Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A2 using cells deficient in the expression of scramblase

Abstract

Summary Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A2 but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt’s lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A2. Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100 s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A2. These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A2, it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine.

Modulated mechanism of phosphatidylserine on the catalytic activity of Naja naja atra phospholipase A2 and Notechis scutatus scutatus notexin

“…Nelson et al (2011) found that A. piscivorus piscivorus PLA 2 showed different activity on hydrolyzing the membrane of ionomycin-, dexamethasone-or thapsigargin-treated cells. Although ionomycin similarly induced PS externalization of normal lymphocytes, Raji human Burkitt's lymphoma cells and S49 mouse lymphoma cells, A. piscivorus piscivorus PLA 2 differently hydrolyzed the membrane of ionomycin-treated lymphocytes, Raji cells and S49 cells (Nelson et al, 2012) Collectively, these findings suggest the notion that membrane compositions and/or membrane-bound conformation of PLA 2 enzymes should be important for the susceptibility of apoptotic cells for PLA 2 . Compared with PS-containing PC/SM/Chol and PE/Chol vesicles, PC/SM/Chol and PE/Chol vesicles show a higher membrane fluidity.…”

“…Previous studies suggested that membrane structure contributed to the level of membrane susceptibility to A. piscivorus piscivorus PLA 2 in addition to PS externalization (Bailey et al, 2007(Bailey et al, , 2009Nelson et al, 2011Nelson et al, , 2012. Nevertheless, the effect of membrane-interacted mode of PLA 2 enzymes and membrane compositions on PLA 2 activity does not explore in the studies of Bailey et al (2007Bailey et al ( , 2009 and Nelson et al (2011and Nelson et al ( , 2012. Nelson et al (2011) found that A. piscivorus piscivorus PLA 2 showed different activity on hydrolyzing the membrane of ionomycin-, dexamethasone-or thapsigargin-treated cells.…”

“…In view of the above results, it is conceivable that PS effect on membrane structure affects membraneinteracted mode of notexin and NnaPLA 2 , leading to PSmodulated effect on PLA 2 activity. Previous studies showed that the binding affinity of Chol for phospholipids is in the order of SM > PS > PC > PE (Ohvo-Rekila et al, 2002;van Dijck, 1979). Competitive binding of PS with Chol might inevitably alter membrane structure of PE/Chol vesicles.…”

“…Previous research has reported that the membranes of cells undergoing apoptosis are susceptible to human secreted PLA 2 enzymatic activity (Atsumi et al, 1997). Bell and his colleagues suggest that PS externalization in concert with alteration of membrane structure increase the susceptibility of apoptotic cells to Agkistrodon piscivorus piscivorus PLA 2 and human secreted PLA 2 s (Harris et al, 2001;Bailey et al, 2007Bailey et al, , 2009Nelson et al, 2011Nelson et al, , 2012. In their studies, ionomycin, dexamethasone or thapsigargin are used to induce apoptosis of cells, and thus the direct effect of these reagents on membrane structure and PLA 2 activity could not exclude out (Harris et al, 2001;Bailey et al, 2007Bailey et al, , 2009Nelson et al, 2011Nelson et al, , 2012.…”

“…Bell and his colleagues suggest that PS externalization in concert with alteration of membrane structure increase the susceptibility of apoptotic cells to Agkistrodon piscivorus piscivorus PLA 2 and human secreted PLA 2 s (Harris et al, 2001;Bailey et al, 2007Bailey et al, , 2009Nelson et al, 2011Nelson et al, , 2012. In their studies, ionomycin, dexamethasone or thapsigargin are used to induce apoptosis of cells, and thus the direct effect of these reagents on membrane structure and PLA 2 activity could not exclude out (Harris et al, 2001;Bailey et al, 2007Bailey et al, , 2009Nelson et al, 2011Nelson et al, , 2012. Our previous studies revealed that Naja naja atra PLA 2 (NnaPLA 2 ) induced apoptosis of human leukemia K562 and U937 cells (Liu et al, 2009;Chen et al, 2010a), while Notechis scutatus scutatus notexin (presynaptic PLA 2 neurotoxin) induced apoptosis of human neuroblastoma SK-N-SH cells (Chen et al, 2010b).…”