1fOO
3.2.2 Prediction ofHLA-type T-cell epitopes in Pc96
Protein GI accession Size (amino acids) Name Molecular weight (Da) hypothetica lprotein(Plasmodiumyoeliiyoelii) 23481602 1523 Pyy178 178052.17
hypothetical protein C0760c-malaria parasite 7494250 3394 Pyy403 403158.04 (Plasmodium jalciparum)
hypothetical protein(Plasmodium yoeliiyoelii) 23484590 713 Pyy84 83779.93 Pc96 antigen(Plasmodium chabaudiadamiy Not deposited 942 Pc96 . 110054.92
The data obtained from the alignments of the BLAST hits, was used to construct a diagram, showing and comparing the sizes and relative positions of the proteins in relation to the Plasmodium falciparum protein Pf403 (Figure 3.1).
Pf403
Pc96
Pyy178
Pyy84
o
I
500
I
10X1
I
a BLAST search for short nearly exact matches of the Plasmodium falciparum malaria genome (PlasmoDB). This identified malaria proteins also containing these specific epitopes. Table 3.6 shows the predicted T cell epitopes for Pc96 and lists the malaria proteins found to contain the same epitope sequences.Itwas interesting to note that peptides(LIKYKNFI) and(LIKYKNFII), which are virtually identical in Pc96, Pf403 and Pyy178, exhibit exact similarity to those contained in a variety of other malaria proteins, as identified by the BLAST search for short nearlyexact matches.
FSVSSDHITL IKCNI LKMFK LGSCYLYIIN RNLKEI KI LF DKINSLEENI QSLNLFINNL 60 KDQNKNNEVI KINNEEQILQ LKNSLQNNEN CINNLNDNLK QKDEMNNSNI KNLI KYKNF I 120 INLVHQTNVF LHIFKTMSTQ QVIQNSEYNQ LTQLLRKELD PYLNDSI MIS ELESKE KSDE 180 ANANNDLLNI PSTFSYENYE HIQIFTNKYN LIIERGQVSI FSDGKIKAPQ NEQTSTFFSN 240 ISSYFHNSNQ YNNTSNSKDA SENTPESPKD SQPQTYQEKE NEEESVRNDN GQMYSVEGNG 300 TDEEDYRDDL EEVEHDEDVE DEEREERDVR VEHDEDDDHD EHEERDEHGE LDEYDDHADH 360 VEHVESDEDD DHDDHDEHEE RDEHDELEEY DDHAGHIEHV EHDED DE KSK ADGDTEVDED 420 ILSNNKEKEN QDVESYNEYN NDREEYENVS DSEKDEERSS IDNNYDEDHR DENETLQRYE 480 ENVIVENVDN LGEKELLPSN VESNVFENNY TSNDVNGNGI VPEVSEQNKV LDGDPI SNI N 540 YENITTDI NI NTSNMQNEEN ISPPFSNKLE NNNSTFVNGT TCSYMSEYEF NFFDKNDHPK 600 MKEAKKRKYH SDSEIVKQNN NNKTGYTKKK IRKID HSYKF NNGD I PTEVV ENQIQ QN INF 660 NNLDNNYTNQ LFNNYYEEQK NKYELLDNI N KINQTYNSNN LDAPNDERND NENGNNSLMK 720 SDKESQNENK DDKASDCYVI SSDDDAEVKD YDEEEDEAGE DDEDDDVKDY DEDESNEEI D 780 EGEEEEDEAD YEQFDSKGEI NSSVADEGEE SNSDDPDKDM I DYDEMDNAN GMKIEMKRTA 840 KLNVTI VNLN VMKAMLIVIK VMLIVMKVMK IVMKVTLI AM KATFDVMKAT LIMMKVI LVM 900 RRMTVVVIV T HI LQATTQMK IIKIKNQLLV LQHQMMVRMM DQ 942
Figure 3.2 Distribution of 17 putative T cell epitopes predicted in the Pc96 amino acid sequence.(Epitopes are highlighted red and overlapping epitopes are highlighted in dark red).
