Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leechHelobdella robusta(Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Abstract

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeria-derived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.