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Supplementary Information

Native enrichment of the Drosophila O-GlcNAc proteome reveals candidate

conveyors of the supersex combs phenotype

Nithya Selvan1,4+

, Ritchie Williamson1,5+

, Daniel Mariappa1+

, David G. Campbell1, Robert Gourlay

1, Andrew T.

Ferenbach1, Tonia Aristotelous

3, Iva Hopkins-Navratilova

3, Matthias Trost

1,6 and Daan M. F. van Aalten

2*

1MRC Protein Phosphorylation and Ubiquitylation Unit,

2Division of Gene Regulation and Expression and

3Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee,

UK

4Present address: Complex Carbohydrate Research Center, University of Georgia, Athens, USA

5Present address: School of Pharmacy, Faculty of Life Sciences, University of Bradford, Bradford, UK

6Institute for Cell and Molecular Biosciences (ICaMB), Newcastle University, Newcastle-upon-Tyne, UK

+These authors contributed equally to this work

*Correspondence to: [email protected]

Nature Chemical Biology: doi:10.1038/nchembio.2404

mailto:[email protected]

Supplementary Results

Supplementary Figures

Supplementary figure 1: Full size blots of Western blot sections depicted in Fig. 1b.

WB: O-GlcNAc RL2

WB: TAB1

55

45

IN IN IN IN

Unmodified TAB1

O-GlcNAcylated TAB1

Unmodified TAB1

CpOGAD298N pull-down

CpOGAD298N, D401A

pull-down

MW (kD)

55

45


Supplementary figure 2: Binding affinity of CpOGAD298N

for an N-linked GlcNAc containing peptide.

Fluorescence polarization assay showing no detectable displacement of a fixed concentration of fluorescent

probe from CpOGAD298N

by increasing concentrations of an N-linked GlcNAc containing peptide derived from

human Cathepsin D. Experiments were performed in triplicate and error bars represent standard error of the

mean. Dose response for the O-linked GlcNAc containing peptide from Drosophila HCF was previously

reported18

and is shown here for comparison.


Supplementary figure 3: SPR sensorgrams for the binding of CpOGAD298N

to GlcNAc and GlcNAc(β-

1,4)GlcNAc. GlcNAc was injected in duplicates at various concentrations between 2-500 μM and GlcNAc(β-

1,4)GlcNAc was injected in duplicates at concentrations between 0.02-6.6 mM. RU, relative units.


Supplementary figure 4: Binding affinity of CpOGAD298N

for Thiamet G. Dose-response curves from

fluorescence polarization assay showing the displacement of a fixed concentration of fluorescent probe from

CpOGAD298N

by increasing concentrations of Thiamet G. Highest amount of probe bound to CpOGAD298N

in the

absence of competing inhibitors was arbitrarily set as 100%. Data points were fitted to a three-parameter

equation for dose-dependent inhibition using Prism (GraphPad). Experiments were performed in triplicate and

error bars represent standard error of the mean. Kd values for peptides were calculated from EC

50 values as

described previously18

.


Supplementary figure 5: Ponceau S stained membrane of the Western blot in Fig. 2b.

Ponceau S

loading control

170130

95

75

55

44

35

26

MW (kDa) IN FT

EL

FT

EL

D401A Halo-CpOGAX : - D298N D298N,

Pull-down


Supplementary figure 6: Eluates of CpOGA pull downs from HeLa cell lysates. Eluates from CpOGAD298N

pull down but not CpOGAD298N,D401A

pull down contain O-GlcNAcylated proteins detected by the anti-O-GlcNAc

antibody RL2.

D401A

Halo-CpOGAX : D298N D298N,

Pull-down

170

130

95

75

MW (kDa)

Eluates

WB: O-GlcNAc

RL2


Supplementary figure 7: Pathway analysis of proteins identified by CpOGAD298N

– only those pathways in

which a total of eight or more proteins were identified are shown. Uniprot accessions of significantly enriched

proteins (in CpOGAD298N

pulldown vs. control pulldown) provided in Supplementary Dataset 3 were used as

input for analysis on PANTHER database.

