Clt,z Biochem, Vo[. 23, pp. 3 4 9 - 3 7 0 , 1990
0009-9120/90 $3.00 - .00 C o p y r i g h t ¢ 1990 T h e C a n a d i a n Society of C l in i c a l C h e m i s t s .
P r i n t e d in C a n a d a . All r i g h t s reserved.
Phospholipases in Biology and Medicine ERICH Department
KAISER,
PETER
of Medical Chemistry,
Phospholipases, a group of enzymes that catalyze the hydrolysis of membrane phospholipids, are classified according to the bond cleaved in a phospholipid into PLA, (EC 3.1.1.3), PLA2 (EC 3.1.1.4), PLB (EC 3.1.1.5), PLC (EC 3.1.4.3), and PLD (EC 3.1.4.4). This paper reviews source and structure of PEA 2 and the involvement of PLA2 and PLC in several biological phenomena, such as, signal transduction, photoreception, biosynthesis of lung surfactant, sperm motility, and fertilization. New assays for PLA2 activity and concentration in biological fluids are discussed. Phospholipases are involved in many inflammatory reactions by making arachidonate available for eicosanoid biosynthesis. The determination of PEA 2 activity and mass concentration in plasma is useful in the diagnosis and prognosis of pancreatitis and of septic shock. Naturally occurring phospholipase inhibitors, such as lipocortins act as second messengers in the anti-inflammatory response to steroids. Lipocortins may be valuable therapeutic agents, because they are more specific in their anti-inflammatory action than glucocorticoids; therefore, they are less likely to produce harmful side effects.
KEY WORDS: alveolar proteinosis; Alzheimer's disease: arachidonate; calcimedin; calelectrin; calmodulin; cardiac arrhythmias; cardiogenic shock; chromobindin; cleft palate; corticosteroids; diacylglycerol; eicosanoids; endonexin; ethanol ingestion; fish eye disease; G-proteins; glucocorticoids; insulin release; lipocortins; lipomodulin; lung surfactant; pancreatitis; phospholipases; photoreception; prostaglandins; protein kinases; psoriasis; rheumatoid arthritis; schizophrenia; seminal plasma; septic shock: signal transduction; snake venoms; sperm motility; streptozotocin-induced diabetes; transducin; tyrosine kinase; uteroglobin; vitamin E. 1. I n t r o d u c t i o n p
hospholipases are enzymes that catalyze the hydrolysis of membrane phospholipids. They are classified according to the bond cleaved in a phospholipid. Thus, phospholipase A 1 (PLA1; EC 3.1.1.3) and phospholipase A 2 (PLA2; EC 3.1.1.4) selectively remove fatty acids from the sn-1 and sn-2 positions, respectively. Phospholipase B (PLB; EC 3.1.1.5) is a lysophospholipase. Phospholipase C (PLC; EC 3.1.4.3) cleaves the bond between glycerol and a phosphate; and phospholipase D (PLD; EC 3.1.4.4) hydrolyzes the amino alcohol moiety from a phospholipid (Figure 1).
Correspondence: Prof. Erich Kaiser, Department of Medical Chemistry, University of Vienna, Vienna, Austria. Manuscript received July 18, 1989; revised March 30,1990; accepted April 5, 1990. CLINICALBIOCHEMISTRY,VOLUME 23, OCTOBER 1990
CHIBA,
and KHALED
ZAKY
U n i v e r s i t y of V i e n n a , V i e n n a , A u s t r i a
Our purpose is not to review the available information on all phospholipases, but to present a survey on PLA 2, because this enzyme has attracted attention due to its biological and medical aspects. Some information on PLC is also given. 2. S o u r c e a n d s t r u c t u r e of PLA2 PLA 2 enzymes are widespread, found in nearly all animal cell types examined, and in bacteria and protozoa. Membrane-associated and soluble PLA 2 are found in m a m m a l i a n cells. Most investigations have been performed on PLA 2 of pancreatic origin and from snake venoms (1). Canine pancreatic PLA 2 is synthesized as a preproenzyme of 146 amino acids containing a 15 residue signal peptide, and a 7 residue activation peptide. The 124 residue mature enzyme shows 79.2c~ homology with the porcine enzyme (2). The complete amino acid sequences of pancreatic PLA 2 from horse t3), dog (4), rat (4), h u m a n (5,6), and from bovine pancreas (7) are now known. The cDNA coding for the porcine pancreatic PLA,~ has been cloned and expressed in E. coli (8). PLA 2 and lecithin: cholesterol acyltransferase (LCAT; EC 2.3.1.43) have a common antigenic determinant which is probably located near or at their esterolytic active site (9). PLA2 is a major component of Elapid and Viperid snake venoms and plays an important role in the pharmacological effects following envenomation. The sequence of many venom enzymes has been determined (10), and it is the basis of their classification ( 11 ). In general, the molecular weights of the purified enzymes are low, within 12-15 kDa. PLA 2 enzymes contain a high degree of disulfide crosslinking and are extremely stable to heat and acid treatment. Such properties are important attributes for an enzyme t h a t is involved in phospholipid degradation. Due to this stability, solubilization of membrane bound enzymes can easily be achieved through mineral acid extraction (12). PLA2 has a higher affinity for aggregated than nonaggregated phospholipids. It is unclear if this is due to a conformational requirement by the enzyme or the substrate. Kinetic studies with PLA 2 are problematic, because classical Michaelis-Menten kinetics do not apply. This has important implications for analysis. Most PLA 2 enzymes need Ca 2÷ ions for full activity (1). 349
KAISER, CHIBA, AND ZAKY
F
16:0 +
OH
--16:0 HO-~
AA 4 L P-CHOLINE
~
16:0
AA 0
+ AA
L'P-CHOLINE
LIPASE
A2I
P-CHOLINE PHOSPHATIDYL CHOLINE I PHOSPHOLIPASE
C ] ~ P-CHOLINE
16:0
AA~ OH
OSPHOLIPASE D I FI6:0 AA10-P
Figure 1--Action of phospholipases on phosphatidyl choline. AA = arachidonic acid.
