DNA AND CELL BIOLOGY Volume 11, Number 3, 1992 Mary Ann Liebert, Inc., Publishers Pp. 233-243

Regulation of Extracellular Phospholipase A2 Activity: Implications for Inflammatory Diseases ANIL B.

MUKHERJEE, ELEONORA CORDELLA-MIELE, and LUCIO MIELE

ABSTRACT

Phospholipases A2 (PLA2s; E.C. 3.1.1.4) are a family of esterases that are involved in myriads of physiological and pathological processes. The involvement of these enzymes, especially the extracellular PLA2s, in the generation of proinflammatory lipid mediators makes them a very important target for investigation. These PLAjS have been suggested to be involved in the pathogenesis of several human inflammatory diseases. Thus, delineating the mechanism(s) of regulation of the activity of these enzymes may provide a better understanding of the pathophysiology of these diseases and allow the rational design and development of novel therapeutic agents. In this article, we provide a brief description of PLA2s in general with a special emphasis on extracellular enzymes, their mechanism(s) of action, and possible role in the pathogenesis of inflammatory diseases. Additionally, we describe: (i) a novel mechanism of activation of extracellular PLA2s by transglutaminases and (ii) the development of one class of antiinflammatory agents, antiflammins, derived from the active site structure of endogenous PLA2-inhibitory proteins.

family of esterases glycerophossee Waite, 1988; Dennis, pholipid (for 1990; Mukherjee, 1990). These enzymes are generally calcium-dependent and have been found both intra- and extracellularly. For the past several years, these enzymes have attracted considerable interest, not only for their involvement in the generation of proinflammatory lipid mediators but also for their possible regulatory role in many (PLA2s) that hydrolyze Phosphol imolecules pases theA2 s«-2-acyl review,

(Burch, 1990) and

in the stimulation of proliferation of Swiss 3T3 cells after binding to a specific site on the cell membrane (Arita et al, 1991). Thus, interest in understanding the mechanism of the regulation of this class of enzymes has enormously increased in recent years. In this paper, we will attempt to present a brief overview of the biochemistry of PLA2s in general with special emphasis on the extracellular PLA2s, as they are implicated in the pathogenesis of several important inflammatory diseases (Table 1). The term "regulation" in this paper is used solely to mean activation and inhibition of PLA2.

are a

ester bond in

vital cellular functions. By hydrolyzing the sn-2 bond in glycerophospholipids, PLA2s release free fatty acids (Fig. 1). If these fatty acids are arachidonic acid, this enzymatic reaction generates the substrate for the initiation of the arachidonic acid cascade that produces various eicosanoids (e.g., prostaglandins, leukotrienes, and thromboxanes). Many of these substances are potent mediators of inflammation. Additionally, hydrolysis of l-alkyl-2-acyl-s«-glycerophosphocholine by PLA2 yields lyso-platelet-activating factor (PAF), the precursor of PAF. PAF is one of the most potent inflammatory lipid mediators known to date and has been implicated in the pathogenesis of such diseases as bronchial asthma. Moreover, PLA2s have been suggested to play a role in cellular signal transduction via the G-protein cycle

STRUCTURE AND DISTRIBUTION OF PLA2s PLA2s have been divided into two major groups: type I and type II (Heinrikson et al, 1977; Heinrikson and Kezdy, 1990). Recently, a third type of PLA2 has been isolated, cDNA cloned and characterized and is now known as cellular PLA2 (cPLA2). Type I and II enzymes are relatively low-molecular-weight proteins (~ 14 kD) that are structurally very similar, except for the fact that type I PLA2s have a disulfide bond between Cys-11 and Cys-76 that is missing in type II enzymes. Type I phospholipases are found primarily in Elapidae snake venoms and in the

Section on Developmental Genetics, Human Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.

233

MUKHERJEE ET AL.