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Supplementary figure 8: Example EThcD fragmentation spectra for HexNAc modified peptides from

Grunge (a) and mop (b). Peptide fragments were assigned using Mascot and Proteome Discoverer 2.0.

Signals of charged reduced species of the precursor and neutral losses associated with it in the centre of the

spectrum were filtered. C and z ions that contain a HexNAc modification are denoted in the spectra by an

asterisk above and to the left of the ion number. Neutral loss of the HexNAc oxonium ion (m/z 204.08) is also

indicated by an asterisk.


Supplementary figure 9: Quantitation of the genetic interaction between OGT/sxcH537A

and Gug

03928 or

mopT482

alleles. Both wings from adult flies (n=124-388) of the respective genotypes were assessed for the

short L5 wing vein phenotype and the percentage of flies with a wing vein defect in at least one wing is

represented in the graph. Fewer of the double heterozygotes, OGT/sxcH537A

/+;Gug03928

/+ or

OGT/sxcH537A

/+;mopT482

/+ display this phenotype as compared to OGT/sxcH537A

;Gug03928

/+ or

OGT/sxcH537A

;mopT482

/+ flies, respectively.


Supplementary Tables

Supplementary Table 1: Proteins from HeLa cell lysates identified by both this study and a previously

published study (Nandi et al., 2006). Proteins identified in only one of the two studies under comparison are not

shown.

Protein name/Fasta header Gene symbol

ATP-citrate synthase ACLY

actin, gamma 1 propeptide ACTG1

Annexin 1 ANXA1

Calregulin CALR

CAPZB CAPZB

beta-coat protein COPB1

carbamoyl-phosphate synthetase 1 CPS1

DEAD (Asp-Glu-Ala-Asp) box polypeptide 1 DDX1

DEAD (Asp-Glu-Ala-Asp) box polypeptide 21 DDX21

eukaryotic translation elongation factor 2 EEF2

eukaryotic translation initiation factor 3 subunit 9 eta EIF3B

eukaryotic translation initiation factor 4A, isoform 1 EIF4A1

eukaryotic translation initiation factor 4 gamma, 1 isoform 4 EIF4G1

alpha enolase ENO1

filamin 1 (actin-binding protein-280) FLNA

Glucose 6 phosphate 1 dehydrogenase G6PD

glucose phosphate isomerase GPI

heterogeneous nuclear ribonucleoprotein K Hnrnpk

heterogeneous ribonuclear particle protein U HNRNPU

heat shock 90kda protein 1, alpha HSP90AA1

heat shock protein HSPA2

heat shock protein 70 HSPA4

heat shock 70kDa protein 8 isoform 2 HSPA8

heat shock 70kDa protein 9B HSPA9

IQ motif containing GTPase activating protein 1 IQGAP1

karyopherin (importin) beta 1 KPNB1

microtubule-associated protein 4 (MAP4) MAP4

P105mcm (MCM6) MCM6

malate dehydrogenase (EC 1.1.1.37), cytosolic MDH1

mitochondrial malate dehydrogenase precursor MDH2

moesin/anaplastic lymphoma kinase fusion protein MSN

myosin heavy chain nonmuscle form A MYH9

splicing factor homolog NONO

poly(rC) binding protein 1 Pcbp1

poly(rC)-binding protein 2 isoform b PCBP2


Chain A, Human Platelet Profilin PFN1

M2-type pyruvate kinase PKM

peroxiredoxin 4 (thioredoxin peroxidase) PRDX4

HMT1 hnRNP methyltransferase-like 2 isoform 3 PRMT1

proteosome beta 2 subunit PSMB2

ribosomal protein L15 RPL15

ribosomal protein S23 RPS23

EBNA-2 co-activator (100kD) SND1

threonyl-tRNA synthetase TARS

elongin C TCEB1

TCP1, subunit 3 (gamma) (HSP60 family) TCP1

tubulin, beta polypeptide 4, member Q TUBB

Tu translation elongation factor, mitochondrial TUFM

tyrosine 3/tryptophan 5-monooxgenase activation protein, gamma polypeptide YWHAG


Supplementary Table 2: Proteins (identified from HeLa cells in this study) with mapped HexNAc sites.

