This project emerged in Emory University laboratory course in Biology 142 during the spring semester of 2015. Newly acquired whale shark proteins of interest were assigned to various student groups in hopes of better understanding the origins of these different proteins. Students were tasked to use different resources to BLAST the assigned protein sequences to better understand the homology of the whale shark proteins as well as determine whether there was an orthologous protein within the whale shark genome.
Background Information
Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) plays a crucial role in the function of the immune system. The NOD2 protein activates in epithelial and immune system cells such as macrophages, dendridic cells and monocytes and is responsible for recognizing bacteria and triggering the immune system to respond (GHR 2015).
Specifically, NOD2 acts in response to the presence of a peptidoglycan-derived muramyl dipeptide in the cell wall of intracellular bacteria (Kaparakis-Liaskos 2015)(Carvalho et al. 2015).This recognition causes NOD2 to activate nuclear factor-kappa-B, a protein complex that in turn regulates additional genes that mediate an inflammatory response through a complex signaling cascade. Figure1. C57BL/6 mice who had M.avium bacteria were tested to determine whether deficiencies in NOD2 production directly leads to decrease in recognition and signaling as well as a decrease in optimal macrophage production. With these mice, the experiment showed that in general deficiencies in NOD2 production are directly related to decreased responses to the M. avium bacteria (Carvalho et al. 2015).
Recent research shows that issues with the NOD2 protein leads to illnesses like Crohn's disease and Blau syndrome. Specifically, single nucleotide polymorphisms in [SNP]8, [SNP]12, and [SNP]13 are evident in people with Crohn’s disease, while NOD/NACHT polymorphisms are found in those with Blau syndrome (Jaskula et al. 2014). Figure 2. NOD2 proteins work in the normal cell when it's in homeostasis (left) by sensing peptidoglycan and producing protective inflammatory programs. NOD2 protein deficiencies (right) lead to loss in producing protective inflammatory programs allowing more infections and antibiotics to come in without supervision causing chronic inflammation issues like Crohn's disease (Philpott et al. 2014).
Methods
Whaleshark Predicted Orthologs The human protein sequence (ENSP00000300589) was used as the query in a Blast against the predicted whale shark protein database using the whaleshark.georgiaaquarium.org Galaxy server. The 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 NOD2 predicted orthologs were identified in species other than whale sharks using the NCBI Blast serves. Protein BLASTs were performed using single species protein databases for mouse, zebra fish, yeast, whale shark, guinea pig, and fruit fly protein databases. The human NOD2 protein (ENSP00000300589) was used as the 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. ClustalW2 with default settings was used to create the alignment and tree.
Searching forNOD2in the whale shark
The human NOD2 protein sequence was used to query the whale shark predicted protein database and results are shown in the table below. The 5 best hits with the lowest E-values were then Blasted against the human protein database using NCBI BLASTp.
Whale Shark ID
E-value
Alignment Length
Predicted Protein Length
% Identity
g44600.t1
1e-36
547
583
24.86
g44890.t1
2e-36
299
359
32.11
g36672.t1
9e-31
358
581
27.93
g46616.t1
3e-30
336
693
28.37
g46384.t1
6e-30
379
648
27.97
Table 1. Best hits for the NOD2 protein in the Whale Shark predicted protein database.
Reciprocal cross and bootstrapping
The top two hits (two lowest E-values) from the whale shark database were blasted against the human protein database using NCBI BLASTp in a reciprocal cross. The top two hits were the human proteins NLRC3 isoform X2 and NLRC3. While not an exact match to NOD2, these both contain a NACHT domain (ABC_ATPase superfamily) shown in blue in Figure 3 below, suggesting functional similarity. In humans, NOD2 is a member of the NLR (nucleotide-binding domain and leucine-rich repeat containing) gene family.
Next, the NOD2 human query was blasted against the elephant shark proteome; a reciprocal cross of this best hit yielded the NOD1 protein, closely related to the NOD2 protein but expressed in different cell types. NOD1 is typically expressed in epithelial cells and fibroblasts in humans, while NOD2 is typically expressed in monocytes and macrophages (Travassos et al, 2009). When compared to the whale shark predicted proteins, the closest found match was the g46616.t1 protein, with an E-value of 5e-25. This suggests that there are several proteins with the NACHT domain present in the whale shark that are a closer match to NOD2 than NOD1. Interestingly, g46616.t1 was the 4th best match for NOD2 in the whale shark protein database.
