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Int J Biol Sci 2022; 18(2):873-888. doi:10.7150/ijbs.68093 This issue Cite

Review

Secreted Phospholipases A₂ - not just Enzymes: Revisited

Adrijan Ivanušec^1,2, Jernej Šribar¹, Igor Križaj¹

1. Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
2. Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia.

✉ Corresponding author: igor.krizajsi

Abstract

Secreted phospholipases A₂ (sPLA₂s) participate in a very broad spectrum of biological processes through their enzymatic activity and as ligands for membrane and soluble receptors. The physiological roles of sPLA₂s as enzymes have been very well described, while their functions as ligands are still poorly known. Since the last overview of sPLA₂-binding proteins (sPLA₂-BPs) 10 years ago, several important discoveries have occurred in this area. New and more sensitive analytical tools have enabled the discovery of additional sPLA₂-BPs, which are presented and critically discussed here. The structural diversity of sPLA₂-BPs reveals sPLA₂s as very promiscuous proteins, and we offer some structural explanations for this nature that makes these proteins evolutionarily highly advantageous. Three areas of physiological engagement of sPLA₂-BPs have appeared most clearly: cellular transport and signalling, and regulation of the enzymatic activity of sPLA₂s. Due to the multifunctionality of sPLA₂s, they appear to be exceptional pharmacological targets. We reveal the potential to exploit interactions of sPLA₂s with other proteins in medical terms, for the development of original diagnostic and therapeutic procedures. We conclude this survey by suggesting the priority questions that need to be answered.

Keywords: Secreted phospholipase A₂, binding protein, promiscuity, cell transport, signalling, phospholipase activity regulation

Introduction

Secreted phospholipases A₂ (sPLA₂s) (EC 3.1.1.4) are a structurally related group of low-molecular-mass enzymes (14-18 kDa) that catalyse the hydrolysis of glycerophospholipids (phospholipids hereafter) at their sn-2 position, to produce lysophospholipids and free fatty acids. sPLA₂s contain 6 to 8 disulphide bonds, a highly conserved His/Asp catalytic dyad, and a Ca²⁺-binding loop [1]. Several sPLA₂ isoforms have been described in mammals. Depending on their structural characteristics, the mammalian sPLA₂s are divided into 11 groups (G): IB, IIA, IIC, IID, IIE, IIF, III, V, X, XIIA and XIIB [2]. Snake venom sPLA₂s are orthologous to mammalian GIIA, GIIB or GIIE sPLA₂s, or they belong to the unique GIA sPLA₂s [3]. sPLA₂s from bee and lizard venoms are homologous to mammalian GIII sPLA₂s. Finally, the further GIX sPLA₂s are found in venom of marine snails, and GXIA and GXIB sPLA₂s are plant proteins.

The importance of sPLA₂s as enzymes

The enzymatic activity of sPLA₂s define their participation in a very broad spectrum of biological processes. Generally, sPLA₂s are secreted from cells and require micromolar to millimolar Ca²⁺ to be catalytically competent. They predominantly target phospholipids in the extracellular space, although they also act intracellularly.

sPLA₂s show distinct substrate specificities in terms of the phospholipid polar headgroups and the fatty acids esterified at the sn-2 position of the glycerol backbone. For instance, GIII, GV and GX sPLA₂s efficiently hydrolyse phosphatidylcholine, while GIIA sPLA₂ has higher activity against negatively charged phospholipid substrates, and in particular, phosphatidylserine, phosphatidylglycerol and phosphatidylethanolamine. For their sn-2 fatty acid tail specificities, GIB, GIIA and GIIE sPLA₂s are promiscuous. GV sPLA₂ preferentially targets sn-2 fatty acyls with low number of double bonds, such as oleyl, while GIID, GIIF, GIII and GX sPLA₂s target polyunsaturated fatty acyls, such as arachidonyl.

sPLA₂ isoforms have unique tissue and cellular distributions, and therefore it is evident that individual sPLA₂s have distinct enzyme-activity-related biological functions. These include the generation of a variety of lipid mediators, along with membrane remodelling, modification of extracellular non-cellular phospholipid components of pulmonary surfactant, microparticles and lipoproteins, and degradation of microbial membranes and dietary phospholipids. The pathophysiological aspects of sPLA₂s as enzymes have been comprehensively reviewed recently (e.g., [2,4]), and therefore we will not focus on these here. Instead, our attention here is focussed on sPLA₂s acting as ligands, where fewer of the details have been described.

The existence of sPLA₂s without enzymatic activity but with pharmacological activity was an early indication that sPLA₂s can participate in physiological settings not just as enzymes, but also as ligands for membrane and soluble receptors. Indeed, in 1982, the first specific sPLA₂-binding protein (sPLA₂-BP) was discovered [5]. Since then, the number of newly characterized sPLA₂-BPs has expanded considerably (Table 1), and due to the development of more sensitive analytical methodologies, this process continues. Several reviews on sPLA₂-BPs have been published over the years [6-9], although the most recent is 10 years old [9]. Therefore, now is an appropriate time to survey the advances in this important area of research, and to critically discuss new insights and suggest research directions.

