Exploring the Lipid Association of ITIM-containing Molecules

Ibrahim VazirabadAugust 23rd, 2011

ECM

Introduction to the Role of Platelets in Hemostasis • Platelets are small anucleate blood cells that travel through the bloodstream.• If the blood vessel wall becomes injured, extra-cellular matrix becomes exposed.• Platelets have many receptors that bind to various constituents of the matrix.

As the platelets pass, they will encounter ECM and adhere to it through their various receptors that recognize matrix proteins.

• Eventually, a thrombus will form at the site of injury.

Proposed Model for Initiation of PECAM-1-Mediated Inhibitory Signaling in Response to GPVI Engagement

6

43

1

2

SS

SS

SS

SS

SS

SS

5

Y663

Y686

SS

SS

PECAM-1

GPVI

Collagen

Kin

ase

SH2

SH3

Fyn/Lyn

FcRg

S-S

CRP

SHP-2

Phos

phot

ase

Phos

phot

ase

SH2

SH2

Ca2+

DAG

IP3

Platelet Activation

Syk

SH2

Kin

ase

SH2

• The cytoplasmic domain of PECAM-1 contains two ITIMs (Immunoreceptor Tyrosine Inhibitory Motifs) which when phosphorylated, recruit phosphatases which oppose platelet activation.

Mori J.,et al., The Journal of Biological Chemistry. 2008; 283: 35419-35427

• One such part of the extra-cellular matrix is collagen.

• A receptor on platelets, GPVI, binds to collagen. GPVI is associated with the FcRγ chain, which has an ITAM (immunoreceptor tyrosine-based activation motif), which positively regulates platelet activation and thrombus formation.

Paddock, C.P., et al., Blood 117:6012, 2011.

PECAM-1 is also known to have a lipid-associated segment which interacts with the plasma membrane. S707 is constitutively phosphorylated in resting platelets, and when S702 is phosphorylated, the PECAM-1 cytoplasmic domain begins to dissociate fromthe plasma membrane. Paddock, C.P.,et al., Blood. 2011; 117: 6012-6023

Activation of a tyrosine kinase will phosphorylate Y686, which then allowsphosphorylation of Y663.

Other ITIM-containing Molecules on Platelets

6

43

1

2

SS

SS

SS

SS

SS

SS

5

Y663

Y686

SS

SS

PECAM-1

GPVI

Collagen

Kin

ase

SH2

SH3

Fyn/Lyn

FcRg

S-S

CRP

Ca2+

DAG

IP3

Platelet Activation

Syk

SH2

Kin

ase

SH2

There are other ITIM-containing molecules expressed on platelets that are suspected to associate with the plasma membrane in an analogous way to PECAM-1:

CEACAM-1G6bBTLT-1

21 S

S

SS

SS

SS

3

Y493

Y520

4 SS

Y211

Y237

1 SS

Y246

Y282

1

CEACAM-1(Carcinoembryonic antigen cell adhesion molecule-1) is a Ig superfamily glycoprotein that also negatively regulates platelet signaling through the GPVI Fc receptor γ chain. Wong, C., et al., Blood. 2009; 113: 1818-1828

G6b-B also negatively regulates platelet activation through the GPVI Fc receptor γ chain, but it also interacts with the C-type lectin-like receptor 2 (CLEC-2).

Mori J.,et al., The Journal of Biological Chemistry. 2008; 283: 35419-35427

TLT-1(TREM-like Transcript-1) is found in the α-granules of platelets and their progenitor, the megakaryocyte. When platelets become activated, TLT-1 migrates to the platelet surface to promote platelet aggregation. Morales, J., et al., Blood Coagulation & Fibrinolysis. 2010;21: 229-236

CEACAM-1

G6bB TLT-1

Evidence for PECAM-like Cytoplasmic Behavior in other ITIM molecules

Immediately after the PECAM C-terminal ITIM are two positively charged residues that are hypothesized to allow the cytoplasmic domain to attach to the plasma membrane.

Therefore, existence of these basic residues in the cytoplasmic domain of the other three ITIM-containing molecules is evidence of similar plasma membrane interaction.