Protein domains
Figure 3. Protein families and domains for the NOD2 human gene. From left to right: Death Domain (DD) superfamily, implicated in apoptosis and inflammatory response; ABC_ATPase superfamily/NACHT protein domain; Leucine-rich repeats, ribonuclease inhibitor (LRR_RI) superfamily. Leucine-rich sequences facilitate protein-protein interactions.
Orthologues
Based on E-value data, there are several predicted orthologs for the NOD2 gene across species: in the mouse (NOD protein 2) the zebrafish (NOD protein 2 isoform X1), and the guinea pig (NOD protein 2). For the whale shark (ref g44600.t1), a reciprocal best hit searches returned the NLRC3 isoform X2 protein with an E-value of 0.0 and a 99% identity match; therefore, it is unlikely that this gene is an ortholog of the NOD2 gene but it is likely an ortholog of a related gene with a NATCH domain.
nucleotide-binding oligomerization domain-containing protein 2 [Mus musculus]
NP_665856.2
0.0
1013
Zebrafish
nucleotide-binding oligomerization domain-containing protein 2 isoform X1 [Danio rerio]
XP_697924.3
0.0
980
Yeast
Mhp1p [Saccharomyces cerevisiae S288c]
NP_012493.1
.085
1398
Whale shark (best hit)
Unknown
g44600.t1
1e-36
583
Guinea pig
nucleotide-binding oligomerization domain-containing protein 2
XP_003477732.2
0.0
1010
Fruit fly
Ran GTPase activating protein, isoform A [Drosophila melanogaster]
NP_476712.1
1e-7
596
Table 2. Data for best protein hits using human NOD2 FASTA sequence and BLASTp for several species.
Phylogeny
Figure 4. Rooted phylogenetic tree with branch length comparing best hit proteins for NOD2 human protein query across species. "Guinea" refers to guinea pig, while "fruit" refers to fruit fly. Relatedness of species can be concluded by proximity on the phylogenetic tree. Guinea pig NOD2 sequence is most closely related to human, followed by mouse, zebrafish, whale shark, yeast, and fruit fly, in that order.
Conclusions
We searched the proteins of whale shark, zebrafish, mouse, guinea pig, fruit fly and human and our group found matches in the NOD2 protein sequence within all of these animals. In the whale shark, the ortholog is likely the related NLRC3 gene in the ATPase superfamily with a NATCH domain. We believe our results to be fairly accurate because the organisms stated above not only had the lowest E-values but also matched when we did a reciprocal test. Since both the whale shark NLRC3 and the NOD2 protein from the other organisms were from the NATCH superfamily, more research should be done to identify the extent of the relationship between the NOD2 protein and the newly sequenced whale shark genome.
References
Carvalho, N. B., Oliveira, F. S., Marinho, F. A., de Almeida, L. A., Fahel, J. S., Báfica, A., ... & Oliveira, S. C. (2015). Nucleotide-binding oligomerization domain-2 (NOD2) regulates type-1 cytokine responses to Mycobacterium avium but is not required for host control of infection. Microbes and Infection.
Jaskula E, Lange A, Mizia S, et al. NOD2/CARD15 Single Nucleotide Polymorphism 13 (3020insC) is Associated with Risk of Sepsis and Single Nucleotide Polymorphism 8 (2104C>T) with Herpes Viruses Reactivation in Patients after Allogeneic Hematopoietic Stem Cell Transplantation. Biology Of Blood & Marrow Transplantation [serial online]. March 2014;20(3):409-414. Available from: Academic Search Complete, Ipswich, MA. Accessed April 14, 2015.
Kaparakis-Liaskos, M. (2015). The intracellular location, mechanisms and outcomes of NOD1 signaling. Cytokine.
National Library of Medicine (US). Genetics Home Reference [Internet]. Bethesda (MD): The Library; 2015 April 13. Cystic fibrosis; [reviewed 2012 July; cited 2015 April 14]; [about 4 screens]. Available from: http://ghr.nlm.nih.gov/condition/cystic-fibrosis
Park HH, Lo Y, Lin S, Wang L, Yang JK, Wu H. The Death Domain Superfamily in Intracellular Signaling of Apoptosis and Inflammation. Annual Review of Immunology. 2007. 25: 561-586.