Protein Accessions Gene/Protein Name

A0A024QZW7 NUP153/Nucleoporin 153kDa, isoform CRA_a

A0A024R899 MNAB/Membrane associated DNA binding protein, isoform CRA_a

A6NEM2 HCFC1/Host cell factor 1

B2RAH5 Protein phosphatase 1 regulatory subunit

D3DUP1 WNK1/WNK lysine deficient protein kinase 1, isoform CRA_b

E7EPN9 PRRC2C/Protein PRRC2C

F8VRH0 PCBP2/Poly(rC)-binding protein 2

O75179 ANKRD17/Ankyrin repeat domain-containing protein 17

O95487-3 SEC24B/Protein transport protein Sec24B

P02545 LMNA/Pre-Lamin A/C

P35658-2 NUP214/Nuclear pore complex protein Nup214

P52594-4 AGFG1/Arf-GAP domain and FG repeat-containing protein 1

P53992 SEC24C/Protein transport protein Sec24C

Q14157-1; Q14157-5 UBAP2L/Ubiquitin-associated protein 2-like

Q14444 CAPRIN1/Caprin-1

Q14686 NCOA6/Nuclear receptor coactivator 6

Q15637-5 SF1/Splicing factor 1

Q2KHR3 QSER1/Glutamine and serine-rich protein 1

Q59FC9; A0A024RDE8 PDLIM5/PDZ and LIM domain 5, isoform CRA_c

Q5JSZ5 PRRC2B/Protein PRRC2B

Q5T6F2 UBAP2/Ubiquitin-associated protein 2

Q8IWZ3-6 ANKHD1/Ankyrin repeat and KH domain-containing protein 1

Q8NDX5-7 PHC3/Polyhomeotic-like protein 3

Q8WU79 SMAP2/Stromal membrane-associated protein 2

Q96KR1 ZFR/Zinc finger RNA-binding protein

Q99700 ATXN2/Ataxin-2

Q9P0H6 AD-012 protein (UBA-like)

Q9ULT8 HECTD1/E3 ubiquitin-protein ligase HECTD1

Q9Y6Y8 SEC23IP/SEC23-interacting protein


Supplementary Table 3: Comparison of proteins with mapped HexNAc sites identified by Hahne et al., 2013

(from HEK293 cells) to proteins with mapped HexNAc sites identified in this study (from HeLa cells). Proteins

in which O-GlcNAc (dehydration due to beta elimination) sites were identified by Hahne et al., 2013 are shaded

in green, those (HexNAc neutral loss by HCD or +HexNAc by ETD) identified in this study are in yellow and

proteins identified in both are shaded in pink. Site mapped proteins from Hahne et al. 2013 which were also

identified (but not site-mapped) in this study are indicated with an asterisk in the first column.

Gene Description Sites Unique peptides Previously reported

AGFG1 Arf-GAP domain and FG repeats-containing protein 1 2 2 dbOGAP

ANKRD17 Ankyrin repeat domain-containing protein 17 1 1 dbOGAP

HCFC1 Host cell factor 1 22 14 dbOGAP

NUP153 Nuclear pore complex protein Nup153 12 7 dbOGAP

NUP214 Nuclear pore complex protein Nup214 7 3 dbOGAP

QSER1 Glutamine and serine-rich protein 1 2 1 dbOGAP

SEC24B Protein transport protein Sec24B 3 2 Hahne et al. 2012 (1)