sPLA₂s bind very structurally diverse proteins

There are two types of sPLA₂-BPs: integral membrane proteins and soluble proteins [9]. The former belong to one of the following protein families: (1) muscle-type sPLA₂ receptors (M-type sPLA₂Rs); (2) heparan sulphate proteoglycans (HSPGs); (3) integrins; (4) vascular endothelial growth factor receptors (VEGFRs); and (5) ion channels. Two new integral membrane sPLA₂-BPs have been described recently, a G-protein-coupled receptor (GPCR), and cytochrome c oxidase (CCOX) [35, 37]. CCOX is the only known intracellular membrane sPLA₂-BP, as all of the other integral membrane sPLA₂-BPs are located in the plasma membrane. On the other hand, soluble sPLA₂-BPs are more frequently found inside cells, in the endoplasmic reticulum (ER), cytosol or nucleus, as well as in the extracellular space. The ER-resident sPLA₂-BPs, disulphide isomerase (PDI), taipoxin-associated 49-kDa Ca²⁺-binding protein (TCBP-49) and crocalbin, and the cytosolic calmodulin (CaM) and 14-3-3 proteins, have been known for some time. Two new soluble sPLA₂-BPs were only described more recently: vimentin [26] and nucleolin [27]. Vimentin is an intermediate filament protein, as a constituent of the cytoskeleton, while nucleolin is a nucleolar protein; however, both of these proteins are also found on the cell surface, which is where they are most likely to interact with sPLA₂s. Extracellular sPLA₂-BPs include pentraxins, α-, β- and γ-type sPLA₂ inhibitors (PLIs), blood coagulation factors, pulmonary surfactant protein A (SP-A), metalloproteinase inhibitor DM64, and a serine protease inhibitor (C1 inhibitor protein). The main characteristics of the sPLA₂-BPs are shown in Table 1.

Structural aspects of sPLA₂ protein-binding-promiscuity

sPLA₂-BPs are structurally so diverse that attempts to define their common sPLA₂-binding attributes have remained unsuccessful. To solve this problem, it would be most helpful to determine the three-dimensional structures of complexes between sPLA₂s and the sPLA₂-BPs, and thus to analyse their interaction areas. However, contemporary methodologies are still not optimal for this kind of approach.

The focus has thus mainly been on well-defined structural elements that are present in more than one type of sPLA₂-BP, such as the C-type carbohydrate recognition domain (CRD) and CRD-like folds (in M-type sPLA₂Rs, SP-A, α-PLIs), the EF-hand Ca²⁺-binding motif (in CaM, crocalbin, TCBP-49), and the immunoglobulin-like (Ig-like) domain (in VEGFRs, DM64) (Figure 1).

Only the CRD fold (Figure 1A) has been confirmed as an sPLA₂-binding structure [10]. Using radio-iodinated snake venom GIA sPLA₂ (¹²⁵I-OS₁), it was established that one of the eight CRDs in M-type sPLA₂R (i.e., CRD5) includes the sPLA₂ binding site. This also revealed that not all of the CRDs can bind sPLA₂s. M-type sPLA₂R bound sPLA₂s only under neutral and basic conditions, which implies that positively charged amino acids have important roles in this interaction. At pH 6 or lower, M-type sPLA₂R also underwent conformational changes that prevented the binding of sPLA₂s [41]. For the other two CRD-fold-containing sPLA₂-BPs, as lung SP-A [13] and soluble α-PLIs from sera [12], the sPLA₂-binding characteristics are not very clear yet. As M-type sPLA₂R and α-PLIs do not require Ca²⁺ to bind sPLA₂s [12, 42], while SP-A does require Ca²⁺, this indicates that CRDs bind sPLA₂s in different ways. To date, Ca²⁺-dependent binding of sPLA₂s has been reported only in a case of a single CRD-containing sPLA₂-BP, SP-A, while Ca²⁺-independent binding of sPLA₂s has been shown for sPLA₂-BPs with multiple CRDs, where more than one CRD constitutes the sPLA₂-binding site. For M-type sPLA₂R, three consecutive CRD repeats (repeats 4, 5, 6) were shown to influence sPLA₂ binding [10], and consistently, in α-PLIs, the trimeric CRD proteins, the central part, where three CRDs are associated together, has been suggested to provide the sPLA₂-binding site [43].

 Figure 1 

Distinct structural elements present in more than one type of sPLA₂-BP. (A) CRD (PDB ID: 6JLI) and the CRD-like fold are structural elements found in M-type sPLA₂Rs, SP-A and α-PLIs. (B) The EF-hand Ca²⁺-binding motif (PDB ID: 1CLL) is found in CaM, crocalbin and TCBP-49, which all bind sPLA₂s. (C) The Ig-like domain (PDB ID: 2X1X) is found in VEGFRs and DM64. To date, only the CRD has been experimentally demonstrated to be a sPLA₂-binding structure. Red, α-helices; violet, β-sheets; grey, loops. The Figure was prepared using UCSF Chimera v1.15.

There are plenty of other CRD-containing proteins, such as the mannose receptor, Endo180, DEC205 and FcRY. At present, only the human mannose receptor has been demonstrated to interact with bee venom GIII sPLA₂ [44], while FcRY did not bind any of the sPLA₂s tested [45].

To recognize sPLA₂-binding attributes of sPLA₂-BPs it is important to also consider the characteristics of the sites on sPLA₂s that interact with sPLA₂-BPs, as these are complementary to the sPLA₂-binding sites on sPLA₂-BPs. It has to be borne in mind, however, that a single sPLA₂ molecule might harbour multiple protein-binding sites [46], and therefore it is reasonable to consider separately binding sites directed towards particular types of sPLA₂-BPs, or those sPLA₂s that induce the same interaction-dependent physiological effects.

The interaction of an sPLA₂ with a CRD fold is primarily governed by the structure of the interfacial binding surface (IBS) of the sPLA₂ [47]. The IBS is defined as the molecular surface with which sPLA₂ contacts a phospholipid membrane - its substrate. The IBS is formed by a collar of hydrophobic amino acids around the entrance to the catalytic site of the enzyme, and by mainly positively charged amino acids in more remote positions. The amino acids within or close to the Ca²⁺-binding loop in sPLA₂s were also shown to be involved in the interactions between sPLA₂s and CRDs [42]; however, not profoundly, consistent with the evidence that the Ca²⁺-binding loop is highly conserved among sPLA₂s that show considerably different affinities for M-type sPLA₂R.

sPLA₂-BPs that contain Ig-like domains, such as VEGFRs and DM64, have been shown to associate with catalytically inactive myotoxic sPLA₂s. VEGFRs are receptor tyrosine kinases, and they interact with sPLA₂s through their extracellular region, which is composed of seven Ig-like domains. Although the interaction of myotoxic sPLA₂s and VEGFRs has not been functionally linked with myotoxic effects yet, the C-terminal region of these sPLA₂s (known as the 'myotoxic site') was shown to also be responsible for binding to VEGFRs [22, 23]. The myotoxic site of the snake venom myotoxic sPLA₂s has been attributed to amino acids 115-129, which form a single α-helical turn at the C-terminus [48-50]. This C-terminal region is firmly attached to the body of the protein by two disulphide bonds, Cys27-Cys126 and Cys50-Cys133, thus constraining its position and orientation. The myotoxicity of sPLA₂s has been associated with a cluster of positively charged amino acids in this region (i.e., conserved Lys115 and Arg118, and possibly also Lys122 and Lys127), and with hydrophobic amino acids at positions 121 and 125 [51, 52].

