Inhibition of Delta-induced Notch signaling using fucose analogs … · 2017-12-12 ·...
Transcript of Inhibition of Delta-induced Notch signaling using fucose analogs … · 2017-12-12 ·...
SupplementaryInformation
InhibitionofDelta-inducedNotchsignalingusingfucoseanalogs
MichaelSchneider1,VivekKumar2#,LarsUlrikNordstrøm3#,LeiFeng3,HideyukiTakeuchi1,4,HuilinHao4,VincentC.Luca5,6†,K.ChristopherGarcia5,6,
PamelaStanley2,PengWu3,7*,RobertS.Haltiwanger1,4*1DepartmentofBiochemistryandCellBiology,StonyBrookUniversity,StonyBrook,
NY11794-52152DepartmentofCellBiology,AlbertEinsteinCollegeofMedicine,1300MorrisPark
Avenue,Bronx,NY104613DepartmentofBiochemistry,AlbertEinsteinCollegeofMedicine,1300MorrisPark
Avenue,Bronx,NY104614ComplexCarbohydrateResearchCenter,UniversityofGeorgia,Athens,GA306025 DepartmentsofMolecularandCellularPhysiologyandStructuralBiology,Stanford
UniversitySchoolofMedicine,Stanford,CA943056HowardHughesMedicalInstitute,Stanford,CA94305
7DepartmentofChemicalPhysiology,TheScrippsResearchInstitute,10550NorthTorreyPinesRoad,LaJolla,CA92037
#Theseauthorscontributedequallytothiswork*Towhomcorrespondenceshouldbesent:[email protected],[email protected]†Presentaddress:DepartmentofDrugDiscovery,H.LeeMoffittCancerCenterand
ResearchInstitute,Tampa,Florida33612
Nature Chemical Biology: doi:10.1038/nchembio.2520
SupplementaryResults
2
SupplementaryFigure1.Additionalperacetylatedfucoseanalogs.(a)StructuresofperacetylatedfucoseanalogsusedinthisstudyinadditiontothoseinFig.1c.(b)StructureofnaturalL-fucose.(c)ModelofcellularuptakeandconversionofperacetylatedfucoseanalogstoGDP-fucoseanalogsincells.
b
SupplementalFig.1a c
Endoplasmic Reticulum
Cytosolic Esterases
Fucose Salvage Pathway
Nature Chemical Biology: doi:10.1038/nchembio.2520
SupplementaryFigure2.MassspectracorrespondingtoEICsinFigure2.Spectraareshownforthetriply-chargedform([M+3H]3+)ofthemolecularionresultingfromthepeptide217LPYVPCSPSPCQNGGTCRPTGDTTHE242frommNotch1EGF6modifiedwithfucoseanalogs.Massspectrometrydataforthispeptidehasbeenpreviouslydescribedwithoutperacetylatedfucoseanalogtreatment31.ToppanelsshowMSspectraat~6.0minandbottompanelsshowMS/MSspectraconfirmingthepeptideassignment.(a)Inthesampletreatedwithcontrolperacetylatedfucose(9),anioncorrespondingtothemassofthispeptideplusthemassofafucosewasidentifiedandtheappropriatepeptideassignmentwasconfirmed.(b-c)Inthesamplestreatedwithtestcompounds10and11weidentifiedionscorrespondingtothemassoftheEGF6peptideplusthemassofthecorrespondingfucoseanalog.Inallcases,fragmentationoftheglycopeptideresultedinneutrallossofthesugar.(d-f)SpectraforpeptidesgrowninthepresenceofLfng.
SupplementalFigure2
-Fng +Lfng a
b
c
d
e
f
Peptide
Fucose
Fucose Analog
GlcNAc
Galactose
Sialic Acid
Nature Chemical Biology: doi:10.1038/nchembio.2520
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SupplementaryFigure3.OtherperacetylatedfucoseanalogsdonotreduceNotchsignaling.(a)EICsshowingrelativeamountsofeachglycoformfromthesamepeptideofmNotch1onEGF6showninFigure2(217LPYVPCSPSPCQNGGTCRPTGDTTHE242)producedbyHEK293Tcellsgrowninthepresenceoftheindicatedperacetylatedfucoseanalog(SeeSupplementaryNote1formassspectra).(b)Co-cultureNotch1reporterassayswereperformedtoevaluatetheeffectsoftheseanalogsonligand-inducedmNotch1signaling.Barsrepresentmean+SEM.Sixbiologicalreplicates(n=6)wereperformedforallexperimentsintwoindependentexperiments.
