This Project
This web page is the product of a project in Emory University's Biology 142 course. Students were assigned human proteins and asked to search for homology between the human protein and the newly analyzed whale shark genome as well as other similar species.

Background Information
IL18The human protein Interleukin 18 (IL18; also known as interferon-gamma inducing factor) is an immuno-modulatory protein that contributes to interferon production in T-helper type cells (IL18 interleukin 18, 2015). T-helper type cells are T cells that aid the adaptive immune system in regulating immune processes. They work to kill infected target cells and microbes by activating B cells and cytotoxic T cells (Alberts et al, 2002). IL18 is also a proinflammatory cytokine that stimulates the production of killer cells in the spleen (IL18 interleukin 18, 2015).
Mutations in IL18In a recent study, it was determined that G/C polymorphisms in the IL18 protein are related to the development and progression of oral squamous cell carcinoma, but that C/A polymorphisms in the gene are not associated with the development of this cancer (Singh et al, 2014). Another recent study showed that IL18 levels in patients with type 1 diabetes are significantly elevated, and as such, regulating IL18 levels might make diabetes therapies more effective (Harms et al, 2015).

MethodsWhale Shark Predicted Orthologs
Ensembl was used to retrieve the fasta sequence of the human protein IL18 ENSP00000280357. This sequence was used as a query against the whale shark predicted proteins database through Galaxy and the Georgia Aquarium. The top hits from this search were recored. The top hits from the first blast searches were run as reciprocal query sequences against the human protein database through NCBI. The Galaxy server was also searched using top hits from predicted matches retrieved from NCBI searches against the elephant shark database using the human protein as the query sequence.Other Predicted Orthologs
The human IL18 sequence was blasted against mouse, hippopotamus, beagle dog, fruit flies, true yeasts, and redeye piranha NCBI databases to identify orthologs in other species using the NCBI BLAST tool.Phylogenetic Tree
The hit with the lowest e-Value from each species was used to create a phylogenetic tree relating whale sharks, humans, and the other species based on the IL18 protein. ClustalW2 was used to generate these data.


Analyzing The Whale Shark GenomeVery little similarity was found between the human protein and any proteins produced in whale sharks.Below is a table of the top three hits returned from the blast.
Sequence ID
Length
% Identical Matches
e-Value
g12092.t1
39
38.46
7x10^-5
g19248.t1
62
33.87
9x10^-5
g18309.t1
51
23.53
9x10^-5
Table 1. The human protein sequence of the IL18 protein was blasted against the Galaxy whale shark database to determine if any homology exists between the human protein and any proteins in the whale shark.
Because the e-Values that were returned from this search are relatively high, it is unlikely that there is any homology in the IL18 protein. The sequences from this search were run back against the human protein database in order to preform a reciprocal search and determine if IL18 is an orthologous protein in humans and whale sharks. Below is the data from this search.
Whale Shark Sequence ID
Protein Name
e-Value
% Identity
Accession
g12092.t1
Nuclear Receptor Corepressor 2 isoform 3
3.2
39%
NP_001193583.1
g19248.t1
hCG1980378
4.8
29%
EAW62213.1
g18309.t1
unnamed protein product
3e-23
56%
BAG37805.1
g18309.t1
fos-related antigen 2
3e-23
56%
NP_005244.1
g18309.t1
unnamed protein product
3e-23
56%
BAG51539.1
Table 2. The whale shark protein sequences that were obtained in the original blast search were blasted against the NCBI human database to determine if any reciprocal hits existed. Because none of the sequences returned the IL18 protein, it is likely that no ortholog exists for IL18 in whale sharks.
Because the search did not return the IL18 human protein, IL18 and the top whale shark hits are not reciprocal hits, implying that the Human IL18 protein does not have a whale shark ortholog. However, the g18309.t1 whale shark protein returned results with such low e-Values that it is potentially orthologous to the human proteins tabulated above that were returned from its search.
To look for homology in a species more closely related to whale sharks, the IL18 protein was blasted against the elephant shark NCBI database. The top hit of this search had an e-Value of 3x10^-8, but it only had a 28% identity. This does not suggest much homology between IL18 and elephant sharks, but to be certain of this, the two top hit elephant shark proteins were blasted against the whale shark Galaxy database to determine if any homology existed between the two. These searches returned equally high e-Values, implying that IL18 does not exist in whale sharks, nor does it exist in elephant sharks.
Protein Domains
Protein Domain.png
Figure 1. IL18 is of the IL1 protein superfamily that contributes to modulating the immune system. None the whale shark sequences obtained from the original blast did not return results in any consistent protein domain, further indicating lack of homology.

