Enzymatic labelling of snake venom phospholipase A2 toxins

Toxicon 170 (2019) 99–107

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Enzymatic labelling of snake venom phospholipase A2 toxins �n Ferna �ndez b, Bruno Lomonte b, Maria Lina Massimino c, Barbara Spolaore a, **, Julia c, * Fiorella Tonello a

Dipartimento di Scienze del Farmaco, Universit� a di Padova, Via F. Marzolo, 5, 35131, Padova, Italy Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San Jos�e, 11501, Costa Rica c Istituto di Neuroscienze, CNR, Viale G. Colombo, 3, 35121, Padova, Italy b

A R T I C L E I N F O

A B S T R A C T

Keywords: Secretory phospholipase A2 Snake venom toxin Protein labelling Enzymatic conjugation Transglutaminase

Almost all animal venoms contain secretory phospholipases A2 (PLA2s), 14 kDa disulfide-rich enzymes that hydrolyze membrane phospholipids at the sn-2 position, releasing lysophospholipids and fatty acids. These proteins, depending on their sequence, show a wide variety of biochemical, toxic and pharmacological effects and deserve to be studied for their numerous possible applications, and to improve antivenom drugs. The cellular localization and activity of a protein can be studied by conjugating it with a tag. In this work, we applied an enzymatic labelling method, using Streptomyces mobaraense transglutaminase, on three snake venom PLA2s: a recombinant neuro- and myotoxic group I PLA2 from Notechis scutatus scutatus, and two myotoxic group II PLA2s from Bothrops asper - one of them a natural catalytically inactive variant. We demonstrate that TGase can be used to produce active mono- or bi-derivatives of these three PLA2s modified at specific Lys residues, and that all three of these proteins, conjugated with fluorescent peptides, are internalized in primary myotubes.

1. Introduction Snake venom phospholipases A2 are globular proteins of about 120 amino acids, 7 disulfide bonds, having a secondary structure composed by three main ɑ-helices and a β-wing region. They are divided in two groups, based on structural characteristics (Arni and Ward, 1996). Group I PLA2 differs from those of the second group for the presence of an insertion of 2–3 amino acids, between the second ɑ-helix and the β-wing, called elapid loop, and for a disulfide bond connecting the central turn region of the β-wing with the first ɑ-helix. They are present in the venoms of Elapidae, Colubridae and Hydrophiidae snakes, and belong to the same group of pancreatic PLA2s present in mammals. PLA2s of group II are characterized by the presence of a longer C-ter­ minal loop (6–7 amino acids), and a disulfide bond connecting a C-ter­ minal cysteine to the second ɑ-helix. They are expressed in venoms of Viperidae and are similar to mammalian non-pancreatic, inflammatory PLA2s (Tonello and Rigoni, 2015; Murakami et al., 2015; Guti� errez and Lomonte, 2013).

Venom PLA2s show a wide range of pharmacological effects. They cause pre- and post-synaptic neurotoxicity, myonecrosis, cardiotoxicity, hemolysis, coagulation alterations, hypotension and edema (Kini, 2003). Some of these effects depend on catalytic activity, others on protein-protein interactions, and in fact some snake venoms include natural catalytically-inactive variants with evident toxic/pharmaco­ logical properties (Lomonte and Rangel, 2012). Their actions take place also at the intracellular level: several secretory PLA2s internalize in different kinds of cells, and localize in the paranuclear/nuclear area (Kudo and Murakami, 2002; Nardicchi et al., 2007; Massimino et al., 2018), or with the mitochondria (Rigoni et al., 2008). The labeling of secretory PLA2s, a modification necessary to study their cellular activity, is particularly complicated as these proteins are very sensitive to molecular alterations. The addition of a single methi­ onine in the N-terminal position greatly dampens their activity (Di Marco et al., 1992; Chiou et al., 2008; Simonato et al., 2014), and even the C-terminal plays a role in the intoxication process (Murakami et al., 1996; Cintra-Francischinelli et al., 2010). For these reasons, the

Abbreviations: ACN, acetonitrile; DC, dansylcadaverine; DMSO, dimethylsulphoxide; DNS, 5-N-(50 -N0 ,N0 - dimethylamino-10 -naphtalenesulfonly)diaminopentane; E:S, enzyme to substrate ratio; RP-HPLC, reverse-phase high-performance liquid chromatography; TFA, trifluoroacetic acid; TGase, transglutaminase; ZQG, carbo­ benzoxy-L-glutaminyl-glycine. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (B. Spolaore), [email protected] (F. Tonello). https://doi.org/10.1016/j.toxicon.2019.09.019 Received 3 July 2019; Received in revised form 2 September 2019; Accepted 25 September 2019 Available online 26 September 2019 0041-0101/© 2019 Elsevier Ltd. All rights reserved.

