Nucleolin internalizes Bothrops asper Lys49 phospholipase A2 forming cell surface amyloid-like assemblies

Phospholipases A2 (PLA2s) are a major component of snake venoms. Some of them cause severe muscle necrosis through a still unknown mechanism. Phospholipid hydrolysis is a possible explanation of their toxic action, but catalytic and toxic properties of PLA2s are not directly connected. In addition, viperid venoms contain PLA2-like proteins, which are very toxic even if they lack catalytic activity due to a critical mutation in position 49. Nucleolin, a main component of the nucleolus, is a disordered protein involved in many protein assembly and phase separation phenomena. In some circumstances nucleolin is exposed on the cell surface from where it is involved in the internalization of many ligands. In this work we demonstrate that Bothrops asper myotoxin II (Mt-II), a Lys49 PLA2-like toxin, interacts with, and is internalized in cells by nucleolin. The internalization process is functional to the toxicity of the protein, as both an antibody and an aptamer specific for nucleolin protect cells from intoxication. We identified central RRM and the C-terminal R/F-GG domain of nucleolin as the regions involved in the interaction with Mt-II. Finally we observed that Mt-II forms, on the cell surface, amyloid-like assemblies that colocalize with nucleolin and that can be involved in the activation of the internalization process. The presence, in the three dimensional structure of Mt-II and related PLA2 homologues, of four exposed loops enriched in prion-like amino acid sequences reinforces this hypothesis. Phospholipases A2 | Lys49 myotoxins | nucleolin | amyloid-like | molecular assemblies SIGNIFICANCE The main finding of this work, the role of nucleolin as Bothrops asper Mt-II receptor, is a remarkable step forward in understanding the mechanism of action of cytotoxic PLA2s. It may suggest new strategies for anti-venom therapies and explain the anti-tumoral and anti-viral pharmacological action of snake PLA2s, since nucleolin is a receptor for many growth factors and virus. The proposed internalization mechanism, via formation of molecular assemblies among Mt-II amyloid-like structures and other proteins, including nucleolin, can be of general validity. Cell surface molecular assemblies couldbepointsofselectionandconcentrationnotonlyofsnake,butalsoofmammaliansecretedPLA2s, proteins involved in different pathologies, and trigger the internalization pathway only when their molarity exceeds a threshold dose.


INTRODUCTION
Snakebites affect every year more people than every other neglected tropical diseases, causing death but also permanent disability and disfigurement (1). Snake venoms consist of a mixture of toxins and enzymes that have evolved mainly to capture and digest the prey. Major clinical effects of envenoming in humans include coagulopathy, neurotoxicity, myotoxicity, and renal impairment, among others (2).
Phospholipases A 2 (PLA 2 s) are major components of snake venoms, acting as hemostasis-impairing toxins, neurotoxins, or myotoxins. They have a high homology with mammalian secreted PLA 2 s, suggesting that they probably share cellular mechanisms and molecular interactors. This is of high relevance, in the light of the emerging involvement of mammalian secreted PLA 2 s in many human disorders (3)(4)(5). Various pharmacological applications have been proposed to exploit the diverse functionalities of snake PLA 2 s, including antiviral and antitumoral activities. However, the basic mechanisms of these activities are not known (6)(7)(8).
Most myotoxic PLA 2 s cause a local myonecrosis at the site of snakebite, but some of them act systemically, causing widespread muscle damage. Systemic myotoxins probably have high specificity for a muscle receptor, while locally-acting myotoxins, which induce myonecrosis only at relatively high doses, appear to interact with low-affinity receptors. Moreover, some local myotoxins also bind to and affect different types of cells, indicating that their receptor(s) is non-muscle-specific. Notwithstanding the many efforts made by several laboratories to identify myotoxic PLA 2 s receptors/acceptors in cell membranes, this search is still ongoing. Moreover, the internalization and possible interaction of these toxins with intracellular targets have not been explored (7).