The sPLA₂-BP DM64 was originally isolated from marsupial (Didelphis marsupialis) blood, and it was shown to inhibit the myotoxic activity of several sPLA₂s [24]. DM64 contains five Ig-like domains. Its myotoxicity-inhibitory activity is most likely due to its binding to the myotoxic site of sPLA₂s, to thus obstruct their association with the 'myotoxicity receptor', which would be VEGFR. Contrary to what is seen for CRD in M-type sPLA₂R, binding of sPLA₂s to DM64 does not inhibit their enzymatic activity. This is in agreement with the involvement of the C-terminal of these sPLA₂s in the interaction with this Ig-like domain-containing sPLA₂-BP, rather than the IBS.

Identification of the PDZ-binding domain at the C-terminus of some myotoxic sPLA₂s [53] suggested that there is another group of sPLA₂-BPs, as PDZ-domain-containing proteins. In muscle cells, these proteins include, e.g., LDB3 and α-1-syntrophin [54, 55]; however, their potential sPLA₂ binding remains to be tested.

The third type of well-defined structural element present in more than one sPLA₂-BP is the EF-hand Ca²⁺-binding motif. These sPLA₂-BPs include CaM, crocalbin and TCBP-49, although the structural aspects of sPLA₂ binding have been studied only for CaM. A three-dimensional model of a complex between CaM and the snake venom GIIA sPLA₂ ammodytoxin (Atx) [16] revealed that CaM 'clamps' Atx between its N-terminal and C-terminal domains (Figure 2A). Atx contacts CaM most extensively through a distinct patch of hydrophobic and charged amino acids at its C-terminus, which interact with the central part of CaM, and through the α-helices C and E, which contact the C-terminal globular domain of CaM. In the complex, Atx is oriented in such a way that the entrance to its enzymatic pocket remains open wide. In the complex, a new larger membrane-contacting area (IBS) is formed; this comprises parts of both Atx and CaM, and explains the increased enzymatic activity of complexed Atx.

 Figure 2 

Secreted PLA₂s are ligands of proteins that are structurally very diverse. The three-dimensional models, generated by molecular docking, are showing complexes between the sPLA₂ Atx and CaM (green, PDB ID: 1CLL) (A), Atx and FXa (blue, PDB ID: 2BOH) (B), CB_b and ΔF508NBD1 of CFTR (PDB ID: 1XMJ) (C), Atx and PDI (purple/pink, PDB ID: 4EL1) (D), and between the sPLA₂ vurtoxin and nAChR (grey, PDB ID: 2BG9) (E). Centre: The sPLA₂ Atx, showing its main structural elements. Red, interfacial binding surface; yellow, disulphide bonds; violet, C-terminal region. Note that the sPLA₂s interact with these different sPLA₂-BPs in very different ways; i.e., they have multiple protein binding sites, as is characteristic of promiscuous proteins. The Figure was prepared by adaptation of Figures from Kovačič et al. (2010) (A), Faure and Saul (2011) (B), Faure et al. (2016) (C), Oberčkal et al. (2015) (D) and Vulfius et al. (2014) (E), using PyMOL.

Considerable structural insight has also been gained for the interactions between the anticoagulant GIIA sPLA₂s Atx, CB (basic sPLA₂ subunit of crotoxin, from venom of the South American rattlesnake [Crotalus durissus terrificus]) and daboxin P (from venom of the Indian viper [Daboia r. russelii]), and human activated blood coagulation factor X (FXa) [40, 56, 57, 58]. The interaction area between the anticoagulant sPLA₂s and FXa is extensive, and encompasses the heavy and light chains of FXa. As evident from the model of the complex between Atx and FXa shown in Figure 2B, Atx interacts with the FXa heavy chain through its Ca²⁺-binding loop, α-helix C, β-wing, and C-terminal region, and with the FXa light chain through its β-wing and α-helices A and B. Electrostatic interactions importantly participate in the efficient binding of sPLA₂s to FXa, including in particular some positively charged amino acids of Atx (e.g., Arg72, Lys74, His76, Arg77 in the β-wing; Arg118, Lys128, Lys127, Lys132 in the C-terminal region). However, not all of the basic sPLA₂s are strong anticoagulants, which are consistent with the importance also of certain hydrophobic and aromatic sPLA₂ amino acids for optimal interactions with FXa (e.g., Atx: Phe124 in the C-terminal region; CB_a2: Trp70 in the 65-72 loop).

Molecular docking allowed to propose a model of the complex between ΔF508-NBD1 (nucleotide-binding domain 1), the sPLA₂-binding domain of ΔF508CFTR (Phe508 deletion mutant of cystic fibrosis transmembrane conductance regulator), and CB_b (Figure 2C) [33]. From the model, which applies also to the wild type NBD1 (i.e. CFTR), it can be seen that the binding interface of CB with ΔF508NBD1/NBD1 is predominately composed of the hydrophobic residues located in its α-helices A and B, Ca²⁺-binding loop and the C-terminal region. Hydrophobic interactions are clearly very important for holding both proteins together, nevertheless, also some polar amino acid residues are involved in the interaction. Forming ionic contacts with ΔF508NBD1/NBD1 of ΔF508CFTR/CFTR, the N-terminal His1 and the Asp112 in the C-terminal region of CB are two examples.

