A Novel O-glycoprotease with Applications in O-glycan Analysis using Mass SpectrometryGENOVIS PRESENTS

AUTHORS

INTRODUCTION

SUMMARY

RESULTS

REFERENCES

The study of O-linked glycosylation within the field of glycomics is known to be a challenging task due to complicated sample preparations and analytical procedures. The field has suffered from lack of O-glycan-

A new endo-O-protease was discovered in Akkermansia muciniphila, a com-mensal bacterium in the human microbiota. The bacteria colonize distal ileum to rectum and degrade and metabolize highly O-glycosylated mucin. The O-protease (OgpA) was recombinantly expressed in E. coli and purified to homogeneity (OpeRATORTM, Genovis). The activity and specificity of the pro-tease was evaluated by digestion of several protein substrates at native con-ditions. The O-protease displayed activity on an asialylated protein, but the activity was markedly increased on O-glycoproteins pretreated with sialidas-es (A. muciniphila). It was found that the protease only digests proteins deco-rated with O-glycans. No activity was observed on non-glycosylated proteins nor on proteins with only N-glycan structures (Fig. 1).

Rolf Lood, Maria Nordgren, Fredrik Leo, Stephan Björk, Malin Mejàre, Helén Nyhlén, Fredrik Olsson | Genovis AB, Lund, Sweden

• Aim: To demonstrate applications of the OgpA and the inactive OgpA in O-glycan analysis using MS

• OgpA is a new, specific endo-O-glycoprotease

1. Valmu L, Alfthan H, Hotakainen K, Birken S, Stenman UH. Site-specific glycan analysis of human chorionic gonadotropin beta-subunit from malignancies and pregnancy by liquid chromatography–electrospray mass spectrometry. Glycobiology. 2006; 16:1207–18.

specific enzymes and efficient enrichment methods. A novel O-glycan-specific endoprotease was discovered in Akkermansia muciniphila and expressed in E. coli. This O-glycoprotease (OgpA) digests the peptide backbone N-

• Immobilized and inactive OgpA selectively binds O-glycosylated proteins and peptides at native conditions

• Selective separation of O-glycan peptides from non- and only N-glycosylated peptides

• Selective enrichment of O-glycoproteins was achieved from human serum

• Site-specific O-glycan characterization of peptides from human protein was achieved after enrichment on the O-glycan binding resin

www.genovis.com

terminally of serine or threonine residues carrying an O-glycan. A catalytically inactive form of OgpA with retained O-glycan binding was also developed. This inactive OgpA was evaluated for selective affinity purification of O-glyco-

proteins from various protein samples. Characterization of O-glycoproteins utilizing both OgpA and the inactive OgpA are demonstrated on both simple and complex protein mixtures.

New O-glycan-specific Protease

OgpA - + - + - + - +

Etanercept Abatacept Fetuin IgA

- + - +

Albumin IgG

Sialidase

Sialidase

OgpA

O- and N-glycosylated Non-

glycosylated N-glycosylated

20

30

40

50

60

kDa

S/T

Figure 1. Specificity for O-glycosylated proteins. The OgpA digests only O-glycosylated proteins. No activity was observed on a non-glycosylated protein, or on a protein with only N-glycans. The proteins were pre-treated with sialidases and incubated O/N with the OgpA. The samples were analyzed by reduced SDS-PAGE.

To further verify the specificity of the O-protease, the O-glycosylated pro-teins erythropoietin (EPO) and human chorionic gonadotropin beta chain (hCGβ) were used as substrates and digested by OgpA at native conditions. EPO is a protein with a single core 1 O-glycan and a specific digestion site N-terminally of the O-glycosylated serine126 was defined (Fig. 2).

hCGβ is a glycoprotein with four O- and two N-glycans, with a constant core 2 at Serine121,1. Digestion of this protein with OgpA was done at native conditions with and without sialidase, and the RP-LC-MS/MS result confirms digestion also at core 2 (Fig. 3).

