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In Silico Characterization and Structural Modeling of Dermacentor andersoni p36 Immunosuppressive Protein.

1. Introduction

Ticks are considered among the most important vectors of livestock diseases worldwide as well as major vectors of pet diseases [1]. In tropical Africa, ticks and the tick-transmissible diseases constitute a major obstacle to livestock development [2]. Like elsewhere in the world, chemical acaricides have been the mainstay of tick control in this region; however, increasing resistance to this group of insecticides threatens livestock production systems, especially small-holding sectors that rely on rearing of exotic cattle breeds that are more susceptible to tick infestation and tick-borne diseases [TBDs] [3]. Integrated tick control incorporating reduced acaricide use, breeding cattle for tick resistance, rotational grazing, and use of vaccines presents a sustainable and long-term strategy to the control of ticks and TBDs in the tropics [4].

Numerous studies have shown the potential of immunological methods to control tick infestation by targeting critical tick physiological processes. Existing antitick vaccines work by eliciting humoral and cellular responses against tick cell membrane antigens [5, 6]. Vaccines capable of quelling both the arthropod vector and disease-causing pathogens are also under development [7]. Despite clear advantages of controlling ticks through vaccination, this strategy is presently hampered by antigenic sequence variations between geographically isolated tick populations and species causing vaccine resistance in some regions [8] and lack of efficacy in others [9]. These limitations necessitate search of alternative antigens for inclusion in the next-generation tick vaccines.

Proteins found in tick saliva play critical roles during blood meal acquisition [10]. The pharmacologically active components secreted in their saliva help ticks circumvent host defenses such as haemostatic and immune responses of the host, thereby enabling blood feeding in hematophagous arthropods [11]. One such class of biological compounds is immunosuppressant proteins, which modulate the host's immune system during tick's blood feeding [12], making them suitable target in the search of novel vaccines against arthropod-transmitted diseases [13]. Low molecular weight proteins 5-36 kDa from tick saliva proteins have been shown to inhibit T-lymphocytes proliferation in vitro [14]. Active immunization of mice with Salp15, a 15 kDa secreted salivary gland protein from I. scapularis, showed substantial protection (60%) from tick-borne Borrelia [15]. Tick subolesin (SUB), the ortholog of insect and vertebrate akirins (AKR), was discovered as a tick protective antigen in Ixodes scapularis [16]. Vaccines containing conserved SUB/AKR protective epitopes have been shown to protect against tick, mosquito, and sandfly infestations, thus suggesting the possibility of developing universal vaccines for the control of arthropod vector infestations [17].

Protein antigens conserved across vector species could be used in developing cross-protective vaccines against multiple arthropod vectors and their associated pathogens [17, 18]. Alarcon-Chaidez et al. [19] cloned and characterized a 36 kDa immunosuppressive protein p36 from the salivary glands of partially engorged, female D. andersoni, that suppressed Con-A induced in vitro proliferation of normal murine T-lymphocytes by more than 90% [20]. Genes related to D. andersoni-derived p36 gene, such as Ra-p36, Av-p36, Hl-p36, and Rhp36, have been reported in A. variegatum [21], R. appendiculatus [22], H. longicornis [23], and R. haemaphysaloides [24]. Most proteins are, however, not sufficiently protective on their own suggesting the need for a multiantigen/chimeric vaccine that incorporates critical tick and pathogen antigenic epitopes [16, 25] to elicit synergistic antipathogen and antitick immune responses. Computational characterization and 3D structure modeling of D. andersoni p36 protein undertaken by this study is an initial step in understanding molecular basis of immune recognition which is a challenge in vaccine development [26]. The p36 conserved antigenic region predicted by this study has binding residues for ligands like glycerol and lactose which are associated with an immunomodulatory role suggesting this site may have a role in suppression of select T-cell receptor induced signaling events of D. andersoni p36 and its related proteins.

2. Methods

2.1. Sequence Characterization of Tick p36 Proteins. All tick proteins deposited in National Centre for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov) protein database were retrieved and deposited in a standalone MySQL based database (https://www.mysql.com). D. andersoni p36 protein was used as reference sequence in conducting homology searches, Blastp [27] and OrthoMCL [28, 29], of tick proteins in the database.