HLA-type and otherP.falciparum proteins containing the peptide.
3.2.3 Multiple sequence alignments using ClustalW of protein regions with high similarity to Pc96
Figure 3.3shows the CLUSTALW alignment of Pc96 and Pf403showing thehigh degree ofsimilarityin theseregions,includingstretches of exactsequence identity.
Pc96 Pf403
Pc9 6 Pf403
Pc 96 Pf403
Pc9 6 Pf403
---- ---- --- FSVSSDH I TLIKCNILKMFKLGSCYLYIINRNL 33 NNI NNVNNI NNVKNI VDINNYLVNNL QLNKDNDNI II I KFNILKLFKLGSCYLYII NRNL 2400
: . ..*:* :** **** : ***************
KEIKILFDKINSLEENIQSLN LFINNLKDQNKNNEVIKINNEEQ ILQLKNSLQNNENCIN 93 KEI QMLKNQILSLEESIKSLNEFINNLKNENEKNELI KINNFEEILKLKNNL QDNESCIQ 2460
***::* ::* *** *.*:*** **** * *:: * ::* *:* * * ** *:** :***.**:**.**:
NLNDNLKQKDEMNNSNI KN LIKYKNFIINLVHQTNVFLHIFKTMSTQQVI QNSEYNQLTQ 153 NLNNYLKKN EELNKINVKN I FKYKGY I I HLIQQSNVFCK IFKHFNENKII DQSI INKL-L 2519
** *: **:: :*: *: *:* * -.** * .* *. *..*.*** :* * * :. :::*::* *:*
LLRKELDPYLNDSIMISELESKE KSDEANANNDLLNI PSTFSYENYEHI QIFTNKYNLII 213 YLKKSFDFYMYDSVIQEIRE- - - -- - -- - -NKNIIINQDFLTDEY FKHIQTFTKTCNVLI 2569
*:*. :* *: **: : . * . :: * ::*** **:. *..*
Pc96 Pf403
ERGQVSIFSDGK-lKAPQNEQT STFFSNI SSYFHNS NQYNNTS NSKDASENTPES PKDSQ 272 QRGYLSILKDTNNDFFIQNKQSNQQGNQNGNHINMCN IYP DDEINVTADQQI FDGTENVQ 2629
:** :* * :. * : **:*:. .: ..::: .* * . *.:: : ..:: *
Pc 9 6 Pf403
PQTYQEKENEEESVRNDNGQMYSVEGNGTDEEDYRDDLEEVEHDE DVEDEEREERDVRVE 332 QSLQNEEDYVNNEEMYTDKMDLDNNMNGDD DDDDD DDDDDDNNNNNNNNNNNNNNNMGDE 2689
:*: : . : ** *: :* ** : : : : : : : ::::. : : . : : *
Pc9 6 Pf403
HDEDDDHDEHEERDEHGELDEYDDHADHVEHVESDEDDDHDDHDEHEERDE HDELEEYDD 392 DNHLVNAFNNHNLLTNGNVKSDQINNETLERYEEN IIQNIYTNDNVDNNQVIENINKILI 2749
:*: : .. : : : :*: * . :*: ::. :
Figure 3.3 Region of ClustalW multiple sequence alignment of Pc96 (Plasmodium chabaudi adami), Pf403 (Plasmodium falciparum).
*
Showsexactamino acid matches,: show conserved substitutions,and. denotes semi-conserved substitutions. Predicted T cell epitopes are highlighted on Pc96).Pc96 aligned with Pf403, shows the first 250amino acids to be homologous, producing a score of 973. To investigate and compare these sequences, pairwise alignments between Pyy178 and Pf403 was performed using CLUSTALW (Figure 3.4). The alignment of Pyy178 with Pf403 calculated an overall score of 2472. In this alignment itis evident that the first 700 amino acids in thePyyl78 sequence containlargeregions of high similarity.
