Insect Molecular Biology (2006) 15(5), 577–581 G Blackwel PublisehingLtd nomic and transcriptional analysis of protein heterogeneity of the honeybee venom allergen Api m 6 N. Peiren, D. C. de Graaf, J. D. Evans* and F. J. Jacobs Laboratory of Zoophysiology, Ghent University, Ghent, Belgium; and *USDA-ARS Bee Research Laboratory, Beltsville, MD, USA Abstract Several components of honeybee venom are known to cause allergenic responses in humans and other vertebrates. One such component, the minor allergen Api m 6, has been known to show amino acid variation but the genetic mechanism for this variation is unknown. Here we show that Api m 6 is derived from a single locus, and that substantial protein-level varia- tion has a simple genome-level cause, without the need to invoke multiple loci or alternatively spliced exons. Api m 6 sits near a misassembled section of the honeybee genome sequence, and we propose that a substantial number of indels at and near Api m 6 might be the root cause of this misassembly. We suggest that genes such as Api m 6 with coding-region or untranslated region indels might have had a strong effect on the assembly of this draft of the honeybee genome. Keywords: allelic variation, allergen, Api m 6, bee venom, signal peptide. Introduction Dangerous allergenic responses can be caused in humans by components of the venom of stinging social insects (Hoffman, 2003). A major research goal remains to predict which fraction of the human population will be likely to react Received 15 March 2006; accepted after revision 10 May 2006. Correspond- ence: Dirk C. de Graaf, Laboratory of Zoophysiology, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium. Tel.: +32 92648732; fax: +32 92645242; e-mail: Dirk.deGraaf@Ugent.be Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2·5, which does not permit commercial exploitation. © 2006 The Authors Journal compilation © 2006 The Royal Entomological Society adversely to speciﬁc venom components received through incidental or occupation-related stinging. Further, an understanding of the components of venoms that induce allergic responses can be used to better tune therapeutic treatments of allergic responses. Allergens from insects and other sources are designated with the terms ‘major’ or ‘minor’ depending on whether more or less than 50% of the hypersensitive patients develops a speciﬁc IgE response against a given allergen (Larsen & Lowenstein, 1996). Honeybee venom contains the major allergens Api m 1 (phospholipase A2), Api m 2 (hyaluronidase), Api m 3 (acid phosphatase), Api m 4 (melittin) and Api m 7 (CUB serine protease), along with at least one minor allergen, Api m 6, which shows 42% IgE responsiveness (Hoffman, 2006). Here we describe the genomic basis for observed protein- level heterogeneity found for Api m 6. Api m 6 migrates as an 8-kDa band in sodium dodecyl sulphate–polyacrylamide gel electrophoresis and its amino acid sequence was determined on high-performance liquid chromatography- puriﬁed preparations (Kettner et al., 2001). It exists as four isoforms of 7190, 7400, 7598 and 7808 Da, respectively, differing in their primary structure at the amino and carboxy terminus by a maximum of six amino acids (Kettner et al., 2001). Allergen heterogeneity can be due to allelic variation at a single allergen gene (Gao et al., 2005), the occurrence of multiple genes of allergens encoding highly homologous proteins (Piersma et al., 2005), or by alternative splicing of a single transcript (Mykles et al., 1998). Some bee venom components are already known to have a rather peculiar gene organization. Indeed, the precursors of bee venom apamin and MCD peptide are encoded by two genes in tandem, which share the same 3′-exon (Gmachl & Kreil, 1995). Here we use new genome-level data for honeybees to determine the cause behind multiple isoforms of Api m 6. We present the complete cDNA sequences for two Api m 6 variants. We then use the latest honeybee assembly to show that these variants arise from a single polymorphic locus. Interestingly, high sequence-level variation at and around this locus appears to be responsible for a break in the honeybee genome assembly. 577