View source for Answers:Malaria Vaccine

'''Answers to case study exercise about malaria vaccines''' (NB: numbers etc. found in the databases 11-10-2023):

== 1 - What exactly is malaria? ==

'''1a)''' If you search for "malaria" on NCBIs Taxonomy page, you find some mosquitoes and some protozoans with the Genus name ''Plasmodium''. Clicking the name of one of these (twice) gets you to a page where you can see the ''lineage'':

* Genus: ''Plasmodium''
* Phylum: ''Apicomplexa'' 
* (Super)Kingdom: ''Eukaryota''


'''1b)''' On NCBI's Taxonomy page is a function named ”[http://www.ncbi.nlm.nih.gov/Taxonomy/CommonTree/wwwcmt.cgi Taxonomy common tree]” which gives a nice overview. Alternatively you can open taxonomy pages for the two organisms to compare, and see on their ''lineages'' how much they have in common. 
* ''Homo sapiens'' and ''Plasmodium'': ''Eukaryota''
* ''Babesia microti'' and ''Plasmodium'': ''Aconoidasida''
Here is the picture you can get from the "Taxonomy common tree" function:
[[Image:Common Taxonomy Tree.png]]



'''1c)''' On [http://www.cdc.gov/dpdx/malaria/ CDC's page about malaria] or on [http://tolweb.org/Plasmodium/68071 Tree of Life's page about Plasmodium] you find:

* ''P. malariae'', ''P. ovale'', ''P. falciparum'' and ''P. vivax''.

By looking up these four species in NCBI Taxonomy and looking at the table to the right, you see that '''all four''' species have a full genome in the databases (see the link named <u>Genome</u> under <u>Entrez records</u>). 

&nbsp;

== 2 - Identification of membrane proteins (potential vaccine targets) ==

===2a)===

'''14''' chromosomes. <!-- Actually, this is not as easy to find as it used to be. Previously, you got a list of the 14 chromosomes just by following the <u>Genome</u> link. Now, you can see the previous Genome page with the chromosomes by following the link labeled "View the legacy Genome page". Alternatively, you can see a list of the chromosomes by clicking the link under "Reference genome" (Genome assembly GCA_000002765). -->

'''5566''' not hypothetical genes (search details below)
 txid36329[Organism:noexp] NOT hypothetical[All Fields] AND alive[prop]

If you instead found 5570 not hypothetical genes, it is because you found the species ''Plasmodium falciparum'' (taxID:5833) in NCBI Taxonomy instead of the specific isolate 3D7 (taxID:36329) as specified in the exercise.

===2b)===

The correct search strings
 (taxonomy_id:5833)
or 
 (organism_name:"Plasmodium falciparum")
both give '''129,611''' hits in total, '''463''' from Swiss-Prot and '''129,148''' from TrEMBL.

If you only found 34,196 hits, it was because you used 
 (organism_id:5833)
which only gives those ''Pf'' proteins that do ''not'' have a specified strain or isolate — cf. question 3.4+3.5 in [[Exercise: The protein database UniProt|the UniProt exercise]].  

If, on the other hand, you found 131,779 hits, it was because you searched in All instead of specifying the search field:
 Plasmodium falciparum
In that case, you will include some proteins that originate from e.g. humans but play a role in ''Plasmodium falciparum'' infection, which may be mentioned in some comment field or reference title.

===2c)===

This can be solved in several ways:
* <tt>(taxonomy_id:36329)</tt> (either selecting the right isolate from the drop-down menu or using the TaxID you found in the Taxonomy database)
* <tt>(taxonomy_id:5833) AND (organism_name:3d7)</tt>
* <tt>(organism_name:"Plasmodium falciparum") AND (organism_name:3d7)</tt>
They all give: '''5,495''' in total, '''275''' from Swiss-Prot and '''5,220''' from TrEMBL.

That corresponds ''approximately'' to the number of genes found in '''2a)'''.

===2d)===

<tt>(taxonomy_id:5833) AND (cc_scl_term:*)</tt>

'''18,931''' ('''370''' from Swiss-Prot and '''18,561''' from TrEMBL).

===2e)===

<tt>(taxonomy_id:5833) AND (cc_scl_term:secreted)</tt>

or

<tt>(taxonomy_id:5833) AND (cc_scl_term:SL-0243)</tt>

'''76''' (39 from Swiss-Prot).