A three-dimensional model of the complex between Atx and PDI has also been reported [20]. PDI consists of multiple domains (i.e., a, a', b, b'), and according to the model shown in Figure 2D, the Atx-binding site on PDI is situated between domains b and b'. Atx interacts with PDI across an extensive area that also includes the Atx IBS. The basic amino acids are important for Atx binding to PDI, especially Arg77 and Arg118, but also Lys69, Arg72, Lys74 and Lys86. However, Atx also contacts PDI with some hydrophobic amino acids, Leu3, Leu19 and Phe24, and through two polar amino acids, Asn17 and Asn119.

Molecular docking has also provided some structural insights into the interactions between vurtoxin, another snake venom GIIA sPLA₂, and the nicotinic acetylcholine receptor (nAChR) [59]. According to their model, vurtoxin binds at the interface of the α and γ subunits of nAChR, and is oriented with its active site towards the lipid bilayer (Figure 2E). Vurtoxin does not occupy the nAChR binding sites for its classical agonists and competitive antagonists; however, it is located very close to these.

Several structural features allow the sPLA₂s to bind to structurally very diverse targets (Table 1; Figure 2), which is characteristic of promiscuous proteins [60, 61]. Despite the generally compact structure of sPLA₂s, the flexibility of the exposed side chains of the amino acids at the IBS promotes their optimal binding to phospholipid aggregates, as their substrates, for efficient hydrolysis [62]. As indicated above, the IBS is critically involved also in the interactions of sPLA₂s with the CRD-containing sPLA₂-BPs and FXa, as also for PDI [20] and vimentin [26]. The association of the sPLA₂ IBS with a variety of different protein surfaces is most likely driven by the same principles as the association of the sPLA₂ IBS with phospholipid membranes. The flexibility of the sPLA₂ β-wing might be another feature that favours their high adaptability for different protein partners. For example, the β-wing of sPLA₂s should be involved in the interactions of sPLA₂s with β-PLIs and γ-PLIs [63]. sPLA₂s are also characterized by patches of basic and hydrophobic amino acids that might also greatly broaden the spectrum of their protein interaction partners. We have already shown that such patches are part of the IBS, but they are also located in other parts of sPLA₂s. Indeed, those at the C-terminus [62] are implicated in the binding of sPLA₂s to CaM and VEGFR.

However, only the atomic structure of an sPLA₂-sPLA₂-BP complex will fully disclose the structural requirements for particular association. The present-day rapid advances in techniques for the determination of the three-dimensional structures of proteins might enable this in the near future; e.g., cryo-electron microscopy.

Pathophysiological significance of sPLA₂ binding to sPLA₂-BPs

The structural diversity of sPLA₂-BPs is consistent with the wide range of pathophysiological activities that have been associated with sPLA₂s. By binding to sPLA₂-BPs, sPLA₂s have been suggested to be involved in inflammation [64, 65], hormone release [66, 67], neurotoxicity [20, 37, 68, 69] and myotoxicity [22]. It has already been confirmed that sPLA₂s are ligands for specific sPLA₂-BPs in cytokine production [70], cell proliferation [71-73], cell migration [74], lipid mediator production [75], antibacterial activity [73] and blood coagulation [40]. These (patho)physiological effects of sPLA₂s might arise through: (1) specific cellular translocation and associated actions of sPLA₂s after their binding to sPLA₂-BPs; (2) triggering of specific signalling following their binding to sPLA₂-BPs; and/or (3) regulation of sPLA₂ enzymatic activity by sPLA₂-BPs (Figure 3). Particular mechanistic possibilities for sPLA₂ actions as ligands for sPLA₂-BPs are exemplified and discussed below.

Binding of sPLA₂s to some sPLA₂-BPs does not have any obvious physiological function. Some functional implications of sPLA₂s acting as ligands certainly remain to be discovered, while others might be latent. When environmental conditions change radically, these interactions might become functional. Promiscuous proteins, which include sPLA₂s, can acquire new functions much more readily than non-promiscuous proteins, which can provide swifter adaptation of the organism to new situations [76, 77].

 Figure 3 

Pathophysiological implications of binding of sPLA₂s to sPLA₂-BPs. Physiological and/or pathological effects of sPLA₂s (red) are also the consequence of their binding to sPLA₂-BPs. (A) By binding to sPLA₂-BPs (blue/cyan), sPLA₂s can be translocated to specific intracellular compartments, such as the endoplasmic reticulum (a), cytosol (b), nucleus (c), mitochondria (d) or lysosomes (e). In each of these compartments, they can act as enzymes or ligands for receptors, or they can undergo proteolytic degradation in the lysosome. (B) As ligands for receptors, sPLA₂s have been specifically implicated in molecular signalling through decreased (1) or increased (2) permeabilities of certain ion channels, inhibition (3) or activation (4) of activities of receptor tyrosine kinases, modulation of activities of GPCR (5), interference in integrin-mediated functions (6), attenuation of ATP production (7) and inhibition of blood coagulation, at different stages (8, 9, 10). (C) Binding of sPLA₂ to a sPLA₂-BP might inhibit or potentiate phospholipolytic activity. The Figure was created with BioRender.com.

Cellular transport of sPLA₂s after binding to sPLA₂-BPs

Although sPLA₂s appear to be secreted into the extracellular space after being synthesized inside the cell, convincing evidence of their intracellular localization and activities has already been provided [69, 71, 78, 79]. sPLA₂s can re-enter cells after their synthesis and secretion, to pass into the cytosol and to different organelles, such as the nucleus and mitochondria [80]. Indeed, several sPLA₂-BPs have been suggested to assist sPLA₂s in their retrograde cellular transport and intracellular translocation (Figure 3A).

The sPLA₂-BP in the lumen of the ER, PDI, has been proposed to be involved in such retro-transport of the Atx snake venom GIIA sPLA₂, which is a neurotoxin from nose-horned viper venom that acts presynaptically [20]. Molecular docking and heterologous competition assays have suggested that PDI acts in a similar way as on Atx also on some mammalian sPLA₂s, such as GIB, GIIA and GV sPLA₂s.