UBAP2 Ubiquitin-associated protein 2 2 1 dbOGAP

UBAP2L Ubiquitin-associated protein 2-like 5 2 dbOGAP

WNK1 Serine/threonine-protein kinase WNK1 1 1 dbOGAP

ZFR Zinc finger RNA-binding protein 2 1 dbOGAP

ANKHD1 Ankyrin repeat and KH domain-containing protein 1 1 1

ATXN2 Ataxin-2 7 1 dbOGAP

CAPRIN1 Caprin-1 8 1 dbOGAP

HECTD1 E3 ubiquitin-protein ligase HECTD1 1 1 Alfaro et al. 2012

LMNA Pre-Lamin A/C 4 1 dbOGAP

MNAB Membrane associated DNA binding protein, isoform CRA_a 2 1 dbOGAP

NCOA6 Nuclear receptor coactivator 6 1 1 Alfaro et al. 2012

PCBP2 Poly(rC)-binding protein 2 3 1 dbOGAP

PDLIM5 PDZ and LIM domain 5, isoform CRA_c 2 1 Alfaro et al. 2012

PHC3 Polyhomeotic-like protein 3 2 1 dbOGAP

PPP1R12A Protein phosphatase 1 regulatory subunit 10 2 dbOGAP

PRRC2B Protein PRRC2B 1 1 Alfaro et al. 2012

PRRC2C Protein PRRC2C 3 1 Alfaro et al. 2012

SEC23IP SEC23-interacting protein 3 1 dbOGAP

SEC24C Protein transport protein Sec24C 2 1 dbOGAP

SF1 Splicing factor 1 2 1 dbOGAP

SMAP2 Stromal membrane-associated protein 2 2 1 Alfaro et al. 2012

AD-012 protein (UBA-like) 4 1

BAT2L2 * Protein BAT2-like 2 1 1 Alfaro et al. 2012

ADRM1* Proteasomal ubiquitin receptor ADRM1 5 1 Trinidad et al. 2012

AHNAK Neuroblast differentiation-associated protein AHNAK 2 1 dbOGAP

AKAP9 A-kinase anchor protein 9 2 1

ARID3B AT-rich interactive domain-containing protein 3B 1 1 Hahne et al. 2012 (1)

BAT2L1* Protein BAT2-like 1 2 1 Hahne et al. 2012 (1)