The identified tick p36-related proteins were subjected to MEME tool search (http://meme-suite.org/tools/meme) to predict conserved motifs characteristic of p36 proteins. Motif search tool (http://www.genome.jp/tools/motif) then searched for function of identified common motifs in the database of known motifs. Tick p36 protein sequences were then aligned by a multiple sequence alignment tool, Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo). Phylogenetic tree construction was by maximum likelihood method [30] and evolutionary distance computed using Poisson correction method [31]. Bootstrap resampling (1000 replicates) assessed robustness of the groupings.

2.2. Identification of Antigenic Determinants in the p36 Proteins. SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP), TMHMM (http://www.cbs.dtu.dk/services/TMHMM), and PredGPI (http://gpcr.biocomp.unibo.it/predgpi) servers were used to determine if tick p36 proteins are preferably secretory, transmembrane, or have a glycosylphosphatidylinositol (GPI) sites, respectively. Antigenic potentials of tick p36 proteins against reference Bm86, a known antitick vaccine antigen, were evaluated by vaxijen tool (http://www.ddgpharmfac.net/vaxijen/VaxiJen/VaxiJen.html); the model selected was parasite whose standard threshold is 0.5000. The antigenic regions of D. andersoni p36 and other p36 proteins predicted with an antigenic score above 0.7000 were mapped by online tools that predict antigenic peptides (Immunomedicine) (http://imed.med.ucm.es/Tools/antigenic.pl) and SVMTrip [32]. Immunogenic segments/residues of the predicted antigenic region were identified by an online Epitopia tool (http://epitopia.tau.ac.il). Sprint-Pep tool (http://sparks-lab.org/server/SPRINT) was then used to predict protein-peptide binding sites while Coach tool (https://zhanglab.ccmb.med.umich.edu/COACH) predicted ligands likely to bind these sites found within the region predicted as a potential p36 protein conserved site.

2.3. Structural Modeling of D. andersoni p36 Protein. Physicochemical properties of D. andersoni p36 protein were analyzed by ExPASyProtParam (https://www.expasy.org) server while its secondary structure was characterized by online tool [Spider.sup.2] (http://sparks-lab.org/yueyang/server/ SPIDER2). The crystal or NMR structure of tick p36 protein is currently not available in the protein data bank (PDB) (https://www.rcsb.org/pdb/). The 3D structure of D. andersoni p36 protein was developed by QUARK ab initio modeling [33] that builds 3D structure from "Scratch," based on physical principles rather than previously solved structures. 10 models, designated as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, were generated and validated by analyzing Verify 3D scores [34] of Ramachandran plots for each model. Based on the scores, models 2 and 9 were selected as likely 3D structures of D. andersoni p36 protein because they scored 81.41% and 88.44%, respectively, meeting Verify 3D validation tool limit of 80% of the amino acids residues scoring >=0.2 in the 3D/1D profile.

The two selected models had their atomic structures refined by ModRefiner [35] after which their respective generated Ramachandran plots were validated by RAMPAGE (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) and ProQ (https://proq.bioinfo.se/ProQ/ProQ.html). The validation scores guided selection of model 2 as the best 3D structure of D. andersoni p36 protein. PDBsum (https://www .ebi.ac.uk/thornton-srv/ databases/cgi-bin/pdbsum/GetPage .pl?pdbcode=index.html) was used to check location of predicted conserved antigenic region in the 3D structure of D. andersoni p36 protein.

3. Results and Discussion

3.1. Identification and Phylogenetic Analysis of p36 Proteins from Ixodid Ticks. The study identified 32 homologs of D. andersoni p36 protein among 6 ixodid (hard) tick species (Table 1). These included p36 genes reported in earlier studies from R. appendiculatus, A. variegatum, H. longicornis, and Rhipicephalus haemaphysaloides tick species as well as those found by this study in Amblyomma sculptum and Amblyomma aureolatum. Among these homologs, 4 co-orthologs which are potential orthologs of D. andersoni p36 protein were identified in R. appendiculatus species (Table 1). Occurrence of p36 protein across a range of tick species may be related to a biological function for this protein in tick feeding [36]. Several p36 immunosuppressant protein sequences were found in a single tick species suggesting functional and structural redundancy in which a tick expresses multiple similar proteins in minute quantities during feeding [37]. Such redundancy may render saliva proteins less immunogenic, as reported with cystatins [38].