25 30 35 40 45 50 55 60 Time [min]

0

10

20

30

Intens.[mAU]

SAAPLR...ACRTGD

APPRLI...SPPDAA

126

1 125

Theor. mass: 13714.0957 Da Meas. mass: 13714.1199 Da ∆ppm: 1.76

Theor. mass: 4900.5546 Da Meas. mass: 4900.5868 Da ∆ppm: 6.57

Figure 2. Specific digestion N-terminally of the O-glycosylation site. N-glycans were removed by PNGaseF and sialic acids were removed by a sialidase mix (A. muciniphila) in parallel to the diges-tion with OgpA. The EPO protein carrying one core 1 O-glycan was hydrolyzed at a single specific site N-terminally of the O-glycosylated Serine126. After digestion the fragments were reduced by DTT and separated on a RP BEH C4 column (Waters) followed by ESI-Q-TOF MS detection.

Applications of the O-protease in LC-MS

The unique specificity for O-glycosylated residues on glycoproteins opens for a variety of applications using this new O-protease (OgpA) in the glycopro-teomic field. The OgpA was used to map glycosylation sites of the biophar-maceutical etanercept, an Fc-fusion protein with a highly core 1 O-glycosyl-ated region. By using the workflow illustrated in Fig. 4, peptides with intact O-glycans, glycopeptides without sialic acids and peptides lacking sugar residues were generated. The intact mass fragments and MS/MS peptides completed the amino acid sequence of etanercept and defined the O-glycosylated serine and threonine residues. A summary of the obtained data is presented in Fig. 5.

+ OgpA+ PNGaseF

+ Sialidase mix

ST

S

T

T

+ O-glycosidase

Separation using HILIC / RP-LC

Mass spectrometry

Figure 4. Workflow from intact glycoprotein to LC-MS and LC-MS/MS. In a native one pot O/N digestion reaction, the glycoprotein is hydrolyzed N-terminally of O-glycosylated sites using the OgpA. Addition of Sialidase mix is optional but improves digestion. In a second step, the remaining GalNAc/Gal can be removed from the asialylated glycopeptides using an O-glycosidase (Streptococcus Oralis). After reduction, the samples were analyzed on RP or HILIC-LC ESI-Q-TOF MS and MS/MS. PNGaseF is added in the O/N digestion step to reduce heterogeneity.

Inactive O-protease

The OgpA protein was engineered for high affinity binding to O-glycosylated proteins and peptides without hydrolyzing the peptide bonds at O-glycosylat-ed serines and threonines.

The inactive OgpA was recombinantly expressed in E. coli, purified to ho-mogeneity and immobilized on agarose beads to create an affinity resin for O-glycoproteins and -peptides (GlycOCATCHTM, Genovis). The specificity and selectivity of the resin were tested by incubation of protein solutions with the resin in presence of the sialidase mix, with end-over-end mixing in mi-crospin columns. After washing the columns, the bound proteins were eluted with 8 M urea. The resin specifically bound proteins carrying O-glycans, sep-arately but also in a mix of proteins. Negligible binding of non- or N-glycosyl-ated proteins were observed (Fig. 6). To study the O-glycan selectivity of the resin at the peptide level, a selection of non-glycosylated peptides and an O-glycosylated peptide were used, and the eluted fraction contained solely the core 1 O-glycosylated peptide (Fig. 7).

Load

260

160

110

80

60

50

40

30

20

15

10

Flowthrough

EluatekDa Ladder

260

160

11080

60

50

40

30

20

15

10

Aflibercept

TNFR

BSA

AGP

ApoE

IgG Fc

kDa Ladder LoadFlowthrough

Eluate

260

160

11080

60

50

40

30

20

15

10

kDa Ladder LoadFlowthrough

Eluate

CetuximabAflibercept

BSAAGP

Carbonic anhydrase

TNFR

a) b) c)

Figure 6. Selectivity of immobilized inactive OgpA for O-glycan proteins. Different proteins and a mix of proteins were tested on the O-glycan-binding resin. Load, flowthrough and eluate were analyzed on SDS-PAGE. The resin with inactive OgpA binds a) heavily glycosylated TNFR, b) spe-cifically O-glycosylated proteins TNFR and Apolipoprotein E (ApoE) in presence of N- and non- gly-cosylated proteins. c) No binding to proteins lacking O-glycans. TNFR: TNF alpha binding domain, BSA: bovine serum albumin, AGP: alpha-1 acid glycoprotein.

