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. Our motivation for researching this protein partially stemmed from it being assigned to us, but we also wanted to see if other species had a similar bacterial immune response. If we could find closely-matched orthologs, this could suggest that other species utilize an innate immune response that involves similar proteins. By synthesizing data from new and old genetic databases, we could find similarities that further research can elaborate on.
Background Information The Lipopolysaccharide Binding Protein (LBP) is found on chromosome 20 in humans (H.M. Kim, B.S. Park, et al.). It plays an important role in the innate immune response (N.J. Gay and M. Gangloff). LBP binds to bacterial lipopolysaccharides (LPS), glycolipids present in the outer membrane of bacteria (B.S. Park, D.H. Song et al.). LBP also functions as an affinity enhancer for CD14, another component of the innate immune response, as well as facilitation for associations with LPS (Gutsmann, Thomas et al.). LBP promotes the release of cytokines in response to LPS. LBP is located on the surface of the cell (Gutsmann, Thomaset al.).
Figure 1. Structure of the Lipopolysaccharide Protein. This figure demonstrates the structure of the LBP binding to LPS (P.S. Tobias et al.). The protein has a crystal structure of murine, causing a negatively charged groove and a hydrophobic core. This crystal structure can lead to frequent mutation that can impair the LBP function of innate immunity (Eckert, Jana K. et al.).
Methods Whale Shark Predicted Orthologs The human protein sequence (ENSP00000217407) was entered as a query in a Blast search against the Predicted Whale Shark Protein database on whaleshark.georgiaaquarium.org. The top predicted protein hits full predicted sequences were in turn used as queries against the NCBI human protein database. The whale shark database was then searched using the elephant shark (closest ortholog) predicted LBP sequence as a query. Predicted Orthologs LBP predicted orthologs were identified in other species using the NCBI Blast server. Protein BLASTs were performed against databases for the mouse, zebrafish, yeast, and elephant shark genomes. The LBP protein was used as query sequence for these searches. Phylogenetic Tree The highest matching ortholog with the lowest E-Value for each species listed above, as well as the closest matches for the whale shark blast hits, were used to make a phylogenetic tree and a multiple sequence alignment among the species.Clustal was used to create both the sequence alignment and tree. Searching for LBP in the Whale Shark The human LBP protein sequence was used as a query against the whale shark predicted protein database. The top five hits are shown below. The lowest e-value was 1e-5 and the highest percent identity was only 40.00%.
Whale Shark ID
E-Value
Alignment Length
Predicted Protein Length
Percent Identity
g29468.t1
1e-5
49
176
30.61
g39413.t1
2e-4
79
118
20.25
g21221.t1
2e-4
76
143
30.00
g41124.t1
2e-4
25
111
40.00
g43785.t1
3e-4
45
130
35.56
Protein Domains The Lipopolysaccharide Binding Protein (LBP) comes from the BPI/LBP/Plunc superfamily. The protein is in the bactericidal permeability-increasing protein, alpha/beta domain. It is part of the lipid-binding serum glycoprotein, C-terminal, as well as the lipid-binding serum glycoprotein, N-terminal.
Figure 2. Putative domains of whale shark best hit predicted proteins. All of the best-hit whale shark predicted proteins contain putative BPI and homeo domains as predicted by NCBI BLAST server analyses. Orthologs
Species
Name
ID
Length
E-Value
Homo sapiens
LBP Precursor
NP_004130.2
481
0.0
Mouse
LBP Precursor
NP_032515.2
481
0.0
Zebrafish
LBP
XP_001342762.2
472
8e-56
Chimpanzee
LBP Isoform
XP_009435501.1
282
0.0
Rhesus-Macaque
LBP
XP_001094451.1
474
0.0
Sperm-Whale
LBP
XP_ 007116789.1
481
0.0
Yangtze River Dolphin
LBP
XP_007447773.1
481
0.0
Western European Hedgehog
LBP
XP_007521714.1
476
0.0
Rabbit
LBP Precursor
NP_001182648.1
482
0.0
Phylogeny
A phylogenetic tree was created using FASTA sequences from best hit orthologs on Clustalw (__www.genome.jp/tools/clustalw/__). Although we were unable to find a best hit within the whale shark genome, there were many found orthologs for the human copy of the LBP protein. Interestingly enough, rodents like rabbits and mice had less matching orthologs than aquatic mammals like the sperm whale and Yangtze River Dolphin. This suggests that speciation may not be closely correlated to the differentiation of the LBP protein between species. Species like dolphins and whales, although still mammals, are generally not as closely related to humans as marsupials and rodents such as rabbits and mice. Conclusions We were not able to securely identify LBP protein in whale sharks; however, we were able to find evidence for several LBP domain transcription factors and structure. The Lipopolysaccharide Binding Protein that is originally found in humans can also be found in chimpanzees. As the comparisons between LBP in species go further, a phylogeny tree could be formed and connections made based on the presence of LBP in these organisms. References Gutsmann, Thomas et al. “Dual Role of Lipopolysaccharide (LPS)-Binding Protein in Neutralization of LPS and Enhancement of LPS-Induced Activation of Mononuclear Cells.” Ed. R. N. Moore. Infection and Immunity 69.11 (2001): 6942–6950. PMC. Web. 31 Mar. 2015.