79 1800
139 1859
170 1919
230 1979 :*:* **:*:
. :: : : *
KDEYVSNLKMNLESSRMQLKDLCDQLEKGNLNEKKCNMKIQMLETKLXKEEKERKRYQIE YQQFIHSLKANLENSRLELKELSNLNEKIQLSDEKNRMKITILEDKLFKNEKDKMKLQQI
. . . ** ***.**::**:*.: ** :*.::* *** :** ** '* : * * : : : * NIRVQKETHLQQQKCIIELR---TKLIDNNMASQ RLKIEKTKINKQEKYIIQLQKDNNLILNDFNSTTTTTNNNNNNDNNNDNNNDNNDNNNDT
.. *
KDNKYLSENLEKEVEISDNNLIIKNRLEQLVEINKDLYKEVQENYNNTEKMKFKIIELKE KDNTYLNDMFKNQINYVDNNLLKN-RLDQLFNINQDLQKHLDTNQKHLEQLKYDYIEIKE
***.**.: :::::: ****: : **:**.:**:** *.:: * :: *::*:. **:**
EYIELLKKEKENVEKKFENTSEKYNEQINLNKKLTDDINLLISTHKEQVKVLNEQINILK DYIKLIKKDKTNIQNEYNDLLEKYNEVVVKNNMLYNDMNVLLKEHKEEIFLLKENIKILQ :**:*:**:* *: :::::: *****: *: -* :*:*:*:. ***:: :*:*:*:**:
IVVKDIKNNMRNEIDKLNNDINEKSYEIKLLKHENNNLINEMNILKNKETENMNIKQKEE :*:.* :. ::**:*:***
Pyy178 Pf403 Pyy178 Pf403 Pyy178 Pf403 Pyy178 Pf403 Pf403
Pyyl78 Pf403
LANKNSTDSISYNKLKTQVEMVTEENKILLLRKECYEKEIEQLKRDSQFFNSTKNNDINI 290 IDDNNKNYMIQYNKLKTNLDMLSEENRMLLLNKEEYEKQIEQLNHDHKLFISTKNNDIQI 2039
: ::* .. *.******:::*::***::***.** ***:****::* ::* *******:*
Pyy178 Pf403
IERDVLKKQVEEHITKINEKDKQIVNLNFEIKKLYNQLEEMKERMNRIETTTP--- 343 I ENEKLQEQVDQYITTINEKDKIIVHLNLQIKKLANQNEHMRSRCDIFNVAHSQDNIKND 2099
**.: *::**:::**.****** **:**::**** ** *.*:.* : ::.: . Pyy178
Pf403
---ILDGPTDEASTLNIDNSKTSQNNQ--- 367 HMVVGEDIMGDTNHDVNKNIDQGTNQHINQGTNQHINQGTNQHDTCDGPNYNYVKVQNAT 2159
: * *:. ***: . . . . : **
Pyy178 Pf403
---LSLEIYKYINENIDLTAELENKNDVIEQMKEDXKNKNKEIAKLNKDVIN 416 NREDNKNKERNLSQEIYKYINENIDLTSELEKKNDMLENYKNELKEKNEEIYKLNNDIDM 2219
** *************:***:***: :*: *:: *:**:** ***:*:
Pyy178 Pf403
LSTNYDKLKESIYMMEKHKTNLNEYIKQKDEIISLLQQKGNNNNNSPTKFCNLVEN---N 473 LSNNCKKLKESIMMMEKYKIIMNNNIQEKDEIIENLKNKYNNKLDDLINNYSVVDKSIVS 2279
**.* ****** ****:* :*: *::***** *::* **: :. . : *: : Pyy178
Pf403
NTNKNGANEDDFDSPIKYNINLKNNNSKEIEEKIMGMEEIMNTSCEEVINTLKKIPQTNV 533 CFEDSNIMSPSCNDILNVFNNLSKSNKKVCTNMDICNENMDSISSINNVNNINNVNNINN 2339
