Involucrin cross-linking by transglutaminase 1 - Binding to membranes directs residue specificity

The transglutaminase 1 (TGase 1) enzyme is essential for the assembly of th e cell envelope barrier in stratified squamous epithelia, It is usually bou nd to membranes, but to date most studies with it have involved solution as says. Here we describe an in vitro model system for characterizing the func tion of TGase 1 on the surface of synthetic lipid vesicles (SLV) of composi tion similar to eukaryote plasma membranes. Recombinant baculovirus-express ed human TGase 1 readily binds to SLV and becomes active in cross-linking a bove 10 mu M Ca2+, in comparison to above 100 mu M in solution assays, sugg esting that the membrane surface is important for enzyme function. Involucr in also binds to SLV containing 12-18% phosphatidylserine and at Ca2+ conce ntrations above 1 mu M. In reactions of involucrin with TGase 1 enzyme in s olution, 80 of its 150 glutamines serve as donor residues. However, on SLV carrying both involucrin and TGase 1, only five glutamines serve as donors, of which glutamine 496 was the most favored, As controls, there was no cha nge in specificity toward the glutamines of other substrates used by free o r SLV-bound TGase 1 enzyme. We propose a model in which involucrin and TGas e 1 bind to membranes shortly after expression in differentiating keratinoc ytes, but cross-linking begins only later as intracellular Ca2+ levels incr ease. Furthermore, the data suggest that the membrane surface regulates the steric interaction of TGase 1 with substrates such as involucrin to permit specific cross linking for initiation of cell envelope barrier formation.