This Project This project is done as a part of Emory University's Biology 142 Laboratory course. The specific analysis conducted was of the human TLR1 (toll-like receptor 1) protein and its potential orthologs in the newly sequenced whale shark genome.
Background Information TLR1 is a gene that encodes for one of the toll-like receptor (TLR) proteins. Toll-like receptors (TLRs) are type 1 transmembrane receptors that are involved in the early immune response to pathogens (Hajjar). The TLR1 protein is 786 amino acids long (Transcript: TLR1-001). This family of proteins plays a crucial role in pathogen recognition and activation of innate immunity (the first line of defense against infection) (TLR1 Toll-like Receptor 1 [Homo Sapiens (humans) ]). The TLR proteins recognize pathogen-associated molecular patterns and play a role in the production of cytokines – necessary for the development of effective immunity (TLR1 Toll-like Receptor 1 [Homo Sapiens (humans) ]).Signals initiated by the interaction of TLRs with specific microbial patterns direct the subsequent inflammatory response (Lien). TLR1 cooperates with TLR2 to mediate the innate immune response to bacterial lipoproteins or lipopeptides and acts via MYD88 and TRAF6. This leads to NF-kappa-B activation, cytokine secretion, and the inflammatory response. It also binds MYD88 (via TIR domain) and interacts with CNPY3 (By similarity) (TLR1, Toll-like Receptor 1). The expression of the gene is widespread, and at higher levels than other TLR genes (TLR1 Toll-like Receptor 1 [ Homo Sapiens (human) ]).
Figure 1. Shows the TLR pathway, the way that TLR proteins mediate innate immune responses. a) shows the TLR 1 and 2 forming a response to lipopeptides, then they are recruited through the TIR domain and TIR interactions will lead to induction of inflammatory cytokines b) shows the Lipopolysaccharide (LPS) binding that induces the formation of a TLR-LPS multimer. c) shows the double stranded RNA from a virus binding to the TLR3 dimer (Hennessy et. al)
Methods:
1) To find an orthologous protein sequence in the whale shark The human protein sequence (ENSP00000421259) was used as a query in the Ensemble server to find the protein sequence. Using the result, TLR1-001 (ENST00000502213), the query was used in BLAST against the predicted whale shark protein database. 2) To make sure the proteins were true orthologues The top predicted hits from the whale shark database (determined by alignment length, % identity, and E-value) were then used as queries to BLAST against the NCBI human protein database. 3) Bootstrapping Next, using the bootstrapping technique, the Human protein sequence for the TLR1-001 protein was used in a BLAST against the elephant shark protein. 4) Using protein domain to find orthologs The human TLR1 protein sequence was used in a query against other specie's genomes (zebrafish, mouse). Orthologues were also searched for using the Ensembl databse and searching for known orthologues of the human TLR1 protein in the "ray finned fishes" category because of ray finned fishes' similarity to the whale shark. 5) Phylogenetic Tree Using the CLUSTALW tool, a phylogenetic tree was assembled using the BLAST searches for TLR1 protein in mice, elephant sharks, zebrafish, and whale sharks (denoted by only their gene name).
Searching for TLR1 in Whale Shark the human TLR1 protein sequence was used in a query to predict the protein that was similar in the Whale shark, results are shown in table 1. The smallest E-value was 4e-40, by far the most significant, the rest varied by magnitudes of 1e10
Whale Shark ID
E-Value
Alignment Length
Predicted protein length
% identity
g48010.t1
1e-31
233
244
34.76
g21305.t1
3e-25
270
290
25.56
g19777.t1
4e-12
125
326
28.80
g36276.t1
4e-40
323
925
31.58
g47253.t1
1e-22
187
218
26.74
Table 1. Human TLR1 best BLASTp results against the whale shark protein database. There semed to only be 2 significant hits with an E-value lower than 1e-30
All 5 were used in a BLAST against the human protein database. results are shown in table 2
Whale shark ID
Human ID matched
E Value
Query match
% identity
g36276.t1
toll-like receptor 3 precursor [Homo sapiens]
2e-58
94%
28%
g21305.t1
Toll-like receptor 7 precurser [homo sapiens]
5e-86
95%
48%
g48010.t1
Toll-like receptor 2 precursor [Homo sapiens]
1e-28
94%
32%
g19777.t1
Leucine-rich repeat transmembrane neuronal protein 3 precursor [Homo sapiens]
4e-166
99%
72%
g47253.t1
Toll-like receptor 5 precursor [Homo sapiens]
7e-76
99%
48%
Table 2. BLAST against the human genome for the most significant match in the whale shark genome revealed many different human proteins, but not TLR1
As none of the predicted proteins from the BLAST of the whale shark proteins using the human TLR1 returned TLR1, we thought that the TLR1 gene was not orthologous with any of the whale shark genes, but because of the presence of other TLR proteins that the whale shark had homologous innate immunity structures. Further comparison of the most significant gene g48010.t1 in the whaleshark genome resume was done using the bootstrap method.