0.0

0.5

1.0

1.5

2.08x10

Intens.

0.0

0.5

1.0

1.5

8x10Intens.

0.0

0.5

1.0

1.5

10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 Time [min]

8x10Intens.

Load

Flowthrough

Eluate

Glycodrosocin H2686H4062 IOB

SialEXO

H8390

TiC

Figure 7. Selective purification of an O-glycosylated peptide. A mix of five synthesized peptides were tested on the O-glycan-binding resin. The data show selective purification of the O-glycan containing peptide. The fractions were analyzed by LC-MS. RP separation was performed on an AdvanceBio Peptide Map C18 column (Agilent) and analyzed by ESI Q-TOF on a Bruker Impact II MS.

Applications of OgpA and O-glycan-binding Resin in LC-MS

The O-glycan affinity columns were used for specific enrichment of O-glycosylated peptides and proteins prior to MS analysis. Using a workflow for tryptic peptides from human IgA, peptides with various degree of glyco-sylation were specifically enriched on the O-glycan-binding resin and detect-ed in the eluate (Fig. 8). The strong binding ability of inactive OgpA to O-gly-can structures was also effectively utilized to enrich O-glycosylated proteins from the complex sample mixture of human serum (Fig. 9).

Further digestion of the enriched proteins with a combination of trypsin and active OgpA generated peptides with N-terminally glycosylated serines and threonines, which enabled site-specific O-glycan characterization (Fig. 10).

E K L G G A E V A V T C T V F Q T Q P V T S Q P Q P E G A N E A V P T P V V D P D A P P S P P L G A P G L P P A G S P P D S H V L L A A P P G H Q L H R A Y H D L R H T F M G V V S L G S P S G E V S H P R K T R T V V Q P V G A A A G P V V P P C P G I E H F K V

240 250 260 270 280 350 360290 300 310 320 330 340S R

Figure 10. O-glycan sites of Alpha-2-HS-Glycoprotein in serum. After enrichment of O-glycoproteins from serum as described above, the sample was in addition digested with both trypsin and active OgpA. Separation, detection and data handling was done according to Fig 9, with the addition of OgpA enzyme in protein search. The OgpA-specific digestion N-terminally of glycosylated serines and threonines pro-vides additional information on the actual O-glycan sites. The figure and table present a selection of the peptides and glycopeptides attained to demonstrate the data this workflow can generate also from very complex sample mixtures. In this test, peptides 270-279, 270-292 and 270-311 carrying 2x HexNAcHex indicate that S280 and S293 is glycosylated.

Range Sequence Measured Mass

z Expected mass Mass ∆ppm Glycosylation site

Indicated Glycosylation site

Glycoform

238 - 255 K.LGGAEVAVTCTVFQTQPV.T 626.3150 3 1875.9400 - 9.08 - T 256 -

256 - 269 V.TSQPQPEGANEAVP.T 895.3940 2 1788.7901 - 9.35 T 256 T 270 1 x HexNAcHex

256 - 269 V.TSQPQPEGANEAVP.T 597.2683 3 1788.7901 - 3.93 T 256 T 270 1 x HexNAcHex

270 - 279 P.TPVVDPDAPP.S 686.8192 2 1371.6293 - 3.97 T 270 S 280* 1 x HexNAcHex

270 - 292 P.TPVVDPDAPPSPPLGAPGLPPAG.S 828.7474 3 2483.2319 - 4.64 T 270 S 293* 1 x HexNAcHex

270 - 311 P.TPVVDPDAPPSPPLGAPGLPPAGSPPDSHVLLAAPPGHQLHR.A 972.6827 5 4858.4189 - 8.59 T 270 S 280* or S 293* 2 x HexNAcHex