Two other candidates for the same function are the ER luminal proteins TCBP-49 and crocalbin, which were discovered through their binding to the hetero-oligomeric snake venom sPLA₂s taipoxin and crotoxin [9]. As for PDI, these two EF-hand Ca²⁺-binding proteins have the characteristic ER-retention motif at their C-termini, which has been suggested to be essential for retention and concentration of sPLA₂s in the ER before they are translocated into the cytosol. Some other EF-hand Ca²⁺-binding proteins might also interact with sPLA₂s. For instance, the Miro proteins are mitochondrial adaptor proteins that promote the transportation of mitochondria by connecting them to motor proteins [81]. Binding of sPLA₂s to the Miro proteins might concentrate the sPLA₂s on the outer mitochondrial membrane, which would explain their colocalization with mitochondria after entering cells [78, 79].

Cellular retro-transport of sPLA₂s might also be associated with two recently discovered sPLA₂-BPs, vimentin and nucleolin. Vimentin belongs to the family of intermediate filaments, although it is also present on the cell surface and in extracellular fluids. Indeed, in recent years, vimentin has been shown to have a much wider role in cell physiology, rather than just being an inert scaffold protein [82]. Vimentin was identified as the receptor for GIIA sPLA₂ on the surface of apoptotic human T lymphocytes [65]. Interactions between these two proteins was also shown in rheumatoid fibroblast-like synoviocytes, which associated rapid internalization of sPLA₂ with arachidonic acid metabolism in synovial inflammation [83]. Recently, vimentin was reported to bind an acidic sPLA₂ (NnPLA₂-I) from venom of the Indian cobra (Naja naja) [26]. Binding of NnPLA₂-I to vimentin resulted in its internalization into partially differentiated myoblasts. The involvement of vimentin in cellular uptake mechanisms has already been shown for C3, a Clostridium botulinum toxin [84, 85], and several viruses, such as human immunodeficiency virus type 1, vaccinia virus and the severe acute respiratory syndrome coronavirus [86]. In all of these cases, vimentin acts as a component of the cellular attachment mechanism, either as a receptor or a co-receptor. In a similar way, vimentin might mediate internalization of sPLA₂s. This is further supported by the similar location of the binding sites on vimentin for dengue virus DENV-2 envelope protein domain III, Clostridium botulinum C3 exoenzyme and NnPLA₂-I, all of which have been proposed to bind to the rod domain of vimentin [26, 84, 87].

Nucleolin was described recently as an sPLA₂-BP due to its binding to the myotoxin MT-II, a Lys49 sPLA₂ from venom of a pit viper, the terciopelo (Bothrops asper) [27]. It appears most likely that nucleolin participates in the toxic mechanism of MT-II through promotion of its translocation from the outside into the perinuclear and nuclear areas of myotubes and macrophages. Nucleolin was previously reported to mediate the internalization of several other molecules from the cell surface to the nucleus through an active Ca²⁺-dependent transport mechanism [88], which might also be the mechanism for sPLA₂ internalization. It would be interesting to investigate the potential involvement of nucleolin in GV sPLA₂ translocation to the nucleus, where this sPLA₂ has been shown to act on the nuclear envelope [89]. Nucleolin has been shown to act as a chaperone for histones and TDP-43 [90, 91], so it might also stabilize sPLA₂s in the reducing environment of the cytosol.

Neuronal pentraxins (i.e., NP1, NP2/Narp) are sPLA₂-BPs that are homologous to acute phase proteins, and these might also function in retrograde transport of sPLA₂s from the cell surface [28, 92]. An sPLA₂-shuttling function has also been suggested for the ubiquitously expressed eukaryotic cytosolic 14-3-3 proteins, which were discovered through their interactions with Atx [29]. The neurotoxicity of Atx also involves its binding to the 14-3-3γ and 14-3-3ε isoforms, to correctly position it at the plasma membrane to hinder the function of amphiphysin, and thus of vesicle endocytosis [69].

HSPGs such as the GPI-anchored glypican I, biglycan, syndecan and perlecan, can bind mammalian sPLA₂s, e.g. GIIA, GIID and GV sPLA₂s. Using transfected human embryonic kidney 293 cells, it was shown that a GPI-anchored HSPG facilitated the shuttling of GIIA sPLA₂ into certain subcellular compartments where the sPLA₂ then released arachidonic acid for prostaglandin synthesis [93]. Binding of human GIIA sPLA₂ to HSPGs was further demonstrated in human primary T-lymphocytes [25]. Recently, it was suggested that GIIA sPLA₂ can increase endothelial cell permeability after binding to HSPGs on the surface of these cells, in a process that is dependent on the sPLA₂ phospholipase activity [39]. In a similar way, HSPGs might be receptors for heparin-binding snake venom sPLA₂s. By binding to HSPGs, these toxins might enhance inflammation and permeability of the endothelium, to allow the venom to spread more efficiently in the tissue.

M-type sPLA₂R can rapidly internalize sPLA₂s and direct them to the lysosomes, where they are then degraded [94, 95]. In this way, M-type sPLA₂R can down-regulate the activity of sPLA₂s. M-type sPLA₂R can also mediate sPLA₂ signalling by transporting sPLA₂s into specific intracellular compartments [96, 97]. Recent structural studies of the human M-type sPLA₂R ectodomain using cryo-electron microscopy showed pH-dependent conformational changes that might have important roles in the control of the functional properties of this receptor, including its sPLA₂ binding [41].

Signalling triggered by sPLA₂s as ligands

As proteins that are secreted into the extracellular space, sPLA₂s encounter various plasma-membrane receptors on target cells. By binding to some of these receptors, sPLA₂s can induce intracellular responses that are associated with different physiological processes, such as vascular permeability, cell growth, migration and senescence, hormone release, cytokine and NO production, inflammation, cell adhesion and angiogenicity (Figure 3B).