ID Sequence pI ++PECAM-1C TVYSEVRKAVPDAVESRYSR 9.1 YesPECAM-1N VQYTEVQVSSAESHKDLGKK 7.5 Yes CEACAM-1C IIYSEVKKQ 9.3 YesCEACAM-1N VTYSTLNFEAQQPTQPTSAS 3.9 No G6bB-C TIYAVVV 5.2 No G6bB-N LLYADLDHLALSRPRRLSTA 9.3 YesTLT1-C VTYATVIFPGGNKGGGTSCG  8.5 NoTLT1-N TTYTSLPLDSPSGKPSLPAP 6.3 No

+ residues ITIM/S-T Other residues

CEACAM and G6bB appear to interact with membrane. TLT-1 does not.

Do CEACAM,TLT-1, and G6b-B associate with the membrane in an analogous way to PECAM-1?

Based on sequencing data, we hypothesize that both G6bB and CEACAM-1 associate with the plasma membrane. TLT-1 will not.

General Project Steps: First, the cytoplasmic domains of the three ITIM-containing molecules

will be cloned into vectors. Then, NMR will be performed on the cytoplasmic domains:

1. In aqueous solution

2. With lipid micelles This will allow understanding whether membrane interaction takes

place.

Step 1:Preliminary Work First two proteins under study: G6bB and TLT-1. We received plasmids from:

1. G6b-B plasmid: Dr. Yotis Senis

2. TLT-1 plasmid: Dr. Valance Washington Dr. Senis sequenced plasmid before sending it, Dr. Washington did not,

so TLT-1 plasmid was sequenced. No surprises. We then transformed E. Coli with the two plasmids and harvested DNA

with the QIAGEN Miniprep Kit.

Step 2: Isolating Cytoplasmic Domains and PCR Primers were designed that would amplify the cytoplasmic domain portion of the

two proteins. RE sites added to allow digest in any vector.

G6bB:Forward: 5’-ATT GGA TCC TGG CTG CAC AGG CGC CTG CCC-3’Reverse: 5’-CCC AGG CTT TCA AAC TAC AAC TGC ATA GAT-3’

TLT-1: Forward: 5’- ATT GGA TCC ATG GCC AAG AAG AAA CAA GGG-3’Reverse: 5’-CCC AGG CTT GCT GGA TGG AGT CTG ATT GTT-3’Red- BamHI site Green-HindIII site

After primers were created, the cytoplasmic domains were amplified via PCR. The product was then ran on a gel.

PCR Product Gel and Extraction

Expected Sizes of Fragments

TLT-1cyto : 390 bp

G6b-Bcyto : 234 bp

Success. TLT-1 and G6BB bands were then removed from the gel according to the QIAGEN gel extraction kit.

100

bp la

dder

TLT-

1 cyto

G6B

b cyto

1kb

ladd

er

Step 3: Ligation of PCR product

The extracted PCR products were then ligated into a new plasmid, called pCR-Blunt II-TOPO.

Used as it is very easy to insert products into and digest with RE.

The plasmids were then transformed into E. Coli, plated on agar.

Ten colonies picked. (6 G6bB, 4 TLT-1)

Step 4: Quality Control The TOPO vector has EcoRI restriction sites on either side of the product insert. Allows for an easy confirmation of successful ligation. All ten clones were digested by EcoRI.

Reaction Mix:

2 ul of NEB buffer 4400 ng plasmid0.5 ul of EcoRI HF (20U/ul)x ul of water (20 ul total)

------------------------------------------------------------------1.5 hr digestion, 37C

Gel was loaded to visualize digest.

EcoRI Digestion Gel

Expected Sizes of Fragments

TLT-1cyto : 408 bp

G6BBcyto : 252 bp

• No 100 bp ladder, mistake. • The fragments “look” correct however.

1kb ladder TLT-1 #1-4 G6Bb #1-6

uncut plasmids

• Next step is to cut with the BamHI and HindIII sites to ligate into another vector

500 bp

Step 5: BamHI and HindIII Double Digestion One G6bB, one TLT-1 clone were double digested by BamHI and HindIII,

along with final plasmid, called pQE30 GB1.

Reaction Mix:

10 ul of NEB buffer 27 ug plasmid DNA2 ul of HindIII (20U/ul)

2 ul of BamHI (20U/ul)

x ul of water (100 ul total)

3 hr digestion, 37C

Agarose gel was loadedto visualize the double digest.

