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

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.     

Exon-intron structure of outlier tick lipocalins indicate a monophyletic origin within the larger lipocalin family

All tick proteins assigned to the lipocalin family lack the structural conserved regions (SCRs) that are characteristic of the kernel lipocalins and can thus be classified as outliers. These tick proteins have been assigned to the tick lipocalin family based on database searches that indicated homology between tick sequences and the fact that the histamine binding protein (HBP2) from the hard tick Rhipicephalus appendiculatus (Ixodidae) shows structural similarity to the lipocalin fold. Sequence identity between kernel and outlier lipocalins falls below 20% and the question raised is whether the outlier and kernel lipocalins are truly homologous. More specifically in the case of the tick lipocalins, whether their structural fold is derived from the lipocalin fold or whether convergent evolution resulted in the generation of the basic lipocalin-like fold which consists of an eight stranded continuous anti-parallel beta-barrel terminated by a C-terminal alpha-helix that lies parallel to the barrel. The current study determined the gene structure for HBP2 and TSGP1, TSGP2 and TSGP4, lipocalins identified from the soft tick Ornithodoros savignyi (Argasidae). All tick lipocalins have four introns (A-D) with conserved positions and phases within the tick lipocalin sequence alignment. The positions and phase information are also conserved with regard to the rest of the lipocalin family. Phylogenetic analysis using this information shows conclusively that tick lipocalins are evolutionary related to the rest of the lipocalin family. Tick lipocalins are grouped within a monophyletic clade that indicates a monophyletic origin within the tick lineage and also group with the other arthropod lipocalins in a larger clade. Phylogenetic analysis of sequence alignments based on conserved secondary structure of the lipocalin fold support the conclusions from the gene structure trees. These results indicate that exon-intron arrangement can be useful for the inclusion of outlier lipocalins within the larger lipocalin family.     

LipocalinPred: a SVM-based method for prediction of lipocalins

Background  

Functional annotation of rapidly amassing nucleotide and protein sequences presents a challenging task for modern bioinformatics. This is particularly true for protein families sharing extremely low sequence identity, as for lipocalins, a family of proteins with varied functions and great diversity at the sequence level, yet conserved structures.     

Assessing the impact of comparative genomic sequence data on the functional annotation of the Drosophila genome

Background

It is widely accepted that comparative sequence data can aid the functional annotation of genome sequences; however, the most informative species and features of genome evolution for comparison remain to be determined.

Results

We analyzed conservation in eight genomic regions (apterous, even-skipped, fushi tarazu, twist, and Rhodopsins 1, 2, 3 and 4) from four Drosophila species (D. erecta, D. pseudoobscura, D. willistoni, and D. littoralis) covering more than 500 kb of the D. melanogaster genome. All D. melanogaster genes (and 78-82% of coding exons) identified in divergent species such as D. pseudoobscura show evidence of functional constraint. Addition of a third species can reveal functional constraint in otherwise non-significant pairwise exon comparisons. Microsynteny is largely conserved, with rearrangement breakpoints, novel transposable element insertions, and gene transpositions occurring in similar numbers. Rates of amino-acid substitution are higher in uncharacterized genes relative to genes that have previously been studied. Conserved non-coding sequences (CNCSs) tend to be spatially clustered with conserved spacing between CNCSs, and clusters of CNCSs can be used to predict enhancer sequences.

Conclusions

Our results provide the basis for choosing species whose genome sequences would be most useful in aiding the functional annotation of coding and cis-regulatory sequences in Drosophila. Furthermore, this work shows how decoding the spatial organization of conserved sequences, such as the clustering of CNCSs, can complement efforts to annotate eukaryotic genomes on the basis of sequence conservation alone.     

A lipocalin sequence signature modulates cell survival

Lipocalins are β-barrel proteins, which share three conserved motifs in their amino acid sequence. In this study, we identified by a peptide mapping approach, a seven-amino acid sequence related to one of these motifs (motif 2) that modulates cell survival. A synthetic peptide based on an insect lipocalin displayed cytoprotective activity in serum-deprived endothelial cells and leucocytes. This activity was dependent on nitric oxide synthase. This sequence was found within several lipocalins, including apolipoprotein D, retinol binding protein, lipocalin-type prostaglandin D synthase, and many unknown proteins, suggesting that it is a sequence signature and a lipocalin conserved property.     

Comparative proteomic analysis of cell lines and scrapings of the human intestinal epithelium

Background

The homologues of human disease genes are expected to contribute to better understanding of physiological and pathogenic processes. We made use of the present availability of vertebrate genomic sequences, and we have conducted the most comprehensive comparative genomic analysis of the prion protein gene PRNP and its homologues, shadow of prion protein gene SPRN and doppel gene PRND, and prion testis-specific gene PRNT so far.