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group II inactive K49 PLA2 homologue, getting the protein labelled with a fluorophore and biotinylated, derivatives that have allowed us to study the cellular localization, and molecular interactions of the protein. In this work, we apply the TGase conjugation reaction, in addition to Mt-II, to two catalytically active snake PLA2s: notexin (Ntx), group I PLA2 from Notechis scutatus scutatus, and myotoxin I (Mt–I), group II PLA2 from Bothrops asper (Fig. 1B). We characterize the sites of derivatization in the reaction products, and their reaction yields, and we determine the toxic and catalytic activity of the derivatives. The results demonstrate that the TGase conjugation method can have a general validity as a system to obtain labelled snake venom PLA2s.

production of these proteins fused to fluorescent chimeras is not conceivable. Chemical methods were used to combine Lys lateral chains of PLA2s, purified from snake venoms, with fluorophores, but this type of reactions are difficult to control, since many Lys residues are present. The products obtained were not homogeneous and the reaction yield was very low (Díaz-Oreiro and Guti�errez, 1997; Rigoni et al., 2008). Streptomyces mobaraense microbial transglutaminase (TGase) is an enzyme that catalyzes the formation of an isopeptide bond between the lateral chains of Gln and Lys residues (Folk, 1983) (Fig. 1A). For the purposes of protein derivatization, TGase can modify a protein either at the level of Gln residues, using an amine containing ligand, or at the level of Lys residues, if a glutamine containing ligand is used. Impor­ tantly, it acts in a site-specific way on a limited number of Lys or Gln residues of a protein, and this characteristic, together with the fact that enzymatic conjugations occur under physiological reaction conditions, makes it appropriate to produce homogeneous derivatives in the biotechnological and pharmaceutical fields (Spolaore et al., 2014; Zhang et al., 2018; Mariniello et al., 2014; Deweid et al., 2019). In our recent work Massimino et al. (2018), we have applied the conjugation reaction with TGase to Bothrops asper myotoxin II (Mt-II), a

2. Materials and methods 2.1. Materials Sequencing grade modified trypsin and sequencing grade endopro­ tease Glu-C were from Promega (Madison, WI, USA). Carbobenzoxy-Lglutaminylglycine (ZQG), dansylcadaverine (DC) where purchased from Sigma-Aldrich (Milwaukee, WI, USA), ZQG- 5-N-(50 -N0 ,N0 -

Fig. 1. (A) TGase catalyzes the formation of an isopeptide bond between the lateral chains of a glutamine and a lysine residue. This reaction can be exploited to conjugate Lys residues of a protein with a ligand containing a Gln-residue (e.g. ZQG), or to conjugate Gln residues of a protein with a primary amine containing ligand (e.g. DC). Chemical structures of DC and ZQG substrates are reported in Fig. S1. (B) Primary, secondary and tertiary structure of Notechis scutatus scutatus Ntx, and Bothrops asper Mt–I (isoform E120 K121), and Mt-II with highlighting of the residues sensitive to reaction with TGase. Sequence alignment was performed on the UniProt website using default settings and discarding the signal peptides. 3D structures were obtained with Cn3D macromolecular structure viewing program from the indicated PDB entries. α-Helices have been highlighted in green and β-sheets in gold. All Lys residues sensitive to reaction with TGase are shown in bold pink in the sequence and with evidenced lateral chains in the 3D structures. The violet circles show the position of Lys residues modified in higher yield (97–99%). Gln residues of these proteins resulted insensitive to the reaction with TGase. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 100

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dimethylamino-10 -naphtalenesulfonly)diaminopentane (ZQG-DNS) and ZQG-TAMRA where purchased from ZEDIRA GmbH (Darmstadt, Ger­ many). TGase from Streptomyces mobaraensis was TGase ACTIVA MP from Ajinomoto Co. (Tokyo, Japan). Stock solutions of TGase were prepared by dissolving the MP powder in 0.1 M sodium phosphate buffer, pH 7.0 in order to obtain a protein concentration of about 1 mg/ mL, as determined by UV spectroscopy. Aliquots of the TGase stock solution were stored at 80 � C until use.

eluted protein was lyophilized, dissolved in 8 M urea and then diluted with 0.1 M phosphate buffer, pH 7.0 in order to obtain a final protein concentration of 0.7–1.0 mg/mL in 0.4 M urea. Protein concentration was measured by UV spectroscopy. For the reactions with DC and ZQG, stock solutions of DC (20 mg/mL) and ZQG (34 mg/mL) in DMSO were added at molar ratios protein/ligand of 1/30 (DC) or 1/50 (ZQG), optimal conditions previously determined for other proteins (Spolaore et al., 2016). TGase was added at an E:S ratio of 1/25 (w/w) for Ntx and Mt–I and of 1/10 for Mt-II and the reaction mixtures were incubated at 37 � C under agitation at 350 rpm in a Thermomixer compact (Eppen­ dorf). Aliquots were collected after 0 min (before enzyme addition), at 1 h and 4 h for Ntx, and Mt–I or 8 h for Mt-II, and reactions were stopped by addition of a 100 molar excess of iodoacetamide in respect to TGase. The reaction times applied to Ntx and Mt–I, where previously found to be optimal to detect the first sites of TGase derivatization (1 h) and to obtain the complete derivatization of a protein (4 h) (Spolaore et al., 2016). Mt-II is not derivatized efficiently by TGase after 4 h, thus we incubated the sample up to 8 h to improve the yield of the reaction. Aliquots of the reaction mixtures were store at 20 � C and then analyzed by RP-HPLC analysis as described above. Fractions collected from RP-HPLC were lyophilized, and used in MS analyses and activity assays (see below). Derivatization of Ntx and Mt-II with ZQG-TAMRA, and of Mt–I with ZQG-DNS, was performed as for the reactions with ZQG but with an incubation time of 4 h for all the proteins. The products of the reactions were purified by RP-HPLC, as described above, and their average mass measured by ESI-MS to confirm the number of ZQG-DNS and ZQGTAMRA moieties conjugated to the proteins (not shown).