Bootstrapping – Elephant Shark
Human protein was run as BLAST against the elephant shark The result was – PREDICTED: toll-like receptor 1 [Callorhinchus milii]. This revealed an orthologous protein in the human and the elephant shark genome, the result was then used in a BLAST against the whale shark genome, the results are shown in table 3.
Whale Shark ID
E-Value
Alignment Length
Predicted protein length
% identity
g21305.t1
1e-31
233
244
34.76
Table 3. Shows the matched protein sequence from the elephant shark in the predicted whale shark genome.
Elephant shark toll-like receptor 1 was blasted against the whale shark, the result was the gene g21305.t1, which was already blasted back against the human and found to be best matching to TLR-7 (figure 2)
Figure 2. NCBI BLAST was used to find potential ortholog of the g21305.t1 protein in the human genome.
Protein Domains TLR1 proteins in the whale shark (from BLAST results) all contain a LRR_8 hit, a LRRCT superfamily, and TIR superfamily (Figure 3).
Figure 3. Shows the protein domain of the TLR1 protein sequence.
LRR-domains are Leucine-Rich Repeats (LRR) and are 22–28 amino acid motifs found in a number of proteins with diverse functions and cellular locations. These repeats are usually involved in protein—protein interactions ("LRR Protein Domains"). The TIR domain consists of three 'boxes' of conserved residues set in a core sequence ranging from 135 to 160 amino acids. Intervening residues may vary, as sequence conservation between domains is only 20–30%. Two interfaces are responsible for mediating TIR domain interactions, which include receptor/adaptor oligomerization and association between receptors and adaptors ("TIR Protein Domains").
Orthologs
When the human TLR 1 protein sequence (ENSP00000421259) was used in a query against other species’ genomes. TLR proteins were found to be present in all genomes examined. With the exception of the zebrafish containing a TLR6 protein, the other queried genomes contained TLR1. This is an indication that the mouse, and elephant shark may have had an orthologous protein to the TLR1 (table 4).
Table 4. shows the orthologues predicted from running a BLAST against other common species for the TLR1 protein.
Next, orthologues were searched for using the Ensembl data base and searching for known orthologues of the human TLR1 protein in "ray finned fishes" which were the closest in phylogeny to the whale shark (Table 5).
Table 5. this table shows that there are othologous proteins sequences, specifically TLR1 protein in many ray finned fish that are closely related to the whale shark. These results would seem to indicate that while ray finned fish contain orthologous innate immunity proteins, the whale shark contains only homologous innate immunity proteins.
Phylogenetic Tree
Five of the best hits from the protein data base searches in the whale shark genome for TLR1 and were used in conjunction with BLAST searches for TLR1 in mouse, elephant shark, and zebrafish genomes. The CLUSTALW was used to align and assemble the tree (figure 4). The tree reveals that the whale shark predicted proteins are similar to each other, but remain divergent from the proteins of the other organisms.
Figure 4. Shows the phylogenetic tree generated from CLUSTALW. There seem to be close matches for the human TLR1 protein in the mouse genome, and some closely related proteins in the elephant shark and zebra fish. All whale shark proteins that seemed to provide a significant match (low E-value, and high alignment) seem to have diverged well before the other species in the tree.
Conclusion
Although it was difficult to find evidence to say that the TLR1 protein had an orthologue in whale shark, given the lack of a matching protein sequence, it seems that the whale shark does exhibit proteins that would give it innate immunity. We know that TLR proteins evolve and are present in most vertebrates, and that sharks diverged earlier than species like mice, humans, and even zebrafish. The fact that the predicted TLR1 orthologue exists in the elephant shark suggests that the protein may have diverged before the whale shark and the elephant shark diverged. To improve on the project, the presumed homologous protein TLR7 could be used in a BLAST against other species more closely related to the whale shark to see if the TLR7 is orthologous in humans and whale sharks.
References
Hajjar, A. M., O’Mahony, D. S., Ozinsky, A., Underhill, D. M., Aderem, A., Klebanoff, S. J., Wilson, C. B. (2001) Cutting edge: functional interactions between Toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin. J. Immunol. 166, 1-3.
Hennessy, Elizabeth J., Andrew E. Parker, and Luke AJ O'Neill. "Targeting Toll-like receptors: emerging therapeutics?." Nature reviews Drug discovery 9.4 (2010): 293-307.