The tick p36 proteins have 3 potential common motifs designated as 1, 2, and 3 with motif 2 being the only one conserved among p36 proteins (Figure 1). Motif 2 is located between amino acid positions "107-127" in the reference D. andersoni p36 protein. This motif 2 may be associated with a functional domain, possibly a role in immunomodulatory activity of tick p36 proteins [39]. The 3 common motifs were not found in the motif database and could be representing an orphan protein family [40].

Alignment of tick p36 proteins revealed a conserved region occurring between amino acid positions "107-115" ("IDKGMLSPF") in the reference D. andersoni p36 protein (Figure 2). This region that coincides with location of conserved motif 2 has polar amino acid residue serine (S) and charged residues lysine (K) and aspartate (D), which are associated with potential active sites [41]. Phylogenetic tree (Figure 3) showed that, among homologs with higher amino acid percentage similarity and E-scores, D. andersoni was closely related to homologs from R. haemophysaloides and R. appendiculatus as compared to homolog from A. variegatum indicating more recent ancestry between Dermacentor and Rhipicephalus than with Amblyomma genera as inferred by phylogeny [42].

3.2. Identification and Characterization of Antigenic Regions in the p36 Proteins. Most tick p36 proteins were predicted as secretory with signal peptide cleavage site at position 21-22 (Supplementary Table S1 and Figure S1). Secretory proteins are favoured candidates for vaccine development as they are easily accessible microbial antigens to the immune system [43]. D. andersoni p36 protein and most p36 variants were predicted as antigenic with several homologs having antigenicity score above 0.7000 (Table 2, Supplementary Table S1), surpassing the vaxijen tool threshold of 0.5000. JAP81944.1, a homolog in R. appendiculatus had antigenicity score of 0.7701, comparably higher than that of Bm86 (0.7681), the constituent antigen of Tickgard and Gavac[TM] commercial tick vaccines. Whether this theoretically predicted immunogenicity can confer protection against tick infestation there is need to be evaluated empirically through an immunization/tick challenge set up.

The potentially conserved motif 2 in p36 protein was predicted as a likely epitope-rich antigenic region with binding residues for glycerol and lactose ligands which are associated with an immunomodulatory role [44, 45]. To facilitate tick feeding a single tick saliva protein ligand may bind receptors on several immune cell types in the vertebrate host; alternatively, multiple tick saliva proteins may bind to a common receptor [37].

3.3. 3D Structure of D. andersoni p36 Protein. D. andersoni p36 protein has an instability index of 35.53 and GRAVY score of -0.324 classifying it as a stable, globular protein [46]. The protein's high aliphatic index of 86.41 is associated with increase in thermostability of globular protein [47]. The stable secondary structures alpha-helix (a) and beta-sheets ([beta]) comprised approximately 55% of D. andersoni p36 protein amino acid sequence (Supplementary Figure S2). The predicted conserved immunogenic region "74-107" in processed secretory D. andersoni p36 protein had several segments within loop regions where epitopes are generally found [48]. The combination of [alpha]-helixes and [beta]-structures through loops with specific geometric arrangements with respect to each is responsible in forming conserved structural motifs [49, 50].

Based on Verify 3D [34] scores of the 10 models designated as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 generated for D. andersoni p36 protein; models 2 and 9 were selected for further validation as they passed tool limit of 80% of the amino acids residues scoring >=0.2 in the 3D/1D profile (Supplementary Table S2). Comparison of validation scores of the selected models 2 and 9 (Table 3, Supplementary Figures S3 and S4) identified model 2 as the best 3D structure of D. andersoni p36 protein.

The predicted 3D structure of D. andersoni p36 protein (Figures 4(a) and 4(b)) is a ball-like structure comprised of 1 alpha-helix and several antiparallel beta-strands. The region predicted as a likely conserved antigenic region "74 ... 107" in D. andersoni p36 protein is not only located in between the alpha-helix and beta-strands but also occurs within the potentially groove region of the predicted 3D structure and further has its loop exposed on the protein surface (Figure 4(c)). Ligands bind in the largest cleft in over 83% of the proteins [51]; thus presence of the predicted conserved antigenic region within this potential groove may be associated with immunosuppressive function of D. andersoni p36 protein, as internal cavities in proteins are important structural elements that may produce functional motions such as ligand binding [52].