SupplementalFig.3a
14
5.2 Time [min]0.0
0.5
1.0
1.5
8x10Intens.
13 5.5 6.0 Time [min]
0.0
0.5
1.0
1.5
8x10Intens.
12 6.0 Time [min]
0
2
4
67x10
Intens.
a
0.4
0.8
1.2
1.6
0.0
17.5 18.0 Time [min]
Intens.
X108
18.5
722.8159, 963.4185, 1444.6238
759.3303, 1012.1045, 1517.6527
766.8330, 1022.1080, 1532.6580L PYVPCS PS PCQNGGT CRPT GDT T HE25 20 15 109 8 7 6 5 4 3 2 1
1 2 3 4 5 6 7 8 910 15 20 25 C C T C
211.1443
b2
374.2070
b2
445.2802
a4
473.2760
b4
487.2128
y4
868.3856
y8
1001.4742
b9
+MS2(1022.1080), 17.9541 min+3
Intens.
X104
3.0
2.0
1.0
0.0
200 400 600 800 1000 1200 m/z
Compound 15
Compound 16
3.0
0.0
17.5 18.0 Time [min]
Intens.
X107
18.5
2.0
1.0
722.8159, 963.4185, 1444.6238
759.3303, 1012.1045, 1517.6527
769.5807, 1025.7716, 1538.1534
E.
L PYVPCS PS PCQNGGT CRPT GDT T HE25 20 15 109 8 7 6 5 4 3 2 1
1 2 3 4 5 6 7 8 910 15 20 25 C C T C
Intens.
X104
4.0
2.0
1.0
0.0
200 400 600 800 1000 1200 m/z
+MS2(1025.7716), 19.37 min
3.0
+3
211.1440
b2
308.0901
y2
374.2072
b3
445.2789
a4
473.2749
b4
487.2122
y4
~y17++
Compound 17
L PYVPCS PS PCQNGGT CRPT GDT T HE25 20 15 109 8 7 6 5 4 3 2 1
1 2 3 4 5 6 7 8 910 15 20 25 C C T C
+MS2(1007.4326), 19.36 min+3
Intens.
X104
4.0
2.0
0.0
200 400 600 800 1000 1200 m/z
6.0
211.1435
b2
487.2157
y4
374.2072
b3
445.2802
a4211.1435
b4
857.3622
y8958.4038
b_(n-1)
1007.4407
M
722.8159, 963.4185, 1444.6238
759.3303, 1012.1045, 1517.6527
755.8264, 1007.4326, 1510.6449
1.6
0.4
Intens.
X108
1.2
0.8
0.0
17.5 18.0 Time [min]18.5
0.4
0.8
1.2
1.6
0.0
17.5 18.0 Time [min]
Intens.
X108
18.5
722.8159, 963.4185, 1444.6238
759.3303, 1012.1045, 1517.6527
766.8330, 1022.1080, 1532.6580L PYVPCS PS PCQNGGT CRPT GDT T HE25 20 15 109 8 7 6 5 4 3 2 1
1 2 3 4 5 6 7 8 910 15 20 25 C C T C
211.1443
b2
374.2070
b2
445.2802
a4
473.2760
b4
487.2128
y4
868.3856
y8
1001.4742
b9
+MS2(1022.1080), 17.9541 min+3
Intens.
X104
3.0
2.0
1.0
0.0
200 400 600 800 1000 1200 m/z
Compound 15
Compound 16
3.0
0.0
17.5 18.0 Time [min]
Intens.
X107
18.5
2.0
1.0
722.8159, 963.4185, 1444.6238
759.3303, 1012.1045, 1517.6527
769.5807, 1025.7716, 1538.1534
E.