Results

While the SPRN and PRNP homologues are present in all vertebrates, PRND is known in tetrapods, and PRNT is present in primates. PRNT could be viewed as a TE-associated gene. Using human as the base sequence for genomic sequence comparisons (VISTA), we annotated numerous potential cis-elements. The conserved regions in SPRNs harbour the potential Sp1 sites in promoters (mammals, birds), C-rich intron splicing enhancers and PTB intron splicing silencers in introns (mammals, birds), and hsa-miR-34a sites in 3'-UTRs (eutherians). We showed the conserved PRNP upstream regions, which may be potential enhancers or silencers (primates, dog). In the PRNP 3'-UTRs, there are conserved cytoplasmic polyadenylation element sites (mammals, birds). The PRND core promoters include highly conserved CCAAT, CArG and TATA boxes (mammals). We deduced 42 new protein primary structures, and performed the first phylogenetic analysis of all vertebrate prion genes. Using the protein alignment which included 122 sequences, we constructed the neighbour-joining tree which showed four major clusters, including shadoos, shadoo2s and prion protein-likes (cluster 1), fish prion proteins (cluster 2), tetrapode prion proteins (cluster 3) and doppels (cluster 4). We showed that the entire prion protein conformationally plastic region is well conserved between eutherian prion proteins and shadoos (18–25% identity and 28–34% similarity), and there could be a potential structural compatibility between shadoos and the left-handed parallel beta-helical fold.

Conclusion

It is likely that the conserved genomic elements identified in this analysis represent bona fide cis-elements. However, this idea needs to be confirmed by functional assays in transgenic systems.     

Structure-Based Phylogenetic Analysis of the Lipocalin Superfamily

Lipocalins constitute a superfamily of extracellular proteins that are found in all three kingdoms of life. Although very divergent in their sequences and functions, they show remarkable similarity in 3-D structures. Lipocalins bind and transport small hydrophobic molecules. Earlier sequence-based phylogenetic studies of lipocalins highlighted that they have a long evolutionary history. However the molecular and structural basis of their functional diversity is not completely understood. The main objective of the present study is to understand functional diversity of the lipocalins using a structure-based phylogenetic approach. The present study with 39 protein domains from the lipocalin superfamily suggests that the clusters of lipocalins obtained by structure-based phylogeny correspond well with the functional diversity. The detailed analysis on each of the clusters and sub-clusters reveals that the 39 lipocalin domains cluster based on their mode of ligand binding though the clustering was performed on the basis of gross domain structure. The outliers in the phylogenetic tree are often from single member families. Also structure-based phylogenetic approach has provided pointers to assign putative function for the domains of unknown function in lipocalin family. The approach employed in the present study can be used in the future for the functional identification of new lipocalin proteins and may be extended to other protein families where members show poor sequence similarity but high structural similarity.     

Conserved signature proposed for folding in the lipocalin superfamily

We systematically identify a group of evolutionarily conserved residues proposed for folding in a model beta-barrel superfamily, the lipocalins. The nature of conservation at the structural level is defined and we show that the conserved residues are involved in a network of interactions that form the core of the fold. Exploratory kinetic studies are conducted with a model superfamily member, human serum retinol-binding protein, to examine their role. The present results, coupled with key experimental studies conducted with another lipocalin beta-lactoglobulin, suggest that the evolutionarily conserved regions fold on a faster folding time-scale than the non-conserved regions.     

Genome-wide identification and comparative analysis of lipocalin families in Lepidoptera with an emphasis on Bombyx mori

Lipocalins exhibit functional diversity, including roles in retinol transport, invertebrate cryptic coloration, and stress response. However, genome-wide identification and characterization of lipocalin in the insect lineage have not been thoroughly explored. Here, we found that a lineage-specific expansion of the lipocalin genes in Lepidoptera occurred in large part due to tandem duplication events and several lipocalin genes involving insect coloration were expanded more via tandem duplication in butterflies. A comparative analysis of conserved motifs showed both conservation and divergence of lepidopteran lipocalin family protein structures during evolution. We observe dynamic changes in tissue expression preference of paralogs in Bombyx mori, suggesting differential contribution of paralogs to specific organ functions during evolution. Subcellular localization experiments revealed that lipocalins localize to the cytoplasm, nuclear membrane, or nucleus in BmN cells. Moreover, several lipocalin genes exhibited divergent responses to abiotic and biotic stresses, and 1 lipocalin gene was upregulated by 300 fold in B. mori. These results suggest that lipocalins act as signaling components in defense responses by mediating crosstalk between abiotic and biotic stress responses. This study deepens our understanding of the comprehensive characteristics of lipocalins in insects.     