234

mammalian pancreas and type II enzymes are found in the of Crotalidae family of snakes. Mammals, including humans, possess both type I and II PLA2s. These are found both intra- and extracellularly, as well as in association with cell membranes (Waite, 1988, 1990). Both type I and type II enzymes contain a total of seven disulfide bonds, despite the lack of the Cys-1 l-Cys-76 bond in type II PLA2s. This is because type II PLA2s have a disulfide bridge between the middle of the C-helix and the carboxyl terminus of an extra long (by 7 residues) tail that also characterizes the type II enzymes (for review, see Ward and Pattabiraman, 1990). The other unique feature of type II PLA2s is the lack of the so called "elapid loop" or D-helix, which is invariably present in type I enzymes. Although these structural differences could be used to distinguish type I from type II enzymes, the overall tertiary structure of these two types of proteins are strikingly similar (Renetseder et al, 1985). As mentioned above, a new family of high-molecularweight (~ 100-110 kD) PLA2s, in addition to the type I and II enzymes, have been discovered within the last few venoms

SITES OF ACTION OF PHOSPHOLIPASES ON

PHOSPHATIDYLCHOLINE Phospholipase Aj

H2COÏ-C-Ri Phospholipase A?

R?-CiOCH 0 Phospholipase D

H?CO j P 0CH2CH2N + (CH3I3

lo-

-

FIG. 1.

years. These enzymes are present in the cytosol of various cells and tissues, including platelets (Kramer et al, 1988),

macrophages (Wijkander and Sundler, 1989), and monomacrophage cell lines (Leslie et al, 1988; Clark et al, 1990; Diez and Mong, 1990; Kramer et al, 1991). These enzymes also preferentially hydrolyze the sn-2 position of phospholipid molecules releasing free arachidonate. More recently, a cDNA coding for a human, Ca2*sensitive, cytosolic PLA2 has been cloned and the recombinant protein expressed in bacteria and eukaryotic cells (Clark et al, 1991; Sharp et al, 1991). It has been suggested that these enzymes may act as novel and important second messengers in cellular signal transduction pathblast and

ways.

ACTIVATION OF PLA2 Various physiological stimuli such as bradykinin, prolactin, angiotensin, and thrombin have been reported to activate cellular PLA2 activity. However, most of these studies used arachidonate release from responsive cells as the criterion for the activation of this enzyme (McGiff et al, 1972; Vargaftig and Dao Hai, 1972; Bills et al, 1977; Rillema and Wild, 1977). As arachidonic acid can also be generated through a non-PLA2 pathway of phospholipid hydrolysis, these studies needed confirmation by more stringent methods. The results of such critical experiments recently have been reported by several investigators (Chang et al, 1986, 1987; Gilman et al, 1986; Burch, 1990). Biochemically, PLA2s and other lipases belong to a unique group of enzymes, as they are water-soluble proteins that must catalyze a reaction involving a lipid substrate, Thus, these enzymes must be activated in a lipidwater interface (Waite, 1988, 1990). All the low-molecularweight PLA2s are more active on phospholipids present in a lipid-water interface than on monomeric substrates. The first step in the initiation of catalysis is what is generally known as "interfacial recognition" (Fig. 2A), which is followed by "interfacial activation" (Heinrikson and Kezdy, 1990; Fig. 2B). Invariably, these processes occur only at phospholipid surfaces, regardless of their micellar status (pure or mixed), monolayers, or bilayers. Interfacial recognition process is marked by a "higher re-

Sites of hydrolysis by phospholipases on a phosphatidylcholine molecule. Note the sn-2 position (middle arrow) which is the hydrolytic site for phospholipase A2. Cleavage of this bond releases a free fatty acid, such as arachidonic acid. (Reprinted, with permission, from Lehninger, 1978.) activity of the enzyme toward a substrate in any of the agTable 1. High PLA2 Activity Associated Diseases

Rheumatoid arthritis Collagen vascular diseases Pancreatitis Peritonitis Septic shock ARDSa Acute renal failure Autoimmune uveitis Bronchial asthma (?)

with

Human Diseases Sites

Serum, synovial fluid, WBC

Serum Serum Peritoneal fluid and cells Serum Serum and alveolar fluid Serum Serum, aqueous humor

(?)

Modified after Pruzanski and Vadas (1990). aAdult respiratory distress syndrome.

EXTRACELLULAR PHOSPHOLIPASES A2

Diagrammatic representation of interfacial recognition and interfacial activation of phospholipase A2. A. Experimental proof that mature pancreatic PLA2 (upper curve), but not its proenzyme (lower curve), shows a dramatic inB. Sugcreases in catalytic activity when the substrate concentration reaches the critical micellar concentration (CMC). gested (Heinrikson and Kezdy, 1990) mechanism of activation by autocatalytic acylation and dimerization of monomeric FIG. 2.