2.2. Purification of PLA2s from Bothrops asper venom Mt–I, and Mt-II were isolated from crude venom of Bothrops asper, collected from specimens kept at the serpentarium of Instituto Clodo­ miro Picado, under authorization of the University of Costa Rica. Lyophilized venom was dissolved in 50 mM TRIS, pH 7 and fractionated by cation-exchange chromatography with a 5 mL HiTrap™ CMFF col­ umn (GE Healthcare Life Sciences), and the gradient 0.1–0.5 M KCl, 60 min, in 50 mM TRIS, pH 7. The collected fractions were further pu­ rified by reverse-phase HPLC on a semi-preparative Jupiter 5u C18 300A 250 � 10 mm column (Phenomenex), applying a two steps gradient of acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) and water, 0.1% TFA (5–30% of ACN in 3 min and 30–50% ACN in 20 min, 2 mL/min). The collected fractions were lyophilized overnight, with a Freeze Dryer Edwards E2-MS (Milano), and stored at 20 � C until use. 2.3. Production and purification of recombinant Ntx LB growth medium (500 mL) containing 50 μg/mL of ampicillin, was inoculated with the E. coli strain BL21(DE3 pLysS) transformed with pET20b-T7-Ntx (Simonato et al., 2014) (Addgene plasmid #58714) and the culture was grown at 37 � C to an OD600 of 0.6. Recombinant protein expression was induced by addition of 1 mM IPTG (4 h at 37 � C). The inclusion bodies were isolated as previous reported (Simonato et al., 2014) and dissolved in 6 M guanidinium (GND), 50 mM TRIS, 5 mM EDTA pH 8 and 10 mM DTT freshly added to the solution. T7-Ntx was folded by dilution at 50 μg/mL protein concentration in 550 mM GND, 440 mM Arg, 55 mM TRIS, 21 mM NaCl, 0.88 mM KCl, 10% glycerol (v/v), 2 mM GSH, 1 mM GSSG, pH 8.2, and kept for 66 h, at 25 � C. After folding, the protein solution was dialyzed against 50 mM ammonium acetate, 50 mM NaCl, pH 5 in SnakeSkin Dialysis Tubing, 3.5 K MWCO (Thermoscientific), loaded in a 5 mL ion exchange column HiTrap SP (GE Healthcare) and the protein mixture eluted with 50 mM ammonium acetate, 500 mM NaCl, pH 5. The folded protein was separated from the partially folded forms by a semi-preparative Jupiter C18 5 μm column 250 � 10 mm (Phenomenex, Torrance, California, USA), applying a two steps gradient of ACN, 0.1% TFA and water, 0.1% TFA (5–20% of ACN in 3 min and 20–50% ACN in 20 min, 2 mL/min). The T7 pre-peptide was removed by cleavage with trypsin, E:S ¼ 1:100, in 50 mM Tris–HCl, 100 mM NaCl, 2 mM CaCl2 pH 8, for 30 min at Tab. The reaction was stopped with a serine protease inhibitor, phenylmethylsulfonyl fluoride, and then the proteins were repurified in RP-HPLC C18. The protein was lyophilized overnight, with a Freeze Dryer Edwards E2-MS (Milano), and stored at 20 � C until use.