L PYVPCS PS PCQNGGT CRPT GDT T HE25 20 15 109 8 7 6 5 4 3 2 1
1 2 3 4 5 6 7 8 910 15 20 25 C C T C
Intens.
X104
4.0
2.0
1.0
0.0
200 400 600 800 1000 1200 m/z
+MS2(1025.7716), 19.37 min
3.0
+3
211.1440
b2
308.0901
y2
374.2072
b3
445.2789
a4
473.2749
b4
487.2122
y4
~y17++
Compound 17
L PYVPCS PS PCQNGGT CRPT GDT T HE25 20 15 109 8 7 6 5 4 3 2 1
1 2 3 4 5 6 7 8 910 15 20 25 C C T C
+MS2(1007.4326), 19.36 min+3
Intens.
X104
4.0
2.0
0.0
200 400 600 800 1000 1200 m/z
6.0
211.1435
b2
487.2157
y4
374.2072
b3
445.2802
a4211.1435
b4
857.3622
y8958.4038
b_(n-1)
1007.4407
M
722.8159, 963.4185, 1444.6238
759.3303, 1012.1045, 1517.6527
755.8264, 1007.4326, 1510.6449
1.6
0.4
Intens.
X108
1.2
0.8
0.0
17.5 18.0 Time [min]18.5
0.4
0.8
1.2
1.6
0.0
17.5 18.0 Time [min]
Intens.
X108
18.5
722.8159, 963.4185, 1444.6238
759.3303, 1012.1045, 1517.6527
766.8330, 1022.1080, 1532.6580L PYVPCS PS PCQNGGT CRPT GDT T HE25 20 15 109 8 7 6 5 4 3 2 1
1 2 3 4 5 6 7 8 910 15 20 25 C C T C
211.1443
b2
374.2070
b2
445.2802
a4
473.2760
b4
487.2128
y4
868.3856
y8
1001.4742
b9
+MS2(1022.1080), 17.9541 min+3
Intens.
X104
3.0
2.0
1.0
0.0
200 400 600 800 1000 1200 m/z
Compound 15
Compound 16
3.0
0.0
17.5 18.0 Time [min]
Intens.
X107
18.5
2.0
1.0
722.8159, 963.4185, 1444.6238
759.3303, 1012.1045, 1517.6527
769.5807, 1025.7716, 1538.1534
E.
L PYVPCS PS PCQNGGT CRPT GDT T HE25 20 15 109 8 7 6 5 4 3 2 1
1 2 3 4 5 6 7 8 910 15 20 25 C C T C
Intens.
X104
4.0
2.0
1.0
0.0
200 400 600 800 1000 1200 m/z
+MS2(1025.7716), 19.37 min
3.0
+3
211.1440
b2
308.0901
y2
374.2072
b3
445.2789
a4
473.2749
b4
487.2122
y4
~y17++
Compound 17
L PYVPCS PS PCQNGGT CRPT GDT T HE25 20 15 109 8 7 6 5 4 3 2 1
1 2 3 4 5 6 7 8 910 15 20 25 C C T C
+MS2(1007.4326), 19.36 min+3
Intens.
X104
4.0
2.0
0.0
200 400 600 800 1000 1200 m/z
6.0
211.1435
b2
487.2157
y4
374.2072
b3
445.2802
a4211.1435
b4
857.3622
y8958.4038
b_(n-1)
1007.4407
M
722.8159, 963.4185, 1444.6238
759.3303, 1012.1045, 1517.6527
755.8264, 1007.4326, 1510.6449
1.6
0.4
Intens.
X108
1.2
0.8
0.0
17.5 18.0 Time [min]18.5
17 16 15
= = =
Peptide
Fucose
Fucose Analog
Dll1
Dll4
Jag1
0.0
0.5
1.0
1.5
Ligand
RLU
EV
9121314151617
ns ns
nsb
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SupplementaryFigure4.FucoseanalogsareeffectiveatnanoMolarconcentrationsandhaveminimaleffectoncellsurfaceexpressionofNotch.(a)Dll1(black)andJag1(grey)expressingcellscauseasimilarfold-increaseinNotch1activationrelativetoemptyvector(EV)controlfortheNotch1co-cultureluciferasereporterassayntheabsenceoffucoseanalogs.(b)InhibitionofDll1-inducedmNotch1signalingwasexaminedatvaryingconcentrationsofperacetylatedfucoseanalogs.(c)CellsurfaceexpressionofmNotch1wasdeterminedwithflowcytometryusingananti-NECDantibodyaftertreatmentoftransfectantswiththeindicatedcompounds.(d)Inhibitionof3T3cellproliferationisnotaffectedbyaninhibitorydoseoffucoseanalogs.Threebiologicalreplicateswereusedforeachconditioninallexperiments(n=3).
