There are thirteen kinds of mammalian phospholipase C that are classified into six isotypes (β, γ, δ, ε, ζ, η) according to structure. Each PLC has unique and overlapping controls over expression and subcellular distribution.
The extensive number of functions exerted by the PLC reaction requires that it be strictly regulated and able to respond to multiple extra- and intracellular inputs with appropriate kinetics. This need has guided the evolution of six isotypes of PLC in animals, each with a distinct mode of regulation. The pre-mRNA of PLC can also be subject to differential splicing such that a mammal may have up to 30 PLC enzymes.[2]
Most of the bacterial variants of phospholipase C are characterized into one of four groups of structurally related proteins. The toxic phospholipases C are capable of interacting with eukaryotic cell membranes and hydrolyzing phosphatidylcholine and sphingomyelin, ultimately leading to cell lysis.[3]
Comparison of C2 domain of mammalian PI-PLC in red and C2-like domain of Bacillus cereus in cyan
In mammals, PLCs share a conserved core structure and differ in other domains specific to each family. The core enzyme includes a split triosephosphate isomerase (TIM) barrel, pleckstrin homology (PH) domain, four tandem EF hand domains, and a C2 domain.[1] The TIM barrel contains the active site, all catalytic residues, and a Ca2+ binding site. It has an autoinhibitory insert that interrupts its activity called an X-Y linker. The X-Y linker has been shown to occlude the active site, and with its removal, PLC is activated.[4]
The genes encoding alpha-toxin (Clostridium perfringens), Bacillus cereusPLC (BC-PLC), and PLCs from Clostridium bifermentans and Listeria monocytogenes have been isolated and nucleotides sequenced. The sequences have significant homology, approximately 250 residues, from the N-terminus. Alpha-toxin has an additional 120 residues in the C-terminus. The C-terminus of the alpha-toxin has been reported as a "C2-like" domain, referencing the C2 domain found in eukaryotes that are involved in signal transduction and present in mammalian phosphoinositide phospholipase C.[5]
Enzyme mechanism
General reaction catalyzed by phospholipase C
The primary catalyzed reaction of PLC occurs on an insoluble substrate at a lipid-water interface. The residues in the active site are conserved in all PLC isotypes. In animals, PLC selectively catalyzes the hydrolysis of the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) on the glycerol side of the phosphodiester bond. There is the formation of a weakly enzyme-bound intermediate, inositol 1,2-cyclic phosphodiester, and release of diacylglycerol (DAG). The intermediate is then hydrolyzed to inositol 1,4,5-trisphosphate (IP3).[6] Thus the two end products are DAG and IP3. The acid/base catalysis requires two conserved histidine residues and a Ca2+ ion is needed for PIP2 hydrolysis. It has been observed that the active-site Ca2+ coordinates with four acidic residues and if any of the residues are mutated then a greater Ca2+ concentration is needed for catalysis.[7]
Small molecule U73122: aminosteroid, putative PLC inhibitor.[10][11] However, the specificity of U73122 has been questioned.[12] It has been reported that U73122 activates the phospholipase activity of purified PLCs.[13]
Autoinhibition of X-Y linker in mammalian cells: It is proposed that the X-Y linker consists of long stretches of acidic amino acids that form dense areas of negative charge. These areas could be repelled by the negatively charged membrane upon binding of the PLC to membrane lipids. The combination of repulsion and steric constraints is thought to remove the X-Y linker from near the active site and relieve auto-inhibition.[1]
Compounds containing the morpholinobenzoic acid scaffold belong to a class of drug-like phosphatidylcholine-specific PLC inhibitors.[15][16][17]
o-phenanthroline: heterocyclic organic compound, known to inhibit zinc-metalloenzymes[18]
EDTA: molecule that chelates Zn2+ ions and effectively inactivates PLC, known to inhibit zinc-metalloenzymes[19]
The two products of the PLC catalyzed reaction, DAG and IP3, are important second messengers that control diverse cellular processes and are substrates for synthesis of other important signaling molecules. When PIP2 is cleaved, DAG remains bound to the membrane, and IP3 is released as a soluble structure into the cytosol. IP3 then diffuses through the cytosol to bind to IP3 receptors, particularly calcium channels in the smooth endoplasmic reticulum (ER). This causes the cytosolic concentration of calcium to increase, causing a cascade of intracellular changes and activity.[23] In addition, calcium and DAG together work to activate protein kinase C, which goes on to phosphorylate other molecules, leading to altered cellular activity.[23] End-effects include taste, tumor promotion, as well as vesicle exocytosis, superoxide production from NADPH oxidase, and JNK activation.[23][24]
Both DAG and IP3 are substrates for the synthesis of regulatory molecules. DAG is the substrate for the synthesis of phosphatidic acid, a regulatory molecule. IP3 is the rate-limiting substrate for the synthesis of inositol polyphosphates, which stimulate multiple protein kinases, transcription, and mRNA processing.[25]
Regulation of PLC activity is thus vital to the coordination and regulation of other enzymes of pathways that are central to the control of cellular physiology.
Additionally, phospholipase C plays an important role in the inflammation pathway. The binding of agonists such as thrombin, epinephrine, or collagen, to platelet surface receptors can trigger the activation of phospholipase C to catalyze the release of arachidonic acid from two major membrane phospholipids, phosphatidylinositol and phosphatidylcholine. Arachidonic acid can then go on into the cyclooxygenase pathway (producing prostoglandins (PGE1, PGE2, PGF2), prostacyclins (PGI2), or thromboxanes (TXA2)), and the lipoxygenase pathway (producing leukotrienes (LTB4, LTC4, LTD4, LTE4)).[26]
The bacterial variant Clostridium perfringens type A produces alpha-toxin. The toxin has phospholipase C activity, and causes hemolysis, lethality, and dermonecrosis. At high concentrations, alpha-toxin induces massive degradation of phosphatidylcholine and sphingomyelin, producing diacylglycerol and ceramide, respectively. These molecules then participate in signal transduction pathways.[5] It has been reported that the toxin activates the arachidonic acid cascade in isolated rat aorta.[27] The toxin-induced contraction was related to generation of thromboxane A2 from arachidonic acid. Thus it is likely the bacterial PLC mimics the actions of endogenous PLC in eukaryotic cell membranes.
^ abSakurai J, Nagahama M, Oda M (November 2004). "Clostridium perfringens alpha-toxin: characterization and mode of action". Journal of Biochemistry. 136 (5): 569–74. doi:10.1093/jb/mvh161. PMID15632295.
^Essen LO, Perisic O, Katan M, Wu Y, Roberts MF, Williams RL (February 1997). "Structural mapping of the catalytic mechanism for a mammalian phosphoinositide-specific phospholipase C". Biochemistry. 36 (7): 1704–18. doi:10.1021/bi962512p. PMID9048554.
^Bleasdale JE, Thakur NR, Gremban RS, Bundy GL, Fitzpatrick FA, Smith RJ, Bunting S (November 1990). "Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils". The Journal of Pharmacology and Experimental Therapeutics. 255 (2): 756–68. PMID2147038.
^Little C, Otnåss AB (June 1975). "The metal ion dependence of phospholipase C from Bacillus cereus". Biochimica et Biophysica Acta (BBA) - Enzymology. 391 (2): 326–33. doi:10.1016/0005-2744(75)90256-9. PMID807246.
^ abcAlberts B, Lewis J, Raff M, Roberts K, Walter P (2002). Molecular biology of the cell (4th ed.). New York: Garland Science. ISBN978-0-8153-3218-3.
