Active human cellular transglutaminase

#Not Applicable

#Transglutaminases are a calcium dependent family of enzymes responsible for post-translational crosslinking of proteins. The crosslinked protein products are large chemically, enzymatically, and mechanically resistant polymers with a variety of functions ranging from wound healing to skin formation (Greenberg et al., FASEB J. 5: 3071-3077 (1991)).

#To date, there are several human transglutaminases which are produced from different genes. The catalytic subunit of blood-clotting factor XIIIa, or plasma transglutaminase, is 80 kDa (Ichinose et al., Proc. Natl. Acad. Sci. USA 85: 5829-5833 (1988)), the epidermal keratinocyte transglutaminase is 106 kDa (Phillips et al., Proc. Natl. Acad. Sci. 87: 933-9337 (1990)), the epidermal and hair follicle transglutaminases are 77 kDa (Kim et al., J. Biol. Chem. 268: 12682-12690 (1993)), the prostate transglutaminase is 78 kDa (Grant et al., Biochem. Biophys. Res. Comm. 203: 117-1123 (1994)), and the cellular transglutaminase is about 80 kDa (Gentile et al., J.Biol.Chem. 266: 478-483 (1991)).

#FIG. 1 illustrates the conversion of the native cellular transglutaminase from human red blood cell membranes. Western blot analysis was performed as described in Example (1). Prestained markers (lane 1), RBC membrane protein (MP) with no trypsin (lanes 2 and 5), MP with 5 mM CaCl2 and trypsin for 1 and 6 min (lanes 3 and 4), MP with 0.3 mM GTP and trypsin for 1 and 6 min (lanes 6 and 7).

#FIG. 2 illustrates the cellular transglutaminase activation by proteolysis: effect of trypsin concentration on transglutaminase activation of membranes from human RBC (A), and human erythroleukemia cells-K562 (B). Insert A) RBC membranes with no trypsin addition (lane 1), transglutaminase digestion by increasing concentrations of trypsin as indicated on the abscissa for each time point assayed and in presence of 5 mM CaCl2 (lanes 7, 6, 8, and 9) or 0.3 mM GTP (lanes 3, 2, 4, and 5). B) K562 membranes in the presence of 5 mM CaCl2 (lanes 1, 2, and 3) or 0.3 mM GTP (lanes 4, 5, and 6), no trypsin (lanes 1 and 4), 2.5 μg/ml trypsin (lanes 2 and 5), and 20 μg/ml trypsin (lanes 3 and 6).

#FIG. 3 illustrates the effect of time on the proteolysis of purified transglutaminase. Immunoblot of purified RBC transglutaminase digested with trypsin for 6, 3, and 1 min in the presence of 5 mM CaCl2 (lanes 1, 2, and 3), or in the presence of 0.3 mM GTP (lanes 4, 5 and 6). Purified RBC transglutaminase without trypsin (lane 7, with calcium and lane 8, with GTP).

#Cellular transglutaminase catalyzes the covalent crosslinking of proteins by forming ε-(γ-glutamyl)lysine isopeptide linkages that are used for different cellular and extracellular functions. The present invention provides isolated nucleotide sequences (cDNAs) which express the active human cellular transglutaminase with a molecular weight of about 55 kDa (TG). The recombinant enzyme was purified to homogeneity and found to be very active in crosslinking reactions. The invention provides that the native cellular transglutaminase with a molecular weight of 80 kDa (TGC) and the transglutaminase homologue with a molecular weight of 63 kDa (TGH) are produced in cells as zymogens and they are inactive in crosslinking reactions. The TGC and TGH forms can be converted proteolytically to the active TG forms.

#The invention also provides the production of recombinant active TG forms in bacterial cells which forms are purified, assayed for crosslinking activity, and shown to be the active species of cellular transglutaminase.

#As will be appreciated by those skilled in the art, the invention also includes those polypeptides having slight variations in amino acid sequences or other properties. Such variations may arise naturally as allelic variations (e.g., due to genetic polymorphism) or may be produced by human intervention (e.g., by mutagenesis of cloned DNA sequences), such as induced point, deletion and insertion mutations.