EVDMSO 9
0
5
10
15
20
25
Dll1 vs. Jag1
Treatment
RLU
Dll1
Jag1
EVDMSO 9 10 11
0.0
0.5
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Treatment
Not
ch1
- PE
*
24 48 720.0
0.5
1.0
1.5
Time (h)
Rel
ativ
e Ab
sorb
ance
(570
)
3T3 Cell Proliferation
DMSO
91011
10 100 1000 100000.0
0.5
1.0
Analog concentration (nM)
RLU
91011
SupplementalFig.4a b
c d
EVDMSO 9
0
5
10
15
20
25
Dll1 vs. Jag1
Treatment
RLU
Dll1
Jag1
Nature Chemical Biology: doi:10.1038/nchembio.2520
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SupplementaryFigure5.LunaticFringepartiallyrescuesDll-mediatedNotchsignalinginhibitionbyfucoseanalogs.Co-cultureassaysrunintheabsence(black)orpresence(grey)ofLunaticFringe(Lfng)showtheeffectsofLfngonligand-inducedNotch1(a-c)andNotch2(d-f)signalingfollowingincorporationofcontrolversusinhibitoryfucoseanalogs.Allbarsrepresentmean±SEM(n=9).*p<0.05,**p<0.01,***p<0.001,Sidak’smultiplecomparisontestadjustedpvalues.
EVDMSO 9 10 11
0.0
0.5
1.0
1.5
2.0
Dll1-N2
Treatment
RLU
nsns
*****
-Fng
+Lfng
EVDMSO 9 10 11
0.0
0.5
1.0
1.5
Dll4-N2
Treatment
RLU
ns ns
*** **
-Fng
+LFng
EVDMSO 9 10 11
0.0
0.5
1.0
1.5
Treatment
RLU
Jag1-N2
*** *** ***
***
-Fng
+LFng
EVDMSO 9 10 11
0.0
0.5
1.0
1.5
2.0
Treatment
RLU
Dll1-N2
* *
*** ***
-Fng
+Lfng
EVDMSO 9 10 11
0
1
2
3
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5
DLL1
Treatment
RLU
*** ***
* *
-Fng
+LFng
EVDMSO 9 10 11
0.0
0.5
1.0
1.5
Dll4-N1
Treatment
RLU
ns ns
*** **
-Fng
+LFng
EVDMSO 9 10 11
0.0
0.5
1.0
1.5
2.0
Jag1
Treatment
RLU
*** *** *** ***
-Fng
+LFng
a b
e
c
f
SupplementalFig.5
d
Nature Chemical Biology: doi:10.1038/nchembio.2520
7
SupplementaryFigure6.LunaticFringepartiallyrescuesDll-Notchbindinginhibitionbyfucoseanalogs.Flowcytometryassaysofcellsincubatedintheabsence(black)orpresence(grey)ofLunaticFringe(Lfng)toexaminetheeffectsofLfngonligand-bindingtoNotch1(a-c)andNotch2(d-f)followingincorporationofcontrolversusinhibitoryfucoseanalogs.Sixexperimentswereperformedforallsamples(n=6,blackbars)andthreeexperimentswereperformedforall+Lfngsamples(n=3,graybars).Allbarsrepresentmean±SEM.*p<0.05,**p<0.01,***p<0.001,Sidak’smultiplecomparisontestadjustedpvalues.