===2f)===

surface:

<tt>(taxonomy_id:5833) AND (cc_scl_term:surface)</tt><br>
or<br>
<tt>(taxonomy_id:5833) AND (cc_scl_term:SL-0310)</tt><br>
'''413''' hits

membrane: 

<tt>(taxonomy_id:5833) AND (cc_scl_term:membrane)</tt><br>
or<br>
<tt>(taxonomy_id:5833) AND (cc_scl_term:SL-0162)</tt><br>
'''11,299''' hits<br>

===2g)===

Potentially useful (found in the cell membrane): 
* Q7KQL9 / ALF_PLAF7 / Fructose-bisphosphate aldolase: ""Host cell membrane"
* A0A2I0BVG8 / CDPK1_PLAFO / Calcium-dependent protein kinase 1: "Cell membrane"
* W7KN63 / W7KN63_PLAFO / Merozoite surface antigen 2: "Cell membrane"
<!--
* Q8IFM5 / RH5_PLAF7 / Reticulocyte-binding protein homolog 5: "Host cell membrane"
* O97364 / SUB2_PLAFA / Subtilisin-like protease 2: "Cell membrane"
* C6KSX0 / PF12_PLAF7 / Merozoite surface protein P12: "Cell membrane"
* Q8I1Y0 / PF41_PLAF7 / Merozoite surface protein P41: "Cell membrane"
* Q8IDN0 / PFS47_PLAF7 / Female gametocyte surface protein P47: "Cell membrane"
* P62343 / CDPK1_PLAFK / Calcium-dependent protein kinase 1: "In the parasite and on erythrocytic membrane at a lower level"
-->

Definitely not useful (found in an inner membrane):
* Q8I6V3 / PLM2_PLAF7 / Plasmepsin II: "Vacuole membrane"
* U3M186 / U3M186_PLAFA	/ Cytochrome c oxidase subunit 1: "Mitochondrion inner membrane"
* O97321 / O97321_PLAF7	/ GlcNAc-1-P transferase: "Endoplasmic reticulum membrane"
<!--
* Q08210 / PYRD_PLAF7 / Dihydroorotate dehydrogenase: "Mitochondrion inner membrane"
* Q8I719 / KGP_PLAF7 / cGMP-dependent protein kinase: "Endoplasmic reticulum membrane"
* Q8IDR3 / MYOA_PLAF7 / Myosin-A: '''NB:''' even though this is found associated with the cell membrane, it is useless, because it is a Peripheral membrane protein bound to the ''Cytoplasmic'' side of the membrane.
* Q08210 / PYRD_PLAF7 / Dihydroorotate dehydrogenase (quinone), mitochondrial: "Mitochondrion inner membrane"
* Q9N623 / CRT_PLAFA / Chloroquine resistance transporter, PfCRT: "Localizes to the parasite digestive vacuole"
* Q9GPP8 / PSD_PLAFA / Phosphatidylserine decarboxylase proenzyme: "Endoplasmic reticulum membrane"
-->
Of course, the actual examples you selected may differ from these!
<!--
'''1699''' of the hits contain the phrase "cell membrane", this can be found by modifying the search to:

<tt>taxonomy:"Plasmodium falciparum [5833]" locations:(location:"cell membrane")</tt>

or, you can find '''1697''' hits with the search string:

<tt>taxonomy:"Plasmodium falciparum [5833]" locations:(location:"Cell membrane [SL-0039]")</tt>
-->
<!--
'''Note:''' UniProt is developing, and not everything we wrote in the exercise guide earlier is still true. In this question, it was stated:
<blockquote>
These should ideally have a "Subcellular location" annotated as "erythrocyte membrane" or "host cell membrane" — but there are no examples of that in your search from the last question (you are welcome to try!). 
</blockquote>
Some of you actually tried this and found three hits with the search string:

<tt>organism:"plasmodium falciparum" locations:(location:"host cell membrane")</tt>

or 

<tt>organism:"plasmodium falciparum" locations:(location:"Host cell membrane [SL-0375]")</tt>

We stand corrected!
-->

===2h)===
'''27''' hits, all from Swiss-Prot.

<tt>(taxonomy_id:5833) AND (cc_scl_term:"host cell membrane")</tt><br>
or<br>
<tt>(taxonomy_id:5833) AND (cc_scl_term:SL-0375)</tt>

===2i)===

<tt>(taxonomy_id:5833) AND (protein_name:erythrocyte)</tt>

'''10,125''', among these only '''4''' from Swiss-Prot.

===2j)===

<tt>(taxonomy_id:5833) AND (protein_name:erythrocyte) AND (protein_name:membrane)</tt>

'''9,533''' hits, all from TrEMBL.

''or''

<tt>(taxonomy_id:5833) AND (protein_name:"erythrocyte membrane")</tt>

'''9,492''' hits, all from TrEMBL.

===2k)===

<tt>(taxonomy_id:5833) AND (protein_name:erythrocyte) AND (protein_name:membrane) AND (fragment:false)</tt>

'''2,386''' (or '''2,384''' if the words "erythrocyte membrane" are combined)

===2l)===

<tt>(taxonomy_id:5833) AND (protein_name:erythrocyte) AND (protein_name:membrane) AND (fragment:false) AND (database:pdb)</tt>

'''6''' hits: '''Q6UDW7''', '''Q8I639''', '''W7K270''', '''Q8IHM0''', '''A0A024V5I6''', and '''I1X0L2'''.