**. : .*.* *:: . *. : : *. : : :: : *
Pyy178 Pf403
ENDDN--NNLNSSKNQFSVN---SDHITLIKCNILKMFKLGSCYLYIINRN 579 VNNINNVNNINNVKNIVDINNYLVNNLQLNKDNDNIIIIKFNILKLFKLGSCYLYIINRN 2399
*:* **:*. ** ..:* .*:* :** ****:**************
Pyy178 Pf403
LKEIKILKDKIHSFEENIQSLNLFINNLKDQHKNNEMIKINNEEQlVELKNNLKNNENCI 639 LKEIQMLKNQILSLEESIKSLNEFINNLKNENEKNELIKINNFEEILKLKNNLQDNESCI 2459
****::**::* *:**.*:*** ******:.::::**:***** *:*::*****::**.**
Pyyl78 Pf403
NNLNDNLKQKDEMNNSNIQNLIKYKSFIINLVYQSNIFFHIFKNINKQKVIQNSIFNQLT 699 QNLNNYLKKNEELNKINVKNIFKYKGYIIHLIQQSNVFCKIFKHFNENKIIDQSIINKLL 2519
:***: **:::*:*: *::*::***.:**:*: ***:* :***::*::*:*::**:*:*
Figure 3.4 Region of ClustalW multiple sequence alignment of Pyy78 (Plasmodium yoelii yoeliis, Pf403 (Plasmodium falciparumy.
*
Shows exact amino acid matches,: show conservedsubstitutions, and . denotes semi-conserved substitutions.
DQMYSVEDDGSEE EEYRFNEEHAEVEIDG KQAEVETDGKHGEVEIDGEQAEVETDRKHGE YDDHADHVEHVES DEDD DHDD HDEHEERDEHDELEEYDDHAGHI EHVEHDEDDEKSKADG
DEYDDVENDDEYDEEYEEEEDDGEMD--EADDGEMDEADDGEMNEADDGEM DEADYDQFD EEIDE GE-EEEDEADYEQFDS KGE INSSVADEGEESNSDDPDKDMIDYDEMDNANGMK I E MKNDKESQNENAKE-SDSCYV I SSDDDNRQIQDFEEDEEDEEDEEDEEDEEDEQDEADEY MKSDKESQNENKDDKASDCYV ISSDDD-AEVKDYDE-EEDEAGEDDEDDDVKDYDEDESN DNNGDNGNN IVSEVGEENKLLGDN SVNHINYENIKTDVAVNVDI DANTSNLQNEEN I S PS ----GNGI VPEVSEQNKVLDGDPISNI NYENITT----- -DI NI NTSNMQNEENISP-
660 113
719 17 3
779 233
83 8 293
898 35 3
958 413
101 8 463
1078 51 6
113 8 563
1198 623
12 5 8 663
1317 718
1376 776
1434 835
14 8 7 894 .... * ....
.. .. ....
:*:**** *
: *: *** ::
*: * : .. *
**: **** :** **** * *
.*: * *:*: .: ** .
**:* * .::** : : * ***:*: ::: *: :* ****
**
:: : * * * .:: *: * ..*.
.. .... *: ..:: ::**.. : :: .: .. : ::.. : : :
** * * * * * ** * * *** * * :* * * *** * *.** * * * *: * * *: ..** *
. . ...*********
**.**.** *-**:* ..: . : . :* * * * * *.*
. * * ..
...... .