Kopp, E. B., and R. Medzhitov. 1999. The Toll-receptor family and control of innate immunity. Curr. Opin. Immunol. 11:13.
This Project
This project is done as a part of Emory University's Biology 142 Laboratory course. The specific analysis conducted was of the human TLR1 (toll-like receptor 1) protein and its potential orthologs in the newly sequenced whale shark genome.
Background Information
TLR1 is a gene that encodes for one of the toll-like receptor (TLR) proteins. Toll-like receptors (TLRs) are type 1 transmembrane receptors that are involved in the early immune response to pathogens (Hajjar). The TLR1 protein is 786 amino acids long (Transcript: TLR1-001). This family of proteins plays a crucial role in pathogen recognition and activation of innate immunity (the first line of defense against infection) (TLR1 Toll-like Receptor 1 [Homo Sapiens (humans) ]). The TLR proteins recognize pathogen-associated molecular patterns and play a role in the production of cytokines – necessary for the development of effective immunity (TLR1 Toll-like Receptor 1 [Homo Sapiens (humans) ]).Signals initiated by the interaction of TLRs with specific microbial patterns direct the subsequent inflammatory response (Lien). TLR1 cooperates with TLR2 to mediate the innate immune response to bacterial lipoproteins or lipopeptides and acts via MYD88 and TRAF6. This leads to NF-kappa-B activation, cytokine secretion, and the inflammatory response. It also binds MYD88 (via TIR domain) and interacts with CNPY3 (By similarity) (TLR1, Toll-like Receptor 1). The expression of the gene is widespread, and at higher levels than other TLR genes (TLR1 Toll-like Receptor 1 [ Homo Sapiens (human) ]).
Figure 1. Shows the TLR pathway, the way that TLR proteins mediate innate immune responses. a) shows the TLR 1 and 2 forming a response to lipopeptides, then they are recruited through the TIR domain and TIR interactions will lead to induction of inflammatory cytokines b) shows the Lipopolysaccharide (LPS) binding that induces the formation of a TLR-LPS multimer. c) shows the double stranded RNA from a virus binding to the TLR3 dimer (Hennessy et. al)
Methods:
1) To find an orthologous protein sequence in the whale sharkThe human protein sequence (ENSP00000421259) was used as a query in the Ensemble server to find the protein sequence. Using the result, TLR1-001 (ENST00000502213), the query was used in BLAST against the predicted whale shark protein database.
2) To make sure the proteins were true orthologues
The top predicted hits from the whale shark database (determined by alignment length, % identity, and E-value) were then used as queries to BLAST against the NCBI human protein database.
3) Bootstrapping
Next, using the bootstrapping technique, the Human protein sequence for the TLR1-001 protein was used in a BLAST against the elephant shark protein.
4) Using protein domain to find orthologs
The human TLR1 protein sequence was used in a query against other specie's genomes (zebrafish, mouse). Orthologues were also searched for using the Ensembl databse and searching for known orthologues of the human TLR1 protein in the "ray finned fishes" category because of ray finned fishes' similarity to the whale shark.
5) Phylogenetic Tree
Using the CLUSTALW tool, a phylogenetic tree was assembled using the BLAST searches for TLR1 protein in mice, elephant sharks, zebrafish, and whale sharks (denoted by only their gene name).
Searching for TLR1 in Whale Shark
the human TLR1 protein sequence was used in a query to predict the protein that was similar in the Whale shark, results are shown in table 1. The smallest E-value was 4e-40, by far the most significant, the rest varied by magnitudes of 1e10
All 5 were used in a BLAST against the human protein database. results are shown in table 2
As none of the predicted proteins from the BLAST of the whale shark proteins using the human TLR1 returned TLR1, we thought that the TLR1 gene was not orthologous with any of the whale shark genes, but because of the presence of other TLR proteins that the whale shark had homologous innate immunity structures. Further comparison of the most significant gene g48010.t1 in the whaleshark genome resume was done using the bootstrap method.
Bootstrapping – Elephant Shark
Human protein was run as BLAST against the elephant shark The result was – PREDICTED: toll-like receptor 1 [Callorhinchus milii]. This revealed an orthologous protein in the human and the elephant shark genome, the result was then used in a BLAST against the whale shark genome, the results are shown in table 3.
Elephant shark toll-like receptor 1 was blasted against the whale shark, the result was the gene g21305.t1, which was already blasted back against the human and found to be best matching to TLR-7 (figure 2)
Figure 2. NCBI BLAST was used to find potential ortholog of the g21305.t1 protein in the human genome.
Protein Domains
TLR1 proteins in the whale shark (from BLAST results) all contain a LRR_8 hit, a LRRCT superfamily, and TIR superfamily (Figure 3).