A large subfamily of natural variants of snake PLA 2 s have no enzymatic activity, since they have a critical mutation at position 49: the aspartic acid is substituted by another amino acid (lysine in most cases), resulting in the impossibility to coordinate the calcium ion essential for catalysis. Despite the lack of catalytic activity, these PLA 2 homologues show a high myotoxic activity and other toxic effects (7,9).
Bothrops asper myotoxin II (Mt-II) is a Lys49 PLA 2 homologue protein acting as a local myotoxin, but also affecting a wide variety of cell types in vitro (10), including macrophages (11). The currently held view is that Mt-II exerts its toxic activity by affecting the plasma membrane integrity, with consequent rapid influx of calcium ions that eventually triggers a series of degenerative events (12). However, this might be a simplistic view since the interaction of Mt-II with cells involves also intracellular signaling pathways: immediately after addition to cell cultures, Mt-II induces calcium release from intracellular stores, followed by opening of potassium and ATP channels, activation of purinergic receptors, and finally massive entry of calcium from extracellular medium (11,13,14).
In this work we conjugated Mt-II, purified from Bothrops asper venom, with a fluorophore to inquire its localization in target cells, and with biotin to use it as bait to isolate its protein interactors. We found that the toxin is rapidly internalized both in myotubes and in macrophages, and transported to the paranuclear and nuclear zone. Among the protein interactors we identified nucleolin (NCL), a multifunctional protein with a high percentage of disordered domains (15) and that is ubiquitously distributed in various eukaryotic cell compartments, such as the nucleolus, the nucleoplasm, the cytoplasm and the cell membrane (16). NCL has been reported to mediate the internalization of different types of molecules (16,17). We verified the involvement of NCL in Mt-II internalization and toxic activity with pull-down experiments, cellular uptake, and cytotoxicity tests in presence of NCL competitors. Moreover, thanks to the different nature of these competitors, we were able to map the NCL domains involved in interaction with Mt-II.
Finally, we found that NCL co-localizes with Mt-II on the cell surface in structures that are sensitive to Congo Red staining. This observation, reinforced by the biochemical properties of NCL as disordered protein, and by the presence of prion-like sequences in the Mt-II structure, led us to hypothesize that Mt-II forms molecular assemblies on the cell surface that could trigger the internalization process and are probably also involved in cell membrane permeabilization.

Mt-II is internalized in myotubes and macrophages
Purified Mt-II was conjugated, by reaction with transglutaminase (18), to TAMRA and DNS fluorophore containing peptides to observe the localization of the toxin in target cells, myotubes and macrophages. The major reaction product was isolated by RP-HPLC and characterized by ESI-mass spectrometry and by cytotoxicity test. The determined molecular masses correspond to that of the mono-conjugated products and the toxic activity is, for more than eighty percent, conserved (see supplementary Fig. S1).
Mt-II resulted to be rapidly internalized (within minutes) both in myotubes and in macrophages in an asynchronous way: some cells are intoxicated before others, and cell death is preceded by the internalization of the toxin ( Fig. 1 and Video S1, S2 and S3). The internalized toxin is transported toward the cell nucleus and in some cases co-localizes with it or with nuclear membrane that appears prominent in dying cells, while no important colocalization with mitochondria was observed (Fig. 1A). Time lapse experiments of macrophages and C2C12 myotubes treated with Mt-II DNS (Video S1 and S3) evidence that the toxin induces membrane blebs typical of macropinocytosis (11,19) and that higher doses of Mt-II cause a rapid detachment of the cells from the substrate (Video S2).