PLA2 molecules.

gregated forms than toward the same substrate in homogeneous solution" (Heinrikson and Kezdy, 1990). This recognition is followed by a second phase of "interfacial activation," which is marked by a lag period between the addition of PLA2 to a solution containing lipid substrate and a measurable catalytic activity. It should be mentioned that all known low-molecular-weight PLA2s demonstrate interfacial recognition, while only monomeric as opposed to dimeric or trimeric PLA2s show interfacial activation (Heinrikson and Kezdy, 1990). It has been suggested that the lag period that characterizes interfacial activation is due to a

slow dimerization of surface-bound monomeric PLA2 molecules (Romero et al, 1987; Cho et al, 1988; Tomasselli et al, 1989; Biltonen et al, 1990; Heinrikson and Kezdy, 1990). The chemical basis of dimerization has been reported to be a self-catalyzed acylation of Lys residue(s) within the PLA2 molecule (Cho et al, 1988; Tomasselli et al, 1989). A schematic diagram illustrating interfacial activation of monomeric PLA2s is presented in Fig. 2. A detailed mechanism of PLA2 catalysis has also been proposed by Scott et al (1990) on the basis of the results obtained from high-resolution crystal structures of Naja naja atra venom PLA2 complexed with a phosphonate transition state analog. This mechanism is also described in an adjoining article. Recently, we have described a novel transglutaminasemediated post-translational modification of porcine pan-

creatic PLA2, which

causes a

(Cordella-Miele (TG; E.C. 2.3.2.13) are enzyme

et

dramatic activation of this

al, 1990). Transglutaminases

a class of Ca2*-dependent acyl transferases which exist both intra- and extracellularly (Folk, 1980). One of the best-characterized extracellular TGs is blood coagulation Factor Xllla, which is activated by the action of thrombin. These enzymes catalyze the formation of e-(7-glutamyl)lysine isopeptide bonds between specific glutamines (acyl donors) and lysine residues (acyl acceptors) located within the same protein or in two different proteins. Thus, TGs can catalyze both intra- and intermolecular covalent cross-linking of specific proteins that are substrates of this enzyme. TGs can also utilize polyamine substrates to cross-link polypeptides. Cordella-Miele et al (1990) have demonstrated that the post-translational modification of PLA2 by TG leads to the formation of an e-(-y-glutamyl)lysine isopeptide formation between Gln-4 and an as yet unknown Lys residue in porcine pancreatic PLA2 causing an intramolecular cross-linking of this enzyme. This intramolecular cross-linking promotes a noncovalent dimerization of PLA2 that leads to a dramatic increase in specific activity. We have also observed similar effects when PLA2 was pretreated with human and rabbit Factor XIHa. When TG-treated PLA2 was analyzed by size-exclusion chromatography on Sephacryl-S200, two peaks of PLA2 activity were observed: a putative dimeric peak of apparent molecular weight 26 kD and a mono-

MUKHERJEE ET AL.

