The 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
Cathepsin S (CTSS) is a lysosomal proteinase among the Cathepsin family that has nervous system functions including possible processing extracellular matrix degradation and endocellular antigen presentation, and is an inactive precursor of 331 amino acids. (1)
CTSS is the only cathepsin that retains proteolytic activity following extensive expose to neutral pH (3). CTSS is activated in lysosomes by a proteolytic cleavage of propeptide, and expressed in antigen presenting cells including dendritic cells, B-cells and macrophages. CTSS is most well known for the proteolytic digestion of the invariant chain chaperone molecules. It also participates in direct "processing of exogenous antigens for presentation(1)" and is involved in "degrading extracellular proteins (at neural pH) after secretion from activated microglia(3)"(1,3,6).
It is possible that in it’s secreted form, it is implicated in degradation of the extracellular matrix, which may contribute to arthritis, atherosclerosis, neurological diseases, and chronic obstructive pulmonary disease. (1)
It was found in nerve-reared rats, in the periphery, that CTSS is important for modulating neuropathic pain and spinal microglia activation. After injury in rat models, CTSS was expressed by activated microglia in the dorsal horn, which directly influences the sensory information from the body to the brain. (2)
Figure 1. (4) Displays steps of nerve injury and response of CTSS (CatS in figure) released into extracellular space, which is then able to separate and free FKN from the dorsal horn neurons.
Cathepsin B
Amongst all species, the orthologous regions were focused in on the Cathepsin B (CTSB) protein. This protein is also found in the lysosome, and is a cysteine peptidase. CTSB is currently being studied as a potential anticancer agent. However, this protein has processing abilities in carboxypeptidase and enopeptidase activities (5).
Methods
Our purpose was to find whale shark predicted orthologous regions:
I. The human protein sequence (ENSP00000357981) was used obtained by using the Ensemble server.
II. A query search in a Blast against the predicted whale shark protein database was performed using the whaleshark.georgiaaquarium.org, Galaxy server.
III. We use the top predicted protein hits as querries (using the full predicted sequence not only the aligned sequence) in protein BLASTs against the NCBI human protein database.
IV. The whale shark predicted protein database was also searched using the elephant shark predicted CTSS protein sequence as a query.
Predicted Orthologs
Orthologs
Cathepsin B ortholog.
Method: Human protein sequence for CTSS was used in a BLAST against the whale shark predicted protein databse, The top 5 results (with regard to e-value) were then used in a BLAST against the human genome. The lowest e-value match out of the reciprocal BLAST against the human genome was then used BLAST against other species (mouse, zebrafish, elephant shark) to construct a set of potential orthologues with the given protein that was potentially orthologous in both the human and the whale shark.
All 5 were used in a BLAST against the human protein database. results are shown in table 2
Table 2. shows the orthologues predicted from running a BLAST against other common species for the cathepsin B preproprotein
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 5 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. These were used along with searches for CTSS in mouse, elephant shark, and zebrafish genomes. The CLUSTALW program was used to align and assemble the tree (figure 2). Figure 3 shows further investigation into CTSB(cathB).
Figure 2. Although the cathepsin protein domain seems relatively conserved over time, it is not the case that CTSS is preserved. That is, we see more relation between the human, mouse, and zebra fish, which are potentially orthologs, which is not the case with the elephant shark and whale shark.
Figure 3. It is important to note, again, that amongst various species, we found the Cathepsin B to be the common ortholog.
Searching for CTSS in the whale shark
The human CTSS protein sequence was used to query the whale shark predicted protein database and results are shown in Table 1. The 5 best hits were then Blasted against the human protein database using NCBI BLASTp. The smallest e-value was 7e-24.