Figure 3. Shows the protein domain of the TLR1 protein sequence.
LRR-domains are Leucine-Rich Repeats (LRR) and are 22–28 amino acid motifs found in a number of proteins with diverse functions and cellular locations. These repeats are usually involved in protein—protein interactions ("LRR Protein Domains").
The TIR domain consists of three 'boxes' of conserved residues set in a core sequence ranging from 135 to 160 amino acids. Intervening residues may vary, as sequence conservation between domains is only 20–30%. Two interfaces are responsible for mediating TIR domain interactions, which include receptor/adaptor oligomerization and association between receptors and adaptors ("TIR Protein Domains").
Orthologs
When the human TLR 1 protein sequence (ENSP00000421259) was used in a query against other species’ genomes. TLR proteins were found to be present in all genomes examined. With the exception of the zebrafish containing a TLR6 protein, the other queried genomes contained TLR1. This is an indication that the mouse, and elephant shark may have had an orthologous protein to the TLR1 (table 4).Next, orthologues were searched for using the Ensembl data base and searching for known orthologues of the human TLR1 protein in "ray finned fishes" which were the closest in phylogeny to the whale shark (Table 5).
Table 5. this table shows that there are othologous proteins sequences, specifically TLR1 protein in many ray finned fish that are closely related to the whale shark. These results would seem to indicate that while ray finned fish contain orthologous innate immunity proteins, the whale shark contains only homologous innate immunity proteins.
Phylogenetic Tree
Five of the best hits from the protein data base searches in the whale shark genome for TLR1 and were used in conjunction with BLAST searches for TLR1 in mouse, elephant shark, and zebrafish genomes. The CLUSTALW was used to align and assemble the tree (figure 4). The tree reveals that the whale shark predicted proteins are similar to each other, but remain divergent from the proteins of the other organisms.Figure 4. Shows the phylogenetic tree generated from CLUSTALW. There seem to be close matches for the human TLR1 protein in the mouse genome, and some closely related proteins in the elephant shark and zebra fish. All whale shark proteins that seemed to provide a significant match (low E-value, and high alignment) seem to have diverged well before the other species in the tree.
Conclusion
Although it was difficult to find evidence to say that the TLR1 protein had an orthologue in whale shark, given the lack of a matching protein sequence, it seems that the whale shark does exhibit proteins that would give it innate immunity. We know that TLR proteins evolve and are present in most vertebrates, and that sharks diverged earlier than species like mice, humans, and even zebrafish. The fact that the predicted TLR1 orthologue exists in the elephant shark suggests that the protein may have diverged before the whale shark and the elephant shark diverged.To improve on the project, the presumed homologous protein TLR7 could be used in a BLAST against other species more closely related to the whale shark to see if the TLR7 is orthologous in humans and whale sharks.
References
Hajjar, A. M., O’Mahony, D. S., Ozinsky, A., Underhill, D. M., Aderem, A., Klebanoff, S. J., Wilson, C. B. (2001) Cutting edge: functional interactions between Toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin. J. Immunol. 166, 1-3.Hennessy, Elizabeth J., Andrew E. Parker, and Luke AJ O'Neill. "Targeting Toll-like receptors: emerging therapeutics?." Nature reviews Drug discovery 9.4 (2010): 293-307.
Kopp, E. B., and R. Medzhitov. 1999. The Toll-receptor family and control of innate immunity. Curr. Opin. Immunol. 11:13.
Lien E, Ingalls RR (January 2002). "Toll-like receptors". Crit. Care Med. 30 (1 Suppl): S1–11. doi:10.1097/00003246-200201001-00001. PMID11782555.
"LRR Protein Domain." Cell Signaling. Web. 7 Apr. 2015. <http://www.cellsignal.com/common/content/content.jsp?id=domains-lrr|>.
"TIR Protein Domain." Cell Signaling. Web. 7 Apr. 2015. <http://www.cellsignal.com/common/content/content.jsp?id=domains-tir>.
"TLR1, Toll-like Receptor 1." CanSAR. N.p., 29 Aug. 2007. Web. 7 Apr. 2015. <https://cansar.icr.ac.uk/cansar/molecular-targets/Q15399/>.
"TLR1 Toll-like Receptor 1 [ Homo Sapiens (human) ]." NCBI. 5 Apr. 2015. Web. 7 Apr. 2015.<http://www.ncbi.nlm.nih.gov/gene/7096|>.
"Transcript: TLR1-001." Ensembl. Mar. 2015. Web. 7 Apr. 2015. <http://useast.ensembl.org/Homo_sapiens/Transcript/ProteinSummary?db=core;g=ENSG00000174125;r=4:38796283-38804376;t=ENST00000502213;redirect=no|>.