236

meric peak of 13 kD. The putative dimeric enzyme manifested a 10-fold higher specific activity as compared with the monomeric enzyme. To determine whether the presumptive dimeric peak was covalently cross-linked PLA2, we performed SDS-PAGE and Western blotting. It was found that in SDS-PAGE under denaturing conditions this peak yielded only a 13-kD band, suggesting that it consists of monomeric PLAa that underwent noncovalent dimerization. The final proof of this was obtained by a chemical analysis and quantitation of e-(7-glutamyl)lysine isopeptide after an exhaustive proteolysis of the material obtained from peak I (putative dimer) and peak II (putative monomer). These data confirmed our hypothesis that TG causes an internal cross-linking in PLA2 and the crosslinked enzyme preferentially forms noncovalent dimers. This dimerization is accompanied by a dramatic activation of this enzyme. TGs are highly selective enzymes in terms of the glutamine residues that can be utilized as acyl donors. In those proteins that are substrates of TG, usually only one specific glutamine residue is active as acyl donor, even if more than one glutamine residue is present in the protein sequence. It has been suggested that this extreme specificity for a glutamine residue indicates that TG-mediated posttranslational modifications in vitro are most likely to be physiologically important in vivo (Davies et al, 1988). Since porcine pancreatic PLA2 contains only one glutamine residue (Gln-4), this residue is the only possible acyl donor for the TG-catalyzed reaction. Gln-4 is a near-invariant residue in the PLA2 family and conserved across phyla. In fact, the type I enzymes of the mammalian pancreatic type, most snake venom enzymes (both type I and II), human lung PLA2 (Seilhamer et al, 1986), and human nonpancreatic PLA2 (Seilhamer, 1989) have a Gin in position 4. This may also be an indication that TG-mediated post-translational modifications of PLA2 has physiological significance. Interestingly, although the rat and human placental/synovial (type II) PLA2s lack Gln-4, they have a Gin at residue 110. Like Gln-4 in type I enzymes, Gln-110 is surrounded by aromatic and positively charged residues, including the two lysines which are present in the flanking region of Gln-4. As yet it has not been established whether TG-catalyzed post-translational modification occurs in human placental/synovial PLA2 and, if so, whether such modification results in the activation of this enzyme. Although we have not determined the specific Lys residue involved in the formation of the intramolecular cross-link in porcine pancreatic PLA2, structural features suggest that it may be Lys-10.

uveitis (see Chan et al, 1990, 1991), septic shock, and possibly bronchial asthma, extracellular PLA2s may play a pathogenic role. The mechanisms of increased PLA2 secretion in these diseases are not yet clearly understood. One possible mechanism may involve cytokines (Pruzanski and Vadas, 1990). It is proposed that various infectious and noninfectious agents can activate cytokine production by monocytes and macrophages. These agents include Epstein-Barr virus (EBV), human T-lymphotropic virus type

1 (HTLV-1), IL-1, lymphotoxin, lipopolysaccharide, phytohemagglutinin (PHA), platelet-derived growth factor, tumor-promoting activity (TPA), tumor necrosis factor (TNF), and interferon-7 (IFN-7). Cytokines, such as IL-6

TNF, can stimulate several cell types to release PLA2. Secreted PLA2 can directly cause cell destruction by hydrolyzing cell membrane phospholipids. Additionally, PLA2generated free arachidonic acid molecules or their metabolites (e.g., prostaglandins, leukotrienes, and thromboxanes) could act as chemoattractant to other immunocytes. These metabolic products of arachidonate can in turn propagate existing inflammation and initiate new ones. It has been shown that IL-6, TNF, and IL-1 stimulate the production of type II extracellular PLA2s in Hep G2 cells (Crowl et al, 1991). Thus, cytokines produced by activated T cells and/or macrophages may stimulate the production of PLA2 from the liver that gets into circulation. The circulating PLA2 may find its way into various tissues and cause inflammation. TG-catalyzed post-translational modifications may cause the secreted monomeric PLA2 to dimerize and may activate this enzyme. Activated macrophages are known to secrete TG (Folk, 1980; Leu et al, 1982) and PLA2 (Vadas and Hay, 1980). Thus, these cells can provide two ingredients necessary for the initiation and propagation of inflammatory reactions (i.e., PLA2 and TG). Moreover, it is well established that inflammation and blood coagulation are interrelated phenomena. Because activation of the coagulation cascade may be triggered by inflammatory processes, it is not unreasonable to speculate that soluble extracellular PLA2 released by activated phagocytes or by necrotic cells could be activated by coagulation Factor XHIa. It would be of interest to delineate whether or not the synovial PLA2 in rheumatoid arthritis is TG modified. It is well known that resolution of an inflammatory reaction may initiate fibrosis in many organs. However, the mechanism by which fibroblasts at a site of inflammation are induced to proliferate is not as yet clear. Arita et al (1991) have reported that type I PLA2s can induce proliferation of Swiss 3T3 cells after a specific binding of PLA2 molecules on the surface of these cells. A high-molecularweight ( 200 kD) cell membrane protein has been shown to be responsible for this specific binding of PLA2. These results may suggest yet another function of PLA2, namely, the stimulation of cellular proliferation. It is yet to be proven experimentally, however, that PLA2 stimulates the proliferation of those fibroblasts that are present at the site of an inflammation before any connection between this enzyme and the development of fibrosis after inflammation could be made. or