* *** :* . : .****.. ::* * * * * **.: * *** :* * *.:::
***** :** : : :*:**
: :: .. : *.. :*
:* *: * ::* : :* * : :..**::
****. : *
**.********
AFSNKLENQNNT FVNDSTRSYLSECE FNSFDKNDNLKIKESKKRKYHSDT EIVKQNNNNK PFSNKLENNNSTFVNGTTCSYMSEYEFNFFDKN DHPKMKEAKKRKYHSD SEIVKQNNNNK
**** * ** : *.****.:* **:** *** *** ** : *:** : * * * *** **: * **** * * * * * VGEHDEDMLNDSKEKESSNI ESYNEYNSNREEYVDVSDN DEEKEGDE FNYSNKEKSS IDN DTEVDEDILSNNKEKENQDVESYNEYNNDREEYENVS DSEKD------ - ---EERSSIDN FKELNENTTEPQKDTQI QTVQI SQEKQNEEENTKNENI I DTQVNNENYQI DALKNEEYND YSVEGNGTDEEDYRD DLEEVEHDEDVEDEEREERDVRVEHDEDDDH DEHEERDEHGELDE
VEYTKKKMRKMNNSHK FTSRGTSDVTVESPTPQKLSEKLSEKLSEKLNGN IN GNINGDDN TGYTKKKIRK IDHSYKFNNGD I PTEVVEN-- - - -- - - -- --- - -Q IQQNIN FNNL
SK- -GELNSS ID DDSNSDDADKDM IDYDDIDNIN ENQNEEDSEVENEDDNNFLDE- - - - - MKRTAKLNVT IVN-LNVMKAMLIVIKVMLIVMKVMK IVMKVTL IAMKAT FDVMKAT LIMM
* .:* * :* * * :*. *
ENEDDENYASESFDNYYDEKK-KYEILEN ISKLNQACNNNNLDATSDGNNYAKKNEHNFL DN- ---NYTNQLFNNYYEEQKNKy ELLDNI NKI NQTy NSNNLDAPNDERN-DNENGNNSL :* **:.: *: *** :* :* ** *: *:**.*: * *: *.***** * * ::* :* * NYDESNCDKSNCEENETLQRYEENVIVENGNN LGEKELLTSNVESNLFENDDNNNDDNND NYDEDHRD- - - - -ENETLQRYEENVI VENVDNLGE KELLPSNVESNVFENNYTSN DVN-- QSENQNEKQNENLS ENLSENLSEKQNENLSEKQNEKSNNFLSN-I SSYFQSSNKYNNAPT TSTFFSNISSYFHNS NQYNNTSNSKDASE NTPESPKDSQPQTYQEKENEEESVRNDNGQM
* .. .0 * :* *: .:: .. . . .. *. . . :. * : :*.
NKEKSNLNN FENNLLNIVS TFSCENYEHIQ I FTNKFNLI I ERGQ FSI LAD SKIQ PSQNEK SKEKS DEANANNDLLNI PS TFSYENYEHIQI FTNKYNLIIERGQVS I FS DGKI KAPQNEQ
****: * : *:**** **** ******* **** *:****** **..**::r •** :....*** ..
IKYKSFIINLVYQSNIFFHIFKNINKQKVIQNSIFNQLTQLR-KELDFYLNDSIMISELE IKYKNFIINLVHQTNVFLHIFKTMSTQQVI QNSEYNQLTQLLRKELDPYLNDS I MI SELE
****.* **** * : * :* :* :****. :..*:***** :* * * *** **** ******* *****
NLFINNLKDQHKNNEM IKINNEEQIV ELKNNLKNNENC INNLNDNLKQKDEMNN SNIQNL NLFINNLKDQNKNNEVIKINNEEQILQLKNSLQNN ENCINNLNDN LKQKDEMNN SNIKNL
**** **** * *: * ***:* * * * **** *::* * *.*:* * * * * ** * * * * **** * * * ** ** * *: * *
***.************************** ***** ~ * ** ***:*:*******
Pyy178 pc 96 Pyy178 pc96
Py y17 8 pc96 Pyy178 pc96 Py y178 pc96
Pyy178 pc96
Py y17 8 pc96
Pyy178 pc96
Pyy178 pc96 Pyy178 pc96 Pyy178 pc9 6 Pyy178 pc96 Pyy 1 78 pc96 Pyy178 pc96 Pyy1 78 pc96 pc9 6
Figure 3.5. Region of ClustalW pairwise sequence alignment of Pyy78 (Plasmodium yoelii yoeliii, Pc96 (Plasmodium chabaudi adamh.
