Jennymchua Week 14 Assignment

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Purpose

The purpose of our final research project is to compare angiotensin-converting enzyme 2 (ACE2) receptors across primates, including homo sapiens, in order to further investigate the structure-function relationship between ACE2 in humans and the SARS-CoV-2 spike protein. Our goal is to compare seventeen primate ACE2 amino acid sequences and determine if there are any "hot spots" in certain regions that could account for why some species, such as humans, are affected by the novel coronavirus and others are not.

Methods/Results

Collecting BLAST sequences

  • Searched on UniProt "ACE2 family" to obtain names of primate species that had known or predicted ACE2 nucleotide sequences. Came up with seventeen results:
    • Homo sapiens (Human)
    • Chlorocebus sabaeus (Green monkey)
    • Gorilla gorilla gorilla (Western lowland gorilla)
    • Macaca fascicularis (Crab-eating macaque)
    • Macaca mulatta (Rhesus macaque)
    • Nomascus leucogenys (Northern white-cheeked gibbon)
    • Pan troglodytes (Chimpanzee)
    • Pan paniscus (Bonobo)
    • Papio anubis (Olive baboon)
    • Aotus nancymaae (Nancy Ma's night monkey)
    • Carlito syrichta (Philippine tarsier)
    • Macaca nemestrina (Southern pig-tailed macaque)
    • Pongo abelii (Sumatran orangutan)
    • Rhinopithecus bieti (Black snub-nosed monkey)
    • Rhinopithecus roxellana (Golden snub-nosed monkey)
    • Saimiri boliviensis boliviensis (Black-capped squirrel monkey)
    • Propithecus coquereli (Coquerel's sifaka)
  • Entered "*species name* ACE2" into NCBI Nucleotide search and downloaded sequences in FASTA format.

Phylogeny.fr

  • Pasted all seventeen ACE2 nucleotide sequences into Phylogeny.fr and generated a phylogenetic tree.

Analysis of conserved sequences

  • Clustally aligned human and primate ACE2 sequences and analyzed in Microsoft Excel.
  • Determined sequences had 94.25% consensus.
    • Residues with different polarities were deemed "non-conservative".
    • Residues with same polarities but different identities were semi-conserved.

Finding relevant residues

  • Retrieved FASTA protein sequences from Pan troglodytes, Pan paniscus, Homo sapiens, and Pongo abelii.
  • Aligned sequences together to see if they have residue differences in the areas highlighted by Yan et al. (2020) due to their close relativities to each other.
CLUSTAL FORMAT: MUSCLE (3.8) multiple sequence alignment

ACE2_Sumat      MSGSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQ
ACE2_Human      MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQ
ACE2_Chimp      MSGSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQ
ACE2_Bonob      MSGSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQ
                **.*********************************************************

ACE2_Sumat      NMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTIL
ACE2_Human      NMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTIL
ACE2_Chimp      NMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTIL
ACE2_Bonob      NMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTIL
                ************************************************************

ACE2_Sumat      NTMSTIYSTGKVCNPNNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLY
ACE2_Human      NTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLY
ACE2_Chimp      NTMSAIYSTGKVCNPNNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLY
ACE2_Bonob      NTMSAIYSTGKVCNPNNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLY
                ****:**********:********************************************

ACE2_Sumat      EEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDSYDYSRGQLIEDVEHTFEEIKPLYEHL
ACE2_Human      EEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHL
ACE2_Chimp      EEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHL
ACE2_Bonob      EEYVVLKNEMARANHYEDYGDYWRGNYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHL
                *************************:*******.**************************

ACE2_Sumat      HAYVRAKLINAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQ
ACE2_Human      HAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQ
ACE2_Chimp      HAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQ
ACE2_Bonob      HAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQ
                ********:***************************************************

ACE2_Sumat      AWDAQRIFKEAEKFFVSVGLPNMTQRFWENSMLTDPGNVQKVVCHPTAWDLGKGDFRILM
ACE2_Human      AWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM
ACE2_Chimp      AWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM
ACE2_Bonob      AWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM
                ************************* ***************.******************