Identification of functionally diverse lipocalin proteins from sequence information using support vector machine

Lipocalins are functionally diverse proteins that are composed of 120–180 amino acid residues. Members of this family have several important biological functions including ligand transport, cryptic coloration, sensory transduction, endonuclease activity, stress response activity in plants, odorant binding, prostaglandin biosynthesis, cellular homeostasis regulation, immunity, immunotherapy and so on. Identification of lipocalins from protein sequence is more challenging due to the poor sequence identity which often falls below the twilight zone. So far, no specific method has been reported to identify lipocalins from primary sequence. In this paper, we report a support vector machine (SVM) approach to predict lipocalins from protein sequence using sequence-derived properties. LipoPred was trained using a dataset consisting of 325 lipocalin proteins and 325 non-lipocalin proteins, and evaluated by an independent set of 140 lipocalin proteins and 21,447 non-lipocalin proteins. LipoPred achieved 88.61% accuracy with 89.26% sensitivity, 85.27% specificity and 0.74 Matthew’s correlation coefficient (MCC). When applied on the test dataset, LipoPred achieved 84.25% accuracy with 88.57% sensitivity, 84.22% specificity and MCC of 0.16. LipoPred achieved better performance rate when compared with PSI-BLAST, HMM and SVM-Prot methods. Out of 218 lipocalins, LipoPred correctly predicted 194 proteins including 39 lipocalins that are non-homologous to any protein in the SWISSPROT database. This result shows that LipoPred is potentially useful for predicting the lipocalin proteins that have no sequence homologs in the sequence databases. Further, successful prediction of nine hypothetical lipocalin proteins and five new members of lipocalin family prove that LipoPred can be efficiently used to identify and annotate the new lipocalin proteins from sequence databases. The LipoPred software and dataset are available at .     

Savicalin,a lipocalin from hemocytes of the soft tick, <Emphasis Type="Italic">Ornithodoros savignyi</Emphasis>

Savicalin, is a lipocalin found in the hemocytes of the soft tick, Ornithodoros savignyi. It could be assigned to the tick lipocalin family based on BLAST analysis. Savicalin is the first non-salivary gland lipocalin described in ticks. The mature sequence is composed of 188 amino acids with a molecular mass of 21481.9 Da. A homolog for savicalin was found in a whole body EST-library from a related soft tick O. porcinus, while other tick salivary gland derived lipocalins retrieved from the non-redundant sequence database are more distantly related. Homology modeling supports the inclusion of savicalin into the lipocalin family. The model as well as multiple alignments suggests the presence of five disulphide bonds. Two conserved disulphide bonds are found in hard and soft tick lipocalins. A third disulphide bond is shared with the TSGP4-clade of leukotriene C4 binding soft tick lipocalins and a fourth is shared with a lipocalin from the hard tick Ixodes scapularis. The fifth disulphide bond is unique and links strands D-E. Phylogenetic analysis showed that savicalin is a distant relative of salivary gland derived lipocalins, but groups within a clade that is possibly non-salivary gland derived. It lacks the biogenic amine-binding motif associated with tick histamine and serotonin binding proteins. Expression profiles indicate that savicalin is found in hemocytes, midgut and ovaries, but not in the salivary glands. Up-regulation occurs in hemocytes after bacterial challenge and in midguts and ovaries after feeding. Given its tissue distribution and up-regulation of expression, it is possible that this lipocalin functions in tick development after feeding or in an anti-microbial capacity.     

Protein Functional Surfaces: Global Shape Matching and Local Spatial Alignments of Ligand Binding Sites

Background

Protein surfaces comprise only a fraction of the total residues but are the most conserved functional features of proteins. Surfaces performing identical functions are found in proteins absent of any sequence or fold similarity. While biochemical activity can be attributed to a few key residues, the broader surrounding environment plays an equally important role.

Results

We describe a methodology that attempts to optimize two components, global shape and local physicochemical texture, for evaluating the similarity between a pair of surfaces. Surface shape similarity is assessed using a three-dimensional object recognition algorithm and physicochemical texture similarity is assessed through a spatial alignment of conserved residues between the surfaces. The comparisons are used in tandem to efficiently search the Global Protein Surface Survey (GPSS), a library of annotated surfaces derived from structures in the PDB, for studying evolutionary relationships and uncovering novel similarities between proteins.