Potentially exposed loop region "87 ... 94" (Figure 4(d)) in predicted 3D structure of D. andersoni p36 protein coincides with its likely conserved alignment region "107 ... 115" after cleavage of signal peptide at amino acid position 21-22. This suggested loop region might be associated with binding site of D. andersoni p36 protein. The ligands predicted with potential to bind on this site include fatty acid glycerol and sugars like lactose. The hydroxyl group of polar amino acid residue serine (S), hydrophobic amino acid residue leucine (L), and charged amino acids lysine (K) and aspartic acid (D) found in this region could, respectively, have a role in binding of these ligands [53]. Immunomodulator ligands predicted with potential of binding at this site include fatty acid glycerol and sugars like lactose. There is need for future studies to evaluate whether immunomodulator ligands have a role in suppression of select T-cell receptor (TCR) induced signaling events in D. andersoni p36 protein mode of action [44, 45].

Collectively results from this in silico study provide further insight into potential characters of p36 protein, which is vital in exploiting the proteins as targets for developing improved next-generation cross-protective tick control approaches. In an effort to determine exact role of these proteins in tick feeding process, it is necessary for future laboratory and animal studies to confirm these preliminary predictive findings.

4. Conclusion

The p36 immunosuppressive proteins from ticks exhibit antigen traits worth evaluating in future experimental in vitro and in vivo trials. This includes potential conservation across several tick species and presence of a likely conserved antigenic region that may be bound by immunomodulator ligands such as glycerol and lactose. A further study is necessary on suitability of this potentially conserved region in development of a multi/chimeric antitick vaccine that incorporates critical antigenic regions. The predicted 3D model of D. andersoni p36 protein may be used as a template to model structures of other orphan proteins related to p36. This work is a step towards developing cross-protective next-generation antitick vaccines, as the results expand our knowledge of p36 tick saliva protein and lay ground for future studies to determine their exact role in tick feeding process, which is useful in designing blockade approaches targeting these proteins.

https://doi.org/10.1155/2018/7963401

Disclosure

This research did not receive any specific grant from funding agencies in the public, commercial, or non-profit sector.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors would like to thank CGIAR Fund Donors (http:// www.cgiar.org/who-we-are/cgiar-fund/fund-donors-2) for supporting the study.

Supplementary Materials

Supplementary Table S1: protein targeting pathway and antigenic potential of tick p36 proteins. Supplementary Table S2: Verify 3D validation scores of models generated for D. andersoni p36 protein. Supplementary Figure S1: D. andersoni p36 protein signal peptide cleavage site location. Supplementary Figure S2: Spider2 tool secondary structure characterization of D. andersoni p36 protein. Supplementary Figure S3: rampage tool assessment of Ramachandran plot for model 2 of D. andersoni p36 protein. Supplementary Figure S4: rampage tool assessment of Ramachandran plot for model 9 of D. andersoni p36 protein. (Supplementary Materials)

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Martin Omulindi Oyugi (ID), (1) Johnson Kangethe Kinyua, (1) Esther Nkirote Magiri, (2) Milcah Wagio Kigoni, (3) Evenilton Pessoa Costa, (4) and Naftaly Wang'ombe Githaka (5)

(1) Department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000, Nairobi 00200, Kenya

(2) Cooperative University of Kenya, P.O. Box 24814, Nairobi 00502, Kenya

(3) Department of Biochemistry, Kenyatta University, P.O. Box 43844, Nairobi 00100, Kenya

(4) Unit of Animal Experimentation, State University of North Fluminense, Centre of Biosciences and Biotechnology, Campos dos Goytacazes, RJ, Brazil

(5) Animal and Human Health Program, International Livestock Research Institute, P.O. Box 30709, Nairobi 00100, Kenya

Correspondence should be addressed to Martin Omulindi Oyugi; omulindimartin@gmail.com

Received 5 November 2017; Accepted 14 February 2018; Published 8 April 2018

Academic Editor: David A. McClellan

Caption: Figure 1: (a) Occurrence of tandem motifs among p36 proteins. Motif 2 is conserved across all homologs; (b) motifs sequence logo analysis.

Caption: Figure 3: Phylogenetic relatedness between p36 proteins. Bootstrap resampling (1000 replicates) was employed to validate the robustness of the groupings yielded.