EVDMSO 9 10 11
0.0
0.5
1.0
1.5
Treatment
Dll4-PE
Dll4-N2 -Fng
+Lfng
ns ns* *
EVDMSO 9 10 11
0.0
0.5
1.0
1.5
2.0
Treatment
Jag1-PE
Jag1-N2 -Fng
+Lfngns ns
nsns
EVDMSO 9 10 11
0.0
0.5
1.0
1.5
2.0
Treatment
Dll1-PE
Dll1-N2 -Fng
+Lfng
* *
* *
EVDMSO 9 10 11
0.0
0.5
1.0
1.5
2.0
Treatment
Dll1-PE
Dll1-N1 -Fng
+Lfng******
*** ***
EVDMSO 9 10 11
0.0
0.5
1.0
1.5
Treatment
Dll4-PE
Dll4-N1 -Fng
+Lfng
ns ns
ns ns
EVDMSO 9 10 11
0.0
0.5
1.0
1.5
Treatment
Jag1-PE
Jag1-N1 -Fng
+Lfng
ns ns
*** ***
a
d
b
e
c
f
SupplementalFig.6
Nature Chemical Biology: doi:10.1038/nchembio.2520
8
SupplementaryFigure7.ModificationofNotchreceptors,notNotchligands,withfucoseanalogsisresponsibleforNotchsignalinginhibition.(a)CellsexpressingfulllengthDll1andgrowninthepresenceoftheindicatedcompoundswereincubatedwithasolubleformofmNotch1EGF1-13-Fc(containingtheligand-bindingdomainofNotch1)andbindingwasevaluatedusingflowcytometry.Dataarerepresentativeofthreeindependentexperiments.(b)Bargraphshowsmeanfluorescentintensities(MFIs)oftheresultsinpanela.(c)SolubleNotchligandsgeneratedintheabsenceoffucoseanalogswereusedtocoatcellculturedishes.Luciferase-basedsignalingassayswereperformedfortransfectantsgrownondishescoatedwithDll1,Dll4,orJag1inthepresenceoftheindicatedcompound.Allbarsrepresentmean±SEM(n=3).*p<0.05,**p<0.01,***p<0.001,Tukeypost-hoctest,adjustedpvalues.
EVDMSO 9 10 11
0.0
0.5
1.0
1.5
Treatment
NEC
D -
PE
ns
EVDMSO 9 10 11 EV
DMSO 9 10 11 EV
DMSO 9 10 11
0.0
0.5
1.0
1.5
2.0
Treatment
RLU
Dll1
Dll4
Jag1
*** ***ns
NECD - PE
EV
DMSO 9 10 11
Cel
l cou
nt
a b
c
SupplementalFig.7
Nature Chemical Biology: doi:10.1038/nchembio.2520
9
SupplementaryFigure8.StructuralmodelingshowsnostericclashbetweeninhibitoryfucoseanalogsandJag1orbetweenfucoseanalogsatEGF12andDll4.(a)FucoseanalogswerealignedwiththenaturallyoccurringNotch1O-linkedfucosemodificationspresentattheNotch1(EGF8-12)-Jag1(N-EGF3)bindinginterface(PDBID:5UK5).ZoomedpanelsdepictselectedJag1residuesadjacenttoO-fucosemodificationsofNotch1EGF8andEGF12.ThedistancebetweentheC7carbonofeachmodeledanalogandthenearestJag1contactisindicatedasadottedgreyline.Notably,noneoftheanalogsarepredictedtoclashwithJag1.(b)FucoseanalogswerealignedwithNotch1O-linkedfucosemodificationswithinthebindinginterfaceoftheNotch1(EGF11-13)-Dll4(N-EGF1)complexstructure(PDBID:4XL1).ZoomedpanelsdepictselectedDll4residuesadjacenttoO-fucosemodificationsNotch1EGF12.ThedistancebetweentheC7carbonofeachmodeledanalogandthenearestDll4contactisindicatedasadottedgreyline.Notably,noneoftheanalogsarepredictedtoclashwithDll4atthissite.