2.5. Digestion of ZQG-modified toxins For trypsin digestion, toxins and their derivatives with ZQG collected in the RP-HPLC analyses were lyophilized and dissolved in 8 M urea, 0.1 M NH4HCO3 pH 8.5 and then diluted with 0.1 M NH4HCO3, pH 8.5 in order to have a final concentration of 2 M urea. Reduction of disulfide bonds was performed by addition of tris(2-carboxyethyl)phosphine to a final concentration of 5 mM and incubation at room temperature for 30 min. Reduced Cys residues were then carbamidomethylated upon addition of iodoacetamide at a final concentration of 10 mM, and in­ cubation at room temperature in the dark for 30 min. The reduced and carbamidomethylated protein samples were diluted two-fold with 50 mM NH4HCO3 pH 8.5. Trypsin was then added at an E:S ratio of 1/ 100, by weight, and the solutions were incubated overnight at 37 � C under agitation. The reaction mixtures were then stopped by acidifica­ tion with an aqueous solution of 5% TFA, and stored at 20 � C before LC-MS analysis. Digestion with endoproteinase Glu-C from Staphylococcus aureus V8 was performed as for the trypsin digestion except that the buffer was 50 mM phosphate, pH 7.8, and the enzyme was added at an E:S of 1/50, by weight. 2.6. Mass spectrometry analyses Mass spectrometry-based analyses of PLA2s and of their derivatives were performed with a Micromass mass spectrometer QTof Micro (Manchester, UK) equipped with an electrospray source. Samples were dissolved in 0.1% formic acid in ACN: water (1:1, v/v) and analyzed in MS positive ion mode. Measurements were conducted at a capillary voltage of 3 kV and at cone and extractor voltages of 35 and 1 V, respectively. Instrument control, data acquisition and processing were achieved with Masslynx software (Micromass). The tryptic digests of toxins and of the same proteins derivatized with ZQG were analyzed using an ACQUITY UPLC H-Class System in line with a Xevo G2-XS QTof (Waters, USA). The UPLC was configured with an AdvanceBio Peptide Map Guard (2.1 � 5 mm, 2.7 μm, Agilent tech­ nologies) and AdvanceBio Peptide Map column (2.1 � 150 mm, 2.7 μm).

2.4. TGase-mediated derivatization of protein toxins with DC, ZQG, and ZQG-TAMRA or ZQG-DNS For the TGase reaction, aliquots of the toxins of about 115 μg were further purified by RP-HPLC on an Agilent series 1100 HPLC with an online UV detection from Agilent Technologies (Waldbroon, Germany) using a C18 Phenomenex column (Jupiter C18, 300 Å, 5 μm, 150 � 4.60 mm, Phenomenex) equipped with a SecurityGuard Cartridge Widepore C18 (4 � 3.0 mm ID, Phenomenex) and applying a two steps gradient of ACN, 0.085% TFA and water, 0.1% TFA from 5 to 30% of ACN in 3 min and from 30 to 50% in 20 min. The column was eluted at a flow rate of 1.0 mL/min and the absorbance was read at 226 nm. The 101

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plate (4 � 104 cells/well), coated with collagen 0.1%. After 48 h of in­ cubation, the proliferation medium was replaced with Dulbeccomodified Eagle’s medium (DMEM) containing 2% (v/v) horse serum (Eurobio), to favour myoblast differentiation. After three days the dif­ ferentiation medium was replaced with 100 μL of 2.5 μM Ntx, 2.0 μM Mt1, or 1.5 μM Mt-II, with or without TGase conjugation, dissolved in a modified Krebs Ringer Buffer (mKRB: 140 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 10 mM Hepes, and 11 mM glucose, pH 7.4). After an incubation (at 37 � C) of 4 h in the case of Ntx, 1 h in the case of B. myotoxins, the necrosis of the myotubes was essayed by measuring the concentration of lactate dehydrogenase (LDH) released in the cell su­ pernatant. Cells release LDH following cell membrane damage, which occurs during necrosis, so its concentration in the supernatant is pro­ portional to the percentage of cell death. We measured the LDH con­ centration in the cell supernatant with the In Vitro Toxicology Assay Kit, Lactic Dehydrogenase based, TOX7 (Sigma), by following the associated instruction manual. Briefly, in presence of LDH, a tetrazolium dye is converted to a soluble, coloured formazan derivative monitored by reading the absorbance at 420 nm.

Mobile phase A was water and 0.1% formic acid, while mobile phase B was 0.1% formic acid in ACN. Peptide separation was performed using a linear gradient from 2% to 65% of B in 36 min at a flow rate of 0.2 mL/ min, with a column temperature set at 30 � C. The Xevo G2-XS QTof was operated in the ESI positive ion, resolution mode and with a detection window between 50 and 2000 m/z. Source parameters were: capillary (kV) 1.5, sampling cone voltage 30 V, source offset of 80 V. Tryptic peptides were analyzed using data-independent MS/MS acquisition (Waters MSE) and data-dependent MS/MS acquisition. MSE acquisition was performed by alternating two MS data functions: one for acquisition of peptide mass spectra with the collision cell at low energy (6 V), and the second for the collection of peptide fragmentation spectra with the collision cell at elevated energy (linear ramp 20–40 V). Analyses were performed with LockSpray™ using a solution of 1 ng/μL LeuEnk in 50:50 ACN/water containing 0.1% formic acid. MSE data were pro­ cessed with the BiopharmaLynx 1.3.4 Software (Waters) setting trypsin as digest reagent and 3 missed cleavages. Applied fixed modification was carbamidomethyl cysteine, while variable modifications were Lys res­ idue modified with ZQG (320.1008 Da as side chain modification of Lys) and methionine oxidation. MS ion intensity threshold was set to 500 counts, and the MSE ion threshold was set at 100 counts. MS mass tolerance was set to 10 ppm and MSE mass tolerance to 15 ppm. The tryptic digests were also analyzed by LC-MS/MS analysis under identical chromatographic conditions with fragmentation of the five most intense precursor ions, except when included masses of conjugated peptides where detected. Conjugated peptides identified in the LC-MSE analyses were indeed listed in an ‘include mass file’ so that priority is given to their MS/MS analysis. Conjugated peptides were considered identified even in the absence of b/y ions only if the MSE spectra or MS/MS spectra show m/z signals characteristic of ZQG fragmentation (Fig. S2). Protein samples digested with endoproteinase Glu-C were analyzed by LC-MSE as for the tryptic digests. MSE data were processed with the BiopharmaLynx 1.3.4 Software (Waters) setting Glu-C (phosphate buffer pH 7.8) as digest reagent and 4 missed cleavages. Applied fixed modi­ fication was carbamidomethyl cysteine, while variable modifications were Lys residue modified with ZQG (320.1008 Da as side chain modi­ fication of Lys) and methionine oxidation. MS ion intensity threshold was set to 250 counts, and the MSE ion threshold was set at 100 counts. MS mass tolerance was set to 10 ppm and MSE mass tolerance to 15 ppm.