Caption: Figure 4: (a, b) D. andersoni p36 protein predicted 3D structure ribbon and space field model; (c) predicted antigenic region "74-107," in 3D structure of D. andersoni p36 protein. (d) Topology of D. andersoni p36 protein showing the likely predicted conserved exposed loop. Yellow: predicted conserved antigenic region "74-107"; red: [alpha]-helix secondary structure; purple: [beta]-strands secondary structure. T: predicted exposed loop region "87-94" ("DKGMLSPF") in D. andersoni p36, showing conservation in alignment of tick p36 proteins.
Table 1: Tick proteins related to D. andersoni p36 protein.

Tick species              NCBI         Sequence similarity searches
                          accession       Blastp homology search
                          number       Protein description% identity
                                          AlignmentE ValueBit score
                                             length (aa)

D. andersoni              AAF03683.1   p36 *
R. appendiculatus         JAP82151.1   Da-p36 family member
R. appendiculatus         JAP81510.1   Da-p36 family member
R. appendiculatus         JAP87204.1   Da-p36 family member
R. appendiculatus         JAP86350.1   Da-p36 family member
A. variegatum             BAD11807.1   Da-p36
R. h. haemaphysaloides    ABB90890.1   Rhh-ISP partial
A. sculptum               JAU03129.1   Hypothetical protein partial
R. appendiculatus         JAP81944.1   Da-p36 family member
R. appendiculatus         JAP88013.1   Da-p36 family member partial
A. sculptum               JAU02613.1   Hypothetical protein partial
R. appendiculatus         JAP85022.1   Hypothetical protein
A. sculptum               JAU02539.1   partial Da-p36 family member
A. variegatum             DAA34595.1   Da-p36 like
A. aureolatum             JAT98922.1   Hypothetical protein partial
A. aureolatum             JAT98921.1   Hypothetical protein
R. appendiculatus         JAP85729.1   Da-p36 family member
R. appendiculatus         JAP85564.1   Da-p36 family member
R. appendiculatus         JAP82143.1   Da-p36 family member
R. appendiculatus         JAP86306.1   Da-p36 family member
R. appendiculatus         JAP81863.1   Da-p36 family member
A. variegatum             DAA34748.1   Da-p36 like
R. appendiculatus         JAP78061.1   Da-p36 family member
R. appendiculatus         JAP86680.1   Da-p36 family member
R. appendiculatus         JAP88255.1   Da-p36 family member
R. appendiculatus         JAP85730.1   Da-p36 family member
H. longicornis            BAG11660.1   Isp-p36
R. appendiculatus         JAP81735.1   Da-p36 family member
R. appendiculatus         JAP86324.1   Da-p36 family member
A. sculptum               JAU02519.1   Hypothetical protein partial
A. variegatum             DAA34145.1   ISP-p36 partial
R. appendiculatus         JAP81446.1   Da-p36 family member
R. appendiculatus         JAP86032.1   Da-p36 family member

Tick species              Sequence similarity searches
                             Blastp homology search
                          Protein description% identity
                             AlignmentE ValueBit score
                                length (aa)

D. andersoni              100 220     1.00E--165   459
R. appendiculatus         37.72 228   2.00E--034   124
R. appendiculatus         37.8 209    3.00E--034   123
R. appendiculatus         37.81 201   3.00E--033   120
R. appendiculatus         35.82 201   6.00E--032   116
A. variegatum             35.04 234   1.00E--029   111
R. h. haemaphysaloides    36.71 158   2.00E--027   103
A. sculptum               35.06 231   1.00E--026   103
R. appendiculatus         32.3 226    2.00E--024   97.4
R. appendiculatus         31.58 228   2.00E--024   97.4
A. sculptum               27.65 217   2.00E--016   74.3
R. appendiculatus         25.11 231   7.00E--016   72.8
A. sculptum               32 175      2.00E--015   70.9
A. variegatum             27.95 229   3.00E--015   70.9
A. aureolatum             31.65 218   4.00E--015   70.5
A. aureolatum             30.19 212   3.00E--013   65.5
R. appendiculatus         26.24 221   4.00E--013   65.1
R. appendiculatus         25.47 212   2.00E--012   63.5
R. appendiculatus         23.31 236   2.00E--011   60.1
R. appendiculatus         25.74 237   3.00E--011   60.1
R. appendiculatus         27.14 210   6.00E--011   59.3
A. variegatum             29.24 171   1.00E--010   57.8
R. appendiculatus         26.23 244   3.00E--009   54.3
R. appendiculatus         27.72 202   7.00E--009   53.5
R. appendiculatus         26.73 202   2.00E--008   52
R. appendiculatus         24.82 141   2.00E--008   51.2
H. longicornis            31.78 107   1.00--007    50.1
R. appendiculatus         24.58 240   6.00E--004   38.9
R. appendiculatus         24.24 198   0.001        38.1
A. sculptum               33.7 92     0.001        36.6
A. variegatum             28.95 76    0.002        36.6
R. appendiculatus         25.74 237   0.011        35
R. appendiculatus         25.18 139   0.019        34.3

