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

Transglutaminases (TGases) are defined as enzymes capable of forming isopep tide bonds by transfer of an amine onto glutaminyl residues of a protein. H ere we show that the membrane-bound form of the TGase 1 enzyme can also for m ester bonds between specific glutaminyl 1 residues of human involucrin an d a synthetic analog of epidermal specific omega-hydroxyceramides. The form ation of a approximate to 5-nm-thick lipid envelope on the surface of epide rmal keratinocytes is an important component of normal barrier function. Th e lipid envelope consists of omega-hydroxyceramides covalently linked by es ter bonds to cornified envelope proteins, most abundantly to involucrin, We synthesized an analog of natural omega-hydroxyceramides, N-[16-(16-hydroxy hexadecyl)oxypalmitoyl]-sphingosine (lipid Z). When recombinant human TGase 1 and involucrin were reacted on the surface of synthetic lipid vesicles c ontaining lipid Z, lipid Z was attached to involucrin and formed saponifiab le protein-lipid adducts. By mass spectroscopy and sequencing of tryptic li popeptides, the ester linkage formation used involucrin glutamine residues 107, 118, 122, 133, and 496 by converting the gamma-carboxamido groups to l ipid esters, Several of these residues have been found previously to be att ached to ceramides in vivo. Mass spectrometric analysis after acetonide der ivatization also revealed that ester formation involved primarily the omega -hydroxyl group of lipid Z. Our data reveal a dual role for TGase 1 in epid ermal barrier formation and provide insights into the pathophysiology of la mellar ichthyosis resulting from defects of TGase 1 enzyme.