2.8.2. Internalization test Primary myoblasts in proliferation medium were seeded in 13 mm coverslips coated with collagen 0.1% in HCl 0.01 M (only for primary myotubes), in a 24 well plate (1.5 � 106 cells/well). After 48 h of incu­ bation, the proliferation medium was replaced with Dulbecco-modified Eagle’s medium (DMEM) containing 2% (v/v) horse serum (Eurobio), to induce myoblasts differentiation to myotubes. After three days, the differentiation medium was replaced 200 μL of 1.5 μM Ntx-ZQGTAMRA, Mt-I-ZQG-DNS or Mt–II–ZQG-TAMRA in mKRB, for 30 min at 37 � C. Coverslips were washed three times with mKRB, fixed with PFA (2% in PBS) for 20 min 4 � C, mounted in 8% Mowiol 40–88 (Sigma) in glycerol and PBS (1:3 ¼ v/v), and then observed with a LEICA CTR6000 inverted epifluorescence microscope. 3. Results and discussion 3.1. TGase modifies Ntx, Mt–I, and Mt-II only at Lys lateral chains First, we conducted an analysis of the PLA2s we intended to modify with TGase: Ntx obtained by expression in E. coli (Simonato et al., 2014), Mt–I, and Mt-II isolated from B. asper venom (Mora-Obando et al., 2014). All three proteins contain residues modifiable by TGase and they display a high number of Lys residues and few Gln residues (Table 1). RP-HPLC and ESI-MS analysis confirmed the homogeneity and the identity of the proteins (Fig. S3, Table 2). The toxins were then subjected to TGase conjugation reactions with two different substrates, carbobenzoxy-L-glutaminyl-glycine (ZQG) specific for Lys residues and dansylcadaverine (DC) specific for Gln residues (Fig. 1A and S1), and for two different times, 1 and 4 h. The RPHPLC chromatograms obtained with the ZQG reactions of Ntx and Mt–I, after 4 h of incubation, showed a decrease in the peak of the starting

2.7. In vitro PLA2 enzymatic activity The PLA2 activity of Ntx and Mt–I, wild type and modified by TGase, was measured with the Cayman secretory PLA2 assay kit that includes a 1,2-dithio analogue of diheptanoyl phosphatidylcholine (Dihepta­ noylThio-PC), as PLA2 substrate, and a positive control, bee venom PLA2. The hydrolysis of DiheptanoylThio-PC sn-2 thioester bond by PLA2s free a thiol that is detected by Ellman’s reagent, monitoring the increase in absorbance at 405 nm. The analysis was performed accord­ ing to the manufacturer’s instructions, with a 1 μM PLA2 concentration. 2.8. Test on mouse primary myotubes

Table 1 Number of Gln and Lys residues present in the sequences of the three PLA2s under study.

Primary cultures of mouse skeletal muscle cells differentiated to myotubes were utilized to test the cytotoxic activity, and the cell internalization of the ZQG-fluorescent-conjugated proteins. Primary myoblasts were obtained from posterior limb muscles of newborn (1–2 days-old) mice (CD1, Charles River). Limb tissue, after a washing in phosphate buffered saline (PBS), was minced and submitted to three successive treatments with trypsin 0.1% (w/v) in PBS, 30 min, 37 � C. Obtained cells were resuspended in Ham’s F12 (Eurobio) supplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (Eurobio) (proliferation medium).

Protein (Uniprot ID, organism)

Number of residues Gln

Lys (isoform)

Notexin (P00608, Notechis scutatus scutatus) Myotoxin I (P20474, Bothrops asper) Myotoxin II (P24605, Bothrops asper)b

3

11

119

3

14 (E120, K121) 13 (D120, P121) 19

122

a

1

a

Total number of amino acids

121

If different isoforms are present in the sample. the sequence of Mt-II corresponds to P24605 L114F natural variant (Lizano et al., 2001).

