Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention.

1.1. Discovery of the Phospholipase A2 Superfamily Phospholipases represent one of the earliest enzyme activities to be identified and studied and the phospholipase A2 (PLA2) superfamily (see defining specificity1 in Figure 1) traces its roots to the identification of lytic actions of snake venom at the end of the 19th century. The enzyme was first purified and characterized from cobra venom and later from rattlesnake venom. As protein sequencing methodologies advanced in the 1970’s, it became apparent that these enzymes had an unusually large number of cysteines (over 10% of the amino acids) and as secreted enzymes, that they were all in the form of disulfide bonds. It was further recognized that in the case of PLA2, cobras and rattlesnakes had six disulfides in common, but one disulfide bond is located in distinctly different locations. This led to the designation of Type 1 and Type 2 for cobras (old world snakes) and rattlesnakes (new world snakes), respectively.2 During that same period, studies on the porcine pancreatic digestive enzyme that hydrolyzes phospholipids led to the determination that this mammalian enzyme (and also the human pancreatic enzyme) had the same disulfide bonding pattern as cobras and hence the designation as IB with the cobra enzyme as IA. Figure 1 The specific reaction catalyzed by phospholipase A2 at the sn-2 position of the glycerol backbone is shown. X, any of a number of polar headgroups; R1, fatty acids, or alkyl, or alkenyl groups and R2, fatty acids or acyl moieties. A dramatic change in the phospholipase A2 field that attracted the attention of the broader scientific community occurred in July, 1988 when at the first FASEB Summer Conference on Phospholipases, Jeffery J. Seilhamer and Lorin K. Johnson from California Biotechnology Inc.3 and Ruth M. Kramer from Biogen Research Corporation4 independently and with much fanfare and excitement reported the purification, sequencing and cloning of the first human non-pancreatic secreted PLA2 which they each had isolated from the human synovial fluid of arthritic knee joints. Since the sequence revealed that the disulfide bond pattern was more like the rattlesnake than the human pancreatic enzyme, this new form of PLA2 was designated IIA. All of these enzymes then became known as secreted or sPLA2s. It wasn’t until the late 1980’s that PLA2-like activities were reported in mammalian cells in contrast to extracellular secreted activities from venom and pancreas. In July, 1992, at the second FASEB Summer Conference on Phospholipases, James D. Clark from the Genetics Institute5 and Ruth M. Kramer (who had moved to Lilly Research Laboratories)6 independently reported the purification, sequencing, and cloning of the first human cytosolic PLA2 (cPLA2) from the U937 macrophage cell line. The sequence was unrelated to those of the secreted enzymes. To track this new enzyme and potentially additional PLA2s, a Group Numbering System7 was established utilizing the preexisting venom designation of I and II and expanding them to include subgroups IA, IB, and IIA (GIA, GIB, GIIA); adding Group III (GIII) for the clearly different PLA2 which had been purified from bee venom; and establishing the Group IV (GIV) designation for the new cytosolic PLA2 (cPLA2). This was fortuitous because soon thereafter a new form of secreted PLA2 was discovered. It was produced by macrophages and it had the same six disulfide bonds as Group I and Group II, but lacked the seventh disulfide bond entirely. To make clear that this sPLA2 was neither GI nor GII, this enzyme was designated as Group V (GV). At the Third FASEB Summer Conference on Phospholipases held in July, 1995, Edward A. Dennis from the University of California, San Diego8 reported on another cystosolic PLA2 purified from macrophages that differed from Group IV cPLA2 in that its activity was not dependent on Ca2+ and Simon S. Jones from the Genetics Institute9 reported that the cloned form from CHO cells had a very different sequence than cPLA2. This new Ca2+-independent PLA2 (iPLA2) was designated as Group VI PLA2 (GVI). 10 Earlier, investigators from the University of Utah11 had isolated an enzyme from human plasma which hydrolyzed platelet activating factor (PAF), a phosphatidylcholine containing an acetate at the sn-2 position, and in 1995 Larry W. Tjoelker from ICOS12 reported its cloning. This enzyme and other related PAF acetyl hydrolases (PAF-AH) were later recognized more broadly as PLA2s with a specificity for a short acyl chain on the sn-2 position and for the plasma one for oxidized lipids for which the same enzyme was independently named lipoproteinassociated phospholipase A2 (Lp-PLA2). These enzymes were designated Group VII and VIII (GVII and GVIII). 13 As additional specific PLA2s were discovered, they were either designated by letters as subgroups of the original Groups indicated above or as additional Groups. Especially noteworthy was the discovery of a number of additional sPla2s in which the sequence and/or disulfide bonding pattern varied significantly from the traditional Groups I, II, III, and V sPla2s. These new forms led to the additional Groups IX, X, XI XII,XIII, and XIV sPla2s representing new human forms (especially Group X, which may have important functions) as well unique enzymes from snail venom, rice shoots, parvovirus, and fungi/bacteria. The only new type of PLA2 reported that did not naturally fit in the four types discussed above (secreted, cytosolic, Ca2+-independent, PAF acetylhydrolases) is the lysosomal PLA2 (LPLA2) which was designated as Group XV (GXV). 14 Recently, a new PLA2 was isolated from adipose tissue and designated as Group XVI (GXVI);15 it appears to be a new type of PLA2 called adipose-PLA2 (AdPLA). The current designations are summarized in Table 1. Table 1 The Phospholipase A2 Superfamily In this review, we will discuss in turn each of the six types of PLA2. For each, we will first discuss the various forms, in terms of groups, subgroups and mechanism of action, their structure and interaction with membranes, their biological activities and role in disease, and the development of selective inhibitors. Of course the commonly used type designation has little meaning today since as we have learned more about these enzymes, it has been recognized that secreted, cytosolic, Ca2+-independent, PAF-AH, and lysosomal make little sense since all four of the later categories are actually intracellular (cytosolic) enzymes, that the secreted ones may occur intracellularly in various vesicles, and that the PAF-AHs, lysosomal and some forms of cPLA2 are also Ca2+-independent. Thus the Group Numbering System designation provides an unambiguous definition of each enzyme form. Over the years, numerous excellent reviews on either the broad family of PLA2s16 or specific types including sPLA2s,17 cPLA2s,18 iPLA2s,19 PAF-AHs20 and LPLA221 have appeared as well as several review articles summarizing the classes of PLA2 inhibitors and their potential role for the treatment of inflammatory diseases.22 We have employed all of these prior reviews heavily in preparing this up-to-date and comprehensive single review covering all aspects of the entire phospholipase A2 superfamily.