The Crystal Structure of Lipopolysaccharide Binding Protein Reveals the Location of a Frequent Mutation that Impairs Innate Immunity Eckert, Jana K. et al. Immunity, Volume 39, Issue 4 , 647 - 660
N.J. Gay and M. Gangloff, Annu Rev Biochem 76, 141 (2007).
H.M. Kim, B.S. Park, J.I. Kim, S.E. Kim, J. Lee, S.C. Oh, P. Enkhbayar, N. Matsushima, H. Lee, O.J. Yoo and J-O. Lee, Cell130, 906 (2007).
B.S. Park, D.H. Song, H.M. Kim, B.S. Choi, H. Lee and J-O. Lee, Nature458, 1191 (2009).
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.
Our motivation for researching this protein partially stemmed from it being assigned to us, but we also wanted to see if other species had a similar bacterial immune response. If we could find closely-matched orthologs, this could suggest that other species utilize an innate immune response that involves similar proteins. By synthesizing data from new and old genetic databases, we could find similarities that further research can elaborate on.
Background Information
The Lipopolysaccharide Binding Protein (LBP) is found on chromosome 20 in humans (H.M. Kim, B.S. Park, et al.). It plays an important role in the innate immune response (N.J. Gay and M. Gangloff). LBP binds to bacterial lipopolysaccharides (LPS), glycolipids present in the outer membrane of bacteria (B.S. Park, D.H. Song et al.). LBP also functions as an affinity enhancer for CD14, another component of the innate immune response, as well as facilitation for associations with LPS (Gutsmann, Thomas et al.). LBP promotes the release of cytokines in response to LPS. LBP is located on the surface of the cell (Gutsmann, Thomaset al.).
Methods
Whale Shark Predicted Orthologs
The human protein sequence (ENSP00000217407) was entered as a query in a Blast search against the Predicted Whale Shark Protein database on whaleshark.georgiaaquarium.org. The top predicted protein hits full predicted sequences were in turn used as queries against the NCBI human protein database. The whale shark database was then searched using the elephant shark (closest ortholog) predicted LBP sequence as a query.
Predicted Orthologs
LBP predicted orthologs were identified in other species using the NCBI Blast server. Protein BLASTs were performed against databases for the mouse, zebrafish, yeast, and elephant shark genomes. The LBP protein was used as query sequence for these searches.
Phylogenetic Tree
The highest matching ortholog with the lowest E-Value for each species listed above, as well as the closest matches for the whale shark blast hits, were used to make a phylogenetic tree and a multiple sequence alignment among the species.Clustal was used to create both the sequence alignment and tree.
Searching for LBP in the Whale Shark
The human LBP protein sequence was used as a query against the whale shark predicted protein database. The top five hits are shown below. The lowest e-value was 1e-5 and the highest percent identity was only 40.00%.
Protein Domains
The Lipopolysaccharide Binding Protein (LBP) comes from the BPI/LBP/Plunc superfamily. The protein is in the bactericidal permeability-increasing protein, alpha/beta domain. It is part of the lipid-binding serum glycoprotein, C-terminal, as well as the lipid-binding serum glycoprotein, N-terminal.
Orthologs
Phylogeny
Conclusions
We were not able to securely identify LBP protein in whale sharks; however, we were able to find evidence for several LBP domain transcription factors and structure. The Lipopolysaccharide Binding Protein that is originally found in humans can also be found in chimpanzees. As the comparisons between LBP in species go further, a phylogeny tree could be formed and connections made based on the presence of LBP in these organisms.
References
Gutsmann, Thomas et al. “Dual Role of Lipopolysaccharide (LPS)-Binding Protein in Neutralization of LPS and Enhancement of LPS-Induced Activation of Mononuclear Cells.” Ed. R. N. Moore. Infection and Immunity 69.11 (2001): 6942–6950. PMC. Web. 31 Mar. 2015.
The Crystal Structure of Lipopolysaccharide Binding Protein Reveals the Location of a Frequent Mutation that Impairs Innate Immunity Eckert, Jana K. et al. Immunity, Volume 39, Issue 4 , 647 - 660
N.J. Gay and M. Gangloff, Annu Rev Biochem 76, 141 (2007).
H.M. Kim, B.S. Park, J.I. Kim, S.E. Kim, J. Lee, S.C. Oh, P. Enkhbayar, N. Matsushima, H. Lee, O.J. Yoo and J-O. Lee, Cell130, 906 (2007).
B.S. Park, D.H. Song, H.M. Kim, B.S. Choi, H. Lee and J-O. Lee, Nature458, 1191 (2009).
http://www.jbc.org/content/272/30/18682.full