270 - 311 P.TPVVDPDAPPSPPLGAPGLPPAGSPPDSHVLLAAPPGHQLHR.A 899.6561 5 4493.2867 - 9.47 T 270 - 1 x HexNAcHex

318 - 337 R.HTFMGVVSLGSPSGEVSHPR.K 694.3419 3 2080.0160 - 5.83 - - -

341 - 361 R.TVVQPSVGAAAGPVVPPCPGR.I 672.6885 3 2015.0622 - 9.22 T 341* - -

341 - 367 R.TVVQPSVGAAAGPVVPPCPGRIRHFKV.- 548.7080 5 2738.5166 - 4.74 T 341* - -

346 - 361 P.SVGAAAGPVVPPCPGR.I 619.6342 3 1855.8986 - 9.64 S 346 - 1 x HexNAcHex

*not previously reported, according to Uniprot entry P02765

0 20 40 60 80 100 120 140

Ig alpha-1 chain C region Apolipoprotein B-100

Kininogen-1 Complement C4-B Complement C4-A

Complement C3 Inter-alpha-trypsin inhibitor heavy chain H4 Inter-alpha-trypsin inhibitor heavy chain H2

Plasminogen Inter-alpha-trypsin inhibitor heavy chain H1

Serum albumin Alpha-2-macroglobulin

Fibronectin Complement C1s subcomponent

N-acetylmuramoyl-L-alanine amidase Plasma protease C1 inhibitor

Prothrombin C4b-binding protein alpha chain

Coagulation factor XII Complement C1r subcomponent

Ig delta chain C region Apolipoprotein F

Complement component C7 Ig mu chain C region

Inter-alpha-trypsin inhibitor heavy chain H3 Histidine-rich glycoprotein

Complement component C9 Alpha-2-HS-glycoprotein

Protein AMBP Ig gamma-1 chain C region

Alpha-1-antitrypsin Plasma kallikrein

Vitronectin Apolipoprotein A-I

Vitamin K-dependent protein S Serotransferrin

Apolipoprotein E von Willebrand factor

Proteoglycan 4

# Peptides other protein# Peptides O-glycosylated protein

260

160110

80

60

50

40

30

20

15

10

kDa Ladder LoadFlowthrough

Eluate+ Sialidase mix

LC-MS/MS

Binding / washing

Urea elution

+ OgpA

S

T

S

T

+ Trypsin

S

T

TS

Figure 9. Enrichment of O-glycoproteins from human serum. a) Workflow for O-glycan characterization in protein samples. b) Human serum (Sigma) and sialidase were incubated on the inactive OgpA resin. Load, flow-through and eluted fractions were visualised on SDS-PAGE. c) The eluted proteins were denatured, reduced, alkylated and trypsin digested. Peptides were analyzed using RP-LC-MS/MS on a C18 column (Advance BioPeptide Plus 2.1x150mm 2.7 µm from Agilent Technologies) in a 0.1%FA in MQ: 0.1% FA in 95% ACN gradient at 45°C and a flow of 0.2 ml/min. Detection was on an ESI-Q-TOF Bruker Im-pact II instrument. Data was converted to mgf format files in Data Analysis Software v 4.4 and searched against the Swiss Prot database. Core-1/ core-2 structures, methionine oxidation and N-terminal acetyla-tion was set as variable modifications and carbamidomethylation as fixed. Identified O-glycosylated proteins and other proteins with >12 matching peptides and a MASCOT score >200 are listed.