His Tag: purification, Ni column. GB-1 Domain: Known chem. shift in NMRTEV: protease which removes first two.

BamHI-HindIII Double Digest Gel

1kb

ladd

erTL

T-1(

uncu

t)G

6Bb(

uncu

t)PQ

E30(

uncu

t)

TLT-

1cyt

o

G6B

bcyt

o

PQE3

0

100

bp la

dder

Expected Sizes of Fragments

TLT-1cyto : 390 bp, 18 bp, 45 bp

G6BBcyto : 234 bp, 18 bp, 45 bp

PQE30 plasmid: 15 bp

• The double digest was unsuccessful. New England Biosciencediscouraged a double digest, so not surprising.

• Ordered the High Fidelity versions of BamHI and HindIII, which are guaranteed to work together.

1 kb

500 bp

200 bp

3 kb

2 kb1.5 kb

Troubleshooting We were unsure of the EcoRI digestion, so

it was repeated, this time with a 100bp ladder.

• Both REs are active.Success.

• Correct bands.Success.

To see whether the BamHI or HindIII

enzymes were inactive, this gel was run.

BamHI-HindIII Double Digest Gel: Repeated

1kb

ladd

er

TLT-

1(un

cut)

G6B

b(un

cut)

PQE3

0(un

cut)

TLT-

1cyt

o

G6B

bcyt

o

PQE3

0

100

bp la

dder

500 bp

200 bp

1000 bp

Expected Sizes of Fragments

TLT-1cyto : 390 bp, 18 bp, 45 bp

G6BBcyto : 234 bp, 18 bp, 45 bp

PQE30 plasmid: 15 bp

• Perfect. A gel with large wells will be loaded to maximize product separated.

Large Well Double Digest Gel

100

bp la

dder

uncu

t TLT

-1 p

lasm

id

TLT-

1 do

uble

dig

est

100

bp la

dder

uncu

t G6b

B pl

asm

id

G6B

b do

uble

dig

est

100

bp la

dder

uncu

t TLT

-1 p

lasm

id

TLT-

1 do

uble

dig

est

Digest Reaction:10 ul of NEB buffer 25 ug plasmid DNA2 ul of HindIII (20U/ul)2 ul of BamHI (20U/ul) x ul of water (100 ul total)

---------------------------------------3 hr digestion, 37C

• G6Bb band did not show up. • G6Bb band runs at ~250 bp, a dark band can be

seen at ~250 bp (marked in red). This is loading dye.

500 bp300 bp

Future Plans1. Ligate G6bB and TLT-1 into PQE30 vector.

2. Cut out to ensure successful ligation.

3. Sequence to make sure no mutations.

CEACAM plasmid has just been sequenced. Repeat process.

Appendix: PCR Protocol5 ul of 2ng/ul DNA

5 ul of forward and reverse primers(5 um solution of each)

1 ul of dNTPs(100 mM, 25 mM of each dNTP)

10 ul 10x PCR buffer

73 ul H2O

0.5 ul PFU Turbo (High Fidelity DNA polymerase)

PCR program:

94°C 5’

65°C 3’

72°C 3’

94°C 1’ Bold repeated for 30 cycles

65°C 3’

72°C 7’

10°C ∞

Appendix: Ligation Reaction4 ul of DNA product

1 ul of provided salt solution

1 ul of PCRII Blunt TOPO plasmid vector

Incubate mixture for 5 minutes at room temperature.

Then transform plasmid into OneShot Chemically Competent cells.

Aliquot 50 ul of competent E.coli, thaw on ice. Add 10 ng of plasmid, let sit for 30 minutes on ice. Heat shock mixture for 30 seconds, then let sit on ice for two minutes. Add 950 ul of LB broth to the mixture. Incubate on shaker at 250 rpm for

one hour at 37 C. Add 100ug/ml ampicillin to the mixture, let incubate for 1 hour. Add to 250 ml flask of LB-amp broth.

Appendix: Transformation Protocol

Appendix: Sequencing ProtocolMixture: 0.5 ug of plasmid DNA2ul of primer stock at 1.6 uMX ul of dH2O2 ul of 5x sequencing buffer4 ul of Big Dye Terminator Kit20 ul total volume

Thermocycler Protocol:96°C 30 sec50°C 15 sec60°C 4 min25 cycles

4°C ∞