Whale Shark ID
E-Value
Alignment Length
Predicted Protein Length
% Identity
g41441.t1
2e-17
148
170
32.43
g32914.t1
1e-20
78
85
44.87
g45165.t1
7e-24
78
170
32.43
g42056.t1
3e-11
153
149
25.49
g10040.t1
1e-05
38
95
36.84
Table 2. Table displays the top hits with name and E-value. Human CTSS best BLASTp best hits against the whale shark predicted protein database. The Galaxy server was used to query the predicted whale shark protein database using the human CTSS protein sequence. The top 5 hits according the E-value are reported here with their database ID and alignment length, as well as their predicted amino acid length. These sequences were also used as querries against the CNBI human protein database.
Protein Domains Resuls showed CTSS proteins in the whale shark from the BLASTp results contain the Inhibitor 29 superfamily. Conclusions Although we were unable to identify a strong CTSS ortholog in the whale shark genome we were able to identify multiple conserved homeodomains — members of the Inhibitor 29 superfamily— within the whale shark predicted protein database and the CTSS protein. During our search we did, however, identify a possible Cathepsin-beta ortholog within the whale shark genome. Cathepsin-b is conserved in all of the species which contained CTSS orthologs and additionally is similar to CTSS in function. Further research into the presence of these domains across different species relative to the whale shark and human genomes might provide insight into the divergence and functioning of this protein. Insight which might shed light onto the connection between the functioning of this protein and neurological pain reception.
References
"Human Cathepsin S / CTSS Protein (His Tag)." Human Cathepsin S / CTSS Protein (His Tag). N.p., n.d. Web. 7 Apr. 2015.
Clark, A. K., P. K. Yip, J. Grist, C. Gentry, A. A. Staniland, F. Marchand, M. Dehvari, G. Wotherspoon, J. Winter, J. Ullah, S. Bevan, and M. Malcangio. "Inhibition of Spinal Microglial Cathepsin S for the Reversal of Neuropathic Pain." Proceedings of the National Academy of Sciences 104.25 (2007): 10655-0660. Web.
Akahoshi, Noriyuki, Yoshiya L. Murashima, Toshiyuki Himi, Yasuki Ishizaki, and Isao Ishii. "Increased Expression of the Lysosomal Protease Cathepsin S in Hippocampal Microglia following Kainate-induced Seizures." Neuroscience Letters 429.2-3 (2007): 136-41. Web.
Clark, Anna K. "Fractalkine/CX3CR1 Signaling during Neuropathic Pain." N.p., n.d. Web.
J, Kos, Mitrovic A, and Mirkovic B. "The Current Stage of Cathepsin B Inhibitors as Potential Anticancer Agents." The Current Stage of Cathepsin B Inhibitors as Potential Anticancer Agents. (n.d.): n. pag. Web.
Nakagawa, Terry Y., et al. "Impaired invariant chain degradation and antigen presentation and diminished collagen-induced arthritis in cathepsin S null mice."Immunity10.2 (1999): 207-217.
The 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
Cathepsin S (CTSS) is a lysosomal proteinase among the Cathepsin family that has nervous system functions including possible processing extracellular matrix degradation and endocellular antigen presentation, and is an inactive precursor of 331 amino acids. (1)
CTSS is the only cathepsin that retains proteolytic activity following extensive expose to neutral pH (3). CTSS is activated in lysosomes by a proteolytic cleavage of propeptide, and expressed in antigen presenting cells including dendritic cells, B-cells and macrophages. CTSS is most well known for the proteolytic digestion of the invariant chain chaperone molecules. It also participates in direct "processing of exogenous antigens for presentation(1)" and is involved in "degrading extracellular proteins (at neural pH) after secretion from activated microglia(3)"(1,3,6).
It is possible that in it’s secreted form, it is implicated in degradation of the extracellular matrix, which may contribute to arthritis, atherosclerosis, neurological diseases, and chronic obstructive pulmonary disease. (1)
It was found in nerve-reared rats, in the periphery, that CTSS is important for modulating neuropathic pain and spinal microglia activation. After injury in rat models, CTSS was expressed by activated microglia in the dorsal horn, which directly influences the sensory information from the body to the brain. (2)
Figure 1. (4) Displays steps of nerve injury and response of CTSS (CatS in figure) released into extracellular space, which is then able to separate and free FKN from the dorsal horn neurons.