Isolation and identification of Mt-II protein interactors from macrophage cell extracts
Mt-II was conjugated to a biotin containing peptide by reaction with transglutaminase. The reaction and the protein purification and characterization were executed as reported in the previous paragraph. Biotinylated Mt-II (Mt-II-B), was combined to streptavidin magnetic beads and utilized as bait to pull-down interacting proteins from a RAW264.7 cell extract. The isolated proteins were eluted with a 5% sodium deoxycholate (SDC) solution, that does not detach the myotoxin from the resin, then digested with trypsin and identified by LC-MS/MS analysis. As controls, the same procedure was performed using beads without Mt-II. The experiment was repeated twice under the same conditions, each time leading to the identification of about one hundred proteins, each of them with a false discovery rate (FDR) less or equal to 0.01, and with at least four sequenced unique peptides (Table   S1 and S2). To identify proteins closer to the bait, the experiment was then repeated a third time by adding a crosslinking step on streptavidin beads, after protein isolation. The crosslinker (DTSSP) contains amine-reactive NHS-ester ends around a 12 Å, 8-atom spacer arm, and a central disulfide bond that can be cleaved with reducing agents. After the crosslinking reaction the non-covalently bound proteins were removed with the 5% SDC solution and finally crosslinked proteins were detached with 50 mM DTT dissolved in the 5% SDC solution.
Also in this case about a hundred proteins were identified but with a lower abundance: only 21 proteins with a sample/control intensity ratio higher than 3 (Table S3), in comparison to 111 and 84 of the first and second experiment. Fifteen proteins were found in common in all three experiments (Fig. 2). NCL was identified in all pull-downs among the proteins with the largest number of unique peptides and with an estimated abundance at least 45 times higher with respect to the control sample. Since NCL is a nucleolar protein known to be present also on the cell surface as well as a receptor or co-receptor of different factors, we decided to enquire the Mt-II/NCL interaction and the role of NCL in Mt-II internalization.

Mt-II pulls down nucleolin from RAW264.7, C2C12 and ex-vivo muscle membrane preparations
NCL is one of the most abundant non-ribosomal proteins of the cell. It is mostly localized in nucleolus (90%) and nucleus (about 5%), but it is present also in cytosol and cell membrane in variable concentrations, depending on cell status (16,20). To confirm the interaction between Mt-II and NCL in cell types other than macrophages, and to verify that Mt-II interacts with NCL present in cell membrane, we prepared an extract of membranes from RAW264.7, C2C12 myotubes and from ex-vivo muscle. The subcellular fractionation was performed with a commercial kit and the fractions were characterized by western blot to verify the efficiency of the protocol (Fig.   S2). Membrane fractions were incubated with Mt-II-B combined with streptavidin magnetic beads, eluted by 5% SDC solution and analyzed by western blot. As observed in Fig. 3 A, Mt-II pulls down NCL from membrane extracts obtained from all three preparations.
NCL is a protein of 710 (human) or 707 (mouse) amino acids, respectively, composed of three domains: an Nterminal disordered domain rich in negatively charged amino acids, a central domain containing four RNA Recognition Motifs (RRMs), and a C-terminal disordered domain containing R/F-GG repeats (Fig. 3 C). Many substances were found to interact with NCL present on the cell surface, in particular on surface of cancer cells where NCL is present at higher concentration, and to trigger an internalization process (17,20). AS1411 is an anticancer aptamer that binds to the central and C-terminal regions of NCL (21), and F3 is a tumor-homing peptide that binds to the N-terminal negatively charged domain of NCL (22). We tested the ability of AS1411, F3 and of an anti-NCL central domain polyclonal antibody to compete for the pull-down of NCL by Mt-II-B.
Interestingly, the antibody and the aptamer inhibit the pull-down of NCL while F3 does not compete (Fig. 3 B), indicating that the central and C-terminal regions of NCL are involved in the interaction with Mt-II.

AS1411 and anti-nucleolin antibody inhibit Mt-II cell internalization and toxic action
To understand if the interaction between NCL and Mt-II has a role in the internalization and toxic activity, we measured the quantity of Mt-II internalized and the cytotoxicity of the protein in the presence of AS1411, a control aptamer (CRO), and an anti-NCL antibody. We performed an internalization assay intoxicating target cells with Mt-II-TAMRA, washing extensively to remove unbound protein, and measuring the fluorescence after cell lysis. For the cytotoxicity assay, cells were incubated with unlabeled Mt-II and the percentage of cell death was measured with a colorimetric assay for assessing cell metabolic activity, in the case or RAW264.7 cells, or by measuring the release of LDH, a plasma membrane damage index, in the case of the myotubes. The results of the two assays are reported in Fig. 4: the two NCL competitors significantly inhibited, though not completely, both the internalization and the toxicity of Mt-II.