CBL* E3 ubiquitin-protein ligase CBL 1 1

CHMP5 Charged multivesicular body protein 5 1 1

CIC* Protein capicua homolog 1 1 dbOGAP

DAZAP1/MEF2D DAZAP1/MEF2D fusion protein 1 1

DKFZp313L0918

Putative uncharacterized protein DKFZp313L0918 (Fragment) 1 1


EAP1 Enhanced at puberty protein 1 2 1 Alfaro et al. 2012

EIF4G1* Eukaryotic translation initiation factor 4 gamma 1 1 1 dbOGAP

ELF2 ETS-related transcription factor Elf-2 5 4 Myers et al. 2011

EMSY* Protein EMSY 3 3 Wang et al. 2010

EP400* E1A-binding protein p400 1 1 Myers et al. 2011

FIP1L1 Pre-mRNA 3'-end-processing factor FIP1 2 1 Wang et al. 2010

FNBP4* Formin-binding protein 4 1 1 Wang et al. 2010

GATAD2A Transcriptional repressor p66-alpha 1 1 Trinidad et al. 2012

HIVEP1* Zinc finger protein 40 1 1 dbOGAP

HNRNPA1L2 Heterogeneous nuclear ribonucleoprotein A1-like 2 2 1

HNRNPF* Heterogeneous nuclear ribonucleoprotein F 1 1 Zaro et al. 2011

HNRNPK* Heterogeneous nuclear ribonucleoprotein K 1 1 dbOGAP

KIAA1310 Uncharacterized protein KIAA1310 1 1 Wang et al. 2010

LIN54* Protein lin-54 homolog 1 1 dbOGAP

MGA* MAX gene-associated protein 4 2 dbOGAP

MLL3 Histone-lysine N-methyltransferase MLL3 1 1

MPP4 MAGUK p55 subfamily member 4 1 1

NCOR1* Nuclear receptor corepressor 1 1 1 dbOGAP

NEFL Neurofilament light polypeptide 4 1 dbOGAP

NEFM Neurofilament medium polypeptide 4 2 dbOGAP

NFRKB* Nuclear factor related to kappa-B-binding protein 2 2 dbOGAP

NONO* Non-POU domain-containing octamer-binding protein 1 1 dbOGAP

NUMA1 Nuclear mitotic apparatus protein 1 1 1 dbOGAP

PCBP1* Poly(rC)-binding protein 1 2 1 dbOGAP

PHF21A PHD finger protein 21A 3 2 dbOGAP

POGZ* Pogo transposable element with ZNF domain 3 1 dbOGAP

POM121* Nuclear envelope pore membrane protein POM 121 1 1 Wang et al. 2010

PRKCSH* Glucosidase 2 subunit beta 1 1 Trinidad et al. 2012

RBM12* RNA-binding protein 12 3 1 Hahne et al. 2012 (1)

RBM14* RNA-binding protein 14 3 3 dbOGAP

RBM16 RNA-binding protein 16 1 2 Hahne et al. 2012 (1)



RFX5 DNA-binding protein RFX5 2 1

RPRD2 Regulation of nuclear pre-mRNA domain-containing protein 2 3 2 dbOGAP

RRP1B Ribosomal RNA processing protein 1 homolog B 1 1 dbOGAP

SAP30BP SAP30-binding protein 10 2 dbOGAP

SCML2 Sex comb on midleg-like protein 2 1 1

SH3RF1* E3 ubiquitin-protein ligase SH3RF1 2 1 Trinidad et al. 2012

SON Protein SON 1 1 dbOGAP

SPEN* Msx2-interacting protein 3 3 dbOGAP

SRCAP* Helicase SRCAP 1 1 dbOGAP

SRRM2 Serine/arginine repetitive matrix protein 2 1 1 Wang et al. 2010

TAB1* TGF-beta-activated kinase 1 and MAP3K7-binding protein 1 5 1 Trinidad et al. 2012

TAF4* Transcription initiation factor TFIID subunit 4 3 1 Wang et al. 2010

TMPO Lamina-associated polypeptide 2, isoforms beta/gamma 1 1 Hahne et al. 2012 (1)

TOX4* TOX high mobility group box family member 4 1 1

UBQLN2* Ubiquilin-2 3 1


VIM* Vimentin 1 1 dbOGAP

YTHDF1* YTH domain family protein 1 2 1 dbOGAP

ZC3H14 Zinc finger CCCH domain-containing protein 14 1 1 dbOGAP

ZHX1* Zinc fingers and homeoboxes protein 1 2 1 dbOGAP

ZMYND8* Protein kinase C-binding protein 1 1 1

ZNF281 Zinc finger protein 281 1 1 dbOGAP

ZNF609* Zinc finger protein 609 1 1 PhosphositePlus

ZYX* Zyxin 1 1 Zaro et al. 2011


Supplementary Table 4: Statistically significantly enriched cellular compartments represented by the

proteins identified (from Drosophila embryos) – output from PANTHER. p < 0.05, with the Bonferroni

correction for multiple testing applied. Fold enrichment values over the Drosophila reference genome (as

annotated in the PANTHER database as of July 2015) are given. The reference genome contains 13690

proteins. No. of proteins identified in the experiments that were recognized by PANTHER; 2040 of 2358.

Uniprot accessions of significantly enriched proteins (in CpOGAD298N

pulldown vs. control pulldown) provided in

Supplementary Dataset 3 were used as input for analysis on PANTHER database.