IL18 is of the IL1 superfamily, meaning that if a whale shark is orthologous to the human IL18 protein, it will contain the IL1 protein domain. The IL1 superfamily contains 11 different proteins, all of which aid the immune system in responses to pathogens ranging from fever production to inflammation to the production of killer cells (Dinarello, 1998), (Smith et al, 2000).When reciprocal searches were performed, none of the whale shark protein hits with relatively significant values returned the IL18 protein as a homologous protein, and additionally, none of them returned the IL1 superfamily as a correlated protein domain. This further indicates the lack of homology between the IL18 human protein and any whale shark proteins.However, in performing this reciprocal search, an interesting discovery was made. When blasted against the human NCBI database, the whale shark protein with sequence ID g18309.t1 returned several human protein hits with e-Values well below 1e-06, which is commonly used as the threshold for determining homology between two proteins. This protein had protein domains in the bZIP superfamily and the DUF3714 superfamily.In comparing IL18 to species other than the whale shark, both the Beagle Dog and the Mouse, that had particularly low e-Values, produced proteins that shared the IL1 protein domain.
OrthologsThe human protein IL18 sequence was used to search for matches in species other than whale sharks on each species' NCBI protein database. The searches returned the following results:
Organism
Protein Name
E-Value
% Identity
Query Coverage
Accession
Beagle Dog
Interleukin 18 precursor
5e-102
74%
99%
NP_001003169.1
Mouse
Interleukin 18 Isoform X3
4e-81
65%
98%
XP_006510087.1
Hippopotamus
taste receptor type 1 member 2
4.5
31%
15%
AIZ35864.1
Fruit Flies
GM13019
5.5
29%
46%
XP_002044311.1
True Yeast
KLTH0F16764p
4.1
29%
26%
XP_002554914.1
Redeye Piranha
40S ribosomal protein S3a
2.7
43%
7%
AGW00214.1
Table 3. The human protein IL18 has orthologous proteins in dogs and mice, however it does not have orthologous proteins in any other species that was searched.

These data indicate orthologous proteins in Beagle Dogs and Mice, but fail to indicate orthologs in any other species that was searched. This suggests a correlation between IL18 and land mammals, rather than between IL18 and other species such as sharks and fish. There is likely a common ancestor shared between dogs, mice, humans, and other land mammals who produced the IL18 protein after mammals and chondrichthyes split.

PhylogenyUsing the sequences found in the human, beagle dog, mouse, whale shark, fruit fly, yeast, hippopotamus, and piranha genome, and the online Clustal tool, a phylogenetic tree was created. According to the table in Orthologs, the greatest match of the whale shark protein was found in human, dog, mouse, and fruit fly organisms. It was surprising to find the lack of similarity between the whale shark protein and the hippopotamus and piranha proteins, however, and the greater similarity of that protein to land mammals. This finding may suggest that IL18 developed a long time ago from a common ancestor that was more related to humans and whale sharks than hippopotami and piranhas.
TREE.jpgFigure 2. Protein sequences of the best returned hit from each species, as well as the top three whale shark hits, were used to generate a phylogenetic tree. Branch length represents time.
ConclusionsUltimately, it can be concluded that the human protein IL18 is not homologous to any proteins in the whale shark genome. The original BLAST of IL18 against the whale shark Galaxy database returned results with e-Values above the standard threshold of 1e-06, indicating that any similarity between the proteins was coincidental. Furthermore, the BLAST of the three top hit whale shark proteins against the NCBI human database did not return the IL18 protein, demonstrating that there are no reciprocal hits. Interestingly, the g18309.t1 whale shark protein returned hits with very low e-Values, which were not reciprocal hits, but could indicate homology between it and the two unnamed human proteins and fos-related antigen that were returned in the search.
References
Alberts, B., Johnson, A., Lewis, J., et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Helper T Cells and Lymphocyte Activation. Available from: http://www.ncbi.nlm.nih.gov/books/NBK26827/

Dinarello C. A. (1998). Interleukin-1, Interleukin-1 Receptors and Interleukin-1 Receptor Antagonist. Int. Rev. Immunol. 16:457–499. Retrieved April 12, 2015, from http://informahealthcare.com/doi/abs/10.3109/08830189809043005

IL18 interleukin 18 [Homo sapiens (human)]. (2015, February 28). Retrieved March 30, 2015, from http://www.ncbi.nlm.nih.gov/gene/3606

Harms, R., Yarde, D., & Guinn, Z. (2015). Increased expression of IL-18 in the serum and islets of type 1 diabetics. <i>Molecular Immunology,</i> 306-312. Retrieved April 1, 2015, from http://www.ncbi.nlm.nih.gov/pubmed/25576800

Singh, P., Ahmad, M., & Kumar, V. (2014). Effects of interleukin-18 promoter (C607A and G137C) gene polymorphisms and their association with oral squamous cell carcinoma (OSCC) in northern India. Tumor Biology, 35(12), 12275-12284. Retrieved March 30, 2015, from http://www.ncbi.nlm.nih.gov/pubmed/25398

Smith, D. E., Renshaw, B, R., Ketchem, R, R., Kubin, M., Garka, K. E., Sims, J. E. (2000). Four new members expand the interleukin-1 superfamily. Journal of Biological Chemistry. 275, 1169-1175. Retrieved April 12, 2015 from http://www.jbc.org/content/275/2/1169.full