Mt-II co-localizes with nucleolin in cell surface assemblies sensitive to Congo Red staining
The interaction between NCL and Mt-II was verified also by co-localization experiments. In developing the experimental protocol, we observed that the amount of NCL present in the cell surface of non-stimulated cells is very low, but it increases in live cells treated with the toxin or with an anti-NCL antibody, at RT or 37°C. In fact NCL is released to the surface through an unconventional secretion that does not pass through the classic ER-Golgi pathway (23). Since we aimed at observing the interaction between NCL and Mt-II on the cell surface, we decided to add the toxin at low temperature (4°C) to prevent its internalization and consequent cell death. Thus, we treated cells with the anti-NCL antibody at RT to stimulate the secretion of NCL and successively with Mt-II-TAMRA at 4°C. Finally, we fixed the cells, treated them with the secondary antibody and we acquired images by confocal microscopy. The obtained images ( Fig. 5 and Fig. S3) show that NCL and Mt-II colocalize in long stretches, fairly thick, on the cell surface. This kind of staining, non-dotted but with bigger areas, is typical of proteins involved in phase transition phenomena that give rise to membrane-less organelles (15,24).
Membrane-less organelles are molecular assemblies that form through multivalent weak interactions and that mediate diverse biological processes. The nucleolus is an example of this kind of organelles and NCL is one of its main components (25). Phase transition phenomena can happen also on the cell membrane, for example to trigger signaling events (26). As secreted PLA 2 s were reported to form amyloid-like fibrils when they come in contact with phospholipid bilayers (27), and prion-like interactions are involved in phase transition phenomena, we decided to assess if Mt-II assemblies on cell membrane are sensitive to Congo Red, an amyloid specific dye.
We observed polymer-like green birefringence on polarized light, on the surface of cells intoxicated with Mt-II ( Fig. 6), indicative of the presence of amyloid-like fibrils, and red coloured zone that co-localize with NCL.
Assemblies formed by Mt-II are not SDS-resistant: SDS-PAGE analysis of extracts from cells incubated with Mt-II-B and a membrane impermeable crosslinking agent shows that Mt-II forms high molecular weight complexes only in crosslinked samples (Fig. S4). Since proteins that form amyloid-like fibrils are characterized by the presence of prion-like domains (15,24), we inspected the primary structure of Mt-II for the presence of prionpromoting amino acids. We found four traits rich in tyrosine, serine and glycine with some asparagine or glutamine, amino acids enriched in human prion-like domains (28), with in addition the presence of proline and charged amino acids that are relevant to modulate the formation of prion aggregates (28,29). Interestingly, these traits are located in exposed loops of Mt-II (Fig. 7 B) and homologous proteins, sites that can undergo conformational changes in an otherwise very stable tertiary structure due to presence of seven disulphide bonds.

DISCUSSION
The first result of this work is the demonstration that B. asper Mt-II is internalized in myotubes and macrophages and that it localizes in perinuclear and nuclear zone (Fig. 1). Other snake PLA 2 s have been reported to be internalized, but only the neurotoxic PLA 2 s, and exclusively in neuronal cells. Notably, notexin, betabungarotoxin and taipoxin have been shown to localize in mitochondria of spinal cord motor neurons and cerebellar granule neurons (30), while ammodytoxin was found into the cytosol, synaptic vesicles, and mitochondria of motoneurons (31,32). Snake cytotoxins, non-enzymatic three-fingered fold proteins, have been reported to be internalized in cells, and to induce cell death mainly by interaction with lysosomes (33).
Importantly, PLA2g2a, the human secreted PLA 2 with higher homology to Mt-II, was also reported to be internalized in monocytes and finally transported into the nucleus (34). Accordingly, the PLA2g2a subcellular localization reported in Human protein atlas (35) is nucleus and nucleolus.