In this context, sPLA₂s have been reported to interact with different ion channels (K⁺, Na⁺ or Ca²⁺). β-Butx is a neurotoxic sPLA₂ from venom of the many-banded krait (Bungarus multicinctus), and it has been shown to interact with voltage-dependent K⁺ channels. Further, MitTx from venom of the Texas coral snake (Micrurus t. tener) was shown to bind to voltage-insensitive Na⁺-conducting acid-sensing ion channels [98]. However, both of these toxins are heterodimers that are composed of the sPLA₂ subunit and a subunit homologous to the Kunitz-type serine protease inhibitor and, with the ion channel binding attributed to the latter. Therefore, these ion channels are not sPLA₂-BPs. Nonetheless, the neurotoxicity with β-Butx and the pain with MitTx did not occur in the absence of the sPLA₂ subunit. Some sPLA₂s can influence the conductance of Ca²⁺ channels, the N-methyl-D-aspartate receptor or L-type voltage-dependent Ca²⁺ channels. Direct binding of sPLA₂s to these channels has, however, not been demonstrated to date [30, 99].

Recently, pentameric ligand-gated ion channels, nAChRs [31, 59] and their bacterial homologue GLIC from the cyanobacterium Gloeobacter violaceus [32], were reported to be sPLA₂-BPs. By binding to nAChRs, sPLA₂s might affect nAChR-related functions, such as neuromuscular transmission and cell proliferation. Interestingly, nAChRs have also been described for the outer mitochondrial membrane, and were associated with regulation of the formation of mitochondrial permeability transition pores, which release pro-apoptotic substances like cytochrome c and reactive oxygen species [100]. Antagonists of nAChR were shown to attenuate the release of cytochrome c. The antagonistic effect of certain sPLA₂s on some nAChRs subtypes might therefore link these sPLA₂s to control of cellular viability through these nAChRs [31, 101].

CFTR is a Cl^- channel that was defined as an sPLA₂-BP using CB, the basic sPLA₂ subunit of crotoxin [33]. CB was shown to bind and allosterically potentiate the activity of CFTR. Of a very high medical significance, CB was found to bind with nanomolar affinity also to ΔF508CFTR mutant, the causative factor of cystic fibrosis, thus augmenting its activity. Importantly, by binding to ΔF508CFTR, CB acts also as a corrector, facilitating trafficking and delivery of the abnormal protein to the plasma membrane.

VEGFRs are receptor tyrosine kinases that can interact with some myotoxic snake venom sPLA₂s. Although some of these toxins are potent antagonists of VEGFRs, it is still not clear whether their binding to VEGFRs is directly involved in their sPLA₂ myotoxicity [22, 23]. Nevertheless, the interactions between snake venom sPLA₂s and VEGFRs might be important to enhance vascular permeability, to facilitate penetration of the snake venoms into tissues.

Epidermal growth factor receptor (EGFR) is another receptor tyrosine kinase that interacts with sPLA₂s. It was reported to interact with human GIIA sPLA₂ [34]. However, in this case, the sPLA₂ acted as an agonist, as it up-regulated HER (human EGFR)/HER2-elicited signalling in lung cancer, thus stimulating cancer cell growth.

M-type sPLA₂R has already been mentioned as an sPLA₂-translocator (see section 4), and it can also act as a signalling receptor to transduce sPLA₂-dependent signals independent of sPLA₂ catalytic activity. Through M-type sPLA₂R, sPLA₂s have been implicated in cell proliferation, migration and senescence, and in hormone release and cytokine and NO production. It was also suggested that human GIB sPLA₂ can induce kidney glomerular podocyte apoptosis via M-type sPLA₂R [102].

PA2-Vb is an acidic sPLA₂ from venom of the Chinese green tree viper (Trimeresurus stejnegeri), and it was shown to induce mouse aorta contraction independent of its enzymatic activity, by acting on a protease-activated receptor (PAR-1), which is a GPCR [35]. GPCRs are the largest and most diverse class of membrane receptors in eukaryotes, and their primary function is to transduce extracellular stimuli into intracellular signals, which lead to different cell responses.

Integrins are extracellular plasma membrane (transmembrane) proteins that are responsible for cell adhesion to the extracellular matrix. They are dimers, as combinations of one of 18 α-subunits and one of 8 β-subunits. Integrins α_vβ₃ and α₄β₁ have been shown to bind mammalian GIIA sPLA₂, while an acidic GIIA sPLA₂ from venom of the viper Macrovipera lebetina transmediterranea was shown to interact with integrins α₅β₁, α_vβ₃ and α_vβ₆, to induce inflammation and inhibition of cell adhesion and migration and angiogenicity [36, 103].

CCOX is an essential constituent of the respiratory chain complex, and was characterized as an sPLA₂-BP due to its binding to Atx [37]. Through binding to subunit II, Atx was shown to inhibit the oxidase activity of CCOX, independent of its phospholipase activity. This might explain the inhibition of ATP production in nerve endings poisoned by the snake venom neurotoxic sPLA₂s [104]. These findings also provide novel indications for the potential functions and malfunctions of the orthologous mammalian GIIA sPLA₂ in mitochondria.

The blood coagulation system consists of a strictly regulated proteolytic cascade of blood coagulation factors that convey signals downstream to fibrinogen, which is then transformed into the insoluble fibrin network, and to activate platelets. Ultimately, a blood clot is formed to prevent blood loss from the injured blood vessel [105]. Several sPLA₂s can affect this process by binding to one of these coagulation factors, to thus inhibit their functions (Figure 3B). Some snake venom sPLA₂s bind to FX, or to its activated form FXa, or to thrombin (FIIa); e.g., crotoxin and Atx can induce anticoagulant effects through binding to FXa, to thus prevent formation of the prothrombinase complex [40, 106]. Human GIIA sPLA₂ acts on blood coagulation in exactly the same way [107]. Daboxin is an anticoagulant sPLA₂ that can bind to both FXa and FX [57]. On the other hand, Nk-PLA₂α and Nk-PLA₂β from venom of the monocled cobra (Naja kaouthia) act as anticoagulants through their binding to thrombin and their consequent inhibition of its proteolytic activity [108].

