Melittin is the main component of the venom of the European honey bee Apis mellifera. Its chemical structure is a linear peptide consisting of 26 amino acid residues. The N-terminal part of the molecule is hydrophobic, while the C-terminal part of the peptide is hydrophilic and positively charged. At physiologic pH, the melittin molecule has a charge of +6, with four charges located at the C-terminus of the molecule (lysine and arginine residues) and two at the N-terminus.

Structurally, melittin is an amphipathic α-helix that is able to interact with the cytoplasmic membrane [1]. When embedded in the erythrocyte membrane, melitin causes hemolysis, first increasing the conductivity for sodium and potassium ions and only after that the release of hemoglobin occurs [2]. The first fast phase of this process corresponds to the binding of the positively charged C-terminal part of the melittin molecule to the membrane, while the slow phase is due to the rearrangement of the lipid bilayer and protein complexes in the membrane.

In addition to the effect on membranes, specific action on proteins has been described for melittin. Apparently, melittin is structurally similar to a module of intracellular proteins that participates in protein-protein interactions. In particular, it mimics the binding site of calmodulin to its protein targets [3]. The catalytic subunits of P-type ATPases are also known to specifically bind melittin [4, 5]. The peptide interacts with the MI/LDPPR sequence, which is located in the α-subunit of H+/K+-ATPase from the gastric mucosa [6]. A similar sequence is present in all isoforms of Na+/K+-ATPase (MI(591)DPPRAA) and in the Ca2+-ATPase of the sarcoplasmic reticulum (M(599)LDPPRKE). A protein with a molecular weight of about 70 kDa that interacts with antibodies against melittin was detected in gastric mucosal cells and in homogenates from rabbit skeletal muscle. This protein directly interacts with the cytoplasmic region of the Na+/K+-ATPase α-subunit [5].

The aim of this work was to identify melittin-like proteins with a molecular mass of about 70 kDa that interact with Na+/K+-ATPase in mouse kidney.

EXPERIMENTAL

Reagents. In this work we used EDTA, sodium deoxycholate, glycine, mouse monoclonal antibodies against Hsp70 (Lot: 029K4845, Sigma-Aldrich, USA), Tris, hydrogen peroxide, protease inhibitor cocktail, CHAPS and PMSF (Sigma-Aldrich); Affi-gel 10, acrylamide, methylene-bis-acrylamide and other reagents for PAGE electrophoresis (Bio-Rad, USA); PVDF-membranes (ICN Biomedical, USA); Тriton X-100, glutaric aldehyde, imidazole (Merck, Germany); Coomassie R-250 (Serva, Germany). Melittin from bee venom (free of phospholipase A2) obtained by HPLC was purchased from Aura (Russia).

Preparation of melittin-modified affinity sorbent. Affi-gel 10 sorbent was used to obtain an affinity sorbent for the isolation of melittin-specific antibodies. First, 5 mL of gel was washed twice with 5-fold volume of cold deionized water and once with 5-fold volume of cold 100 mM MOPS solution (pH 7.4). Melittin (75 mg) was dissolved in 10 mL of 100 mM MOPS, pH 7.4 (at a rate of 15 mg melittin per 1 mL gel). The melittin solution (10 mL) was added to 5 mL of sorbent and incubated under stirring at 4°C for 2.5 h. The liquid was decanted, a twofold volume of 100 mM MOPS solution (pH 7.4) was added, and 0.5 mL of 1 M ethanolamine solution (pH 8.0) was added to block free succinimide groups (100 μL of solution per 1 mL of sorbent). The resulting suspension was incubated for 1 h at 4°C.

Production of polyclonal antibodies against melittin. Polyclonal antibodies against cross-linked with 3% glutaric aldehyde (to enhance immunogenicity) melittin were produced by immunizing rabbits as previously described [5]. Antibodies were purified using melittin-modified Affi-gel 10. The prepared affinity sorbent with a volume of 5 mL and a capacity of 15 mg of protein per 1 mL was placed in a chromatographic column into which partially purified antibodies were added at a rate of 20 mL/h. The sorbent was washed sequentially with 10-fold volume of PBS (pH 7.2) with 0.1% Tween-80 (PBST), 5-fold volume of 100 mM Gly-HCl buffer (pH 2.5), and 10-fold volume of PBST to zero optical density. The fraction of proteins not sorbed on the column was eluted with PBST containing 0.2% NaN3 and then with the same solution containing an additional 300 mM NaCl. For elution of antibodies from the column we used 100 mM Gly-HCl buffer, pH 2.5. In the resulting antibody preparation, the pH was adjusted to neutral values using 1 M NaHCO3 solution (pH 8.0) and dialyzed twice against PBS containing 0.2% NaN3.

Protein concentration in fractions obtained by affinity chromatography was determined by the Lowry method [7]. BSA solution (Thermo Fisher Scientific, USA) with a concentration of 2 mg/mL was used as a standard.

