Sphingosylphosphocholine reduces the calcium ions requirement for activating tissue transglutaminase

Tissue transglutaminase (tTG) catalyzes a Ca+2-dependent transglutaminase (TGase) reaction resulting in the formation of γ-glutamyl-ε-lysine bonds between proteins. tTG is present in the cytoplasm of most cells and must remain latent to prevent the cross-linking of the membrane, cytoplasmic and cytoskeletal proteins under physiologic conditions. The enzyme is activated during programmed cell death forming apoptotic body to prevent the release of intracellular contents. Recent studies suggested that lipids could modulate TGase activity. In this study, we investigated whether lipid molecules that are components of membrane and involved in cell signaling could modify tTG function. We found that sphingosylphosphocholine (lyso-SM) was the only lipid molecule to activate TGase activity at low Ca+2 concentrations. In the presence of 125 μM of lyso-SM, the TGase activity was detectable at 10 μM Ca+2, whereas similar activity was obtained at 160 μM of Ca+2 in the absence of lyso-SM. TGase activity obtained at 100 μM Ca+2 was 20-25 fold lower than that activity measured in the presence of 125 μM lyso-SM. Kinetic studies demonstrated that lyso-SM did not significantly change the Km for the glutamyl and primary amine substrates. However the Kact for Ca+2 was reduced from 300 μM to 90 μM. Lyso-SM bound specifically to tTG as demonstrated by altering the sensitivity to trypsin degradation. The specificity was demonstrated by using lyso-SM analogs including sphingomyeline, lysophosphocholine, lyso-platelet activating factor and dihydro lyso-SM. These results indicate that phosphocholine on C1, the unsubstituted amino group at C2, a C3-C4 double bondt are critical for the activation of TGase activity.