Juruin: an antifungal peptide from the venom of the Amazonian Pink Toe spider, Avicularia juruensis, which contains the inhibitory cystine knot motif

Abstract

The aim of this study was to screen the venom of the theraposid spider Avicularia juruensis for the identification of antimicrobial peptides (AMPs) which could be further used as prototypes for drug development. Eleven AMPs, named juruentoxins, with molecular weight ranging from 3.5 to 4.5 kDa, were identified by mass spectrometry after the soluble venom was separated by high performance liquid chromatography. Juruentoxins have a putative inhibitory cystine knot (ICK) motif, generally found in neurotoxins, which are also resistant to proteolysis. One juruentoxin that has 38 amino acid residues and three disulfide bonds were characterized, to which we proposed the name Juruin. Based on liquid growth inhibition assays, it has potent antifungal activity in the micromolar range. Importantly, Juruin lacks haemolytic activity on human erythrocytes at the antimicrobial concentrations. Based on the amino acid sequence, it is highly identical to the insecticidal peptides from the theraposid spiders Selenocosmia huwena, Chilobrachys jingzhao, and Haplopelma schmidti from China, indicating they belong to a group of conserved toxins which are likely to inhibit voltage-gated ion channels. Juruin is a cationic AMP, and Lys22 and Lys23 show maximum positive charge localization that might be important for receptor recognition. Although it shows marked sequence similarity to neurotoxic peptides, Juruin is a novel exciting molecule with potent antifungal activity, which could be used as a novel template for development of drugs against clinical resistant fungi strains.

Keywords: Avicularia juruensis; Juruin; Theraphosidae venom; antimicrobial peptides; inhibitory cystine knot motif; juruentoxins.

Figure 1

Adult female Avicularia juruensis (Theraphosidae, Mygalomorphae). Photo: Ayroza, G.

Figure 2

Purification and covalent structure of Juruin. (A) 2.5 mg of soluble venom from A. juruensis was separated by HPLC using a C18 reverse-phase column, eluted with a linear gradient from solution A to 80% solution B run for 50 min. The fractions labeled with the asterisk exhibited antimicrobial activity and were eluted at 11.2, 40.6, and 42.0 min, respectively. The fraction labeled with an arrow was eluted at 40.0 min, and was rechromatographed in the same system and run from 30% to 40% solution B in 60 min (inset). The major component is pure Juruin. (B) Complete amino acid sequence of Juruin was obtained by mass spectrometry fragmentation of several peptides obtained by enzymatic hydrolysis of Juruin, as indicated by the segments underlined by dotted lines. Solid lines linking the cysteine residues indicate the disulfide bridges in positions Cys³ to Cys²⁴, Cys⁷ to Cys³⁰, and Cys¹⁵ to Cys³⁵. The N-terminal phenylalanine and the C-terminal amidated valine were determined by mass spectrometry (MS/MS) fragmentation.

Figure 3

Mass spectroscopic analysis of peptides. (A–D) are the mass spectra of the peaks obtained by HPLC with retention times 11.2, 40.0, 40.6, and 42.0 min, respectively. (B) Correspond to the observed mass of native Juruin (4005.83) and reduced Juruin (inset).

Figure 4

Mass spectrometry analysis of Juruin peptides. (A) Collision-induced dissociation spectrum from mass/charge (m/z) 1211.3 generated by trypsin digestion after analysis by LC/MS, showing the dominant fragment KIDCS with a m/z of 621.26, which corresponds to an N-terminal segment. (B) Collision-induced dissociation spectrum from m/z 2579.8, showing the b and y ion series that corresponds to the partial sequencing of the tryptic peptide between residues Phe¹ to Lys³⁰, which allowed the assignment of four cysteines, Cys³, Cys⁷, Cys²⁴, and Cys³⁰, as well as the lysine rich region Lys²²-Lys²³. (C) MS/MS spectrum from the precursor ion at m/z 1679.69 which corresponds to the C-terminus of Juruin, lacking the amidated valine at the end.

Figure 5

Amino acid sequence comparision of Juruin (Aju1a) to other ICK-containing toxins. (A) Multiple sequence alignement of Juruin to selected toxins. Amino acid sequences of toxins were retrieved from public databases and aligned with Muscle (Egdar, 2004). Cysteines are in yellow. To minimize confusion, all sequences are referred to by their UniProt accession numbers (http://www.uniprot.org/). Juruin (B3EWQ0), U3-theraphotoxin-Cj1a (B1P1A5), U3-theraphotoxin-Cj1a (B1P1A6), U3-theraphotoxin-Cj1b (B1P1A8), U3-theraphotoxin-Cj1b (B1P1A7), U3-theraphotoxin-Cj1c (B1P1A9) from Chilobrachys jingzhao. U1-theraphotoxin-Ba1b (P85504), U1-theraphotoxin-Ba1a (P85497) from Brachypelma ruhnaui. U1-theraphotoxin-Bs1a (P49265) from Brachypelma smithi. U1-theraphotoxin-Asp1b (P61510), U1-theraphotoxin-Asp1a (P61509) from Aphonopelma californicum. Putative mature sequence toxin-like RFEC (D5J6X7), putative mature peptide toxin-like LFEC (D5J6X5) from Pelinobius muticus. U1-theraphotoxin-Lp1b (P61506) from Lasiodora parahybana. Hainantoxin-II-17 (D2Y2D4), Hainantoxin-II-15 (D2Y226) from Haplopelma hainanum. Huwentoxin-7 (P68421), U1-theraphotoxin-Hh1a (P82959), HWTX-VIII (B5U1K0), HWTX-II (B3FIU5), HWTX-VIIIa (B3FIT5), HWTX-IIb (B3FIS9), HWTX-IIa (B3FIS8), U1-theraphotoxin-Hh1f (P68422), Huwentoxin-2a (Q86C49), HWTX-VIIb (B3FIT3), HWTX-VIIa (B3FIT2) from Haplopelma schimidti. (B) Amino acid occurrence within selected toxins. (C) Structural similarity of Juruin to that of U3-theraphotoxin-Cj1a, from Chilobrachys jingzhao, to which Juruin shares 80% sequence similiarity. Conserved residues are shown in black boxes.

Figure 6

Homology modeling of Juruin. Comparison of Juruin and U1-theraphotoxin-Ba1a (PDB ID: 2KGH), from Brachypelma ruhnaui, structures. (A,B) Juruin (blue) and U1-theraphotoxin-Ba1a (green) ribbon structures were superimposed over the backbone atoms. (C) Ribbon structure of Juruin in a different view related by ~90° rotation. (D,E) Molecular surface of Juruin highlighted to show electrostatic potential, surfaces with positive, negative, and neutral electrostatic potentials are drawn in blue, red, and white, respectively. (F) The Lys²²-Lys²³ segment shows maximum positive charge localization represented by the intense blue color. Model pairs show the sides of the protein rotated by ~180°.