2.8.1. Cytotoxicity test Primary myoblasts in proliferation medium were seeded in a 96 well

b

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Table 2 Measured molecular masses of the reaction products of Ntx, Mt–I, and Mt-II with ZQG in presence of the TGase enzyme. Protein

Isoform

Modification (number)

React. Time, h

Ret. Time, min

Molecular mass, Da Found

Calculateda

Notexin

– – –

– ZQG (1) ZQG (2) ZQG (2)

0 1 1 4

10.6 11.6 12.9 12.9

13,579.60 � 0.06 13,899.45 � 0.14 14,219.40 � 0.14 14,219.70 � 0.23

13,579.42 13,899.72 14,220.02 14,220.02

Miotoxin I

E120; K121 D120; P121 E120; K121 D120; P121 E120; K121 D120; P121

– – ZQG ZQG ZQG ZQG

(1) (1) (1) (1)

0 0 1 1 4 4

18.7 18.7 19.7 19.7 19.7 19.7

13,967.68 � 0.09 13,922.74 � 0.07 14,287.80 � 0.13 14,242.84 � 0.19 14,287.75 � 0.17 14,242.72 � 0.14

13,967.23 13,922.15 14,287.53 14,242.45 14,287.53 14,242.53

L114F L114F L114F

– ZQG (1) ZQG (2)

0 8 8

13.0 13.9 14.7

13,759.99 � 0.02 14,079.77 � 0.09 14,400.85 � 0.10

13,759.04 14,079.34 14,399.64

Miotoxin II

a Average molecular masses calculated for the three PLA2s and their ZQG derivatives. The conjugation of the protein to ZQG corresponds to an average mass increment of 320.30 Da.

protein and the formation of new species with a higher retention time, as expected since ZQG modification increases protein hydrophobicity (Fig. 2A, C). ESI-MS analysis of the collected peaks (Table 2) showed that Ntx reaction with ZQG leads to the formation of two main derivatives, an intermediate mono-derivative (Ntx1ZQG, RT 11.6 min, Fig. 2A), which is the main product after 1 h of incubation (Fig. S4), and a bi-derivative (Ntx2ZQG, RT 12.9 min, Fig. 2A and Fig. S4), which is the main prod­ uct after 4 h of reaction. The disappearance of the starting Ntx peak in the RP-HPLC analysis indicates that the reaction was complete. The reaction of Mt–I is also almost quantitative, with formation of a single derived species (Mt–I1ZQG, RT 19.7 min) for both isoforms of the toxin (Fig. 2C, Table 2). Mt-II reaction with ZQG shows a different trend since

under the same conditions used for the other two PLA2s, the yield is too low (data not shown). The reported chromatographic profile (Fig. 2E) was obtained after incubation of Mt-II with TGase at a higher enzyme/ substrate (E:S) ratio (1:10 vs 1:25) and for a longer time (8 h) and it shows two derivative products: a mono- (Mt-II1ZQG, RT 13.9 min), pre­ sent in higher percentage, and a bi-derivative (Mt-II2ZQG, RT 14.7 min). However, even under these conditions, an important proportion of the starting protein remains unmodified (about 30%), maybe because, in solution, Mt-II forms dimers or multimers that limit the accessibility of some sites (Tonello and Rigoni, 2015; Angulo et al., 2005). The reaction patterns were found to be reproducible. We obtained a maximum re­ action yield of 75% for Mt–I1ZQG, 58% for Ntx2ZQG, and of 48% for

Fig. 2. RP-HPLC analysis of the TGase-mediated conjugation of Ntx, Mt–I, and Mt-II with ZQG, specific for Lys residues, and with DC, specific for Gln residues. Reaction mixtures of Ntx (A, B) and of Mt–I (C, D) with TGase and ZQG (A, C) or DC (B, D) after 4 h of incubation. Reaction mixtures of Mt-II with TGase and ZQG (E) or DC (F) after 8 h and 4 h of incubation, respectively. A dashed line indicates the chromatograms at time 0 h of reaction, while a solid line is used for the chro­ matograms after 8 or 4 h of reaction. 103