Philpott, D. J., Sorbara, M. T., Robertson, S. J., Croitoru, K., & Girardin, S. E. (2014). NOD proteins: regulators of inflammation in health and disease. Nature Reviews Immunology, 14(1), 9-23. doi:10.1038/nri3565
Travassos et al. Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry. Nature Immunology 2010. 11: 55-62.
Zurek, B., Proell, M., Wagner, R. N., Schwarzenbacher, R., & Kufer, T. A. (2012). Mutational analysis of human NOD1 and NOD2 NACHT domains reveals different modes of activation. Innate immunity, 18(1), 100-111.
This Project
This project emerged in Emory University laboratory course in Biology 142 during the spring semester of 2015. Newly acquired whale shark proteins of interest were assigned to various student groups in hopes of better understanding the origins of these different proteins. Students were tasked to use different resources to BLAST the assigned protein sequences to better understand the homology of the whale shark proteins as well as determine whether there was an orthologous protein within the whale shark genome.
Background Information
Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) plays a crucial role in the function of the immune system. The NOD2 protein activates in epithelial and immune system cells such as macrophages, dendridic cells and monocytes and is responsible for recognizing bacteria and triggering the immune system to respond (GHR 2015).
Specifically, NOD2 acts in response to the presence of a peptidoglycan-derived muramyl dipeptide in the cell wall of intracellular bacteria (Kaparakis-Liaskos 2015)(Carvalho et al. 2015).This recognition causes NOD2 to activate nuclear factor-kappa-B, a protein complex that in turn regulates additional genes that mediate an inflammatory response through a complex signaling cascade.
Figure1. C57BL/6 mice who had M.avium bacteria were tested to determine whether deficiencies in NOD2 production directly leads to decrease in recognition and signaling as well as a decrease in optimal macrophage production. With these mice, the experiment showed that in general deficiencies in NOD2 production are directly related to decreased responses to the M. avium bacteria (Carvalho et al. 2015).
Recent research shows that issues with the NOD2 protein leads to illnesses like Crohn's disease and Blau syndrome. Specifically, single nucleotide polymorphisms in [SNP]8, [SNP]12, and [SNP]13 are evident in people with Crohn’s disease, while NOD/NACHT polymorphisms are found in those with Blau syndrome (Jaskula et al. 2014).
Figure 2. NOD2 proteins work in the normal cell when it's in homeostasis (left) by sensing peptidoglycan and producing protective inflammatory programs. NOD2 protein deficiencies (right) lead to loss in producing protective inflammatory programs allowing more infections and antibiotics to come in without supervision causing chronic inflammation issues like Crohn's disease (Philpott et al. 2014).
Methods
Whaleshark Predicted Orthologs
The human protein sequence (ENSP00000300589) was used as the query in a Blast against the predicted whale shark protein database using the whaleshark.georgiaaquarium.org Galaxy server. The 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
NOD2 predicted orthologs were identified in species other than whale sharks using the NCBI Blast serves. Protein BLASTs were performed using single species protein databases for mouse, zebra fish, yeast, whale shark, guinea pig, and fruit fly protein databases. The human NOD2 protein (ENSP00000300589) was used as the 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. ClustalW2 with default settings was used to create the alignment and tree.
Searching for NOD2 in the whale shark
The human NOD2 protein sequence was used to query the whale shark predicted protein database and results are shown in the table below. The 5 best hits with the lowest E-values were then Blasted against the human protein database using NCBI BLASTp.
Reciprocal cross and bootstrapping
The top two hits (two lowest E-values) from the whale shark database were blasted against the human protein database using NCBI BLASTp in a reciprocal cross. The top two hits were the human proteins NLRC3 isoform X2 and NLRC3. While not an exact match to NOD2, these both contain a NACHT domain (ABC_ATPase superfamily) shown in blue in Figure 3 below, suggesting functional similarity. In humans, NOD2 is a member of the NLR (nucleotide-binding domain and leucine-rich repeat containing) gene family.
Next, the NOD2 human query was blasted against the elephant shark proteome; a reciprocal cross of this best hit yielded the NOD1 protein, closely related to the NOD2 protein but expressed in different cell types. NOD1 is typically expressed in epithelial cells and fibroblasts in humans, while NOD2 is typically expressed in monocytes and macrophages (Travassos et al, 2009). When compared to the whale shark predicted proteins, the closest found match was the g46616.t1 protein, with an E-value of 5e-25. This suggests that there are several proteins with the NACHT domain present in the whale shark that are a closer match to NOD2 than NOD1. Interestingly, g46616.t1 was the 4th best match for NOD2 in the whale shark protein database.
