A novel function for transglutaminase 1: attachment of long-chain omega-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 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.

Figure 1

Representative structure of a natural ω-hydroxyceramide (A) and the structure of the artificial substrate analog lipid Z (B). The natural and synthetic lipids have similar chain length and differ only by the presence of an (unreactive) ether bond.

Figure 2

Separation by HPLC of lipopeptide adducts from the tryptic digest of involucrin. Involucrin was reacted with SLV containing lipid Z in the presence of TGase 1. Lipid-attached hydrophobic peptides were selectively retained on a C₄ column under strongly desorbing solvent conditions (black line). Numbering of peaks refers to the corresponding lipopeptides in Fig. 3 and Tables 1 and 2. In controls for the TGase reaction, no lipopeptides were recovered in the presence of 5 mM EDTA (green line), and lipopeptide amounts were significantly reduced by putrescine (see Fig. 5) or by 20 mM (red line) of the generic TGase inhibitor cystamine.

Figure 3

Analysis of peak 1 of Fig. 2 by electrospray ionization mass spectrometry before (A) and after (B) saponification. Peaks with corresponding masses marked by arrows denote multiple charge states of the peptides used for mass determination. Charge states correspond to m/z = (m + n)/n atomic mass units (n, number of charges). Saponification reduces the molar mass of peptides by 776 amu, indicating the loss by hydrolysis of 1 mol of lipid Z (lipid Z has a mass of 794.3 amu). Deconvoluted masses for all five lipopeptides are listed in Table 1.

Figure 4

Mass spectrometry of lipid Z after acetonide formation of its peptide adducts and subsequent alkaline hydrolysis. Most of the lipid Z (M + H⁺ = 795 amu) was recovered as its acetonide derivative (M + H⁺ = 835 amu), indicating that the hydroxyl groups of the sphingosine moiety in lipid Z are not involved in ester bond formation with involucrin.

Figure 5

Inhibitory effect of putrescine on glutaminyl ester formation by TGase 1. Amounts of individual lipopeptides were quantitated by amino acid analysis after isolation by C₄ HPLC chromatography. Yields were related to the total of reactive Gln residues. (A) Ester modification of reactive glutamines without inhibitor. (B) Putrescine (1 mM) inhibited the formation of lipid adducts. (C) Putrescine (20 mM) significantly (P < 0.01) reduced the amount of ester linkages when added to the reaction at 45 min, showing the availability of ester linkages for TGase-mediated aminolysis. Values (mean ± SEM) are from five separate experiments.

Figure 6

Model for the reaction of the ω-hydroxyl group of epidermal ceramides. The TGase 1 enzyme (green) and involucrin (red) are bound to cellular membrane bilayers (brown) by acyl lipid adducts and Ca²⁺ ions, respectively (Left). The catalytic membranes might be either the plasma membrane or the limiting membranes of lamellar bodies (33). The weak nucleophil ω-hydroxyl group is exposed adjacent to the TGase 1-involucrin acyl-enzyme intermediate (Center), resulting in a high likelihood of nucleophilic attack of the thioester bond (Right), resulting in the release of TGase 1 and formation of an ester linkage.