The preparation of antibodies binding to melittin was used as a ligand for preparation of affinity sorbent (according to the method given above for melittin) for the isolation of melittin-like proteins from mouse kidney homogenate (6 mg of antibodies were added to 1 mL of sorbent).

Fig. 1.
figure 1

Elution profile of melittin-immunised rabbit serum proteins from a column with melittin-conjugated Affi-gel 10 sorbent. The dotted line indicates the pH gradient.

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Fig. 2.
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Analysis of protein composition of melittin-immunised rabbit serum by affinity chromatography and analysis of the specificity of the antibodies obtained. Analysis of protein composition of affinity chromatography eluate fractions by electrophoresis in SDS-PAGE (a) and identification of fractions containing rabbit IgG by immunoblotting using HRP-conjugated mouse antibodies of appropriate specificity (b). PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific, USA) was applied to lanes 1a and 1c; Spectra Multicolor Low Range Protein Ladder (Thermo Fisher Scientific)—1b; antibody fractions corresponding to their elution peak were applied to lanes 2−4. Specificity analysis of antibodies (c) in fractions obtained from affinity chromatography by immunoblotting using melittin (indicated by arrow) as an antigen.

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Fig. 3.
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Elution profile of proteins of supernatant obtained from mouse kidney homogenate from Affi-gel 10 sorbent on which antibodies against melittin are immobilised.

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Fig. 4.
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Analysis of melittin-like proteins fraction obtained by affinity chromatography of mouse kidney homogenate (see Fig. 3): (a) electrophoresis in 10% SDS-PAGE with Coomassie R-250 staining and (b) immunoblotting using antibodies against melittin obtained from rabbit serum (see Fig. 2) and secondary antibodies—HRP-conjugated mouse antibodies against rabbit IgG. The PageRuler Plus Prestained Protein Ladder kit (Thermo Fisher Scientific) was used for protein molecular weight labelling.

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Fig. 5.
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Analysis of melittin-like proteins with molecular weight ~70 kDa by immunoblotting using antibodies against melittin (1) and Hsp70 (2). The Blue Plus® V Protein Marker kit (TransGen Biotech Co., China) was used to mark the molecular weight of proteins.

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Table 1.   Melittin-like proteins from mouse kidney with a molecular weight of ~70 kDa
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Protein electrophoresis in SDS-PAGE and immunoblotting. The eluate fractions obtained by affinity chromatography were analyzed by electrophoresis in 10% SDS-PAGE. Electrotransfer of proteins from the gel to the PVDF membrane was carried out for 1 h 30 min at a current strength of 200 mA. After the electrotransfer was completed, the membrane was blocked in 5% milk powder (Valio, Finland) prepared with PBST for 1 h at room temperature and constant stirring. Melittin-specific polyclonal antibodies purified by affinity chromatography were then added at a dilution of 1 : 1 000 and the membrane was incubated for 1 h at room temperature and stirring. The membrane was washed with PBST from unbound primary antibodies 3 times for 10 min each and incubated with horseradish peroxidase (HRP)-conjugated mouse antibodies against rabbit IgG (Sigma-Aldrich) at a dilution of 1 : 5000 for 1 h at room temperature. For visualization of antigen-antibody complexes a commercial ECL-kit SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and a ChemiDoc XRS+ Molecular Imager (Bio-Rad) were used.

Purification of melittin-like proteins. A fraction of melittin-like proteins with a molecular mass of ~70 kDa was purified from mouse kidney homogenate using an affinity chromatography method [8]. All operations for homogenate preparation were performed at 4°C. Kidneys from two mice totaling 0.93 g were minced with scissors, and a 10-fold volume of extraction medium (30 mM imidazole, pH 7.4, 130 mM NaCl, 5 mM EDTA, 1.1% Triton X-100, 0.25% CHAPS, 2 μM PMSF, protease inhibitor cocktail) was added. The protease inhibitor cocktail and PMSF were added immediately before isolation. Tissue was homogenized using a Potter-Elvehjem homogenizer (Kleinfeld, United States). The homogenate was centrifuged for 10 min at 13 000 g, the precipitate was removed, and the supernatant was passed through a paper folded filter. Next, the supernatant in a volume of 8 mL was applied to an Affi-gel 10 column with immobilized melittin-specific antibodies. Serum components not bound to the sorbent were washed with PBST with 0.2% NaN3 and then with the same solution containing additional 300 mM NaCl. For elution of melittin-like proteins, 100 mM Gly-HCl buffer (pH 2.5) was used. In fractions containing melittin-like proteins, the pH was adjusted to neutral values with 1 M NaHCO3 solution (pH 8.0). The obtained preparation of melittin-like proteins was analyzed by Lammli electrophoresis [9] and immunoblotting methods.

Identification of melittin-like proteins. The target fractions of affinity chromatography were analyzed by electrophoresis in 10% SDS-PAGE and immunoblotting methods using melittin-binding fraction of rabbit serum (see above) as primary antibodies and HRP-conjugated mouse antibodies against rabbit IgG as secondary antibodies.

