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Short Linear Motifs Characterizing Snake Venom and Mammalian Phospholipases A2

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Short Linear Motifs Characterizing Snake Venom and Mammalian Phospholipases A2 toxins Article Short Linear Motifs Characterizing Snake Venom and Mammalian Phospholipases A2 Caterina Peggion 1 and Fiorella Tonello 2,* 1 Department of Biomedical Sciences, University of Padova, Via U. Bassi, 58/B, 35131 Padova, Italy; [email protected] 2 CNR of Italy, Neuroscience Institute, viale G. Colombo 3, 35131 Padova, Italy * Correspondence: fi[email protected] Abstract: Snake venom phospholipases A2 (PLA2s) have sequences and structures very similar to those of mammalian group I and II secretory PLA2s, but they possess many toxic properties, ranging from the inhibition of coagulation to the blockage of nerve transmission, and the induction of muscle necrosis. The biological properties of these proteins are not only due to their enzymatic activity, but also to protein–protein interactions which are still unidentified. Here, we compare sequence alignments of snake venom and mammalian PLA2s, grouped according to their structure and biological activity, looking for differences that can justify their different behavior. This bioinformatics analysis has evidenced three distinct regions, two central and one C-terminal, having amino acid compositions that distinguish the different categories of PLA2s. In these regions, we identified short linear motifs (SLiMs), peptide modules involved in protein–protein interactions, conserved in mammalian and not in snake venom PLA2s, or vice versa. The different content in the SLiMs of snake venom with respect to mammalian PLA2s may result in the formation of protein membrane complexes having a toxic activity, or in the formation of complexes whose activity cannot be blocked due to the lack of switches in the toxic PLA2s, as the motif recognized by the prolyl isomerase Pin1. Keywords: snake venom phospholipases A2; neurotoxins; myotoxins; secretory phospholipases A2; Citation: Peggion, C.; Tonello, F. PLA2G1B; PLA2G2A; sequence alignment; short linear motifs; prolyl isomerase; glycogen synthase Short Linear Motifs Characterizing kinase 3 Snake Venom and Mammalian Phospholipases A2. Toxins 2021, 13, Key Contribution: Comparison of sequence alignments of snake and mammalian phospholipases 290. https://doi.org/10.3390/ A2 revealed the presence of short linear motifs potentially involved in protein–protein interactions toxins13040290 and modified by enzymes with a central role in many diseases. Received: 8 March 2021 Accepted: 16 April 2021 Published: 20 April 2021 1. Introduction Publisher’s Note: MDPI stays neutral Phospholipases (PLs) are fundamental lipolytic enzymes for living organisms. They are with regard to jurisdictional claims in classified in different families (A1, A2, B, C and D), distinguishable by the position where published maps and institutional affil- they induce lipid hydrolysis [1,2]. The PLA2 family is involved in the cleavage of an iations. sn-2 ester bond of the glycerophospholipid substrate, with the consequent generation of 1-acyl-lysophospholipids and free fatty acids, in particular polyunsaturated acids that are metabolized to eicosanoids and bioactive lipid mediators [3,4]. Their key role in in- flammation processes and in the arachidonic acid metabolism in mammals is what made such enzymes the most studied PLs. Inside the PLA2 family, it is possible to discriminate Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. different subfamilies based on their structure, localization, and catalytic mechanism [5]. This article is an open access article One of these subfamily comprises the secretory PLA2s (sPLA2s) (EC 3.1.1.4) which is distributed under the terms and subdivided in seventeen groups [6], according to their molecular structures. Interestingly, conditions of the Creative Commons sPLA2s are well-conserved from eubacteria to mammals, suggesting an early appearance Attribution (CC BY) license (https:// during evolution [4,6,7]. Conventional sPLA2s (groups I, II, V and X) are extracellular 2+ creativecommons.org/licenses/by/ small-molecular-weight (13–15 kDa) enzymes, requiring millimolar Ca concentrations 4.0/). for their catalytic activity, principally targeting phospholipids in the extracellular milieu. Toxins 2021, 13, 290. https://doi.org/10.3390/toxins13040290 https://www.mdpi.com/journal/toxins Toxins 2021, 13, x FOR PEER REVIEW 2 of 15 Toxins 2021, 13, 290 2 of 15 for their catalytic activity, principally targeting phospholipids in the extracellular milieu. sPLA2ssPLA2s are are widely widely distributed distributed in in different different body body fluids fluids (e.g., (e.g., snake snake and and other other venoms, venoms, plasma,plasma, pancreatic pancreatic juice, juice, tears, tears, seminal seminal fluid) fluid) and and are are involved involved in in a a variety variety of of processes, processes, fromfrom snakebite snakebite envenomation envenomation to to signal signal tran transduction,sduction, to to lipid lipid mediator mediator production production and and dietarydietary lipids digestiondigestion inin mammals mammals [6 ,[6,8,9].8,9]. Moreover, Moreover, it is it important is important to consider to consider that snake that snakevenom venom PLA2s PLA2s toxins toxins are potential are potential therapeutic therapeutic drugs drugs against against many many pathophysiological pathophysiolog- icalconditions conditions [10 ].