3. Phospholipases in biology 3.1. PHOSPHOLIPASESIN SIGNALTRANSDUCTION Cells respond to their environment through cell surface receptors that bind specific ligands. These "signals" are transduced into changes in cellular metabolism and function. One system that mediates this transduction process involves a ubiquitous family of guanosine triphosphate (GTP)-binding proteins (G-proteins). These proteins transduce signals generated by ligand interactions with specific cell surface receptors into changes in intracellular levels of cyclic nucleotides (13,14). A variety of hormones, neurotransmitters, and growth factors induce the rapid catabolism of phosphatidyl inositol-4,5-biphosphate (PIP 3) via a receptor-mediated process (15-18). Membrane receptors are thought to be coupled with PLC via a G-protein (13,14). This conclusion is based on the observation that GTP and stable GTP analogues promote PIP 3 hydrolysis in permeabilized cells (19,20), broken cell preparations (21), and isolated cell membranes (22-30). The hydrolysis of PIP 3 by an activated inositol-specific PLC generates two putative second messengers:inositol-l,4,5-triphosphate (IP~)aand diacylglycerol. IP a promotes the release of 2+ ions from intracellular stores by opening Ca2+-channels (31-34) which mediate the transient increase in cytosolic Ca 2÷ ions after binding of the agonist to the receptor. Diacylglycerol activates protein kinase C, resulting in the phosphorylation of a num-
350
ber ofintracellular proteins (14,35-38). The activated protein kinase C phosphorylates the lipocortin moiety of the lipoprotein-PLA 2 complex and PLA 2 is liberated. In the presence of elevated cytosolic Ca 2+ ions, PLA 2 is activated and splits phospholipids into lysophospholipid and arachidonic acid. These events are illustrated in Figure 2. Table 1 gives some examples of effects mediated by the synergistic action o f I P 3 (release of Ca 2+ ions from intracellular stores) and diacylglycerol (activation of protein kinase C) (39-53). 3.2. PLA 2 IN GLUCOSE-INDUCEDINSULINRELEASE Glucose-induced insulin release is coupled with high turnover of membrane phospholipids in islet cells (54,55) due to PLA 2 release (56). Both arachidonate and lysophospholipids have membrane-disrupting properties; high concentrations induce morphologic alterations at the periphery of islet cells (57,58). A blockade of glucose-induced insulin release is effected by PLA 2 inhibitors (58,59). Glucose may activate PLA 2 in the islet, leading to the generation of lysophospholipids. The latter may couple energy production to insulin release, at least partially through the promotion of Ca 2+ translocation (59). 3.3. PHOSPHOLIPASESIN PHOTORECEPTION In the rod outer segments of the retina, a Gprotein, transducin, is localized (60). Transducin,
CLINICAL BIOCHEMISTRY,VOLUME 23, OCTOBER 1990
PHOSPHOLIPASES IN BIOLOGYAND MEDICINE
PHOSPHATIDYL INOSITOL 2 ATP
~
2 ADP PHOSPHATIDYL INOSITOL-415-BIPHOSPHATE (PIP3)
PHOSPHOLIPASE C (INACTIVE)
AGONIST
> RECEPTOR
'1
j
PHOSPHOLIPASE C (ACTIVE)
INOSITOL-3,4,5-TRISPHOSPHATE (IP3)
DIACYLGLYCEROL
|
PROTEIN KINASE C
0009-9120/90 $3.00 - .00 C o p y r i g h t ¢ 1990 T h e C a n a d i a n Society of C l in i c a l C h e m i s t s .
