TLR5
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: The TLR5, or toll-like receptor 5, gene is one of the TLR genes. This gene is found on chromosome 1 in humans. This gene is shared with Drosophila (fruit flies) and other animals. This gene is important for the activation of the natural immune system. TLR5 works by recognizing the flagellum in bacteria and recognizes it as a foreign cell. Another function of the TLR5 gene is that it recognizes pathogen-associated molecular patterns (PAMs). After recognizing these PAMs, the gene regulates production of cytokines. Cytokines are proteins for cell signaling and they affect the behavior of other cells. Past studies done with mice have shown that the TLR5 gene is related to metabolic syndromes since TLR5-defficient mice showed high metabolic syndromes.
TLR5.jpeg
Figure 1: The physical location of the TLR5 gene on chromosome 1


Figure 1: Physical Location of the TLR5 gene on chromosome 1

Methods
Whale shark predicted homologues
The TLR5 human protein sequence (ENSP00000540964) 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) in protein BLASTs against the NCBI human protein database.
Predicted orthologs
TLR5 predicted orthologuess were identified in species other than whale sharks using the NCBI Blast server. Protein BLASTs were performed using single species protein databases for mouse, zebra fish, rabbit, whale shark and human protein databases. The human TLR5 protein (ENSP00000540964) 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 4 whale shark BLAST hits were used to create a multiple sequence alignment and phylogenetic tree using ClustalW.


Protein Domains:
TLR5 belongs to the TLR_2 super family. This family is known as the classical TLRs as they perform the basic functions of Toll-like receptors. Proteins belonging to this domain are characterized for binding to molecules of microbial origin.
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Figure 2: NCBI BLASTed protein family: TLR_2 superfamily

Searching for TLR5 in the Whale Shark:

Species
Sequence ID
Percent Identity
e-value
alignment length
Homo Sapien
ENSP 00000540964
100%
0.0
858
Mouse
NP058624.2
72%
0.0
704
Cow
NP001035591.1
79%
0.0
750
Zebra Fish
NP001124067.1
38%
6e-176
478
Arabidopsis
NP199445.1
25%
5e-10
213
Elephant Shark
calMil1_genscan_KI635879.230_1
26.05%
2e-52
902
Whale Shark
Top Hit
g19777.t1
24.86%
1e-23
366
Whale Shark
Second
g29067.t1
28.57%
1e-22
553
Whale Shark
Third
g48010.t1
23.97%
3e-22
242
Whale Shark
Fourth
421305.t1
23.44%
2e-20
273
Whale Shark
Fifth
g45743.t1
27.32%
1e-18
549
Fruit Fly
NP524056.3
28%
1e-24
182
Clawed Frog
XP002940742.2
47%
0.0
559
Table 1: Table of significant values from NCBI BLASTs, using multiple genomic sequences as databases, against the query sequence (ENSP00000540964)

The NCBI BLAST results indicated a high chance of homology for several species including the mouse, cow, zebra fish, clawed frog and potentially even the elephant shark (whose genome is believed to be highly similar to that of the whale shark). Results for the whale shark protein sequence BLASTs were too insignificant to make conclusions alone, and so a reciprocal search would have to be conducted.

Reciprocal search:

Whale Shark Sequence
Reciprocal Search Result ID
Protein Description
e-value
Percent Identity
g19777.t1
NP821079.3
leucine-rich transmembrane neuronal protein 3
4e-166
72%
g29067.t1
XP011520778.1
insulin-like growth factor-binding protein complex acid labile subunit isoform X1
2e-59
34%
g48010.t1
NP003255.2
toll-like receptor 2 precursor
1e-28
32%
g21305.t1
NP057646
toll-like receptor 7 precursor
5e-86
48%
g45743.t1
NP057646.1
toll-like receptor 7 precursor
0.0
56%

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

Results from the reciprocal search prove that unfortunately, there is a high chance that the TLR5 gene is NOT orthologous to the whale shark genome. However its similarity to many genes suggests that it is instead a paralogue.

