This Project:
This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.

Background Information:
TLR10 is the toll-receptor 10 protein. This gene is found in chromosome 4 and is a protein-coding gene.


Figure 1.   The physical location of the TLR10 gene on chromosome 4.
Figure 1. The physical location of the TLR10 gene on chromosome 4.


Figure 1. The physical location of the TLR10 gene on chromosome 4

Toll-like receptors are proteins that are part of the innate immune system. The innate system responds to common structures that many pathogens share. These are called PAMPs, or pathogen associate molecular patterns. The toll-like receptors, including TLR10, recognize these PAMPs and then initiate inflammatory responses to eliminate the potential dangers that these PAMPs may represent.

external image page2b.jpg
Figure 2. This image is a representation of the job of TLRs to recognize PAMPs.Methods
Whale shark predicted homologues
The TLR10 human protein sequence (ENSP00000308925) was used as query in a Blast against the predicted whale shark protein database using the whaleshark.georgiaaquarium.org Galaxy server. Top predicted protein hits were then used as queries (using the full predicted sequence not only the aligned sequence) in protein BLASTs against the NCBI human protein database. The whale shark predicted protein database was also searched using the elephant shark predicted TLR10 protein sequence as query.
Predicted orthologs
TLR10 predicted orthologues were identified in species other than whale sharks using the NCBI Blast server. Protein BLASTs were performed using single species protein databases for the mouse, cow, zebra fish, arabidopsis, elephant shark, fruit fly, and clawed frog protein databases. The human TLR10 protein (ENSP00000308925) was used as query sequence in these searches with default settings.
Phylogenetic tree
The hit with the lowest E-value for each non-whale shark species search (using the human protein as query) along with the top whale shark BLAST hit were used to create a multiple sequence alignment and phylogenetic tree. ClustalW with default settings was used to create the alignment and tree.


Protein Domains:
This protein belongs to the LRR family. This family of proteins is known to have protein-to-protein interactions. The proteins belonging to this family also have characteristically distinct functions and cellular locations. Also, they have substrate binding sites.

TIR10 belongs to the TIR_2 super family. Proteins belonging to this domain are characterized for binding to molecules of microbial origin. This is a family of bacterial toll-like receptors.


*PROTEIN DOMAIN GOES HERE*
Figure 3. NCBI BLASTed protein families: LRR super-family and TIR super-family.



Searching for TLR10 in the Whale Shark:
Species
Sequence ID
Percent Identity
e-value
alignment length
Homo Sapien
ENSP00000308925
100%
0.0
811
Cow
NP001070386.1
81%
0.0
721
Mouse
NP109607.1
50%
0.0
534
Zebra Fish
NP001124065.1
34%
2e-126
423
Arabidopsis
NP177295.1
28%
7e-06
110
Elephant Shark
calMil1_genscan_KI635971.4_1
32.72%
3e-112
764
Whale Shark
Top Hit
g36276.t1
31.23%
3e-34
317
Whale Shark
Second
g48010.t1
31.17%
6e-26
231
Whale Shark
Third
g21305.t1
34.23%
9e-24
149
Whale Shark
Fourth
g47253.t1
25.34%
4e-20
221
Whale Shark
Fifth
g48273.t1
32.81%
4e-15
128
Fruit Fly
NP001246845.1
31%
3e-28
115
Clawed Frog
XP002938695.1
44%
0.0
503

Table 1: Table of significant values from NCBI BLASTs, using multiple genomic sequences as databases, against the query sequence (ENSP00000308925)

The NCBI BLAST results indicated that there is a high chance of orthology with the cow, mouse, clawed frog, zebra fish, and elephant shark, a possibility for homology to the whale shark and fruit fly, and a skeptical homology to arabidopsis. Before definitive conclusions can be made of the whale shark protein sequences, a reciprocal search will have to be conducted to further prove, or disprove, similarity to the human protein sequence.

Reciprocal search:

Whale Shark Sequence
Reciprocal Search Result ID
Protein Description
e-value
Percent Identity
g36276.t1
NP84079.3
leucine-rich repeat transmembrane neuronal protein 3
0.0
70%
g48010.t1
NP003259.2
toll-like receptor 5 precursor
7e-76
48%
g21305.t1
NP057646.1
toll-like receptor 7 precursor
5e-86
48%
g47253.t1
NP003255.2
toll-like receptor 2 precursor
1e-28
32%
g48273.t1
NP003256.1
toll-like receptor 3 precursor
2e-58
28%

Table 2: Reciprocal search results using top whale shark hits for ENSP00000540964 against human genome database.

The reciprocal search hits were within the correct protein domain, but none of the hits directly matched TLR10. Because of this conclusion, it is evident that the whale shark hits are merely similar in sequence to that of the human protein but likely not strongly enough to retain similarity of protein function.

Orthologs:
Finding Whale Shark Orthologues

*PUT CLUSTAL DATA HERE*

Figure 4. ClustalW comparisons from the first thirteen alignments. Alignment scores indicate the proportion of positive matches to total amino acids.

The alignment scores of the ClustalW program reinforce the assumption of homology between the human genome, cow, mouse, and clawed frog. Meanwhile, the most significant whale shark score, with reciprocal search results considered as well, likely represents a paralogue because of difference in function but structural similarity.

Phylogeny:


*PHYLOGENETIC TREE GOES HERE*

Figure 5: Phylogenic tree depicting the genetic relationship between the thirteen independent sequences.

The phylogenetic tree generated by the ClustalW program depicts an evolutionary flow that should agree with intuition. Arabidopsis, whale shark, and fruit fly genomes are least related to the others, following the elephant shark, zebra fish, and clawed frog whose genomes slowly become more similar to the human genome, and finally the cow, mouse, and human genomes expressed the most orthology.

Conclusion:
From the information gathered from research, it was concluded that homology to the TLR10 gene for the whale shark is likely, although probably through paralogues rather than orthologues. The similarity in reciprocal search values to TLR10 suggest that it is more likely the gene duplicated and continued to mutate and diverge from its counterpart. Orthology was found in the cow genome which came back as a direct match while there was some dispute between the mouse results. The other genomes expressed enough similarity to suggest paralogues, but enough divergence to reject orthology.
References:
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  • "TLR10 Gene." GeneCards: The Human Gene Compendium. GeneCards Suite. Web. 31 Mar. 2015.
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