Inability of keratinocytes lacking their specific transglutaminase to form cross-linked envelopes: absence of envelopes as a simple diagnostic test for lamellar ichthyosis

Abstract

Epidermal keratinocytes, late in their terminal differentiation, form cross-linked envelopes resistant to ionic detergent and reducing agent. Because the cross-linking process is catalyzed by the keratinocyte transglutaminase, the absence of active transglutaminase should result in failure of the keratinocyte to form a cross-linked envelope. Three keratinocyte strains bearing mutations in the keratinocyte transglutaminase were examined: two contained no detectable transglutaminase mRNA and none contained active enzyme. All three were unable to form cross-linked envelopes, either spontaneously in stratified cultures or upon induction with Ca2+. Although stratum corneum of normal humans and scales from patients with different ichthyotic diseases contain cross-linked envelopes, those from patients with transglutaminase-negative lamellar ichthyosis do not. Therefore, the disease due to the absence of transglutaminase may be readily distinguished from other ichthyotic disease by a simple test for cross-linked envelopes.

Figure 1

Expression of TGase1 in wild-type strains (YF29 and KACO) and mutant strains (KALE, TAYA, and LEMA). (A) Northern blots hybridized with a TGase1-specific cDNA probe (Upper) and with an oligonucleotide probe specific for 28S rRNA (Lower). Positions of RNA molecular weight markers are shown on the right (Upper). The autoradiograph is overexposed to demonstrate clearly the absence of TGase1 mRNA in KALE and TAYA. (B) Western blot probed with a rabbit polyclonal antibody raised against a C-terminal peptide of TGase1. Arrow indicates the position of the 92-kDa TGase1. A protein extract prepared from 3T3-J2 cells was included as a negative control. Neither KALE nor TAYA produced an enzyme band but a faint band is present in extract of LEMA cells, which contain a missense mutation.

Figure 2

Spontaneous envelope formation in wild-type and mutant strains. After heating to 100°C in the presence of SDS and 2-mercaptoethanol envelopes were counted. The numbers obtained are expressed as percentage of total number of cells and plotted as a function of time after confluence. Virtually no envelopes were produced by any of the three mutant strains.

Figure 3

Envelope formation after induction by calcium ionophore A23187. (A) Number of cross-linked cell envelopes as a function of incubation time in the presence of A23187 (100 μg/ml). There was virtually no envelope formation by any of three mutant strains. (B) The effect of TGase inhibitor RS48373 on envelope formation in wild-type cells treated with ionophore.

Figure 4

Absence of cornified cell envelopes in scales from a patient with TGase-negative lamellar ichthyosis. (A) Cornified cell envelopes in corneocytes scraped from normal human. (B) Absence of envelopes in corneocytes from a patient with lamellar ichthyosis. (C) Presence of dense envelopes in corneocytes of a patient with Netherton syndrome. Assays for TGase in the insoluble fraction of these corneocytes gave values 10.6 times higher than those of B, whose small amount of enzyme activity we attribute to TGase2.