Tick species              OrthoMCL          Reference
                          Search

D. andersoni              Reference         Bergman et al., 2000
R. appendiculatus         Co-ortholog       De castro et al., 2016
R. appendiculatus         Co-ortholog       De castro et al., 2016
R. appendiculatus         Co-ortholog       De castro et al., 2016
R. appendiculatus         Co-ortholog       De castro et al., 2016
A. variegatum             In-paralog        Roller et al., 2004
R. h. haemaphysaloides    In-paralog        Xiang et al., 2005
A. sculptum               In-paralog        Eliane et al., 2016
R. appendiculatus         In-paralog        De castro et al., 2016
R. appendiculatus         In-paralog        De castro et al., 2016
A. sculptum               In-paralog        Eliane et al., 2016
R. appendiculatus         In-paralog        De castro et al., 2016
A. sculptum               In-paralog        Eliane et al., 2016
A. variegatum             In-paralog        Ribeiro et al., 2011
A. aureolatum             In-paralog        Martins et al., 2016
A. aureolatum             In-paralog        Martins et al., 2016
R. appendiculatus         In-paralog        De castro et al., 2016
R. appendiculatus         In-paralog        De castro et al., 2016
R. appendiculatus         In-paralog        De castro et al., 2016
R. appendiculatus         In-paralog        De castro et al., 2016
R. appendiculatus         In-paralog        De castro et al., 2016
A. variegatum             In-paralog        Ribeiro et al., 2011
R. appendiculatus         In-paralog        De castro et al., 2016
R. appendiculatus         In-paralog        De castro et al., 2016
R. appendiculatus         In-paralog        De castro et al., 2016
R. appendiculatus         In-paralog        De castro et al., 2016
H. longicornis            In-paralog        Nakajima et al., 2008
R. appendiculatus         In-paralog        De castro et al., 2016
R. appendiculatus         In-paralog        De castro et al., 2016
A. sculptum               In-paralog        Eliane et al., 2016
A. variegatum             In-paralog        Ribeiro et al., 2011
R. appendiculatus         In-paralog        De castro et al., 2016
R. appendiculatus         In-paralog        De castro et al., 2016

(aa) Amino acid; * reference p36 protein in D. andersoni.

Table 2: Conserved motif 2 of tick p36 proteins mapped as a potential
antigenic/binding site region.

Tick species           NCBI       Antigenic    Conserved    Antigenic
                     accession      score       Motif 2      region
                       number                   location    antigenic
                                                             peptide
                                                            tool/SVM
                                                            Trip tool

D. andersoni         AAF03683.1     0.5880      107-127       95-128
R. appendicu-latus   JAP81944.1     0.7701      117-137      108-137
R. appendicu-latus   JAP88013.1     0.7379      113-133      104-133
R. appendicu-latus   JAP81510.1     0.7258      102-122       94-133
R. appendicu-latus   JAP86350.1     0.7072       78-98        74-110

Tick species           Mapped immunogenic
                      segments of Motif 2
                          Epitopia tool

D. andersoni         IDKGMLSPFNLSATVKFPLIP #
R. appendicu-latus   IDNGIKTPFHLKAVFSFPITG #
R. appendicu-latus   IDNGIKTPFHLKAVFSFPITG #
R. appendicu-latus   IDDSMYSPFNIMTTVAFPLIG #
R. appendicu-latus   IDYGIYSPFNLVTPVQFPLMG #

