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:pengwu@scripps.edu,rhalti@uga.edu†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

4

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

Nature Chemical Biology: doi:10.1038/nchembio.2520

5

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

1.0

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

6

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

4

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