C7
T466 E81
Fuc466 (alkene)
Y82 3.5Å
C7
T466 E81
Fuc466 (alkyne)
Y82 4.5Å
Fuc466
C6
E81
Y82
T466
Jag1 Notch1
Fuc311
T311
Q304 N298
N314
Fuc311 (alkyne)
T311
Q304 N298
N314
2.8Å C7
Fuc311 (alkene)
T311
Q304 N298
N314
3.6Å
C7
Fucose analogs were aligned with Notch1 O-linked fucose modifications within the binding interface of the Notch1(EGF8-12)-Jag1(N-EGF3) complex structure (PDB ID: 5UK5). Zoomed panels depict selected Jag1 residues adjacent to O-fucose modifications Notch1 EGF8 and EGF12. The distance between the C7 carbon of each modeled analog and the nearest Jag1 contact is indicated as a dotted grey line. Notably, none of the analogs are predicted to clash with Jag1.
EGF8
EGF12
C6
DLL4
Notch1
EGF12
Y65
H64 T466
Fuc466
C6
Fuc466 (alkene)
C7 3.3Å 4.3Å C7
Y65
H64
Y65
H64 T466 T466
Fuc466 (alkyne)
Fucose analogs were aligned with Notch1 O-linked fucose modifications within the binding interface of the Notch1(EGF11-13)-DLL4(N-EGF1) complex structure (PDB ID: 4XL1). Zoomed panels depict selected DLL4 residues adjacent to O-fucose modifications Notch1 EGF12. The distance between the C7 carbon of each modeled analog and the nearest DLL4 contact is indicated as a dotted grey line. Notably, none of the analogs are predicted to clash with DLL4.
a
b
Nature Chemical Biology: doi:10.1038/nchembio.2520
10
SupplementaryFigure9.Gatingstrategiesforflowcytometryexperiments:(a)GatingstrategyfortheligandbindingexperimentsinHEK293Tcells.LivecellswereselectedusingSSCandFSC.GFPexpresserswereselectedtoisolatecellsexpressingthehighestlevelsoftransfectedNotch.TypicalhistogramsusedtocalculateMFIsinmaintextandsupplementaryfiguresareshowninthefarrightpanel.(b)GatingstrategyforpurificationofLin-Sca-1+cKit+(LSK)cellsfrombonemarrow.BonemarrowcellswerepreparedandincubatedwithantibodiesforFlowcytometryasdescribedinonlinemethods.SSC-AandFSC-Agateswereusedforlymphocytes(P1).SSC-W,FSC-H,FSC-W,andSSC-Hwereusedtogateoutdoubletcells.DeadcellswereexcludedwithDAPI.Livecells(P4)weretakenforlineagenegativegating(StreptavidinPE-Cy7lo).cKitAPChiandSca-1PEhigates(P6)wereusedforthedetectionandisolationofLSKcells.IsolatedLSKcellswere>90%pureandwereusedforLSKdifferentiationassay.(c)GatingstrategyforLSKcelldifferentiationassay.PurifiedLSKcellsfrombonemarrowwereco-culturedwithOP9-GFP,OP9-Dll1andOP9-Dll4stromalcells.Onday8,co-cultureswereharvestedandfixedwith4%paraformaldehyde(PFA).Fixedcellswereusedforflowcytometry.GFP-positive(stromal)cellsweregatedout.OnGFP-negative(non-stromal)cells,SSCandFSCwereusedtogatelymphocytes.FSC-AandFSC-Wwereusedtogatesingletcells.CD44-PEloandCD25-PerCPCy5.5higateswereusedforthedetectionofdifferentiatedCD25+T-cells.
EV
9
10
a
SSC-A(x1000)
FSC-A(x1000)
LymphocyteGate
SSC-W(x1000)
FSC-H(x1000)
SSCSinglet
FSC-W(x1000)
SSC-H(x1000)
FSCSinglet
DAPI
FSC-A(x1000)
LivecellGate
SSC-A(x1000)
StreptavidinPE-Cy7
Lineage(-)Gate
cKitAP
C
Sca-1PE
LSKGate(P6)
#Ce
lls
GFP
GFP(-)Gate
SSC
FSC
LymphocyteGate
FSC-A
FSC-W
SingletGate
CD44
-PE
CD25-PerCPCy5.5
b
c
Nature Chemical Biology: doi:10.1038/nchembio.2520