The second result of this work is the isolation of putative cellular proteins interacting with B. asper Mt-II. NCL, one of the proteins identified in all pull-down/mass spectrometry experiments, is known to be present also on the plasma membrane and acts as a receptor or co-receptor for several factors (16,17). The interaction of Mt-II with this protein was confirmed by pull-down/western blot experiments from cell membrane extracts and by the competition exerted by an anti-NCL antibody and an aptamer specific for NCL. The inhibition of cytotoxicity and internalization by these competitors was partial, but this result was expected considering that AS1411 and anti-NCL antibody, after internalization, induce the secretion of further NCL in a continuous process (23,36).
The Mt-II/NCL interaction is remarkable for several reasons. First, NCL is the first Mt-II interacting protein connected to the toxic mechanism. An interaction of Mt-II with the KDR/VEGF receptor 2 has been previously reported (37,38) but no evidence for a functional role of this interaction was found by using a blocking monoclonal antibody to the receptor or the inhibitor tyrphostin (9). Second, the interaction of Mt-II with NCL may explain pharmacological properties of snake myotoxins. The preferential activity of these toxins against cancer cells (6,39,40) can be correlated to the fact that NCL is more abundant on the surface of these cells (16,41,42). Moreover, some snake PLA 2 s have been reported to have antiviral activity (43,44) and accordingly NCL is involved in internalization of many viruses (16,17). Third, several phenomena observed in cells following Mt-II intoxication can be explained by the interaction My-II/NCL: calcium entry is triggered also by NCL (45); the internalization of AS1411 takes place through macropinocytosis (46); NCL is involved also in cell adhesion and migration (42) and this could explain the rapid detachment of myotubes intoxicated with high doses of Mt-II (Video S2).
In the last part of this work we verified the colocalization of NCL and Mt-II in large areas of the cell surface and we observed that Mt-II forms amyloid-like structures in contact with cells. This suggests that the NCL-Mt-II interaction is not one-to-one but a multi-molecular assembly where other components are likely to be present.
Nucleophosmin for example, a known partner of NCL, was identified among proteins co-precipitated by Mt-II (Table S4). However transmembrane proteins also can be involved, and they may not have been detected in our pull-down because they are more difficult to isolate and identify by mass spectrometry. In this regard it is worth remembering that both NCL and PLA2g2a interact with integrins and EGFR (47)(48)(49)(50). Since NCL is not a membrane protein, it will have to interact with transmembrane proteins to communicate with the cell interior and trigger the internalization process.
NCL sequence (Fig. 3C), rich in disordered regions and low complexity domains, is typical of proteins participating in multimolecular interactions and phase transition phenomena, that is, transitions from dispersed protein solutions to liquid-like phase-separated compartments or to solid protein aggregates (15,24). Mt-II, like other secreted PLA 2 s, has a well-defined and compact 3D structure stabilized by seven disulphide bonds; as a consequence, one would not expect it to be inclined to form multimolecular interactions. However, the fact that Mt-II forms amyloid-like structures, when in contact with cell surface, changes the view. Several proteins involved in phase transition phenomena possess prion-like domains, and prion structures contribute to the protein assembly. Moreover, many disordered proteins are thought to recognize and interact with amyloid structures, and NCL could be one of them. In fact, NCL is also a receptor of amyloid beta peptide 1-42 (51) and the R/FGG domain, that we found to be involved in interaction with Mt-II, is similar to the glycine rich domains of the chaperones involved in yeast prion propagation (52).