Conclusion

We provide an assessment of our method using library retrieval experiments for identifying functionally homologous surfaces binding different ligands, functionally diverse surfaces binding the same ligand, and binding surfaces of ubiquitous and conformationally flexible ligands. Results using surface similarity to predict function for proteins of unknown function are reported. Additionally, an automated analysis of the ATP binding surface landscape is presented to provide insight into the correlation between surface similarity and function for structures in the PDB and for the subset of protein kinases.     

Genomic and gene regulatory signatures of cryptozoic adaptation: Loss of blue sensitive photoreceptors through expansion of long wavelength-opsin expression in the red flour beetle Tribolium castaneum

Background

Hox genes are expressed in specific domains along the anterior posterior body axis and define the regional identity. In most animals these genes are organized in a single cluster in the genome and the order of the genes in the cluster is correlated with the anterior to posterior expression of the genes in the embryo. The conserved order of the various Hox gene orthologs in the cluster among most bilaterians implies that such a Hox cluster was present in their last common ancestor. Vertebrates are the only metazoans so far that have been shown to contain duplicated Hox clusters, while all other bilaterians seem to possess only a single cluster.

Results

We here show that at least three Hox genes of the spider Cupiennius salei are present as two copies in this spider. In addition to the previously described duplicated Ultrabithorax gene, we here present sequence and expression data of a second Deformed gene, and of two Sex comb reduced genes. In addition, we describe the sequence and expression of the Cupiennius proboscipedia gene. The spider Cupiennius salei is the first chelicerate for which orthologs of all ten classes of arthropod Hox genes have been described. The posterior expression boundary of all anterior Hox genes is at the tagma border of the prosoma and opisthosoma, while the posterior boundary of the posterior Hox genes is at the posterior end of the embryo.

Conclusion

The presence of at least three duplicated Hox genes points to a major duplication event in the lineage to this spider, perhaps even of the complete Hox cluster as has taken place in the lineage to the vertebrates. The combined data of all Cupiennius Hox genes reveal the existence of two distinct posterior expression boundaries that correspond to morphological tagmata boundaries.     

Multiple molecular recognition properties of the lipocalin protein family

The lipocalins, a diverse family of small extracellular ligand proteins, display a remarkable range of different molecular properties. While their binding of small hydrophobic molecules, and to a lesser extent their binding to cell surface receptors, is well known, it is shown here that formation of macromolecular complexes is also a common feature of this family. Analysis of known crystallographic structures reveals that the lipocalins process a conserved common structure: an antiparallel β-barrel with a repeated +1 topology. Comparisons show that within this overall similarity the structure of individual proteins is specifically adapted to bind their particular ligands, forming a binding site from an internal cavity (within the barrel) and/or an external loop scaffold, which gives rise to different binding modes that reflects the need to accommodate ligands of different shape, size, and chemical structure. The architecture of the lipocalin fold suggests that the both the ends and sides of this barrel are topologically distinct, differences also apparent in analyses of structural and sequence variation within the family. These different can be linked to experimental evidence suggesting a possible functional dichotomy between the two ends of the lipocalin fold. The structurally invariant end of the molecule may be implicated in general binding small ligands and forming macromolecular complexes via an exposed binding surface.     

Chromothripsis is a common mechanism driving genomic rearrangements in primary and metastatic colorectal cancer

Background

Structural rearrangements form a major class of somatic variation in cancer genomes. Local chromosome shattering, termed chromothripsis, is a mechanism proposed to be the cause of clustered chromosomal rearrangements and was recently described to occur in a small percentage of tumors. The significance of these clusters for tumor development or metastatic spread is largely unclear.

Results

We used genome-wide long mate-pair sequencing and SNP array profiling to reveal that chromothripsis is a widespread phenomenon in primary colorectal cancer and metastases. We find large and small chromothripsis events in nearly every colorectal tumor sample and show that several breakpoints of chromothripsis clusters and isolated rearrangements affect cancer genes, including NOTCH2, EXO1 and MLL3. We complemented the structural variation studies by sequencing the coding regions of a cancer exome in all colorectal tumor samples and found somatic mutations in 24 genes, including APC, KRAS, SMAD4 and PIK3CA. A pairwise comparison of somatic variations in primary and metastatic samples indicated that many chromothripsis clusters, isolated rearrangements and point mutations are exclusively present in either the primary tumor or the metastasis and may affect cancer genes in a lesion-specific manner.

Conclusions

We conclude that chromothripsis is a prevalent mechanism driving structural rearrangements in colorectal cancer and show that a complex interplay between point mutations, simple copy number changes and chromothripsis events drive colorectal tumor development and metastasis.     

