Abstract
Transglutaminase 1 (TGase1) is one of three known enzymes involved in terminal differentiation in stratified squamous epithelia, possibly in the formation of a cornified cell envelope. Because the intact enzyme is particularly difficult to isolate in quantity from keratinocytes for characterization, comparatively little is known about its properties. We have expressed the full-length as well as a series of deletion forms of this enzyme in a bacterial system and analyzed their enzymatic properties. The specific activity of the full-length enzyme isolated and purified from the bacterial lysate was comparable to that of the native enzyme of keratinocytes. Analysis of several deletion constructs demonstrated that removal of the first 60-109 residues, which include sequences involved in membrane association, results in upwards of a 10-fold increase in the specific activity. Deletions beyond residue 109, into sequences conserved within the TGase family of proteins, result in loss of activity. Similarly, as many as 240 residues can be removed from its carboxyl-terminal end before activity is lost. Thus, a molecule of 466 residues, containing virtually only the conserved core sequences of TGases, retains a specific activity comparable to the intact enzyme. In addition, the various deletion forms display wide variations in substrate specificity toward a series of synthetic peptide substrates, designed from possible target TGase1 substrate proteins of epithelia. The data show that sequences between residues 62 and 92 are important in defining the substrate specificity of the TGase1 enzyme system. Furthermore, it may now be possible to design an enzyme with a defined substrate specificity. Together, these data suggest TGase1 has recruited additional sequences on its amino terminus in relation to other members of the TGase family, which have the net effect of permitting sequestration onto membranes, changing its specific activity and modifying its likely substrate specificities.
Highlights
From the $Skin Biology Branch, NIAMS, and the Laboratory of Cellular Development and Oncology, NIDR, National Institutes of Health, Bethesda, Maryland 20892-2755
Because the intact enzyme is the TGase family consists of six known members to date: a difficult to isolate in quantity from kerati- membrane-associated enzyme first discoveredin keratinocytes nocytes for characterization, comparatively little is known about its properties
We have shown recently that three of the active TGases are expressed in the epidermis, TGasel, TGase2, and TGase3 [34, 35]. These are thought t o be involved in the formation and assembly of the cornified cell envelope(CE) of terminally differentiating epidermis and of other stratified squamous epithetually only the conserved core sequences of TGases, re- lia [4, 5, 36,37,38], critically involved in barrier functions
Summary
Kinetic Studies-Rate studies were carried out in 0.1 M "is acetate buffer (pH 8.0) containing 10 mM CaCl,, 0.1 m EDTA, 5 mM dithiothreitol, varying concentrations of succinylatedHammerstein casein or peptide substrates, three different levels of ["Clputrescine (0.03 mM, 0.06 mM, and 1.2 m;specific activity 110 Ci/mol), and with an appropriate amount of enzyme at 37 "C.In thecase of the casein substrate, the reaction was terminated by the addition of a 10-fold volumeof 7.5% cold trichloroacetic acid. Each peptide sample was separated from the unreacted radiolabeled putrescine by gel filtration on a Sephadex G-25 SF (Pharmacia) high performance liquid chromatography column. Following boosting to achieve maximum titer, the goat serum was purified by passing antiserum through an immunoabsorbent column containing antigen chemicallycoupled to Reacti-Gel (HW-65)using the manufacturer's protocol (Pierce Chemical Co.). To reduce the risk of denaturation due to chaotropic salts, pooled fractions of eluted specific antibody were immediately desalted with a Speedy desalting column (10 ml) (Pierce)and stored at -20 "C
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