Regulation of sPLA₂ enzymatic activity by sPLA₂-BPs

sPLA₂s participate in many important physiological processes through their enzymatic activity [2]. These range from those dependent on the composition and flexibility of biological membranes, to those regulated by the products of the sPLA₂ phospholipolytic activity, which include lysophospholipids, free fatty acids and their metabolites - the whole range of signalling hormones [109,110]. Regulation of the enzymatic activity of sPLA₂s is thus extremely important for the correct functioning of an organism, and this regulation is also mediated through the binding of sPLA₂s to particular sPLA₂-BPs (Figure 3C).

M-type sPLA₂R has been shown to regulate the activity of sPLA₂s in two ways: through acting as an sPLA₂ inhibitor; and through mediating sPLA₂ endocytosis, which leads to sPLA₂ degradation in lysosomes [111]. Soluble form of M-type sPLA₂R that is detected in the blood can function only in the first way [8]. Pulmonary SP-A is structurally and functionally similar to the soluble form of M-type sPLA₂R. SP-A belongs to the CRD-containing family of proteins, and it inhibits the enzymatic activity of GIIA and GX sPLA₂s in pulmonary surfactant [9].

Soluble proteins that inhibit sPLA₂s have been isolated from the blood of snakes and some other animals. These are ecologically connected with venomous snakes, and are known as the α-PLIs, β-PLIs and γ-PLIs [9].

sPLA₂ inhibitors have also been found in plants. Withania somnifera glycoprotein (WSG) is an acidic glycoprotein that is similar to the α-chain of the γ-PLIs, and it was isolated from the medicinal plant known variously as Ashwagandha, Indian ginseng and winter cherry. WSG inhibits the enzymatic activity and toxicity of NN-XIa-PLA₂, an sPLA₂ myotoxin from venom of the Indian cobra [112-114].

Interestingly, sPLA₂-BPs have also been shown to increase the enzymatic activity of sPLA₂s. One such sPLA₂-BP is the EF-hand Ca²⁺-binding intracellular protein CaM. When sPLA₂s are in a complex with CaM, they also become more resistant to chemical denaturation [16, 17]. Two other sPLA₂-BPs that can potentiate the catalytic activity of sPLA₂s are vimentin [65] and GLIC [32]. The potentiating effect appears to occur because the interactions with these three sPLA₂-BPs position the complexed sPLA₂ on the membrane in a way that provides more efficient catalytic function.

Medical potential of sPLA₂s as ligands for receptors

sPLA₂-BPs have diverse physiological functions, and therefore their interactions with sPLA₂s can be insightful and potentially helpful for medical applications. Important roles of M-type sPLA₂R in cancers have been outlined recently, with a tumour suppressive role demonstrated [74, 115]. As ligands of M-type sPLA₂R, sPLA₂s can be used to study or to regulate processes in which M-type sPLA₂R is involved. By binding to M-type sPLA₂R, sPLA₂s have already been indicated to have roles in cell proliferation, migration and senescence, hormone release, and cytokine and NO production. sPLA₂s were also demonstrated to induce kidney glomerular podocyte apoptosis via M-type sPLA₂R [102]. A recently generated conditional transgenic mouse that expresses human M-type sPLA₂R1 will certainly facilitate future investigations into this sPLA₂-BP under different pathophysiological conditions [116].

VEGFRs are sPLA₂-BPs that have also been shown to be involved in cancer development. By binding to VEGFRs, sPLA₂s might inhibit angiogenesis, which is an essential process for cancer metastasis formation [117]. Therefore, the use of sPLA₂s in chemotherapy has been proposed. In addition, the angiogenic pathways of VEGFRs and endothelial nAChRs have been shown to have cross-talk, which suggests that as antagonists of nAChRs, sPLA₂s might further inhibit angiogenesis in this way [31, 118]. In contrast to the antagonistic effects on VEGFRs and nAChRs, GIIA sPLA₂ can activate EGFRs in lung cancer cells [34]. This leads to elevated HER /HER2-elicited signalling, which contributes to overexpression of GIIA sPLA₂. Plasma concentration of GIIA sPLA₂ might therefore serve as a biomarker for lung cancer, and GIIA sPLA₂ might represent a therapeutic target to treat patients with lung cancer.

The integrin binding of snake venom sPLA₂s has also been exploited for development of new anti-cancer agents that target cell proliferation and migration [36, 103]. MT-II binding to nucleolin appears to explain the higher toxicity of MT-II against cancer cells, as nucleolin is more abundant on the surface of cancer cells than of normal cells [90]. Nucleolin also participates in internalization of many viruses, which suggested that the anti-viral activity of some sPLA₂s might be due to their nucleolin binding [27].

The involvement of GIIA sPLA₂ in synovial inflammation through liberation of arachidonic acid for production of inflammatory eicosanoids has long been known. Efforts have been made to develop inhibitors of GIIA sPLA₂ to attenuate rheumatoid arthritis and sepsis, but without substantial success so far [119, 120]. Colocalization of GIIA sPLA₂ and vimentin is, however, associated with phospholipase-activity-independent mechanisms of signalling through arachidonic acid metabolism [83], which provides the way for targeted studies of GIIA sPLA₂ signalling, and for the development of new therapeutic strategies based on inhibition of GIIA sPLA₂. Lee et al. (2013) [83] also identified vimentin as an interesting player in some other diseases where the involvement of GIIA sPLA₂ has been indicated, and particularly in cancers. GIIA sPLA₂ is overexpressed in many types of cancers, while vimentin is one of the signature biomarkers of tumour dedifferentiation through epithelial-mesenchymal transition [121, 122].

Abundant expression of vimentin has also been observed in adult neurons as a response to injury, such as in Alzheimer's disease [123]. As vimentin might assist the internalization of GIIA sPLA₂ into neurons, this suggests why GIIA sPLA₂ is involved in the aetiology of Alzheimer's disease. GIIA sPLA₂ might directly damage neurons or boost inflammation by releasing excessive arachidonic acid. Targeting of vimentin is, therefore, potentially interesting as a new therapeutic approach to treat patients with Alzheimer's disease.