Protein domains
Figure 3. Protein families and domains for the NOD2 human gene. From left to right: Death Domain (DD) superfamily, implicated in apoptosis and inflammatory response; ABC_ATPase superfamily/NACHT protein domain; Leucine-rich repeats, ribonuclease inhibitor (LRR_RI) superfamily. Leucine-rich sequences facilitate protein-protein interactions.
Orthologues
Based on E-value data, there are several predicted orthologs for the NOD2 gene across species: in the mouse (NOD protein 2) the zebrafish (NOD protein 2 isoform X1), and the guinea pig (NOD protein 2). For the whale shark (ref g44600.t1), a reciprocal best hit searches returned the NLRC3 isoform X2 protein with an E-value of 0.0 and a 99% identity match; therefore, it is unlikely that this gene is an ortholog of the NOD2 gene but it is likely an ortholog of a related gene with a NATCH domain.
Phylogeny
Figure 4. Rooted phylogenetic tree with branch length comparing best hit proteins for NOD2 human protein query across species. "Guinea" refers to guinea pig, while "fruit" refers to fruit fly. Relatedness of species can be concluded by proximity on the phylogenetic tree. Guinea pig NOD2 sequence is most closely related to human, followed by mouse, zebrafish, whale shark, yeast, and fruit fly, in that order.
Conclusions
We searched the proteins of whale shark, zebrafish, mouse, guinea pig, fruit fly and human and our group found matches in the NOD2 protein sequence within all of these animals. In the whale shark, the ortholog is likely the related NLRC3 gene in the ATPase superfamily with a NATCH domain. We believe our results to be fairly accurate because the organisms stated above not only had the lowest E-values but also matched when we did a reciprocal test. Since both the whale shark NLRC3 and the NOD2 protein from the other organisms were from the NATCH superfamily, more research should be done to identify the extent of the relationship between the NOD2 protein and the newly sequenced whale shark genome.
References
Carvalho, N. B., Oliveira, F. S., Marinho, F. A., de Almeida, L. A., Fahel, J. S., Báfica, A., ... & Oliveira, S. C. (2015). Nucleotide-binding oligomerization domain-2 (NOD2) regulates type-1 cytokine responses to Mycobacterium avium but is not required for host control of infection. Microbes and Infection.
Jaskula E, Lange A, Mizia S, et al. NOD2/CARD15 Single Nucleotide Polymorphism 13 (3020insC) is Associated with Risk of Sepsis and Single Nucleotide Polymorphism 8 (2104C>T) with Herpes Viruses Reactivation in Patients after Allogeneic Hematopoietic Stem Cell Transplantation. Biology Of Blood & Marrow Transplantation [serial online]. March 2014;20(3):409-414. Available from: Academic Search Complete, Ipswich, MA. Accessed April 14, 2015.
Kaparakis-Liaskos, M. (2015). The intracellular location, mechanisms and outcomes of NOD1 signaling. Cytokine.
Marchler-Bauer A et al. (2015), "CDD: NCBI's conserved domain database.", Nucleic Acids Res. 43(Database issue):D222-6.
National Library of Medicine (US). Genetics Home Reference [Internet]. Bethesda (MD): The Library; 2015 April 13. Cystic fibrosis; [reviewed 2012 July; cited 2015 April 14]; [about 4 screens]. Available from: http://ghr.nlm.nih.gov/condition/cystic-fibrosis
Park HH, Lo Y, Lin S, Wang L, Yang JK, Wu H. The Death Domain Superfamily in Intracellular Signaling of Apoptosis and Inflammation. Annual Review of Immunology. 2007. 25: 561-586.
Philpott, D. J., Sorbara, M. T., Robertson, S. J., Croitoru, K., & Girardin, S. E. (2014). NOD proteins: regulators of inflammation in health and disease. Nature Reviews Immunology, 14(1), 9-23. doi:10.1038/nri3565
Travassos et al. Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry. Nature Immunology 2010. 11: 55-62.
Zurek, B., Proell, M., Wagner, R. N., Schwarzenbacher, R., & Kufer, T. A. (2012). Mutational analysis of human NOD1 and NOD2 NACHT domains reveals different modes of activation. Innate immunity, 18(1), 100-111.