~

POSSIBLE MECHANISM(S) OF EXTRACELLULAR PLA2 ACTIVATION AND ITS PATHOGENIC ROLE There is

compelling evidence to suggest that in several inflammatory diseases (for review, see Pruzanski and Vadas, 1990; Table 1) such as rheumatoid arthritis, autoimmune

EXTRACELLULAR PHOSPHOLIPASES A2

DEVELOPMENT OF BIOLOGICALLY ACTIVE PLA2 INHIBITORY PEPTIDES FROM THE ACTIVE SITE STRUCTURES OF AN ENDOGENOUS PLA2-INHIBITORY PROTEIN, UTEROGLOBIN

Uteroglobin (UG; Beier, 1968) or blastokinin (Krishnan and Daniel, Jr., 1967) is a 15.8-kD steroid-dependent, secretory protein, first detected in the uteri of rabbits during early pregnancy. Subsequently, this protein was found to be synthesized in the tracheobronchial, prostatic, seminal vesicular, and oviductal epithelia. This protein has potent antiinflammatory and immunosuppressive properties. It has been suggested that the immunomodulatory and antiinflammatory properties of this protein may, at least in part, stem from its ability to inhibit PLA2 activity (for review, see Miele et al, 1987; Mukherjee et al, 1988). Structurally, UG is a homodimer in which each monomer consists of 70 amino acids, oriented antiparallely and connected by two disulfide bridges (Mornon et al, 1980). The disulfide bonds are formed between Cys residues at 3 and

237 69' and 69 (Fig. 3a). Each monomer consists of 4 a-helices and a 0-turn between a-helix 2 and 3 (Fig. 3b). Most interesting of all, the crystallographic surface structure of UG bears a striking similarity to that of PLA2 (Morize et al, 1987). Additionally, there is also a similarity in the hydropathy profiles of UG and PLA2 (Fig. 4b) and local amino acid sequence similarity between these two proteins (Facchiano et al, 1991). These structural similarities, together with the finding that UG inhibits PLA2 activity, led us to determine whether or not there is a specific region of this protein that is active in the inhibition of PLA2. Our approach to resolve the structure-function relationship of UG as an inhibitor was based primarily on two different experimental approaches: (i) structural comparison of UG with those of other PLA2 inhibitory proteins to identify a putative active site of this protein followed by oligopeptide design and synthesis and (ii) bacterial expression of recombinant UG and site-directed mutagenesis of the putative active site to identify the specific amino acid residues involved in the inhibition of PLA2. Both of these approaches were potentially problematic. In fact, no dimeric protein

FIG. 3. Crystallographic structure of UG. a. a-Carbon structure of UG dimer projected along the ö-axis. Shown are the two interchain disulfide bonds between Cys-3 and -69' and -3' and -69. Crystallographic resolution 2.2 À. (Reproduced, with permission, from Mornon et al, 1980.) b. a-Carbon structure of UG monomer. The numbers within circles represent the a-helices. Note the 0-turn between a-helices 2 and 3. Also note that the residues 39-47 (i.e., AF-1) with PLA2 inhibitory activity, are part of an exposed helix. Crystallographic resolution 1.34 Á. (Reproduced, with permission, from Morize et al, 1987.)

MUKHERJEE ET AL.

10

20

40

30

70

60

50

GICPRFAHVIENLLLGTPSS-YETSLKEFEPODTMKDAGMQMKKVLDSLPQTTRENIMKLTEKIVKSPLCM • • *••



*

*

*

*

*

•*

* *

*

*

*•••••***••*

GLGTDEDTL I E I LASRTNKE I RD I NRVYREELKRDLAKDI TSDTSGDFRNALLSLAKGDRSEDFGVNÉDLADSDARA .

