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Ixodes ricinus tick lipocalins: identification, cloning, phylogenetic analysis and biochemical characterization.

Beaufays J, Adam B, Decrem Y, Prévôt PP, Santini S, Brasseur R, Brossard M, Lins L, Vanhamme L, Godfroid E - PLoS ONE (2008)

Bottom Line: This 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Biology of Ectoparasites, IBMM, Université Libre de Bruxelles, Gosselies, Belgium.

ABSTRACT

Background: During 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 findings: Screening 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/significance: This 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.

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Related in: MedlinePlus

Refined alignment of representative LIRs.The numbering of 1QFT (Ra-HBP2) is that described in [18]. The colors of secondary structures are blue: coil; red: helix; green: beta. The Cys implicated in disulfide bridges for Ra-HBP2 are indicated by a bold “C”. Cys conserved only in LIRs are also indicated. The residues belonging to the structural core of lipocalins and present in Ra-HBP2 are bold and underlined.
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pone-0003941-g001: Refined alignment of representative LIRs.The numbering of 1QFT (Ra-HBP2) is that described in [18]. The colors of secondary structures are blue: coil; red: helix; green: beta. The Cys implicated in disulfide bridges for Ra-HBP2 are indicated by a bold “C”. Cys conserved only in LIRs are also indicated. The residues belonging to the structural core of lipocalins and present in Ra-HBP2 are bold and underlined.

Mentions: We have aligned LIRs representatives of each group (LIR1, LIR2, LIR6, LIR7, LIR8 and LIR11) with Ra-HBP2 on the basis of the secondary structure predictions and some important residues conserved in all lipocalins such as the residues 48 or 80 and Cys 75, involved in a disulfide bridge conserved in arthropod lipocalins (numbering for RaHBP2 as described in reference 18 and noted in figure 1) [18]. We also used most of the residues typically found in the structural core composed of two clusters of residues (one internal and one external to the β-barrel) [18]; the clusters of Ra-HBP2 were taken as references. The alignment is shown in figure 1; most residues of both clusters are well conserved in LIRs (Tables 2 and 3). However, some discrepancies are observed: i) positions 52, 156, 168 and 220 are the most divergent; for Ra-HBP2, those residues are also somewhat different in other lipocalins, where they are all hydrophobic [18]; ii) residues 38 and 39 of helix H1 are hydrophobic for LIRs, except LIR6 which has a Ser and an Asn, respectively, and LIR8 which has a Thr and an Ile, like Ra-HBP2; iii) the internal cluster for LIR6 contains more hydrophilic residues than Ra-HBP2, and the LIR7'one is richer in aromatic residues.


Ixodes ricinus tick lipocalins: identification, cloning, phylogenetic analysis and biochemical characterization.

Beaufays J, Adam B, Decrem Y, Prévôt PP, Santini S, Brasseur R, Brossard M, Lins L, Vanhamme L, Godfroid E - PLoS ONE (2008)

Refined alignment of representative LIRs.The numbering of 1QFT (Ra-HBP2) is that described in [18]. The colors of secondary structures are blue: coil; red: helix; green: beta. The Cys implicated in disulfide bridges for Ra-HBP2 are indicated by a bold “C”. Cys conserved only in LIRs are also indicated. The residues belonging to the structural core of lipocalins and present in Ra-HBP2 are bold and underlined.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2601031&req=5

pone-0003941-g001: Refined alignment of representative LIRs.The numbering of 1QFT (Ra-HBP2) is that described in [18]. The colors of secondary structures are blue: coil; red: helix; green: beta. The Cys implicated in disulfide bridges for Ra-HBP2 are indicated by a bold “C”. Cys conserved only in LIRs are also indicated. The residues belonging to the structural core of lipocalins and present in Ra-HBP2 are bold and underlined.
Mentions: We have aligned LIRs representatives of each group (LIR1, LIR2, LIR6, LIR7, LIR8 and LIR11) with Ra-HBP2 on the basis of the secondary structure predictions and some important residues conserved in all lipocalins such as the residues 48 or 80 and Cys 75, involved in a disulfide bridge conserved in arthropod lipocalins (numbering for RaHBP2 as described in reference 18 and noted in figure 1) [18]. We also used most of the residues typically found in the structural core composed of two clusters of residues (one internal and one external to the β-barrel) [18]; the clusters of Ra-HBP2 were taken as references. The alignment is shown in figure 1; most residues of both clusters are well conserved in LIRs (Tables 2 and 3). However, some discrepancies are observed: i) positions 52, 156, 168 and 220 are the most divergent; for Ra-HBP2, those residues are also somewhat different in other lipocalins, where they are all hydrophobic [18]; ii) residues 38 and 39 of helix H1 are hydrophobic for LIRs, except LIR6 which has a Ser and an Asn, respectively, and LIR8 which has a Thr and an Ile, like Ra-HBP2; iii) the internal cluster for LIR6 contains more hydrophilic residues than Ra-HBP2, and the LIR7'one is richer in aromatic residues.

Bottom Line: This 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Biology of Ectoparasites, IBMM, Université Libre de Bruxelles, Gosselies, Belgium.

ABSTRACT

Background: During 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 findings: Screening 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/significance: This 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.

Show MeSH
Related in: MedlinePlus