Analysis Type: PANTHER Overrepresentation Test (release 20150430)

Annotation Version and Release Date:

PANTHER version 10.0 Released 2015-05-15

Reference List: Drosophila melanogaster (all genes in database-REFLIST)

Bonferroni correction: TRUE

PANTHER GO-Slim Cellular Component

Drosophila melanogaster - REFLIST (13690)

Input (2040) Input (expected)

Input (over (+)/underrepresented(-))

Input (fold Enrichment)

Input (P-value)

extracellular matrix (GO:0031012)

56 31 8.34 + 3.71 5.17E-08

vesicle coat (GO:0030120) 24 13 3.58 + 3.64 3.77E-03

extracellular region (GO:0005576)

266 85 39.64 + 2.14 6.74E-09

cytoplasm (GO:0005737) 454 116 67.65 + 1.71 1.32E-06

macromolecular complex (GO:0032991)

544 136 81.06 + 1.68 3.07E-07

protein complex (GO:0043234) 438 107 65.27 + 1.64 3.56E-05

intracellular (GO:0005622) 1378 308 205.34 + 1.5 3.24E-11

cell part (GO:0044464) 1548 337 230.67 + 1.46 5.53E-11

organelle (GO:0043226) 992 205 147.82 + 1.39 8.44E-05

Unclassified (UNCLASSIFIED) 11807 1597 1759.41 - 0.91 0.00E+00


Supplementary Table 5: Statistically significantly enriched protein classes represented by the proteins

identified (from Drosophila embryos) – output from PANTHER. p < 0.05, with the Bonferroni correction for

multiple testing applied. Fold enrichment values over the Drosophila reference genome (as annotated in the

PANTHER database as of July 2015) are given. The reference genome contains 13690 proteins. No. of

proteins identified in the experiments that were recognized by PANTHER; 2040 of 2358. Uniprot accessions of

significantly enriched proteins (in CpOGAD298N

pulldown vs. control pulldown) provided in Supplementary

Dataset 3 were used as input for analysis on PANTHER database.

Analysis Type: PANTHER Overrepresentation Test (release 20150430)

Annotation Version and Release Date:

PANTHER version 10.0 Released 2015-05-15

Reference List: Drosophila melanogaster (all genes in database)

Bonferroni correction: TRUE

PANTHER GO-Slim Cellular Component

Drosophila melanogaster - REFLIST (13690)

Input (2040) Input (expected)

Input (over (+)/underrepresented(-))

Input (fold Enrichment)

Input (P-value)

vesicle coat protein (PC00235) 25 13 3.73 + 3.49 2.57E-02

immunoglobulin superfamily cell adhesion molecule (PC00125)

32 15 4.77 + 3.15 2.52E-02

membrane-bound signaling molecule (PC00152)

69 31 10.28 + 3.01 2.45E-05

cysteine protease (PC00081) 34 15 5.07 + 2.96 4.77E-02

cell adhesion molecule (PC00069)

143 63 21.31 + 2.96 2.57E-11

glycosidase (PC00110) 44 19 6.56 + 2.9 1.03E-02

extracellular matrix protein (PC00102)

151 63 22.5 + 2.8 2.54E-10

metalloprotease (PC00153) 143 58 21.31 + 2.72 5.67E-09

extracellular matrix glycoprotein (PC00100)

47 19 7 + 2.71 2.38E-02

RNA helicase (PC00032) 57 22 8.49 + 2.59 1.41E-02

translation initiation factor (PC00224)

66 23 9.83 + 2.34 4.24E-02

helicase (PC00115) 106 36 15.8 + 2.28 1.53E-03

membrane traffic protein (PC00150)

150 48 22.35 + 2.15 2.68E-04

chaperone (PC00072) 108 34 16.09 + 2.11 1.17E-02

glycosyltransferase (PC00111) 141 44 21.01 + 2.09 1.33E-03

cation transporter (PC00068) 187 53 27.87 + 1.9 2.34E-03

ligase (PC00142) 252 64 37.55 + 1.7 8.42E-03

RNA binding protein (PC00031)