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Mt-II1ZQG, calculated from the RP-HPLC analyses considering the area of the peak of the derivative in respect to that of the protein at 0 h of incubation. The RP-HPLC elution profiles of the reaction mixtures with DC showed no difference with the chromatograms of the starting material (Fig. 2B, D, F). ESI-MS analysis of the collected protein peaks after 4 h of incubation with DC and TGase demonstrated that conjugation at the level of Gln residues did not occur (data not shown). 3.2. Identification of all Lys residues derivatized by TGase in Ntx, Mt–I, and Mt-II In order to identify which Lys residues are modified by TGase, we digested the native and ZQG conjugated PLA2s with trypsin. Trypsin is an endoprotease that cleaves peptide bonds in which the carbonyl group is contributed either by an Arg or by a Lys residue. If the Lys lateral chains are modified by ZQG, the hydrolysis does not occur. We did a LCMSE analysis of the digestion products, obtaining a full sequence coverage (except for the last two amino acids of Ntx) with an accuracy of 10 ppm (Table S1). The identified sites of conjugation were validated by LC-MS/MS analyses, and the MS/MS spectra of the main modified peptides are shown in Fig. S5-7. MSE or MS/MS spectra of modified peptides showed signals that can be assigned to typical fragmentations of ZQG: the presence of an ion at 91.05 m/z assigned to the benzyl group, or the neutral loss of the carbobenzoxy- ( 134.04 Da) or ZQG( 320.1 Da) moieties from the peptide ion, or from b- and y-ions (Fig. S2). These signals were used as an indication of the presence of the derivatization, especially when the coverage in b and y ions was low. Several Lys residues were found to be reactive to TGase in the different PLA2s (Fig. 1B) and in particular the bi-derivative Ntx was modified at Lys residues 63, 82, 83, 84, 115, 116, Mt–I1ZQG at Lys 60, 69, 104, 105 and 121 and Mt-II1ZQG at Lys 60, 105, 106, 112, 116 and 117. The mono- and biderivatives of PLA2sZQG thus appeared as a nonhomogeneous mixture of proteins modified in different positions. 3.3. TGase mainly modifies one or two specific Lys residues in every tested snake PLA2 Proteolysis with trypsin is not ideal to calculate the yield of modi­ fication with TGase at each Lys residue. Since trypsin does not act on peptide bonds with modified Lys, the tryptic peptides produced from modified PLA2s are different from those of the control, and a quantita­ tive comparison would not be precise. Using Glu-C in phosphate buffer at pH 7.8, which is suitable for hydrolysing peptide bonds at the C-ter­ minal side of Glu and Asp residues, we obtained peptides of the de­ rivatives small enough to be analyzed by LC-MSE (Table S2) and useful to estimate the derivatization yields. Mass data were analyzed with BiopharmaLynx and a sequence coverage of 100% was obtained for the three proteins with an accuracy of 10 ppm. The ZQG modified peptides identified in the Glu-C proteolytic digest mixtures are those expected on the basis of data obtained with the corresponding tryptic digests. In Fig. 3 (and Fig. S8 for Ntx1ZQG) the Extracted Ion Chromatograms (EICs) of peptides that encompass Lys residues modified by TGase are overlaid for the LS-MSE analyses of the native and of the modified proteins. Table 3 shows the derivatization yields of the different Lys residues calculated from the same peptides. Importantly, each PLA2s derivative shows one main site of derivatization (two in Ntx2ZQG), which are modified to a 97–99 percent, while the other modified Lys residues show a low derivatization yield.

Fig. 3. Extracted Ion Chromatograms (EICs) from LC-MSE analyses of the Glu-C digests of Ntx (A), Ntx2ZQG (B), Mt–I (C), Mt–I1ZQG (D), Mt-II (E) and Mt-II1ZQG (F). The elution profiles of each modified and unmodified peptides are overlaid in the same chromatogram for each digest and the species eluting in the most intense signals are indicated.

proteins (Fig. 1B). A first region is the loop, between the second α-helix and the β-turn, that contains the Lys residues of Ntx and Mt-II with the higher percentage of modification. A second region concerns only Ntx: it is a loop connecting the β-turn with the third α-helix, containing a stretch of three Lys, not present in B. asper myotoxins, and very sensitive to TGase reaction. A third region is in the C-terminal side of the proteins, after the third α-helix, where there are the Lys residues of Mt–I modified with the higher percentage. In general, the higher yield of derivatization is observed for Lys residues located in flexible regions of the proteins, confirming that this is the main factor affecting TGase specificity (Spo­ laore et al., 2012). An interesting aspect of this in depth analysis of the sites of Lys derivatization by TGase is that even though the position of the modified residues are quite conserved among the three sequences, they are

3.4. Correlations between sites of TGase-derivatization in Ntx, Mt–I, and Mt-II Comparison of the position of the modified Lys residues on the aligned sequences of the three PLA2s shows that the sites of derivati­ zation are located on three main regions on the C-terminal half of the 104

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Table 3 Yields of derivatization at different Lys residues of Ntx, Mt–I, and Mt-II. The quantification is based on the MS signal intensities of ZQG-modified peptides obtained by proteolysis with endoprotease Glu-C of ZQG-derived proteins. Protein species Ntx

1ZQG

Ntx2ZQG

Mt–I1ZQG Mt-II1ZQG

Site of derivatizationa

Peptide

Intensity –ZQG (counts)b

Intensity þ ZQG (counts)b

Yield of derivatization (%)c

K63 K82, K83 K115 K63 K82, K83, K84

55–73 74–95 96–119 55–73 74–95

500,000 10,600 1,610,000 9750 326

K115, K116 K69 K104, K105 K121 K60 K105, 106, 112, 116, 117

96–119 63–77 99–112 113–122 13–62 99–121

935,000 272,000 1280 762,000 2950 2,060,000

41,200 972,000 10,200 563,000 445,000 (1ZQG) 3770 (2ZQG) 30,800 1290 799,000 1580 114,000 173320d

7.6 98.9 0.6 98.3 99.1 0.8 3.2 0.5 99.8 0.2 97.5 7.8

a

Derivatization sites identified in the LC-MSE and -MS/MS analyses of the tryptic digests of the ZQG modified toxins. The derivatization of Mt–I K60 is not reported in the table because it was not detected in the digest with Glu-C likely due to the very low conjugation yield. b Intensity counts corresponds to the intensity of the same charge state signal for the peptide with or without derivatization with ZQG. c Yields of derivatization were calculated based on the percent intensity of the modified peptide in respect to the sum of the intensities of the modified and nonmodified peptide. d The modified peptide eluted at different retention times (17.9, 18.2 and 18.4 min) likely due to the presence of the derivatization at different sites (Table S2). The reported intensity is the sum of the intensities of the signal at each RT.