In addition, mass spectrometric analysis of the resulting proteins was performed. Preparations of melittin-like proteins for mass spectrometric analysis were prepared as follows. Reagents for electrophoresis were filtered through filters with a pore size of 0.45 μm in a laminar flow box. Protein fraction samples were applied to PAGE and electrophoresis was performed at a current strength of 25 mA in the concentrating and 50 mA in the separating gels according to the Laemmli method. The gel was stained with Coomassie R-250 solution. In a laminar flow box, protein bands were cut from the gel, ground into 1 mm × 1 mm cubes, and placed in 100 μL of deionized water. The obtained protein samples were analyzed at the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences (Moscow) using tandem mass spectrometry coupled to HPLC with nanoflow (LC-MS/MS).

RESULTS AND DISCUSSION

Figure 1 shows the elution profile of immunized rabbit serum (30 mL with protein concentration of 3 mg/mL) on an Affi-gel 10 column with immobilized melittin.

The polyclonal antibody fraction removed from the column after an abrupt pH change was analyzed by immunoblotting with staining with secondary antibodies—HRP-conjugated mouse anti-rabbit IgG antibodies. Figure 2 shows the results of electrophoresis of this fraction (2a) and subsequent immunoblotting (2b) using secondary antibodies, as well as melittin detection on PVDF membrane using the obtained serum fraction of antimellitin antibodies (2c).

As shown in Fig. 2b, mouse antibodies against rabbit IgG bind to two protein bands, with apparent molecular masses of ~55 and ~25 kDa, which correspond to the heavy and light chains of IgG, respectively. The combined sample of fractions 2−4 was shown to bind to melittin (Fig. 2c), i.e. the target antibodies were present in the affinity chromatography-purified rabbit serum.

Having proved that the antibodies we obtained and purified by affinity chromatography bind melittin, we used them as a ligand for affinity chromatography to obtain proteins with melittin-like determinants. The antibodies in the combined fraction were immobilised on Affi-gel 10 sorbent. The elution profile of the supernatant obtained after centrifugation of mouse kidney homogenate and plated on Affi-gel 10 with immobilised melittin antibodies is shown in Fig. 3. Proteins that came off the column at pH of elution buffer 2.5 were analysed by PAGE electrophoresis and immunoblotting. The results of the analyses are presented in Fig. 4.

Proteins in the protein band with a molecular mass of ~70 kDa were analysed using tandem mass spectrometry coupled to nanoflow HPLC (LC-MS/MS) technology. The results of the analyses are presented in Table 1.

Thus, mass spectrometric analysis showed that the melittin-like protein fraction we obtained contains seven proteins, three of which are molecular chaperones from the Hsp70 superfamily: mtHsp70 (mortalin), Hsp73 and Grp78.

To confirm these data, we performed immunoblot analysis of the melittin-like protein fraction using antibodies not only against melittin but also against Hsp70. As can be seen from the results presented in Fig. 5, proteins interacting with antibodies against melittin also bound to antibodies against Hsp70.

Previously, we found that immunoprecipitation of Na+/K+-ATPase from mouse kidney homogenate using antibodies against the α1-subunit co-precipitated proteins with a molecular mass of about 70 kDa that stained with antibodies against melittin. Cross-immunoprecipitation using antibodies against melittin detected α1-subunit Na+/K+-ATPase in the precipitate. Moreover, pretreatment of the homogenate with the specific Na+/K+-ATPase inhibitor ouabain at a concentration of 0.5 mM resulted in an increase in the amount of melittin-like protein with a molecular weight of ~70 kDa in the immunoprecipitate [8]. Based on these data, we hypothesised that Na+/K+-ATPase interacts with this protein and that binding of Na+/K+-ATPase to ouabain enhances this interaction.

In the study now performed, proteins interacting with antibodies against melittin were obtained, and mass spectrometry identified three chaperone proteins from the Hsp70 superfamily in this fraction. So far we have no information about which of them interacts with Na+/K+-ATPase. Let us try to hypothetically explain the fact that the amount of melittin-like protein binding to Na+/K+-ATPase increases in the presence of ouabain. Most likely, ouabain puts Na+/K+-ATPase into a conformation that interacts better with the chaperone, resulting in the removal of the Na+/K+-ATPase–ouabain complex from the membrane. It is logical to assume that this occurs under the action of the chaperone Hsp70, which functions in protein folding as well as in solubilisation of aggregated proteins [10].

Two other chaperones found in the melittin-like protein fraction (mortalin and Grp78) may also be related to the removal of Na+/K+-ATPase from the plasma membrane. However, they belong to chaperones of the endoplasmic reticulum and are rather relevant to the folding and delivery of Na+/K+-ATPase to the plasma membrane. We have shown that the chaperone that interacts with Na+/K+-ATPase contains a melittin-like motif, but the answer to the question of its functional significance remains open. Other questions raised by the results of this study remain to be clarified: which chaperone interacts with Na+/K+-ATPase, whether this interaction leads to the removal of the enzyme from the membrane, and whether a melittin-like motif is required for this.