Pilosulin 2

Background: The 'Jack Jumper Ant' (JJA; Myrmecia pilosula species complex) is the major cause of ant sting anaphylaxis in Australia. Our aims were to determine the allergenicity of previously described venom peptides in their native forms, identify additional allergens and if necessary, update nomenclature used to describe the allergens according to International Union of Immunological Societies criteria. Methods: Various polyacrylamide gel electrophoresis methods were used to separate JJA venom. Gel resolved venom was Western-blotted and probed with individual sera taken from patients with a history of JJA sting anaphylaxis and immunoglobulin E radioallergosorbent test (IgE RAST) tracer uptakes of >1% to whole venom. Results: Of 67 available sera, 54 had RAST uptakes >1%. Thirteen IgE binding bands were identified using these sera. Pilosulin 3, [Ile(5)]pilosulin 1, and pilosulin 4.1 were recognized by 42 (78%), 18 (33%) and nine (17%) of the 54 sera that were tested. Immunoglobulin E-binding proteins with estimated molecular masses of 6.6, 22.8, 25.6, 30.4, 32.1, 34.4 and 89.8 kDa were each recognized by three or more individual sera. Two of these (25.6 and 89.8 kDa) were recognized by 46% and 37% of sera, respectively. Conclusion: Nomenclature used to describe JJA venom allergens has been revised. Pilosulin 3 (Myr p 2) is the only major allergen, whilst [Ile(5)]pilosulin 1 (Myr p 1), and pilosulin 4.1 (Myr p 3) are minor allergens. There are an additional five IgE-binding proteins that require further characterization before they can be named as allergens. These findings provide a framework for standardizing venom extracts for diagnosis and immunotherapy.

The jack jumper ant, Myrmecia pilosula, is endemic to South-Eastern Australia, where around 2.7% of the population has a history of systemic allergic reactions (anaphylaxis) to its venom. Previous work had indicated that there were several allergenic peptides derived from the cDNA Myr p 1, the major expressed allergenic product being a 56-residue peptide (Myr p 1 57-->112, 'pilosulin 1', approximately 6052 Da). Another major allergen had been described as a 27 residue peptide derived from the cDNA Myr p 2 (Myr p 2 49-->75, 'pilosulin 2', approximately 3212 Da), possibly existing as part of a disulfide complex. As a preliminary step in detailed stability studies of a pharmaceutical product used for venom immunotherapy, LC-MS and Edman sequencing analysis of venom collected from various locations by both electrical stimulation and venom sac dissection was undertaken. More than 50 peptides in the 4-9 kDa range were detected in LC-MS analyses. A subsequence of Myr p 2 was found as part of the major peptide present in all samples; this was a bis-disulphide linked, antiparallel aligned heterodimer consisting of Myr p 2 49-->74, (des-Gly(27)-pilosulin 2, approximately 3155 Da) and a previously unreported peptide of approximately 2457 Da. Pilosulin 1 was found by a combination of tandem mass spectrometry and Edman sequencing to exist mainly, and sometimes exclusively, as a previously unreported approximately 6067 Da variant, in which the valine at residue 5 is replaced by isoleucine. A range of hydrolysis products of [Ile(5)]pilosulin 1 and pilosulin 1 were also detected in partially degraded venom. Further IgE-binding studies using these peptides are warranted and a revision of the nomenclature of allergenic components of M. pilosula venom may be required to conform with established IUIS guidelines.

Venom of the Australian ant species Myrmecia pilosula contains a number of allergenic peptides including pilosulins. To obtain novel cDNA clones that encode the pilosulin-related bioactive peptides, mRNA of M. pilosula species complex was subjected to RT-PCR in which the forward primer corresponds to a nucleotide sequence in the leader sequences of pilosulins. We isolated a cDNA clone encoding the novel bioactive peptide pilosulin 5. Tandem mass analysis was entirely consistent with the cDNA derived sequence, and indicated that pilosulin 5 is connected by a single disulfide bridge to create a dimmer peptide of 8546Da. Synthetic pilosulin 5 peptide caused a significant histamine release in a dose-dependent manner, and the mastoparan homologous region of pilosulin 5 was responsible for the activity.