Tick species         Mapped Binding sites of     Predicted ligands
                      Motif 2 Sprint pep tool       for Motif 2
                                                     Coach tool

D. andersoni          IDKGMLSPFNLSATVKFPLIP #    Lactose, Glycerol,
                                                   NAG-(4-l)GAL,
                                                       Sucrose
R. appendicu-latus    IDNGIKTPFHLKAVFSFPITG #             --
R. appendicu-latus    IDNGIKTPFHLKAVFSFPITG #             --
R. appendicu-latus    IDDSMYSPFNIMTTVAFPLIG #     B-Octylglucoside
R. appendicu-latus    IDYGIYSPFNLVTPVQFPLMG #     Alpha-D-Mannose,
                                                 Alpha- D- Lactose,
                                                  B-Octylglucoside

Bold sections: tick p36 conserved region residues mapped as
potentially antigenic/binding sites.

Bold sections: tick p36 conserved region residues mapped as
potentially antigenic/binding sites is indicated with a # sign.

Table 3: Validation of 3D structures for models 2 and 9 of D.
andersoni p36 protein.

Number   Validation        Parameter monitored
         tool

 (1)      ProQPsiPred             LGscore

                                  MaxSub

 (2)      ProQJPred               LGscore

                                  MaxSub

                         Favoured region residues
(3)      Rampage tool    Allowed region residues
                         Outlier region residues

Number                 Limit                        Results

                                               Model 2       Model 9

         LG score > 1.5 fairly good model
          LG score > 2.5 very good model      LGscore       LGscore
(1)      LG score > 4 extremely good model      2.797         2.366
          MaxSub > 0.1 fairly good model
           MaxSub > 0.5 very good model        MaxSub        MaxSub
              MaxSub > 0.8 extremely            0.208         0.055
                    good model

         LG score > 1.5 fairly good model
          LG score > 2.5 very good model      LGscore       LGscore
(2)      LG score > 4 extremely good model      2.914         2.231
          MaxSub > 0.1 fairly good model
           MaxSub > 0.5 very good model        MaxSub        MaxSub
              MaxSub > 0.8 extremely            0.219         0.046
                    good model

                                             156 (79.2%)   152 (77.2%)
(3)                                          26 (13.2%)    24 (12.2%)
                                              15 (7.6%)    21 (10.7%)

Figure 2: Multiple alignment of p36 homologous amino acid sequences
showing likely conservation region. In the case of reference D.
andersoni p36 protein, the conserved region "IDKGMLSPF" is located at
positions "107/115." (:) and (.): marks conservation between groups
of strongly or weakly similar properties, respectively. Note. Amino
acids colour according to physicochemical properties: red is for
small hydrophobic, blue for acidic, magenta for basic, and green for
hydroxyl/sulfhydryl/amine amino acid residues. Highlighted region
shows conservation in tick p36 proteins.

