eacri summer 2015 internship
TRANSCRIPT
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Requirements for EGFRvIII-mediated enhancement of SecA2-dependent
secretion from Listeria monocytogenes
Shane Klumpp EACRI Summer 2015 project
Bahjat Lab
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Epidermal Growth Factor Receptor(EGFR) Protein
• EGFR: Epidermal Growth Factor Receptor
• EGFRvIII: EGFR with 801 bp deletion• Novel Glycine at junction
EGFRvIII acts as an oncogene. An EGFRvIII-expressing L. monocytogenes clinical strain has been constructed
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Efficient intracellular secretion of 5xEGFRvIII in J774
macrophages
! " ! #$%& '( )* !" #$%&' ( ( !) * %
+++++++! " ! #$%& '( )*
+++++ ++++++! " ! #$%& '( )* !+, - . / 0 12223( !4(
+++++++ + ++++! " ! #$%& '( )* !' , - . / 0 12223( !4(
) * % +, - . / 0 12223( !4(
' , - . / 0 12223( !4(
0 56" $7158- , 9:5; ; 7<=
' 4>?
!>( '
@+>A
3>(
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4B
our “gold-standard”
for intracellular protein
secretion...
Hypothesis: EGFRvIII when included in a construct should help secretion
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Objectives
• Determine the important residues in the EGFRvIII epitope that are relevant for SecA-dependant secretion.
• Construction of L. monocytogenes strains containing vaccinia and EGFRvIII epitopes• Test for secretion and presentation of constructs
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QVACNEW Lm-ΔactA-ΔgcpE Strain
Strain Name Characteristics Strain image
QVACNEW ActAN100, New vaccinia virus epitopes (Vac epitopes), SIINFEKL, EGFRvIII
ActA-N100 Vac epitopes SIINFEKL EGFRvIII
Strain will be a standard we can measure against when testing EGFRvIII mutations
A3L A23R B16RB2R
VAC Epitope
Amino Acid sequence
A3L KSYNYMLL
A23R IGMFNLTFI
B2R YSQVNKRYI
B16R ISVANKIYM
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Protocol
Construct in Lm ΔactA-ΔgcpE
Macrophage Infection/Lysis
Western Blot
B3Z Assay
3) Ligation to pPL2e
2) Digestion with SalI-NotI
Construct in pPL2e
Use of QVACNEW construct as a template
Construct in Top10
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B3Z assay
SIINFEKL in QVACNEW is being presented by dendritic cells
24 hours
Ab
so
rba
nc
e
Clo
ne 5
Clo
ne 6
Clo
ne 9
+ Qva
c Lm
ΔactA
- Lm
ΔactAΔgcpE
0.00
0.05
0.10
0.15
0.20
0.25
Qvac Lm-ΔactA does not contain EGFRvIII
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QVACNEW Lm-ΔactA-ΔgcpE Western
α-ActA
α-P60
α-β-Actin
1 4 5 6 9 + - No
α-ActA: Targets ActA proteinα-P60: Targets secreted Lm proteinα-β-Actin: Targets actin protein
+: QV LmΔactA-: LmΔactAΔgcpE
The QVACNEW protein is secreted into the cytoplasm of RAW264.7 macrophages
p60
ActA-QVACNEW
ActA-Qvac
β-Actin
20KDa
25KDa
55KDa
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Constructed strains
Strain Name Characteristics Strain image
QVACNEW ACTAN100, New vaccinia virus epitopes (Vac epitopes), SIINFEKL, EGFRvIII
QVACNEW-ΔGSC QVACNEW epitopes + GSC deletion in EGFRvIII
QVACNEW-GSC-AAA QVACNEW epitopes,GSC replaced with AAA
QVACNEW-ΔTDH QVACNEW epitopes, TDH deleted
QVACNEW-ΔEGFR QVACNEW epitopes, without EGFRvIII
E-QVACNEW-S QVACNEW strainrearranged to EGFRvIII-QVAC-SIINFEKL
QVACNEW-E-S QVACNEW strain rearranged to QVAC-EGFRvIII-SIINFEKL
ActA-N100 Vac epitopes SIINFEKL EGFRvIII
ActA-N100 Vac epitopes SIINFEKL EGFRvIII-ΔGSC
ActA-N100 Vac epitopes SIINFEKL EGFRvIII-GSC-AAA
ActA-N100 Vac epitopes SIINFEKL EGFRvIII-ΔTDH
ActA-N100 Vac epitopes SIINFEKL
ActA-N100 Vac epitopes SIINFEKLEGFRvIII
ActA-N100 Vac epitopes SIINFEKLEGFRvIII
EGFRvIII epitope: PASRALEEKKGNYVVTDHGSC
A3L A23R B16RB2R
A3L A23R B16RB2R
A3L A23R B16RB2R
A3L A23R B16RB2R
A3L A23R B16RB2R
A3L A23R B16RB2R
A3L A23R B16RB2R
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The different strains showed various levels of SIINFEKL presentation by B3Z assay
24 hoursA
bso
rban
ce
GSC-A
AA
ΔTD
H
ΔG
SC
ΔEG
FR
E-Q
VACNEW
-S
QVACNEW
-E-S + -
0.0
0.1
0.2
0.3GSC-AAA
DTDH
DGSC
DEGFR
E-QVAC-S
QVAC-E-S
+ QVAC Lm DactA
- Lm ΔActAΔgcpE
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Constructs in Western Blot α-ActA Replicate 1
20KDa
25KDa
37KDa
1 42 3 65 7 8 9 10 11 No
M
20KDa
25KDa
37KDa
Lane Strain
1 QVACNEW-ΔEGFR
2 QVACNEW-E-S
3 E-QVACNEW-S
4 QVACNEW
5 QVACNEW-ΔGSC
6 QVACNEW-GSC-AAA
7 QVACNEW-ΔTDH
8 Lm-ΔactA-OVA
9 Lm-ΔactA-Qvac
10 EGFRvIII 5x
11 Lm ΔactA-ΔgcpE
No No Lm
M Marker
α-β-actin
*
*: Unspecific Protein
*
α-P60
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1 42 3 65 7 8 9 10 11 No
Constructs in Western Blot α-ActA Replicate 2
MLane Strain
1 QVACNEW-ΔEGFR
2 QVACNEW-E-S
3 E-QVACNEW-S
4 QVACNEW
5 QVACNEW-ΔGSC
6 QVACNEW-GSC-AAA
7 QVACNEW-ΔTDH
8 Lm-ΔactA-OVA
9 Lm-ΔactA-Qvac
10 EGFRvIII 5x
11 Lm ΔactA-ΔgcpE
No No Lm
M Marker
20KDa
25KDa
37KDa
20KDa
25KDa
37KDa
α-β-actin
*
*
*: Unspecific Protein
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Conclusions
• Location of EGFRvIII does not make a difference in secretion
• Deleting EGFRvIII entirely does effect secretion
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Further studies
• Lysis of QVAC-ΔEGFR Lm-ΔactAΔgcpE strain and western blot to confirm construct translation
• Constructing strains with EGFR5x in different locations to see effect on expression
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Acknowledgments
Keith Bahjat
Alejandro Alice
Gwen Kramer
Shelly Bambina
EACRI staff