140

130

II

160

150



170

200

190

GICPRFAHVIENLLLGTPSSYETSLKEFEPDDTMKDAGMQMKKVLDSLPOTTRENIMKLTEKIVKSPLCM • * *** *+** ** * •• *• * • •* • * * * » • * * ADSDARALYEAGERRKGTOVNVFNTILTTRSYPGLRRVFQKY-T-KYSKHDMNKVLDLËLKGDIEKC--LTA-IVKCATSKP •

III



-ICPRFAHVIENLLLGTPSSYETSLKÉFEPDDTMKDAGMQMK-K-VLDS * *. *

GTD E DS L I

I

II

IV

*•••

*

*

*

***

*

*

*•*

L PQTTR E N I M-KLTEKIV-KSPLC ** **•**

ICSRTNQELOEINRVYKEMYKTDLEKDIISDTSGDFRKLMVALAKGRRAEDGSVIDY-ELIDQDARDLYDAGVKRKGTD--VPKWISI

EXTRACELLULAR PHOSPHOLIPASES A2

239

with interchain disulfide bonds had ever been expressed in E. coli in its natural quaternary conformation. Since then, Miele et al. (1990) have successfully expressed recombinant UG in its natural dimeric form in E. coli. In addition, the only mammalian PLA2 inhibitory protein with biological properties similar to those of UG was lipocortin I. However, the crystal structure of lipocortin I had not been determined and very little structural data were available on this protein. Lipocortin I, in addition to having PLA2 inhibitory activity, has also been shown to share antichemotactic effects in vitro with UG. Subsequently, like UG, lipocortin I has also been demonstrated to be a substrate of TG (Ando et al., 1991). Our primary approach to gener-

antiinflammatory/immunomodulaprimary structures of UG and lipocortin I by several computer-based algorithms. Using this approach, we were able to identify a general sequence similarity between the UG monomer and the 70amino-acid nonidentical "repeat" of lipocortin I, which represents the main recognizable structural unit of this protein. More specifically, by using the program PRTALN, UG was aligned to repeats 2 and 3 of human lipocortin I and to repeat 2 of lipocortin II. Lipocortin I repeat 3 (residues 199-275) manifested the highest similarity with UG (43% similarity, including identities and conservative substitutions). Additionally, a distinct region of local similarity was identified between UG residues 40-46 and lipocortin I repeat 3 residues 247-253. In UG, this reate

oligopeptides

tory activity

was

with

to compare the

Table 2. Peptides

Peptide AF-1 AF-2 2-Asn-AF-1 ¡» AF-2a AF-2n AF-2ns LC5 204-212 LC5 206-212 Consensus

gion corresponds to the carboxy-terminal half of a-helix 3 (residues 32-47). This region of a-helix 3 of UG is exposed to the solvent and there was no evidence that exposed amino acid residues in this region are involved in interchain interactions. The general structural similarity between UG monomer and the lipocortin repeat was also confirmed by hydropathy analysis according to Kyte and Doolittle (1982). Although there is a generalized similarity in the hydropathy profile of these two protein sequences, the most remarkable is in the amphipathic region of UG including residues 37-52 (Fig. 4b). Interestingly, the hydropathy profile of porcine pancreatic PLA2 shows a striking similarity to those of UG and the corresponding regions of lipocortin I and II (Fig. 4b). This finding is consistent with the apparent similarity between UG and PLA2 in their three-dimensional structure. The above findings were further supported by a sequence similarity between UG and porcine pancreatic PLA2 identified by using a different protein alignment program (Facchiano et al, 1991). These studies also revealed a high local amino acid sequence similarity between a region of type I PLA2s and UG residues 40-47. Based upon these results, we synthesized oligopeptides corresponding to the region of highest local similarity between UG and lipocortin I repeat 3. The nonapeptides corresponding to UG residues 39-47, lipocortin I residues 246-254, and a chimeric peptide in which UG Lys-42 was replaced by an Asn residue were synthesized (see Table 2). All three of these peptides subsequently

of the

Antiflammin Family

Amino acid sequence

Reference

Met-Gln-Met-Lys-Lys-Val-Leu-Asp-Ser His-Asp-Met-Asn-Lys-Val-Leu-Asp-Leu Met-Gln-Met-Asn-Lys-Val-Leu-Asp-Ser His-Asp-Ala-Asn-Lys-Val-Leu-Asp-Leu His-Asp-Nle-Asn-Lys-Val-Leu-Asp-Leu His-Asp-Nle-Asn-Lys-Val-Leu-Asp-Ser Ser-His-Leu-Arg-Lys-Val-Phe-Asp-Lys Leu-Arg-Lys-Val-Phe-Asp-Lys Xxx-Fil-Fob-Fil-Lys-Val-Fob-Asp-Xxx

Miele et al. (1988) Miele et al. (1988) Miele et al. (1988) Tetta et al. (1991) Tetta et al. (1991) Tetta et al. (1991) Peretti et al. (1991) Perretti et al. (1991)

Xxx, Indeterminate residue; Fil, hydrophilic residue; Fob, hydrophobic residue. aChimeric peptide (see text for explanation).