AAF03683.1   --HKRGHPIKATVKRLSCVPGYKLDK-PLRMS-
               QDYKCESELSLFIDKGMLSPFNLSATV

JAP81944.1   -QSTIGQPVKAKVGTIRCVDNVNND-PLQNSV-
              ERGTCSQSFKLSIDNGIKTPFHLKAVF

JAP88013.1   -QSTIGQPVKAKVGTIRCVDNVNND-PLQNSV-
              ERGTCSQSFKLSIDNGIKTPFHLKAVF

BAD11807.1   -FTDGERPVVAAVMKPECRLKEEQE--
              IVQSV-ELNDCNEIFTWNISHGIKSPFQLLVNV

JAU03129.1   -FTQGEKPIEAKVSDLKCDNVEGKN---METK-
              QVIGCNETFIWEFTHGLQSPFELLVNV

ABB90890.1   -------PISVHVESLKCASTDDGD---
             QVGV-RYPKCEEDLGLFITHGLQAPFDLNTTF

JAP81510.1   -QSIQGKLITASVPNLECIPHQSNKGSVQRLR-
              RQSKCTEEFILFIDDSMYSPFNIMTTV

JAP87204.1   -QSIQGKLITASVPNLECIPHQSNKGSVQRLR-
              RQSKCTEEFILFIDDSMYSPFNIMTTV

JAP82151.1   -QGDLGFPISTRVDKLECVPEDANRDGVQKMR-
              RTSSCTEEFTLDIDRGMYIPFTLSTVV

JAP86350.1   -QGDVGLPITTSVDKLECISEDTRG---
              QKMD-YKYRCTEDFMLHIDYGIYSPFNLVTPV

BAG11660.1   -IGGNRRPISTYAQAMQVGKMKVLRKPVKFSPPQ-
              DLVCHLNLTWNFTRHLLSPFPTYLNL

DAA34748.1   -APNGLYPVKAKLQNVT CEPPFHDYKEMSAEV---
              Q--MINYFLKLKNGIYCPFALSLNL

JAU02519.1   -LANDAVPITATVGAINFSSPPADLIPLSEDL---
              QFMTVAHLFSLKKSICGPFRLPINV

DAA34145.1   -NLSGVLPLTAKLENVSFNPPLEEFSPFVTSV---
              EMWAIHHLWNLTKWILCPMALPVNM

JAU02613.1   -QQDEVYPIRAHISTITCEPPVDAYDSINSDA---
               GLLLITYVWNLTKQIYCPFLVSAKV

DAA34595.1   -DRPNVSPITVKVADVT CHPAPLEYERLDSNF---
              GVKRIVYVWNITSRIWSPFKLNINV

JAP85564.1   -NLFGQQPISAHAGSFNCDKELLD----YSDL---
              DVKLVMCLWYIPQKICSPVGIYVNV

JAT98922.1   -NMTNEHSIEAKVGKMKCADVRYFG---TGDS---
              RGTMALYIWNFRQSIQSPFPLFTTV

JAU02539.1   -NKSNEHPIVANVDGMRCKSIIPDN---NRYP---
              KVVMALYIWRFNHSIRSPFELPVNV

JAT98921.1   -NKSNEHPIVAKANEMKCNNIVTNR---YDYP---
              KGVMALYIWHFNHSIRSPFHLFVRV

JAP85022.1   --YPEKHPVKARVVSTTYTQCQGTS---
               TLNA-REGHCKGSFYWNIKRGISSSFSIKAEE

JAP86306.1   --PQQIQPVKAEVESMDYSMCFGAD---Y-QY-
               EEQDCTGLFSWSVNGGITSPFSAKVEI

JAP82143.1   --SAEIPPVTAKVAWMIYGDCHPHT---
               RREI-PKMNCSGHFSWAFHEGIVCPFHLQYNT

JAP88255.1   RGGVKKSPVSAEVDWINYEKCNETK---YNES-
             QTKNCTGYFKWSLVAGVNSSFSIQQFT

JAP85729.1   --GATGHPVSAEVDWYGYEKCNETE---NLTR-
               PAEDCMGYFKWSLSEGINGPFSFKQFT

JAP85730.1   --GATGHPVSAEVDWYGYEKCNETE---NLTR-
               PAEDCMGYFKWSLSEGINGPFSFKQFT

JAP81863.1   -KRKNKHPITASVSEITYHGDCSYG----RDF-
              QSKICNDFFQWYIYSSIVSPFSLLVNL

JAP81735.1   --NGESPTLTAKAGQLAFGNGCEKR----L-S-
               TTKECSDLYMFYIYNGTITPFNLTIDL

JAP86032.1   ---NEWPTVKAKTGELVYGDQCKKV----
                TYF-PDMDCSDYYIYYIYNGISSPFDLPINL

JAP78061.1   --LSYEHAVTSKVSPLSYGTGCEDT----SPF-
              QNSKCKEMFNWQIDNGIVTPLDLLVNV

JAP86680.1   --LPSEPPVNAVVSPLIYEANCTYT----AEF-
               DYSKCKDMFTWDIDYGIFSPLSLPVNV

JAP86324.1   --LKDEHPVEGKVQEFEYQNECERT----NSS-
               PTSKCFDSYTFYFSKSILTPFDLKANI

JAP81446.1   --PGHERIVTASVGQVHYNGDCYHS----KRF-
               DYKNCKETYVWLMLKSILTPFRLPISV
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Title Annotation:Research Article
Author:Oyugi, Martin Omulindi; Kinyua, Johnson Kangethe; Magiri, Esther Nkirote; Kigoni, Milcah Wagio; Cost
Publication:Advances in Bioinformatics
Date:Jan 1, 2018
Words:5994
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