[10]. MoreMore than than 400 400 unique unique snake snake venom venom PLA2s PLA2s toxins toxins are arereported reported in the in UniProtKB the UniProtKB da- tabase,database, with with an overall an overall conserved conserved tertiary tertiary stru structurecture [11]. [ 11All]. these All these sPLA2s sPLA2s toxins toxins represent repre- optimalsent optimal models models for the for study the study of sPLA2 of sPLA2 functi functionon and mechanism and mechanism of action. of action. Based Based on pri- on maryprimary structure structure similarity, similarity, position position of ofdisulfide disulfide bridges, bridges, and and loop loop insertion, insertion, snake snake venom venom PLA2sPLA2s are are separated separated in in two two groups. Those Those derived derived from from Elapidae Elapidae and and Hydrophiidae Hydrophiidae snakessnakes belong belong to to the the group group I IPLA2s, PLA2s, more more similar similar to to the the mammalian mammalian pancreatic pancreatic phospho- phospho- lipaseslipases (PLA2G1B), (PLA2G1B), while while those those deriving deriving from from Viperidae Viperidae and and Crotalidae Crotalidae snakes snakes belong belong to groupto group II, given II, given their their high high homology homology with with the mammalian the mammalian phospholipases phospholipases enriched enriched in syn- in ovialsynovial fluids fluids and tears and tears(PLA2G2A) (PLA2G2A) [10,12]. [10 Snake,12]. Snake venom venom PLA2s PLA2s are also are subdivided also subdivided in three in differentthree different categories categories based based on their on their toxic toxic effe effects:cts: myotoxins, myotoxins, targeting targeting skeletal skeletal muscles; muscles; neurotoxins,neurotoxins, that that act act on on pre- pre- or or post-synapti post-synapticc elements elements of of the the neuromuscular neuromuscular junction; junction; and and hemostasis-impairinghemostasis-impairing toxins toxins [12,13]. [12,13]. sPLA2ssPLA2s of of both both groups groups share share a a highly highly conserved conserved amino amino acidic acidic sequence sequence and and a a well- well- conservedconserved tertiary tertiary structure structure (Figure (Figure 11),), that was unveiled by the crystallization of various sPLA2ssPLA2s isolated isolated from from snake snake venoms, venoms, humans, humans, an andd bovines bovines [4,6,7]. [4,6,7]. Although Although the the majority majority of snake venom PLA2s exist as monomers, some of them have acquired a quaternary of snake venom PLA2s exist as monomers, some of them have acquired a quaternary structure by a non-covalent interaction between two or more PLA2s [12]. structure by a non-covalent interaction between two or more PLA2s [12]. FigureFigure 1. 1. ConservationConservation of of the the structures structures of of snake snake venom venom and and mammalian mammalian sPLA2s. sPLA2s. (A (A) )3D 3D structures structures of of a agroup group I Ineuro- neuro- myotoxinmyotoxin (notexin (notexin of of NotechisNotechis scutatus scutatus scutatus scutatus, ,PDB PDB entry entry AE7) AE7) and and of of human human PLA2G1B PLA2G1B (PDB (PDB entry entry 3ELO) 3ELO) and and their their superposition.superposition. (B (B) )3D 3D structures structures of of a a group group II II myotoxin myotoxin (bothropstoxin-I (bothropstoxin-I of of BothropsBothrops jararacussu jararacussu, ,PDB PDB entry entry 3I3I) 3I3I) and and of of humanhuman PLA2G2A PLA2G2A (PDB (PDB entry entry 1BBC) 1BBC) and and their their superposition. superposition. The The calcium calcium binding binding loop loop region region is is colored colored in in grey. grey. PLA2G1B PLA2G1B has an extra loop, named the pancreatic loop, represented in magenta. The lateral chains of the amino acids involved in has an extra loop, named the pancreatic loop, represented in magenta. The lateral chains of the amino acids involved in the the active site are represented in ball and sticks: H48 in yellow, D49 in orange. Bothropstoxin-I is a PLA2-like toxin (not- active site are represented in ball and sticks: H48 in yellow, D49 in orange. Bothropstoxin-I is a PLA2-like toxin (not-D49) D49) with a lysine (in green) in the place of the glutamic acid 49. with a lysine (in green) in the place of the glutamic
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