derivatized with different yields in each PLA2s. The 3-D structure of the three proteins is very similar as indicated by X-ray analyses (Arni and Ward, 1996) (Fig. 1B), however it is likely that differences in the overall dynamics of protein conformation can favour one site of derivatization over the others. Moreover, the reactivity of Lys and Gln residues toward TGase is also determined by nearby residues, as the presence of a Pro residue at the C-terminus of a Gln or Lys residue inhibits TGase deriv­ atization (with the exception of Lys112 in Mt-II which, however, is derivatized in low yield) (Spolaore et al., 2012; Piersma et al., 2002). Even the presence of acidic amino acids such as Asp and Glu residues, at the C-terminus of Gln and Lys, interferes with TGase derivatization (Ohtsuka et al., 2000), and in the case of Gln119 of Ntx, the presence of the negatively charged C-terminus likely prevents its modification (Ando et al., 1989). None of the Gln or Lys residues present in the N-terminal side of the proteins (1–59), are derivatized, probably because they are positioned in rigid, buried or structured areas of the polypeptide chain (Gln4 and 10 in Ntx, Gln84 in Mt–I, and Gln11 in Mt-II) or near residues that impair the interaction with the enzyme (see above). 3.5. The enzymatic and toxic activities of the TGase modified snake PLA2s are conserved We tested Ntx1ZQG, Ntx2ZQG, and Mt–I1ZQG in vitro enzymatic activity on an artificial substrate (Table 4) and Ntx1ZQG, Ntx2ZQG, Mt–I1ZQG, MtII1ZQG cytotoxicity on mouse primary myotubes (Fig. 4). Moreover, we prepared toxin derivatives with ZQG peptides conjugated with a fluo­ rophore (Ntx2ZQG TAMRA, Mt–I1ZQG DNS, Mt-II1ZQG TAMRA) and we observed, by fluorescence microscopy, their internalization into primary mouse myotubes (Fig. 5). These tests confirmed that both the toxic and

Fig. 4. Percentage of toxicity of Ntx, Mt–I, and Mt-II ZQG derivatives on mouse primary myotubes compared to the activity of the unmodified proteins. The cytotoxicity was measured by an LDH release assay. The bars represent the average values obtained from six independent experiments.

enzymatic activity of Ntx and Mt–I, and the cytotoxicity of Mt-II are not altered by modification with TGase to one or two specific lysine resi­ dues. Fluorescence microscopy images show that all three toxins pene­ trate inside the cells, and localize in para-nuclear/nuclear area as already reported for Mt-II (Massimino et al., 2018).

Table 4 Phospholipase A2 enzymatic activity of Ntx and Mt–I and of their ZQG derivatives. Protein

Phospholipase activitya

Ntx Ntx1ZQG Ntx2ZQG Mt-1 Mt-1ZQG

305 � 55 298 � 61 293 � 57 131 � 33 117 � 38

4. Conclusions The results obtained from the derivatization of Ntx, Mt–I, and Mt-II with TGase have shown that this enzyme can be used as an effective method to produce active derivatives of these PLA2 toxins. In fact, monoor bi-toxin derivatives are obtained at specific Lys residues, with a good reaction yield and a high reproducibility, and importantly with

a Phospholipase A2 activity (μM/min/mg) was measured on the substrate diheptanoylthio-PC (Cayman). Values are mean � S.D. of at least three independent experiments.

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Fig. 5. Fluorescence microscopy of primary myotubes incubated for 30 min at 37 � C, with 1.5 μM Ntx (A), Mt–I (B), or Mt-II (C) conjugated with synthetic peptides containing a fluorophore: ZQG-TAMRA (Red) in the case of Ntx and Mt-II, ZQG-DNS (Blue) in the case of Mt–I. Fluorescence images were superimposed (Sup) to images acquired with a Nomarski interference contrast (DIC) to evidence the cell borders, and nuclei (marked with an asterisk). Cells in panel D were treated under the same conditions, 30 min, 37 � C, with a control buffer. The white line correspond to 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

preservation of enzymatic, cytotoxic and cell penetrating activities. We think that the method described here may be useful to study the inter­ nalization and cellular activity of Ntx and B. asper myotoxins, and that it could also be applied to other snake venom PLA2s to clarify their pharmacological activity. Unravelling the mechanism of action of these toxins will hopefully pave the way to the development of novel thera­ peutic strategies against snakebites.

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