660 165 98.35 + 1.68 3.27E-08

protease (PC00190) 464 113 69.14 + 1.63 9.33E-05

hydrolase (PC00121) 1183 287 176.28 + 1.63 8.41E-14

transporter (PC00227) 703 162 104.76 + 1.55 1.05E-05

receptor (PC00197) 675 150 100.58 + 1.49 2.51E-04

transferase (PC00220) 776 172 115.63 + 1.49 4.78E-05

DNA binding protein (PC00009)

407 90 60.65 + 1.48 3.80E-02

nucleic acid binding (PC00171)

1342 289 199.98 + 1.45 4.27E-08

Unclassified (UNCLASSIFIED) 7506 769 1118.5 - 0.69 0.00E+00

G-protein coupled receptor 207 9 30.85 - 0.29 6.48E-04


(PC00021)

homeobox transcription factor (PC00119)

85 1 12.67 - < 0.2 7.84E-03

helix-turn-helix transcription factor (PC00116)

85 1 12.67 - < 0.2 7.84E-03


Supplementary Table 6: Total number of high confidence Peptide Sequence Matches (PSMs), with and

without HexNAc, (from Drosophila embryos) identified in each of the three replicate experiments.

Sample PSMs [Total ] PSMs [with HexNAc] PSMs [by ETD, with HexNAc]

Ctrl1 CpOGAD298N,D401A

4289 1 0


5647 2 0


4435 0 0

Total Ctrl 14371 3 0

Test1 CpOGAD298N

16345 94 17

Test2 CpOGAD298N

18988 103 7

Test3 CpOGAD298N

21399 89 8

Total Test 56732 268 32


Supplementary Table 7: Proteins (from Drosophila embryos) with mapped HexNAc sites.

Protein Accession Protein names

Q8SWR8 Ataxin-2 homolog (Datx2)

Q7KMM4 BcDNA.GH04962 (CG14476, isoform A) (CG14476, isoform B) (CG14476, isoform C) (CG14476, isoform D) (CG14476, isoform E) (EC 3.2.1.20)

Q9Y0Z1 BcDNA.LD23876 (CG11970, isoform A) (CG11970, isoform B)

Q9VCI4 CG10217, isoform A (CG10217, isoform B) (LD32918p)

Q9VMM2 CG11034, isoform B (CG11034, isoform C)

Q9VGL0 CG14712 (LD43047p)

Q9VW34 CG32209-PB (FI03450p) (Serpentine, isoform C) (EC 3.5.1.41)

Q8INH6 CG32473, isoform C (EC 3.4.11.-) (FI18373p1)

Q6NR09 CG33993, isoform A (RE08107p)

Q7K3E2 CG5080, isoform A (CG5080, isoform B) (CG5080, isoform C) (LD34147p)

B7Z0D3 CG7546, isoform D

Q9VAU1 CG9990, isoform A (EC 3.6.1.3)

Q9W3Q5 Checkpoint suppressor homologue, isoform A (Checkpoint suppressor homologue, isoform B) (Checkpoint suppressor homologue, isoform D)

Q24368 Chromatin-remodeling complex ATPase chain Iswi (EC 3.6.4.-) (CHRAC 140 kDa subunit) (Nucleosome-remodeling factor 140 kDa subunit) (NURF-140) (Protein imitation swi)

Q9V9X0 FI18240p1 (Nyobe, isoform C) (Nyobe, isoform D)

Q7JXF5 GH12838p (Nucleoporin 62kD, isoform A) (Nucleoporin 62kD, isoform B)

Q8MLT4 Gp150, isoform D

Q86BH2/M9NE54 Grunge, isoform C/G (EC 3.5.1.-)

A0A0B4KFJ8 Histone demethylase 4B, isoform E (EC 1.14.11.27)

Q9V4C8 Host cell factor (dHcf) [Cleaved into: HCF N-terminal chain; HCF C-terminal chain]

P12080 Integrin alpha-PS2 (Position-specific antigen subunit alpha-2) (Protein inflated) [Cleaved into: Integrin alpha-PS2 heavy chain; Integrin alpha-PS2 light chain]

