No 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...

Transglutaminase (TGase) enzymes catalyze the formation of covalent cross-links between protein-bound glutamines and lysines in a calcium-dependent manner, but the role of Ca 2+ ions remains unclear. The TGase 3 isoform is widely expressed and is important for epithelial barrier formation. It is a zymogen, requiring proteolysis for activity. We have solved the three-dimensional structures of the zymogen and the activated forms at 2.2 and 2.1 A Ê resolution, respectively, and examined the role of Ca 2+ ions. The zymogen binds one ion tightly that cannot be exchanged. Upon proteolysis, the enzyme exothermally acquires two more Ca 2+ ions that activate the enzyme, are exchangeable and are functionally replaceable by other lanthanide trivalent cations. Binding of a Ca 2+ ion at one of these sites opens a channel which exposes the key Trp236 and Trp327 residues that control substrate access to the active site. Together, these biochemical and structural data reveal for the ®rst time in a TGase enzyme that Ca 2+ ions induce structural changes which at least in part dictate activity and, moreover, may confer substrate speci®city.