ACE2_Sumat      CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKS
ACE2_Human      CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKS
ACE2_Chimp      CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKS
ACE2_Bonob      CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKS
                ************************************************************

ACE2_Sumat      IGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEM
ACE2_Human      IGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEM
ACE2_Chimp      IGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPEDQWMKKWWEM
ACE2_Bonob      IGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEM
                *************************************************:**********

ACE2_Sumat      KREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLH
ACE2_Human      KREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLH
ACE2_Chimp      KREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLH
ACE2_Bonob      KREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLH
                ************************************************************

ACE2_Sumat      KCDISNSTEAGQKLLNMLRLGKSEPWTLALENVVGAKNMNVRPLLDYFEPLFTWLKDQNK
ACE2_Human      KCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNK
ACE2_Chimp      KCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNK
ACE2_Bonob      KCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNK
                **************:******************************:**************

ACE2_Sumat      NSFVGWSTDWSPYADQSIKVRISLKSALGNKAYEWNDNEIYLFRSSVAYAMRKYFLEVKN
ACE2_Human      NSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKN
ACE2_Chimp      NSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKN
ACE2_Bonob      NSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKN
                *****************************:*********:************:***:***

ACE2_Sumat      QMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDN
ACE2_Human      QMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDN
ACE2_Chimp      QMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRKSRSRINDAFRLNDN
ACE2_Bonob      QMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRKSRSRINDAFRLNDN
                ********************************************* **************

ACE2_Sumat      SLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVVLIFTGIRDRKKKNKARNEENP
ACE2_Human      SLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSGENP
ACE2_Chimp      SLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSEENP
ACE2_Bonob      SLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSEENP
                **************************************:****************. ***

ACE2_Sumat      YASIDISKGENNPGFQNTDDVQTSF
ACE2_Human      YASIDISKGENNPGFQNTDDVQTSF
ACE2_Chimp      YASVDTSKGENNPGFQNTDDVQTSF
ACE2_Bonob      YASVDTSKGENNPGFQNTDDVQTSF
                ***:* *******************
  • Used highlighted sequences in Yan et al. (2020) to determine if all sequences were the same by highlighting residues in yellow.
  • Determined all species had the same residues in the same locations highlighted by Yan et al. (2020).
  • Aligned other primate ACE2 sequences to human ACE2 to see if any of the primates had unique changes at relevant positions.

Predicting secondary structures

  • Aligned Phylogeny.fr Homo sapiens ACE2 sequence with primate ACE2 sequences
  • Analyzed clustal alignment results in Microsoft Word to look for identity differences in Y41, Q42, K353, and R357.
  • Visualized SARS-CoV-2 and hACE2 from Yan et al. (2020) in UCSF's Chimera and changed residue identities to see how structure changed.

Results

  • His in Y41H alteration in S. and A. is likely not positioned to interact with N501 in SARS-CoV-2 (Yan et. al., 2020)
  • Glu in Q42E alteration is not likely positioned to interact with Q498 (Yan et. al., 2020)
  • Altered residues also have different chemical characteristics
    • Y41H
    • A42E

Data and files

All seventeen primate ACE2 nucleotide sequences
Phylogenetic tree of primate ACE2 nucleotide sequences

Scientific conclusion

Through our research, we found that many primates are probably susceptible to the SARS-CoV-2 virus based on similarities in their ACE2 nucleotide sequences when compared to huamns, specifically their binding residues. This information is helpful, as it could launch further protection of vulnerable species, such as orangutans and chimpanzees, and possibly could be critical in further drug development and testing for humans.

Acknowledgments

  • I worked with my homework partners Nathan and Carolyn throughout the week via Zoom and text to discuss our final project and check-in with our individual progress.
  • We corresponded with Dr. Dahlquistto refine our final project question and go over methods to answering it.
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Jennymchua (talk) 23:08, 29 April 2020 (PDT)

References

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