FIG. 4. Similarities in amino acid sequences and hydropathy profiles, a. Alignment of UG sequence with lipocortin I repeat two (I), lipocortin I repeat three (II), and lipocortin II (III). Identities are indicated by asterisks and conservative substitutions by dots. b. Hydropathy profiles of UG (I), lipocortin I repeat two (II), lipocortin I repeat three (III), lipocortin II repeat two (IV), and porcine pancreatic PLA2, residues 24-92 (V). These profiles were obtained by using a segment of seven residues. Positive peaks are hydrophobic and negative peaks are hydrophilic regions. (Reproduced, with permission, from Miele et al, 1988).

240

found to be very potent inhibitors of PLA2 activity by using a mixed micellar assay. A longer peptide corresponding to the entire a-helix 3 of UG was also found to be active, but less potent than the three nonapeptides described above. The removal of two amino-terminal residues and one carboxy-terminal residue rendered the peptides totally inactive as PLA2 inhibitors. The core tetrapeptide Lys-Val-Leu-Asp, which is conserved in the three active peptides, was by itself inactive. When the UG-derived peptides were preincubated with the lipid substrate of PLA2 instead of incubating them with the enzyme, no PLA2-inhibitory activity was apparent. A control peptide, corresponding to UG residues 1-10, was inactive as PLA2 inhibitor. Both parent proteins, UG and lipocortin I respectively, were active as PLA2 inhibitors in this assay. The PLA2 inhibitory peptides were tested for their biological activity as antiinflammatory agents in vivo using the classic model of carrageenan-induced rat footpad edema assay. In this assay system, both UG-derived and lipocortin I-derived peptides manifested very potent antiinflammatory activity in a dose-dependent manner. As expected, this antiinflammatory activity of the peptides was totally overcome by a concomitant administration of free arachidonic acid. The parent proteins of these peptides also demonstrated antiinflammatory effects in this system while nonspecific proteins and peptide controls were ineffective in suppressing the inflammatory process induced by carrageenan. The results of these experiments suggested that one of the predominant mechanisms by which these peptides were able to suppress inflammation, at least in part, has to do with their PLA2 inhibitory activity (Miele et al, 1988). Because of their profound antiinflammatory, activity these peptides are now known as "antiflammins" were

measured

(AFs). OTHER PROPERTIES OF ANTIFLAMMINS We have recently discovered that antiflammin peptides may directly interact with a type I PLA2 (porcine pancreatic PLA2) and may prevent the activation of this enzyme (Facchiano et al, 1991). As mentioned earlier, PLA2 activation involves surface recognition, acylation of Lys residue 56, and interfacial activation. At present, we do not have any experimental evidence to suggest which phase or phases of PLA2 activation is inhibited by the antiflammins. In addition to the antiinflammatory activity, the AFs also inhibit platelet aggregation. Vostal et al (1989) have demonstrated that AFs inhibit thrombin and ADP-induced platelet aggregation and ADP-induced serotonin secretion in human platelets in vitro. Interestingly, the core tetrapeptide (Lys-Val-Leu-Asp), which does not inhibit PLA2 activity, is a potent inhibitor of ADP-induced platelet aggregation (Vostal et al, 1989). This effect is due to a different mechanism than that of the AFs, namely, competition with the fibrinogen receptor on the platelet surface. Recently, Camussi et al (1990a,b) have demonstrated that the AFs exhibit yet another property. They inhibit the synthesis of PAF, one of the most potent lipid mediators of inflammation discovered so far.

MUKHERJEE ET AL.

