Identification, expression and characterization of the recombinant Sol g 4.1 protein from the venom of the tropical fire ant Solenopsis geminata

Abstract

Background: Fire ant venom is a complex mixture consisting of basic piperidine alkaloids, various biologically active peptides and protein components, including a variety of major allergenic proteins. Tropical fire ant Solenopsis geminata is an important stinging ant species that causes anaphylaxis and serious medical problems. Although the biological activities of allergenic venom proteins that are unique to ant venom, particularly Solenopsis 2 and 4, are still unknown, these proteins are believed to play important roles in mediating the effects of the piperidine derivatives in the venom.

Methods: In the present study, the cDNA cloning, sequencing and three-dimensional structure of Sol g 4.1 venom protein are described. The recombinant Sol g 4.1 protein (rSol g 4.1) was produced in E. coli, and its possible function as a hydrophobic binding protein was characterized by paralyzing crickets using the 50% piperidine dose (PD₅₀). Moreover, an antiserum was produced in mice to determine the allergenic properties of Sol g 4.1, and the antiserum was capable of binding to Sol g 4.1, as determined by Western blotting.

Results: The molecular weight of Sol g 4.1 protein is 16 kDa, as determined by SDS-PAGE. The complete cDNA is 414 bp in length and contains a leader sequence of 19 amino acids. The protein consists of six cysteines that presumably form three disulfide bonds, based on a predicted three-dimensional model, creating the interior hydrophobic pocket and stabilizing the structure. The rSol g 4.1 protein was expressed in inclusion bodies, as determined by SDS-PAGE. Dialysis techniques were used to refold the recombinant protein into the native form. Its secondary structure, which primarily consists of α-helices, was confirmed by circular dichroism analysis, and the three-dimensional model was also verified. The results of allergenic analysis performed on mice showed that the obtained protein was predicted to be allergenically active. Moreover, we report on the possible role of the Sol g 4.1 venom protein, which significantly reduced the PD₅₀ from 0.027 to 0.013% in paralyzed crickets via synergistic effects after interactions with piperidine alkaloids.

Conclusions: The primary structure of Sol g 4.1 showed high similarity to that of venom proteins in the Solenopsis 2 and 4 family. Those proteins are life-threatening and produce IgE-mediated anaphylactic reactions in allergic individuals. The possible function of this protein is the binding of the interior hydrophobic pockets with piperidine alkaloids, as determined by the analysis of the structural model and PD₅₀ test.

Keywords: Allergen; Fire ant; Sol g 4.1 protein; Stinging ant; Venom protein.

Fig. 1

Full-length DNA sequence and translation of the region encoding the Sol g 4.1 protein. Yellow-shaded areas were verified by LC-MS/MS of a partial amino acid sequence. The leader sequence is underlined. The 5΄ and 3΄UTRs are indicated by small letters, and the poly (A) tail initiation signal is double underlined. The boxed residue was determined by automated Edman degradation sequencing. The red letters represent cysteines residues, and the termination codon is indicated by *

Fig. 2

Alignment of the deduced amino acid sequences of the Sol g 4.1 protein with other Solenopsis 2 and 4 venom proteins from S. invicta, S. geminata, S. saevissima, S. xyloni and S. richteri: conserved (red letters, green region), identical (yellow region), and groups of similar (turquois region) or non-similar (black letters, no color region) residues are shown. The end of the signal sequence is indicated by a blue triangle (). Alignment of the six cysteines (red stars) between all Solenopsis 2 and 4 genes and the alignment of the seventh cysteine in the Sol 2 genes (pink star). Residues lining the interior face of the Sol g 4.1 protein are indicated by x. The sequences were submitted to GenBank with the following accession numbers: Solenopsis 2 proteins: P35775 for Sol i 2, P35776 for Sol r 2, ABC58726 for Sol s 2, ALM98859 for Sol × 2, AAY32928 for Sol i 2q and AAY32926 for Sol g 2q; and Solenopsis 4 proteins: AAC97369 for Sol i 4.01, AAC97370 for Sol i 4.02, AAF65312 for Sol g 4.01, AAF65313 for Sol g 4.02, AAY32927 for Sol g 4q and AAY32929 for Sol i 4q

Fig. 3

Determination of the overexpression of rSol g 4.1 protein by SDS-PAGE and Western blotting. a Protein expression patterns in BL21(DE3) pLysS competent cells cultured under optimal conditions obtained using SDS-PAGE. Lanes: M – molecular weight standards; 1 – expression without IPTG; 2 – culture grown in the presence of 0.4 mM IPTG for 8 h.; 3 – cell extract in solution; 4 – cell extract in pellet. b Western blot of the rSol g 4.1 protein using an anti-His tag antibody; lane 1 – cells lacking the rSol g 4.1 protein and lane 2 – expression of the rSol g 4.1 protein

Fig. 4

SDS-PAGE analysis of the purified rSol g 4.1 protein and product after cleavage of the N-terminal tag: lane M – molecular weight standards; lane 1 – purified rSol g 4.1 protein; lane 2 – cleavage of the tagged protein by one unit of enzyme for 7 h; and lane 3 – Sol g 4.1 protein after tag deletion and purification

Fig. 5

A ribbon diagram of the three-dimensional model of the predicted structure of the Sol g 4.1 protein constructed using S. invicta venom allergen Sol i 2 dimer (PDB accession no. 2ygu) as a template. The disulfide bonds are highlighted in tan. a Structural features of the Sol i 2 template dimerized by a disulfide bond on symmetrical residues Cys22. b The three-dimensional homology model of the predicted structure of the Sol g 4.1 protein revealed a structure stabilized by three disulfide bonds, and the molecular view is the same as the view shown for the right molecule in A. c The surface of Sol g 4.1 is marked according to amino acid residue properties: red – acidic residues; blue – basic residues; gray – apolar residues; green – polar residues; and yellow – aromatic residues. The molecule in the top view is the same as the molecule shown in B and has been rotated by 180° along the horizontal axis to show the bottom view. The model was obtained using Swiss-Model and was visualized with UCSF Chimera

Fig. 6

Allergenic analysis of native and recombinant Sol g 4.1 with anti-Sol g 4.1 IgE antibody. a Crude venom expression pattern, as determined by SDS-PAGE. b Determination of the allergenic properties of the Sol g 4.1 protein by producing an antiserum in mice and analyzing the product using Western blotting. Recognition of native Sol g 4.1 and rSol g 4.1 proteins by serum IgE in Sol g 4.1 protein-sensitized mice. Serum samples: P1-P3 = individual sera of Sol g 4.1 protein-sensitized mice; N1-N3 = serum from mice injected with PBS, acrylamide gel and adjuvant, respectively, as controls

Fig. 7

PD₅₀ values for crickets injected with piperidine alone (control) and piperidine plus the rSol g 4.1 protein (treatment). The graph shows the means ± SEM for different percent dilutions of piperidine in the PD₅₀ assay. *Values were significantly different from the control at p < 0.05