&nbsp;

== 3 - Analysis of membrane protein domain structure ==


'''3a)''' 

InterPro identifier and name: '''IPR008602, Duffy-antigen binding'''<br>
Pfam identifier and name: '''PF05424, Duffy binding domain'''<br>
If we limit the analysis to the three hits whose accession codes begin with "Q" (as mentioned in the announcement from October 10), we find that it is found '''4''' times in Q8IHM0 and '''6''' times in each of Q8I639 and Q6UDW7.


'''3b)'''

The transmembrane segments are in the following positions:
* Q6UDW7: 2653-2674
* Q8I639: 2650-2667 
* Q8IHM0: 2695-2717

The extracellular parts are the N-terminal parts (all the positions ''before'' the transmembrane segments), and the intracellular (cytoplasmic) parts are C-terminal (positions ''after'' the transmembrane segments).  


'''3c)'''

The following positions are structurally determined by X-ray in the three proteins:
* Q6UDW7: 
** 1215-1950, covering Duffy_binding domain 3 and 4
** 1218-1577 or 1220-1580, covering Duffy_binding domain 3 
** 2326-2631 covering Duffy_binding domain 6
* Q8I639: 2333-2634, covering Duffy_binding domain 6
* Q8IHM0: 728-1214, covering Duffy_binding domain 2


'''3d)'''

Yes! 
* Biological process: cytoadherence to microvasculature, mediated by symbiont protein
* Biological process: pathogenesis
* Cellular component: host cell plasma membrane
* Cellular component: infected host cell surface knob
* Molecular function: cell adhesion molecule binding
* Molecular function: host cell surface receptor binding
All these examples support that these proteins are involved in binding the infected erythrocytes to the endothelial cells (as described in the exercise).

'''Tip:''' You can click <u>View the complete GO annotation on QuickGO</u> in UniProt.

&nbsp;

== 4 - Prediction of B-cell epitopes in a membrane protein ==

'''4a)''' The PDB entry is '''2WAU''' and it's a crystal structure (X-ray).

'''4b)''' 
FASTA sequence for the Duffy Binding domain covered by the 3D structure:
 >2WAU_1|Chains A, B|ERYTHROCYTE MEMBRANE PROTEIN 1 (PFEMP1)|PLASMODIUM FALCIPARUM (36329)
 ICNKYKNINVNMKKNNDDTWTDLVKNSSDINKGVLLPPRRKNLFLKIDESDICKYKRDPKLFKDFIYSSAISEVERLKKV
 YGEAKTKVVHAMKYSFADIGSIIKGDDMMENNSSDKIGKILGDGVGQNEKRKKWWDMNKYHIWESMLSGYKHAYGNISEN
 DRKMLDIPNNDDEHQFLRWFQEWTENFCTKRNELYENMVTACNSAKCNTSNGSVDKKECTEACKNYSNFILIKKKEYQSL
 NSQYDMNYKETKAEKKESPEYFKDKCNGECSCLSEYFKDETRWKNPYETLDDTEVKNNCMCK


'''4c)''' 
The sequence interval was 2333-2634. This means that the '''first''' postion in the new FASTA file corresponds to position '''2333''' in the original sequence.

'''4d)''' It's possible to convert from the coordinates in the FASTA files to the full length sequence by adding '''2332'''. In the table below the epitopes have been named by their starting position as well as numbered.

 EPITOPE     POSITIONS    LENGTH     ORIG_POSITIONS
 #1 ep_5       5 to  29       25     2337 to 2361
 #2 ep_49     49 to  57        9     2381 to 2389
 #3 ep_107   107 to 114        8     2439 to 2446
 #4 ep_153   153 to 172       20     2485 to 2504
 #5 ep_209   209 to 218       10     2541 to 2550
 #6 ep_249   249 to 258       10     2581 to 2590
 #7 ep_273   273 to 294       22     2605 to 2626

&nbsp;

== 5 - Visualization of epitopes ==

'''5a)''' 
Invisible positions:

 Chain A: 2333-2349 and 2540-2546
 Chain B: 2333-2348 and 2535-2549

This means that the first epitope (pos 5-29, orig pos 2337 to 2361) and the 5th epitope (pos 209 to 218, orig pos 2541 to 2550) are partially invisible. 

'''5b)''' 
<!--
Overview figure with SURFACE visualization and indication of epitopes in Chain B. Notice that epitope #1 is partly hidden and epitope #6 is fully hidden (as expected from '''Q5b''' - here its also directly seen by the grey positions in the sequence). Note that if chain A is chosen a few amino acids of epitope #6 will be visible.
-->
[[Image:Epitopes_PyMol_figure.pptx.png|thumb|center|800px|Click to zoom]]

<!--
[[Image:2wau_figure_for_wiki1.png|thumb|center|1000px|Click to zoom]]

Same as above, but with MESH + CARTOON visualization (for a combined overview of surface + interior)
[[Image:3d_mesh_view3.png|thumb|center|1000px|Click to zoom]]
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