Ixodes ricinus Tick Lipocalins: Identification, Cloning, Phylogenetic Analysis and Biochemical Characterization

Abstract

BackgroundDuring their blood meal, ticks secrete a wide variety of proteins that interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response.Methodology/Principal FindingsScreening a cDNA library in association with RT-PCR and RACE methodologies allowed us to identify 14 new lipocalin genes in the salivary glands of the Ixodes ricinus hard tick. A computational in-depth structural analysis confirmed that LIRs belong to the lipocalin family. These proteins were called LIR for “Lipocalin from I. ricinus” and numbered from 1 to 14 (LIR1 to LIR14). According to their percentage identity/similarity, LIR proteins may be assigned to 6 distinct phylogenetic groups. The mature proteins have calculated pM and pI varying from 21.8 kDa to 37.2 kDa and from 4.45 to 9.57 respectively. In a western blot analysis, all recombinant LIRs appeared as a series of thin bands at 50–70 kDa, suggesting extensive glycosylation, which was experimentally confirmed by treatment with N-glycosidase F. In addition, the in vivo expression analysis of LIRs in I. ricinus, examined by RT-PCR, showed homogeneous expression profiles for certain phylogenetic groups and relatively heterogeneous profiles for other groups. Finally, we demonstrated that LIR6 codes for a protein that specifically binds leukotriene B4.Conclusions/SignificanceThis work confirms that, regarding their biochemical properties, expression profile, and sequence signature, lipocalins in Ixodes hard tick genus, and more specifically in the Ixodes ricinus species, are segregated into distinct phylogenetic groups suggesting potential distinct function. This was particularly demonstrated by the ability of LIR6 to scavenge leukotriene B4. The other LIRs did not bind any of the ligands tested, such as 5-hydroxytryptamine, ADP, norepinephrine, platelet activating factor, prostaglandins D2 and E2, and finally leukotrienes B4 and C4.

Ancestral reconstruction of tick lineages

“…Mans, personal observation). In the hard tick I. ricinus, LTB 4 scavenging by a lipocalin was observed (Beaufays et al 2008a), but these does not show monophyletic clustering with the Ornithodoros lipocalins (Beaufays et al 2008b), suggesting independent origins for this function.…”

“…This included studies of the BTSP family , the Kunitz/BPTI family (Dai et al 2012;Louw et al 2013;Mans and Neitz, 2004b;Mans et al , 2003b2002a;Schwarz et al 2014b), the lipocalin family (Beaufays et al 2008b;Mans, 2005;Mans and Neitz, 2004c;Ribeiro, 2008a, 2008b;Mans et al , 2008cMans et al , 2003a) and the apyrases (Stutzer et al 2009). These studies suggested that argasid BPTI anti-hemostatic factors share a common evolutionary pathway; that biogenic amine binding lipocalins and TXA 2 /LTB 4 scavenging lipocalins are closely related; that the biogenic amine binding site is probably ancestral; that tick lipocalins share a monophyletic origin with lipocalins from other metazoan and that apyrase activity probably evolved a single time in the ancestral tick lineage.…”

Antigen Fingerprinting of Polyclonal Antibodies Raised in Immunized Chickens with Tick Total Proteins: A Reservoir for the Discovery of Novel Antigens

“…interestingly, both GP80 41 and metalloproteases have shown protective effects against ticks. 42 For GP80, it is assumed that critical protective epitopes have been associated with the folding of the protein and/or the oligosaccharides attached to it. 41 it is interesting to note that eight of nine mimotopes (18, 19, 20, 21, 22, 26, 32, and 37) that shared the consensus sequence Sc1 were used in a preliminary vaccine trial in bovine, and the cattle immune response induced specific changes in the teleogine physiology, manifested as a hemorrhagic event in the tick gastrointestinal and reproductive tracts.…”

Savicalin, a lipocalin from hemocytes of the soft tick, Ornithodoros savignyi

“…However, a number of lipocalins have been identified in whole-body derived EST libraries from B. microplus and O. porcinus, and has the potential to function in other facets of tick biology ). In addition, salivary glandderived lipocalin transcripts were also detected in midguts from the hard tick Ixodes ricinus (Beaufays et al 2008). However, this study is the first to assign a lipocalin-like molecule, savicalin, uniquely to non-salivary gland derived tissues that includes hemocytes, midgut and ovaries of the soft tick O. savignyi to the exclusion of the salivary gland.…”