Th2 cytokines through STAT-6

“…IVL is an also common component of the CE and provides a scaffold to which other proteins subsequently become cross-linked [8]. In the CE structure, IVL is adjacent to the cell membrane and form the exterior surface of the CE [18].…”