The formation of amyloid structures and multimolecular assemblies explains many characteristics of Mt-II and similar proteins. One is the need for a relatively high concentration of protein for toxic activity: locally-acting PLA 2 myotoxins act at a concentration of micromolar order, while neurotoxic PLA 2 s and systemic myotoxins at a concentration even 1000-fold lower (12). Another is the tendency of Mt-II and other PLA 2 s to be unstable in vitro: when dissolved, we keep the protein in 50% glycerol otherwise it slowly forms aggregates, even at low temperatures. Finally, we should consider the antimicrobial capacity of Mt-II, and other snake PLA 2 s (8, 53) and the fact that several antimicrobial peptides are amyloid-like and vice versa amyloid peptides have antimicrobial properties. Both classes of peptides exhibit membrane-interaction and disruption ability with common mechanisms (54,55). Interestingly, antimicrobial peptides and PLA 2 enzymes can form amyloid-type co-fibrils that require the presence of the PLA 2 lipid hydrolytic product, so the PLA 2 catalytic activity synergizes with the propensity to form amyloid fibril (56). This can contribute to explain the synergy between catalytic and noncatalytic PLA 2 s, as observed for B. asper myotoxins I and II (57).
Why should secreted PLA 2 s form these complexes in membrane? Code et al (27) proposed that amyloid-type formation of PLA 2 is functional to the control of enzyme action. We add that the function, on cell surface, could be also of triggering the endocytic process, only when protein concentration exceeds a threshold value. Phase transition in cell surface could be useful to select and concentrate secreted protein factors with consequent activation of a signaling cascade, membrane movements and internalization mechanisms. Different compositions of this multimolecular assembly could explain the various actions and specificities of secreted PLA 2 s.
In conclusion, with this work, we have identified, for the first time, a functional interaction between Mt-II and a cell surface protein, NCL. Our result definitively excludes that Mt-II interacts only with membrane lipids but also excludes interaction with a single protein receptor and indicates that this PLA 2 -like toxin, due to its propensity to form amyloid-like fibrils, probably participates in a multi-molecular assembly with many actors. We observed that secreted PLA 2 s possess prion-like sequences, in exposed loops, very similar to the low complexity domains interacting with NCL in phase transition phenomena inside the cell. We think therefore that the observed Mt-II/NCL protein assembly can be considered a phase separation on the cell surface, functional to the internalization of external factors. This internalization pathway, found for a snake toxin, may be the one followed by the human homologous protein, PLA2g2a, and this would explain its localization in nucleus and nucleolus. If so, it will be interesting to understand why so similar proteins, despite following the same path, cause such a different effect in cells.

Isolation of Mt-II from Bothrops asper venom and modification with transglutaminase
Mt-II was isolated from the crude venom of Bothrops asper, a pool obtained from at least 30 specimens kept at the serpentarium of Instituto Clodomiro Picado, University of Costa Rica, as described in a previous work (57). Reactions were stopped by addition of iodoacetamide (100 μM final concentration). The fraction of mono-labeled Mt-II was purified from the reaction mixture by RP-HPLC with a C18 column (150 × 4.6 mm, 5 m particle size; Phenomenex). The obtained product was lyophilized with a Freeze Dryer Edwards E2-MS (Milano) and analysed by a Q-Tof Micro mass spectrometer (Micromass, Manchester, UK). The toxic activity of the modified Mt-II was analyzed by a vitality test in RAW 264.7 cells and verified to be conserved (Fig. S1).
For the cytotoxicity assay macrophages and differentiated myotubes were grown in 96-well plates and then

Mass spectrometry analysis
Samples isolated from the pull-down experiments performed as described above were loaded in a 4-12% SDS-   • The authors declare no conflict of interest.   (Table S1-S4). The experiment was repeated two times with the same conditions, and a third with the addition of a crosslinking step to isolate proteins closer to the bait. (Right) Venn diagram of the number of proteins identified in the three pull down experiments. Among the fifteen proteins present in all three experiments NCL was identified with at least 13 unique peptides and an intensity ratio, sample over control, higher than 45.     Secondary structure elements were underlined and traits rich in prion-like amino acids were evidenced in green.
(Down) Mt-II 3D model (PDB: 1CLP) where the traits rich in prion-like amino acids (see main text for definition) were colored in green. The picture was obtained with the Cn3D macromolecular structure viewing program.