One of the initial steps in the synthesis of PAF is the activation of a PLA2. The peptides inhibit PLA2 activity in neutrophil lysates and prevent the release of arachidonic acid induced by TNF or by phagocytosis (Camussi et al, 1990a,b). Additionally, the same authors (Camussi et al, 1990a,b; Tetta et al, 1991) found that AFs also inhibit the activation of acetyl CoA-lyso PAF acetyltransferase in intact cells. The continued presence of AFs in the cell culture medium was necessary to have an effect on PAF synthesis because washing the cells with fresh AF-free medium reversed these effects. The IC50 of AFs for these effects ranged from 4 x 10"8 to 10"7 M. Both AFs 1 and 2 are highly effective inhibitors of neutrophil aggregation and chemotaxis. Also, these peptides inhibited the increase in vascular permeability, neutrophil infiltration, and Arthus reaction induced by intradermal injection of C5a or TNF. Since UG is a potent inhibitor of neutrophil chemotaxis and phagocytosis, we speculate that the antiinflammatory properties of AFs may be due, at least in part, to an impairment of the function of phagocytes. The observations of Camussi et al (1990a,b) and Tetta et al (1991) are consistent with this hypothesis. The effects of AFs on acute inflammation in rats were also evaluated by Ialenti et al (1990) and DiRosa et al (1990). These authors have demonstrated that AF-2 action is on the late phase of carrageenan-induced edema which is mediated by the release of eicosanoids. The early phase of carrageenan- and dextran-induced edema are marked by the release of histamine and 5-hydroxytryptamine rather than by arachidonate metabolism and were not inhibited by AFs. These activities are similar to those observed with recombinant lipocortins I and V and are consonant with the hypothesis that AFs exert their pharmacological effects by inhibiting arachidonate release and PAF biosynthesis. The results of these and other studies reported so far, demonstrate that AFs are pharmacologically active in controlling inflammation if such processes involve PLA2 activation and/or PAF formation. A second-generation of AF peptides and AF analogs have also been described. Tetta et al (1991) recently described two AF-2 derivatives in which the Met in position 3 was replaced with Nie. These peptides are not susceptible to oxidative degradation and have biological activities apparently identical to those of AF-2. By aligning UG with lipocortin V, Perretti et al. (1991) identified an AF-like sequence in lipocortin V. A nonapeptide and a heptapeptide derived from this region have been shown to inhibit arachidonate and prostaglandin E2 release from macrophages and fibroblasts in vitro as well as preventing porcine pancreatic PLA2-induced contractions of rat stomach strips. The lipocortin-derived peptides show antiinflammatory effects identical to those of AFs in carrageenan-induced rat paw edema. The structures of all biologically active peptides of the AF family described so far are shown in Table 2. Based upon the results reported above, Chan et al (1990, 1991) assessed the AFs for their ability to inhibit the inflammation in autoimmune uveitis in rats, a model for human anterior uveitis. This disease is quite prevalent and one of the major causes of blindness in man. It has also been demonstrated that the inflammation in this disease is

EXTRACELLULAR PHOSPHOLIPASES A2 initiated and

propagated by PLA2 activation. Thus, Chan

et al (1990, 1991) used AFs topically to treat this disease in rats and were highly successful. Based upon the results of

these studies, a clinical trial of the AFs on human autoimmune uveitis has ben initiated by Chan et al at the National Eye Institute. In conclusion, our studies involving the structure-function relationship of UG as a PLA2 inhibitor have resulted in the design and synthesis of a new family of bioactive peptides that show promise as potent therapeutic agents for a variety of inflammatory diseases including autoimmune uveitis. These peptides also provide a biochemical tool for investigating the mechanism(s) of inhibition of PLA2 activity. The results of these and other ongoing studies may further our understanding of the mechanism of activation of this class of enzymes as well as the development of peptide mimetic pharmacological agents with high efficacy and potency for the therapy of inflammatory diseases.

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EXTRACELLULAR PHOSPHOLIPASES A2

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publication January 17, 1992; accepted January 27,

Regulation of extracellular phospholipase A2 activity: implications for inflammatory diseases.

Phospholipases A2 (PLA2s; E.C.3.1.1.4) are a family of esterases that are involved in myriads of physiological and pathological processes. The involve...
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