P r i n t e d in C a n a d a . All r i g h t s reserved.
Phospholipases in Biology and Medicine ERICH Department
KAISER,
PETER
of Medical Chemistry,
Phospholipases, a group of enzymes that catalyze the hydrolysis of membrane phospholipids, are classified according to the bond cleaved in a phospholipid into PLA, (EC 3.1.1.3), PLA2 (EC 3.1.1.4), PLB (EC 3.1.1.5), PLC (EC 3.1.4.3), and PLD (EC 3.1.4.4). This paper reviews source and structure of PEA 2 and the involvement of PLA2 and PLC in several biological phenomena, such as, signal transduction, photoreception, biosynthesis of lung surfactant, sperm motility, and fertilization. New assays for PLA2 activity and concentration in biological fluids are discussed. Phospholipases are involved in many inflammatory reactions by making arachidonate available for eicosanoid biosynthesis. The determination of PEA 2 activity and mass concentration in plasma is useful in the diagnosis and prognosis of pancreatitis and of septic shock. Naturally occurring phospholipase inhibitors, such as lipocortins act as second messengers in the anti-inflammatory response to steroids. Lipocortins may be valuable therapeutic agents, because they are more specific in their anti-inflammatory action than glucocorticoids; therefore, they are less likely to produce harmful side effects.
KEY WORDS: alveolar proteinosis; Alzheimer's disease: arachidonate; calcimedin; calelectrin; calmodulin; cardiac arrhythmias; cardiogenic shock; chromobindin; cleft palate; corticosteroids; diacylglycerol; eicosanoids; endonexin; ethanol ingestion; fish eye disease; G-proteins; glucocorticoids; insulin release; lipocortins; lipomodulin; lung surfactant; pancreatitis; phospholipases; photoreception; prostaglandins; protein kinases; psoriasis; rheumatoid arthritis; schizophrenia; seminal plasma; septic shock: signal transduction; snake venoms; sperm motility; streptozotocin-induced diabetes; transducin; tyrosine kinase; uteroglobin; vitamin E. 1. I n t r o d u c t i o n p
hospholipases are enzymes that catalyze the hydrolysis of membrane phospholipids. They are classified according to the bond cleaved in a phospholipid. Thus, phospholipase A 1 (PLA1; EC 3.1.1.3) and phospholipase A 2 (PLA2; EC 3.1.1.4) selectively remove fatty acids from the sn-1 and sn-2 positions, respectively. Phospholipase B (PLB; EC 3.1.1.5) is a lysophospholipase. Phospholipase C (PLC; EC 3.1.4.3) cleaves the bond between glycerol and a phosphate; and phospholipase D (PLD; EC 3.1.4.4) hydrolyzes the amino alcohol moiety from a phospholipid (Figure 1).
Correspondence: Prof. Erich Kaiser, Department of Medical Chemistry, University of Vienna, Vienna, Austria. Manuscript received July 18, 1989; revised March 30,1990; accepted April 5, 1990. CLINICALBIOCHEMISTRY,VOLUME 23, OCTOBER 1990
CHIBA,
and KHALED
ZAKY
U n i v e r s i t y of V i e n n a , V i e n n a , A u s t r i a
Our purpose is not to review the available information on all phospholipases, but to present a survey on PLA 2, because this enzyme has attracted attention due to its biological and medical aspects. Some information on PLC is also given. 2. S o u r c e a n d s t r u c t u r e of PLA2 PLA 2 enzymes are widespread, found in nearly all animal cell types examined, and in bacteria and protozoa. Membrane-associated and soluble PLA 2 are found in m a m m a l i a n cells. Most investigations have been performed on PLA 2 of pancreatic origin and from snake venoms (1). Canine pancreatic PLA 2 is synthesized as a preproenzyme of 146 amino acids containing a 15 residue signal peptide, and a 7 residue activation peptide. The 124 residue mature enzyme shows 79.2c~ homology with the porcine enzyme (2). The complete amino acid sequences of pancreatic PLA 2 from horse t3), dog (4), rat (4), h u m a n (5,6), and from bovine pancreas (7) are now known. The cDNA coding for the porcine pancreatic PLA,~ has been cloned and expressed in E. coli (8). PLA 2 and lecithin: cholesterol acyltransferase (LCAT; EC 2.3.1.43) have a common antigenic determinant which is probably located near or at their esterolytic active site (9). PLA2 is a major component of Elapid and Viperid snake venoms and plays an important role in the pharmacological effects following envenomation. The sequence of many venom enzymes has been determined (10), and it is the basis of their classification ( 11 ). In general, the molecular weights of the purified enzymes are low, within 12-15 kDa. PLA 2 enzymes contain a high degree of disulfide crosslinking and are extremely stable to heat and acid treatment. Such properties are important attributes for an enzyme t h a t is involved in phospholipid degradation. Due to this stability, solubilization of membrane bound enzymes can easily be achieved through mineral acid extraction (12). PLA2 has a higher affinity for aggregated than nonaggregated phospholipids. It is unclear if this is due to a conformational requirement by the enzyme or the substrate. Kinetic studies with PLA 2 are problematic, because classical Michaelis-Menten kinetics do not apply. This has important implications for analysis. Most PLA 2 enzymes need Ca 2÷ ions for full activity (1). 349
KAISER, CHIBA, AND ZAKY
F
16:0 +
OH
--16:0 HO-~
AA 4 L P-CHOLINE
~
16:0
AA 0
+ AA
L'P-CHOLINE
LIPASE
A2I
P-CHOLINE PHOSPHATIDYL CHOLINE I PHOSPHOLIPASE
C ] ~ P-CHOLINE
16:0
AA~ OH
OSPHOLIPASE D I FI6:0 AA10-P
Figure 1--Action of phospholipases on phosphatidyl choline. AA = arachidonic acid.