Structural, phylogenetic and docking studies of D-amino acid oxidase activator (DAOA), a candidate schizophrenia gene

Background

Schizophrenia is a neurodegenerative disorder that occurs worldwide and can be difficult to diagnose. It is the foremost neurological disorder leading to suicide among patients in both developed and underdeveloped countries. D-amino acid oxidase activator (DAOA), also known as G72, is directly implicated in the glutamateric hypothesis of schizophrenia. It activates D-amino acid oxidase, which oxidizes D-serine, leading to modulation of the N-methyl-D-aspartate receptor.

Methods

MODELLER (9v10) was utilized to generate three dimensional structures of the DAOA candidate gene. The HOPE server was used for mutational analysis. The Molecular Evolutionary Genetics Analysis (MEGA5) tool was utilized to reconstruct the evolutionary history of the candidate gene DAOA. AutoDock was used for protein-ligand docking and Gramm-X and PatchDock for protein-protein docking.

Results

A suitable template (1ZCA) was selected by employing BLASTp on the basis of 33% query coverage, 27% identity and E-value 4.9. The Rampage evaluation tool showed 91.1% favored region, 4.9% allowed region and 4.1% outlier region in DAOA. ERRAT demonstrated that the predicted model had a 50.909% quality factor. Mutational analysis of DAOA revealed significant effects on hydrogen bonding and correct folding of the DAOA protein, which in turn affect protein conformation. Ciona was inferred as the outgroup. Tetrapods were in their appropriate clusters with bifurcations. Human amino acid sequences are conserved, with chimpanzee and gorilla showing more than 80% homology and bootstrap value based on 1000 replications. Molecular docking analysis was employed to elucidate the binding mode of the reported ligand complex for DAOA. The docking experiment demonstrated that DAOA is involved in major amino acid interactions: the residues that interact most strongly with the ligand C₂₈H₂₈N₃O₅PS₂ are polar but uncharged (Gln36, Asn38, Thr 122) and non-polar hydrophobic (Ile119, Ser171, Ser21, Ala31). Protein-protein docking simulation demonstrated two ionic bonds and one hydrogen bond involving DAOA. Lys-7 of the receptor protein interacted with Lys-163 and Asp-2037. Tyr-03 interacted with Arg-286 of the ligand protein and formed a hydrogen bond.

Conclusion

The predicted interactions might serve to inhibit the disease-related allele. It is assumed that current bioinformatics methods will contribute significantly to identifying, analyzing and curing schizophrenia. There is an urgent need to develop effective drugs for schizophrenia, and tools for examining candidate genes more accurately and efficiently are required.     

Structure-Function Analysis of Diacylglycerol Acyltransferase Sequences from 70 Organisms

Background

Diacylglycerol acyltransferase families (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Understanding the roles of DGATs will help to create transgenic plants with value-added properties and provide clues for therapeutic intervention for obesity and related diseases. The objective of this analysis was to identify conserved sequence motifs and amino acid residues for better understanding of the structure-function relationship of these important enzymes.

Results

117 DGAT sequences from 70 organisms including plants, animals, fungi and human are obtained from database search using tung tree DGATs. Phylogenetic analysis separates these proteins into DGAT1 and DGAT2 subfamilies. These DGATs are integral membrane proteins with more than 40% of the total amino acid residues being hydrophobic. They have similar properties and amino acid composition except that DGAT1s are approximately 20 kDa larger than DGAT2s. DGAT1s and DGAT2s have 41 and 16 completely conserved amino acid residues, respectively, although only two of them are shared by all DGATs. These residues are distributed in 7 and 6 sequence blocks for DGAT1s and DGAT2s, respectively, and located at the carboxyl termini, suggesting the location of the catalytic domains. These conserved sequence blocks do not contain the putative neutral lipid-binding domain, mitochondrial targeting signal, or ER retrieval motif. The importance of conserved residues has been demonstrated by site-directed and natural mutants.

Conclusions

This study has identified conserved sequence motifs and amino acid residues in all 117 DGATs and the two subfamilies. None of the completely conserved residues in DGAT1s and DGAT2s is present in recently reported isoforms in the multiple sequences alignment, raising an important question how proteins with completely different amino acid sequences could perform the same biochemical reaction. The sequence analysis should facilitate studying the structure-function relationship of DGATs with the ultimate goal to identify critical amino acid residues for engineering superb enzymes in metabolic engineering and selecting enzyme inhibitors in therapeutic application for obesity and related diseases.