*
Shows exact amino acid matches, : show conserved substitutions,and.denotes semi-conserved substitutions.(Figure 3.6). Itcan beseen that the entire length of the Pc96sequence is similarto a portion of the Pyy178 protein. This indicates a high probability of these sequences beinghomologous. The alignment shows several extended regions of sequence similarity. The full length of the Pc96 sequenceshows highsimilarity to Pyy78 from residues546 to 1523.
Pyy178 Pc96 Pf403
--VENDDNNNLNSSKNQEFSVNS DHI TLIKCNILKMFKLGSCYLYI INRN 58 0 ---FSVSSDHI TLI KCNILKMFKLGSCYLYIINRN 32 NNVKNIV DI NNYLVNNLQLNKDNDN III IKFNILKLFKLGS CYLYIINRN 23 9 9
: . . .*:* .* * ****.** ********* ***
Pyy178 Pc96 Pf403
Pyy178 Pc96 Pf403
LKEIKILKDKIHSFEE NI QSLNLFI NNLKDQHKNNEMIKI NNEEQI VELK LKEI KILFDK I NSLEENI QSLNLFI NNL KDQNKNN EVI KI NNEEQILQLK LKEIQMLKNQILSLEES IKSLNEFI NNLKNENEKNELI KI NNFEEIL KLK
****..* ..* *.** *-*** ** * * * *: :: : :* *:***** *.*. .**
NNLKNNENCINNLNDNLKQK DEMNNSNIQNLIKYKS FIINLVYQSN I FFH
NSLQNNENCINNLNDN LKQK DEMNNSNIKNLI KYKNFIINLVHQTNVFLH NNLQDNESCI QNLNNY LKKNEELNKINVKN I FKYKGY I I HLIQQSNVFCK
*.*: :** .* * : * * * : **: ::*:* : *::*: :*** . :**:*: *.* .*
630
82 24 49
68 0 132 2499
Pyy178 Pc96 Pf403
IFKNINKQKVI QNS IFNQLTQLR-KELDFYLNDS I MISELENKEKSNLNN 729 IFKTMS TQQVIQN SEYNQLTQLLRKELD PYLNDS I MIS ELESKEKSDEAN 182 IFKHFNENKII DQSII NKLLYLK-KSFDFYMYDSVI QEI RENK- - -- - - - 254 2
** * :. :: :* : :* *:* * * .* *. **:: . *.*
Pyy178 Pc96 Pf403
FENNLLNIVSTFS CENYEHI QIFTNKFNLIIERGQFSILAD SK-IQPSQN ANNDL LNIPSTFSYENYEHIQIFTNKYNLIIERGQVSIFSDGK-lKAPQN -- - NI I INQDFLTDEYFKHIQTFTKTCNVLI QRGYLSILKDTNNDFFI QN
778 231 2589
* ::*** ** . *: :*:** **: * **
Figure 3.6 ClustalW multiple sequence alignment ofPyy178 (Plasmodium yoelii yoelil), Pca96 (Plasmodium chabaudi adamiy and Pf403 (Plasmodium falciparum).
*
Shows exact amino acidmatches,: showconserved substitutions,and.denotes semi-conservedsubstitutions.The alignment score for these three proteins was 5462. Pc96 shows homology from initial aminoacidsin the sequence,while the similar region in Pyy78 spans from amino acids 532-729.
Pf403, being a much larger protein, is similar across a 221 amino acid region, from residues 2367 to 2589. From this multiple sequence alignment it can be seen that in these regions,there
more similar to Pyy78 than Pc96. The putative T cell epitopes, predicted in Section 2.1.2, are indicated in the alignments, along with the casein kinase II phosphorylation site, shared between, Pf403 and Pc96.