3. Phospholipases in biology 3.1. PHOSPHOLIPASESIN SIGNALTRANSDUCTION Cells respond to their environment through cell surface receptors that bind specific ligands. These "signals" are transduced into changes in cellular metabolism and function. One system that mediates this transduction process involves a ubiquitous family of guanosine triphosphate (GTP)-binding proteins (G-proteins). These proteins transduce signals generated by ligand interactions with specific cell surface receptors into changes in intracellular levels of cyclic nucleotides (13,14). A variety of hormones, neurotransmitters, and growth factors induce the rapid catabolism of phosphatidyl inositol-4,5-biphosphate (PIP 3) via a receptor-mediated process (15-18). Membrane receptors are thought to be coupled with PLC via a G-protein (13,14). This conclusion is based on the observation that GTP and stable GTP analogues promote PIP 3 hydrolysis in permeabilized cells (19,20), broken cell preparations (21), and isolated cell membranes (22-30). The hydrolysis of PIP 3 by an activated inositol-specific PLC generates two putative second messengers:inositol-l,4,5-triphosphate (IP~)aand diacylglycerol. IP a promotes the release of 2+ ions from intracellular stores by opening Ca2+-channels (31-34) which mediate the transient increase in cytosolic Ca 2÷ ions after binding of the agonist to the receptor. Diacylglycerol activates protein kinase C, resulting in the phosphorylation of a num-
350
ber ofintracellular proteins (14,35-38). The activated protein kinase C phosphorylates the lipocortin moiety of the lipoprotein-PLA 2 complex and PLA 2 is liberated. In the presence of elevated cytosolic Ca 2+ ions, PLA 2 is activated and splits phospholipids into lysophospholipid and arachidonic acid. These events are illustrated in Figure 2. Table 1 gives some examples of effects mediated by the synergistic action o f I P 3 (release of Ca 2+ ions from intracellular stores) and diacylglycerol (activation of protein kinase C) (39-53). 3.2. PLA 2 IN GLUCOSE-INDUCEDINSULINRELEASE Glucose-induced insulin release is coupled with high turnover of membrane phospholipids in islet cells (54,55) due to PLA 2 release (56). Both arachidonate and lysophospholipids have membrane-disrupting properties; high concentrations induce morphologic alterations at the periphery of islet cells (57,58). A blockade of glucose-induced insulin release is effected by PLA 2 inhibitors (58,59). Glucose may activate PLA 2 in the islet, leading to the generation of lysophospholipids. The latter may couple energy production to insulin release, at least partially through the promotion of Ca 2+ translocation (59). 3.3. PHOSPHOLIPASESIN PHOTORECEPTION In the rod outer segments of the retina, a Gprotein, transducin, is localized (60). Transducin,
CLINICAL BIOCHEMISTRY,VOLUME 23, OCTOBER 1990
PHOSPHOLIPASES IN BIOLOGYAND MEDICINE
PHOSPHATIDYL INOSITOL 2 ATP
~
2 ADP PHOSPHATIDYL INOSITOL-415-BIPHOSPHATE (PIP3)
PHOSPHOLIPASE C (INACTIVE)
AGONIST
> RECEPTOR
'1
j
PHOSPHOLIPASE C (ACTIVE)
INOSITOL-3,4,5-TRISPHOSPHATE (IP3)
DIACYLGLYCEROL
|
PROTEIN KINASE C