A0A0B4KEI6 Jun-related antigen, isoform C

Q00174 Laminin subunit alpha (Laminin A chain)

Q9VXM5 LD22609p (Tay bridge, isoform A) (Tay bridge, isoform B) (Tay bridge, isoform C)

Q9V3N1 LD24467p (MIP27581p) (Serpin 27A) (Serpin 27A, isoform A) (Serpin 27A, isoform B)

Q7K0D8 LD27030p (Nucleoporin 50kD)

A0A0B4KFC5 Lingerer, isoform K

Q9W2U7 LP18708p (No circadian temperature entrainment, isoform A) (No circadian temperature entrainment, isoform B) (No circadian temperature entrainment, isoform C)

A1Z877 LP19846p (Nidogen/entactin, isoform A) (Nidogen/entactin, isoform C)

Q9VUH6 Myopic (EC 3.1.3.48)

Q7KTF8 Nicotinic acetylcholine receptor alpha6, isoform E

A8JV18 Nucleoporin 153kD, isoform D

Q9VDV3 Probable nucleoporin Nup58

Q9VWN5 Protein FAM188A homolog (Protein CARP homolog)

Q9W2E7 Rae1

A1Z6S6 RE54322p (Visceral mesodermal armadillo-repeats, isoform B)

Q9VWL7 Rho GTPase activating protein at 18B, isoform C

Q9VCD1 Rootletin, isoform D (Rootletin, isoform E) (Rootletin, isoform F)

P13607 Sodium/potassium-transporting ATPase subunit alpha (Na(+)/K(+) ATPase alpha subunit) (EC 3.6.3.9) (Sodium pump subunit alpha)

Q9VI72 Spindle assembly abnormal 4 ortholog

Q7JQR3 Sulfhydryl oxidase (EC 1.8.3.2)

B7Z060 TBP-associated factor 4, isoform E

E1JI40 Vermiform, isoform I (EC 3.5.1.41)


Supplementary Table 8: Genetic interaction between OGT/sxcH537A

and Gug03928

or mopT482

. The number of

flies scored for the short L5 wing vein phenotype in each of the genotypes is shown with the percent of flies

displaying the phenotype in one or both the wings listed.

Genotype Number of flies Percent Short L5 wing vein defect in:

(n) 1 wing 2 wings

CRISPR control 388 0.0 0.0

sxcH537A

/sxcH537A

302 0.0 0.0

Gug03928

/+ 124 0.0 0.0

sxcH537A

/ +; Gug03928

/+ 180 1.7 0.6

sxcH537A

/ sxcH537A

; Gug03928

/+ 232 9.1 4.7

mopT482

/+ 139 0.0 0.0

sxcH537A

/ +; mopT482

/+ 250 0.8 0.0

sxcH537A

/ sxcH537A

; mopT482

/+ 162 7.4 0.6


Supplementary Dataset legends (Datasets provided in the attached Excel (.xlsx) files)

Supplementary Dataset 1: All proteins (from HeLa cells) identified in this study. Label-free mass

spectrometry quantitation of three independent replicates using MaxQuant. Proteins significantly enriched (4-

fold, p<0.05; t-test) in the CpOGAD298N

pull down compared to the control CpOGAD298N,D401A

pulldown are indicated

with a ‘+’ in the first column (T-test significant).

Supplementary Dataset 2: HexNAc peptides identified (from HeLa cells) in this study.

Supplementary Dataset 3: All proteins (from Drosophila embryos) identified in this study. Label-free

mass spectrometry quantitation of three independent replicates using MaxQuant. Proteins significantly

enriched (4-fold, p<0.05; t-test) in the CpOGAD298N

pull down compared to the control CpOGAD298N,D401A

pulldown

are indicated with a ‘+’ in the first column (T-test significant).

Supplementary Dataset 4: HexNAc peptides identified (from Drosophila embryos) in this study.

Potential NXS/T motifs within the peptides are shown in red font.


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