Sphingosylphosphocholine Reduces the Calcium Ion Requirement for Activating Tissue Transglutaminase

Abstract

Tissue transglutaminase (tTG) catalyzes a Ca2؉ -dependent transglutaminase reaction resulting in the formation of ␥-glutamyl-⑀-lysine bonds and is activated during apoptosis to catalyze the formation of apoptotic body. We investigate whether lipids that are membrane components and involved in cell signaling could modify the Ca 2؉ -dependent activation of tTG. We found that sphingosylphosphocholine (lyso-SM) was the only lipid to activate transglutaminase at low Ca 2؉ concentrations. In the presence of lyso-SM (125 M), transglutaminase was detectable at 10 M Ca 2؉ , whereas in the absence of lyso-SM, similar activity was obtained at 160 M Ca 2؉. Furthermore, in the presence of lipid vesicles lyso-SM retained the ability to enhance the Ca 2؉ -dependent activation of tTG. Lyso-SM did not significantly change the K m for the glutamyl and primary amine substrates. However, the K act for Ca 2؉ was reduced from 300 M to 90 M. Structure-function studies of lyso-SM analogs indicate that phosphocholine group on C1, the free amino group at C2 and a C4 -C5 double bond are critical for the activation of transglutaminase activity. This is the first demonstration that a specific sphingolipid could enhance the activity of tTG and could play a role in vivo in activation of the tTG at physiologic Ca 2؉ levels.Tissue transglutaminase (tTG) 1 is a calcium-dependent enzyme that plays an important role in many different intracellular and extracellular processes ranging from apoptosis to extracellular matrix formation (1-3). Calcium ions are required for the enzyme to adopt the proper conformation to bind glutamine and lysine residues within proteins or peptides (1-3). The intracellular levels of calcium are much lower than the reported calcium levels required to obtain measurable transglutaminase (TGase) activity in vitro. This raises the question of whether there are intracellular cofactor(s) that can modify the sensitivity of tTG to Ca 2ϩ . The tTG first forms a calciumdependent thioester bond with select protein bound glutamines and releases ammonia (1-4). The thioester is a reactive enzyme-substrate intermediate that can then react rapidly with the primary amine group of either a lysine within a protein or a polyamine (1-4). The final reaction product contains an isopeptide bond that stabilizes inter-and intramolecular protein structure or generates a protein-polyamine conjugate that has unique biologic properties (1-4).The binding of calcium ions reduces the affinity of tTG for GTP (5). GTP binding to tTG causes a conformational change in the protein which leads to a reduction in TGase activity (1-3). Based upon the affinity of the tTG for both calcium ions and GTP as well as the intracellular concentrations of these cofactors, the majority of the tTG in vivo is predicted to remain latent (1, 2, 6).tTG is reported to associate with phospholipid bilayers in vitro (7,8) and with cell membranes (9, 10). The recent discovery that a portion of the intracellular tTG is associated with the cytoplasmic domain of the cell membrane a...

Functional characterization of naturally occurring transglutaminase 2 mutants implicated in early-onset type 2 diabetes

“…These results are in line with earlier reports on the reduction of insulin secretion from rat b-cells treated with inhibitors of TG2 transamidation activity (Bungay et al 1986, Driscoll et al 1997. However, the increase in Ca 2C level that leads to insulin secretion in b-cells in response to glucose is much lower (w150-250 nM) than the level of Ca 2C (w150-500 mM) required for TG2 transamidation activity (Lai et al 1997, Parkash et al 2002. In addition, intracellular levels of GTP, which is known to inhibit transamidation activity, are many fold higher (w100 mM) than Ca 2C levels (Siegel & Khosla 2007).…”

Tissue transglutaminase is a caspase substrate during apoptosis. Cleavage causes loss of transamidating function and is a biochemical marker of caspase 3 activation

“…23,28,50 Conformational changes able to modulate tTG activity are also described following binding of sphingosylphosphocholine. 47 In any situation involving conformational changes, cryptic epitopes may be exposed which make tTG a better target for select monoclonal antibodies while other epitopes may be hidden. This seems the likely explanation of the lack of reactivity with the purified thymic TG/F8-tTG by the CUB 7401 anti tTG mAb, as well as of other observations made possible by the TG/F8 scFv.…”

“…45 Several in vitro studies describe in detail the inhibitory effect of GTP binding on tTG activity 23,46 and its interplay with other regulatory elements. 47 In addition, GTP binding causes conformational changes in tTG that affect both enzymatic activity 23,48,49 and interactions with other proteins including proteolytic enzymes. 23,28,50 Conformational changes able to modulate tTG activity are also described following binding of sphingosylphosphocholine.…”

Roles of transglutaminases in cardiac and vascular diseases

“…The activation of TG2 during the increase in intracellular calcium and the reduced GTP generation that occurs with apoptosis leads to cross-linking of cellular proteins stabilizing the apoptotic body, thereby potentially limiting the release of damaging intracellular contents to neighboring cells and tissues (141). The sensitivity of TG2 activity to inhibition by GTP is increased by nitrosylation of cysteine residues (97) whereas the calcium-dependent transglutaminase activity of TG2 is increased by sphingosylphosphocholine, which is increased in cells undergoing apoptosis (142). The calcium binding protein calreticulin inhibits both the GTP binding and transglutaminase activity of TG2 (143,144).…”