Orthologs:
Finding Whale Shark Orthologues

*PUT CLUSTAL DATA HERE*

Figure 3. ClustalW comparisons from the first thirteen alignments. Alignment scores indicate the proportion of positive matches to total amino acids.

Orthology was determined using reciprocal searches (for the whale shark sequences) and also alignment scores from the ClustalW program. Significant scores, such as those of the Mouse and Cow, suggest orthology. Scores that are higher than average but not definitively significant like those of the zebra fish and clawed frog would require more research (such as reciprocal searches against their databases) to make a more accurate prediction.


Phylogeny:
The best hits of individual species acquired through BLAST protein database searches using the TLR5 protein sequence were used to create a phylogenetic tree. The phylogenetic tree interestingly depicts a slow transition from distantly related species (fruit fly, arabidopsis, and whale shark) to marine life (whale shark, elephant shark, zebra fish), apmhibians (clawed frog), and finally land-dwelling mammals (mouse, cow, human). This tree mimics the theoretical assumptions of the species' relative similarities. These results helped to reassure and reinforce our conclusions.

*PHYLOGENETIC TREE GOES HERE*

Figure 4. Phylogenic Tree outlining the relationship betweeen the 5 seperate species, connected by their likelihood of orthology to one another.

Conclusion:
In conclusion, we determined that the whale shark genome is distantly related to the human genome and that TLR5 (ENSP00000540964) is a paralogue, having developed mutations over time from evolution that caused the genetic similarity once expressed to fade. Analysis of alternative genomes allowed the production of a phylogenic tree, which depicts the hypothesized evolutionary change over time, and the conclusion that the mouse and cow are certainly orthologs (their top hits were both significant in e-value and were the TLR5 gene) while the zebra fish and clawed frog require more information to accurately determine homology (though the homology of the two species is certain, the degree of which cannot be confirmed).

References:

1.Rock, F. L. "A Family of Human Receptors Structurally Related to Drosophila Toll." Proceedings of the National Academy of Sciences (1997): 588-93. Print.


2.Hayashi, Fimitaka, Kelly D. Smith, Adrian Ozinsky, Thomas Hawn, Eugene C. Yi, David R. Goodlett, Jimmy K. Eng, Shizuo Akira, David M. Underhill, and Alan Aderem. "The Innate Immune Response to Bacterial Flagellin Is Mediated by Toll-like Receptor 5." Nature 410 (2001): 1099-103. Web. 31 Mar. 2015. <http://www.nature.com/nature/journal/v410/n6832/full/4101099a0.html>.

3.Vijay-Kumar, Matam, Jesse D. Aitken, Frederick A. Carvalho, Tyler C. Cullender, Simon Mwangi, Shanthi Srinivasan, Shanthi V. Sitaraman, Rob Knight, Ruth Ley, and Andrew T. Gerwirtz. "Metabolic Syndrome and Altered Gut Microbiota in Mice Lacking Toll-Like Receptor 5." Science Magazine 9 Apr. 2010: 228-31. Print.

4. Oh, Jason Z., Rajesh Ravindran, Benoit Chassaing, Frederic A. Carvalho, Mohan S. Maddur, Maureen Bower, Paul Hakimpour, Kiran P. Gill, Helder I. Nakaya, Felix Yarovinsky, R. Balfour Sartor, Andrew T. Gewirtz, and Bali Pulendran. "TLR5-Mediated Sensing of Gut Microbiota Is Necessary for Antibody Responses to Seasonal Influenza Vaccination." Immunity: 478-92. Print.


5."TLR5 Gene." Genetics Home Reference. 6 Apr. 2015. Web. 8 Apr. 2015. <http://ghr.nlm.nih.gov/gene/TLR5>.


6. Smith, Kelly D, Erica Andersen-Nissen, Fumitaka Hayashi, Katie Strobe, Molly A Bergman, Sara L Rassoulian Barrett, Brad T Cookson, and Alan Aderem. "Toll-like Receptor 5 Recognizes a Conserved Site on Flagellin Required for Protofilament Formation and Bacterial Motility." Nature Immunology: 1247-253. Print.