Cathepsin B
Amongst all species, the orthologous regions were focused in on the Cathepsin B (CTSB) protein. This protein is also found in the lysosome, and is a cysteine peptidase. CTSB is currently being studied as a potential anticancer agent. However, this protein has processing abilities in carboxypeptidase and enopeptidase activities (5).
Methods
Our purpose was to find whale shark predicted orthologous regions:
I. The human protein sequence (ENSP00000357981) was used obtained by using the Ensemble server.
II. A query search in a Blast against the predicted whale shark protein database was performed using the whaleshark.georgiaaquarium.org, Galaxy server.
III. We use the top predicted protein hits as querries (using the full predicted sequence not only the aligned sequence) in protein BLASTs against the NCBI human protein database.
IV. The whale shark predicted protein database was also searched using the elephant shark predicted CTSS protein sequence as a query.
Predicted Orthologs
Orthologs
Cathepsin B ortholog.
Method:
Human protein sequence for CTSS was used in a BLAST against the whale shark predicted protein databse, The top 5 results (with regard to e-value) were then used in a BLAST against the human genome. The lowest e-value match out of the reciprocal BLAST against the human genome was then used BLAST against other species (mouse, zebrafish, elephant shark) to construct a set of potential orthologues with the given protein that was potentially orthologous in both the human and the whale shark.
All 5 were used in a BLAST against the human protein database. results are shown in table 2
Human gene Cathepsin B preproprotein was BLASTed against the whale shark genome
Presumably, this demonstrates that g42056.t1, and the cathepsin B preproprotein in the human genome is orthologous.
Orthologues for cathepsin B preproprotein in other species
Table 2. shows the orthologues predicted from running a BLAST against other common species for the cathepsin B preproprotein
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 5 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. These were used along with searches for CTSS in mouse, elephant shark, and zebrafish genomes. The CLUSTALW program was used to align and assemble the tree (figure 2). Figure 3 shows further investigation into CTSB(cathB).
Figure 2. Although the cathepsin protein domain seems relatively conserved over time, it is not the case that CTSS is preserved. That is, we see more relation between the human, mouse, and zebra fish, which are potentially orthologs, which is not the case with the elephant shark and whale shark.
Figure 3. It is important to note, again, that amongst various species, we found the Cathepsin B to be the common ortholog.
Searching for CTSS in the whale shark
The human CTSS protein sequence was used to query the whale shark predicted protein database and results are shown in Table 1. The 5 best hits were then Blasted against the human protein database using NCBI BLASTp. The smallest e-value was 7e-24.
Table 2. Table displays the top hits with name and E-value. Human CTSS best BLASTp best hits against the whale shark predicted protein database. The Galaxy server was used to query the predicted whale shark protein database using the human CTSS protein sequence. The top 5 hits according the E-value are reported here with their database ID and alignment length, as well as their predicted amino acid length. These sequences were also used as querries against the CNBI human protein database.
Protein Domains
Resuls showed CTSS proteins in the whale shark from the BLASTp results contain the Inhibitor 29 superfamily.
Conclusions
Although we were unable to identify a strong CTSS ortholog in the whale shark genome we were able to identify multiple conserved homeodomains — members of the Inhibitor 29 superfamily— within the whale shark predicted protein database and the CTSS protein. During our search we did, however, identify a possible Cathepsin-beta ortholog within the whale shark genome. Cathepsin-b is conserved in all of the species which contained CTSS orthologs and additionally is similar to CTSS in function. Further research into the presence of these domains across different species relative to the whale shark and human genomes might provide insight into the divergence and functioning of this protein. Insight which might shed light onto the connection between the functioning of this protein and neurological pain reception.
References