A phospholipase-activity-independent mode of action is also characteristic for snake venom catalytically inactive sPLA₂s. These can induce inflammation [124]; e.g., MT-II can stimulate the production and release of inflammatory mediators, such as interleukin-6 [125], interleukin-1, tumour necrosis factor α, leukotriene B4, thromboxane A2 and prostaglandins E2 and D2 [126-129]. Indeed, this activity of MT-II was recently used to establish a new experimental model of acute arthritis [130].

Mammalian GIB, GIIA, GV and GX sPLA₂s induce the production of pro-inflammatory cytokines and chemokines, whereby these sPLA₂s increase inflammation after binding to HSPGs or to M-type sPLA₂R on macrophages, neutrophils, eosinophils, monocytes and endothelial cells [131]. This binding to HSPGs also participates in clearance of these sPLA₂s and in reduction of their enzymatic activity towards low-density lipoprotein, which is an important factor in atherosclerosis [3, 132].

sPLA₂s also mediate pro-inflammatory actions via integrins, as binding of GIIA sPLA₂ to human integrins α_vβ₃, α₄β₁ and α₅β₁ trigger signalling that leads to inflammation [36, 133]. The effects of sPLA₂s on haemostasis have great potential for medical applications as well. The induction of vasoconstriction by PA2-Vb binding to PAR-1 makes PA2-Vb interesting for the development of new therapeutic approaches against hypertension, atherosclerosis and diabetes-related vascular problems [35]. Moreover, snake venom sPLA₂s can induce strong anticoagulant effects through competitive binding to constituents of the prothrombinase complex, which suggests their great potential for the development of therapeutic procedures to attenuate or prevent blood-clot formation [108].

As already mentioned, GIIA sPLA₂ has been associated with the aetiology of some neurodegenerative diseases, such as Alzheimer's disease [134-137]. A hallmark of the induction of Alzheimer's disease is the elevated expression of GIIA sPLA₂ in the affected tissue, with concomitant dysfunction of the neuronal mitochondria. As the pathological effects of presynaptically neurotoxic sPLA₂s from snake venoms and GIIA sPLA₂ on mitochondria are similar, a description of the mode by which neurotoxic sPLA₂s encounter and affect neuronal mitochondria at the molecular level is expected to advance the study of the role of endogenous GIIA sPLA₂ in this and other related destructive diseases.

These findings indicate a way to the development of original diagnostic and therapeutic solutions. An important breakthrough in this direction was the recent identification of CCOX as the mitochondrial receptor for Atx [37]. Application-wise, a very promising discovery was also the binding of crotoxin to ΔF508CFTR, responsible for cystic fibrosis. The sPLA₂ subunit of crotoxin has been used as a template to develop a new line of anti-cystic fibrosis agents [33].

Conclusions and outlook

As well as participating in a very broad spectrum of biological processes through their enzymatic activity, sPLA₂s participate in many physiological settings as ligands for membrane or soluble receptors. As new and more sensitive analytical tools are developed, the number of newly discovered sPLA₂-BPs is growing. Further technical advances will promote the discovery of even more sPLA₂-BPs.

sPLA₂-BPs are structurally diverse integral membrane and soluble proteins. Despite many attempts, their common sPLA₂-binding attributes remain largely obscure. The solution to this puzzle will enable targeted searches of additional sPLA₂-BPs. A straightforward approach to solve this problem would be the determination of the three-dimensional structures of complexes between sPLA₂s and their sPLA₂-BPs. With the development of powerful new technologies, such as cryo-electron microscopy, this is becoming more and more realistic.

sPLA₂s are very useful molecules from the evolutionary point of view. Particular structural features have made the sPLA₂s promiscuous, and promiscuous proteins can acquire new functions more readily than other proteins. In this way, these can enable organisms to adapt more successfully to environmental changes.

Finally, due to their enzymatic activity and extensive interactomes, sPLA₂s are exceptional pharmacological targets. Detailed descriptions of their actions at the molecular level will initiate the development of a plethora of original diagnostic and therapeutic approaches.

Abbreviations

Atx: sPLA₂ from Vipera a. ammodytes venom; CaM: calmodulin; CB: basic sPLA₂ subunit of crotoxin; CCOX: cytochrome c oxidase; CFTR: cystic fibrosis transmembrane conductance regulator; CRD: carbohydrate recognition domain; DM64: metalloproteinase inhibitor from Didelphis marsupialis blood; ECD: extracellular domain; EGF(R): epidermal growth factor (receptor); ER: endoplasmic reticulum; FX/FXa/FIIa: blood coagulation factors; GLIC: proton-gated ion channel from Gloeobacter violaceus; GPCR: G-protein-coupled receptor; HER: human EGFR; hGIIA: human group IIA sPLA₂; HSPGs: heparan sulphate proteoglycans; IBS: interfacial binding surface; Ig-like: immunoglobulin-like; M-type sPLA₂Rs: muscle-type sPLA₂ receptors; nAChR: nicotinic acetylcholine receptor; NBD: nucleotide-binding domain; NP: neuronal pentraxin; OS₁: sPLA₂ from Oxyuranus s. scutellatus venom; PAR: protease-activated receptor; PDI: protein disulphide isomerase; PLIs: sPLA₂ inhibitors; SP-A: pulmonary surfactant protein A; sPLA₂: secreted phospholipase A₂; sPLA₂-BP: sPLA₂-binding protein; TCBP-49: taipoxin-associated 49-kDa Ca²⁺-binding protein; VEGFR: vascular endothelial growth factor receptor; WSG: Withania somnifera glycoprotein.

Acknowledgements

This work was supported by grants from the Slovenian Research Agency, a programme grant P1-0207 (to I.K.) and a Young Researcher grant 1000-17-0106-6 (to A.I.).

Competing Interests

The authors have declared that no competing interest exists.

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Corresponding author: igor.krizajsi

Received 2021-10-15
Accepted 2021-12-2
Published 2022-1-1

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