• The OgpA and the O-glycan-binding resin are useful tools for O-glycan analysis using MS

8 -

8 -

8 - 185

8 - 183

1 - 185

1 - 183 226 - 467

L P A Q V A F T P Y -- T S P T R S M A P G A V H L P Q P V S T R S Q H T Q P T P E P S T A P S T S F L L P M G P S P P A E G S T G D E P K S C D K T H T C P P -- P G K

1 8 11-180 184 186 199 200 202 208 212 216 217 226 243 245 467

N-terminal , TNF receptor O-glycosylated region C-terminal, Fc fusion

249-464

215

205

199

---

---

---

---

---

---

---

---

---

Inta

ct m

ass

on

larg

er fr

agm

ents

MS/

MS

on p

eptid

es

( ) ( ) ( )

Figure 5. OgpA digestion in LC-MS analysis of etanercept. a) Both larger C- and N-terminal frag-ments as well as smaller O-glycosylated fragments were generated using the OgpA. The hetero-geneity in O-glycosylation led to overlapping peptides. b) Selected mass data of peptides cover-ing the entire amino acid sequence. c) Typical MS/MS spectrum with the HexNAc still attached to the peptide (in ESI-Q-TOF-MS, the glycan is often lost from the peptide before fragmentation and found as oxonium ions). RP separation was performed on an AdvanceBio Peptide Map C18 col-umn from Agilent and samples were desalted in-line prior to ESI Q-TOF on a Bruker Impact II MS. HILIC separation was performed on a Waters Acquity BEH Amide column after buffer exchange on Graphite carbon.

Inte

nsity

pep

Range Sequence G. CompositionMeasured

MassExpected

Mass ∆ppm

1-7 -.LPAQVAF.T - 744.412 744.417 - 7.39

1-185 -LPA...PVS.R - 19842.873 19842.895 - 1.07

184 - 198 P.TRSMAPGAVHLPQPV.S - 1559.813 1559.824 - 7.12

186 - 199 R.SMAPGAVHLPQPVS.T - 1405.699 1405.702 - 2.13

186 - 204 R.SMAPGAVHLPQPVSTRSQH.T 2729.256 2729.27 - 5.06

200 - 207 S.TRSQHTQP.T - 953.47 953.468 + 1.99

205 - 207 H.TQP.T - 344.172 344.17 + 5.81

208 - 216 P.TPEPSTAPS.T 1980.796 1980.805 + 4.24

212 - 216 P.STAPS.T - 461.213 461.212 + 1.95

217 - 225 S.TSFLLPMGP.S 1618.7012 1617.693 +17.62

217 - 244 S.TSFLLPMGPSPPAEGSTGDEPKSCDKTH.T - 2885.309 2885.321 - 4.16

226 - 242 P.SPPAEGSTGDEPKSCDK.T - 1703.726 1703.731 - 2.93

226-467 P.SPP..PGK- 27210.356 27210.461 - 3.87

2 x

3 x

a)

b)

c)

+ Sialidase mix

LC-MS/MS

T

Binding / washing

Urea elution

T

S

S

Figure 8. Specific enrichment of O-glycosylat-ed peptides from IgA. a) IgA was trypsin digest-ed, reduced, denatured and alkylated. Trypsin was inhibited, and the sample was incubated on the inactive OgpA resin whilst being desialylated. The resin was washed, and the bound pep-tides were eluted with 8 M urea. Separation was performed on a RP-LC C18 column (Advance BioPeptide Map 2.1x100 2.7µm from Agilent) and detection on an ESI-Q-TOF Bruker Impact II mass spectrometer. b) The generated data confirm that the O-glycosylated tryptic hinge peptide of human IgA was straightforwardly en-riched on the resin and eluted free from N-gly-can carrying peptides.

0.0

0.2

0.4

0.6

0.8

1.0

1.28x10

Intens.

0.0

0.2

0.4

0.6

0.8

0.0

0.5

1.0

1.5

2.0

2.5

15 20 25 30 35 40 45 Time [min]

Load

Flowthrough

Eluate

8x10Intens.

7x10Intens.

12

3Peak

Theoretical monoisotopic mass Mass determined Mass diff. ppm Peptide

1 5799.491 5799.497 0.006 0.984 89-126 5x GalNAc 4gal 3x alkylation2 5596.411 5596.419 0.007 1.288 89-126 4x GalNAcGal 3x alkylation3 5434.359 5434.358 0.001 0.183 89-126 4x GalNAc 3x Gal 3x alkylation

TiCa) b)

a)

b)

c)

a)

b)

S K P L R P R C R P C G G P K D H P L T C D D P R F Q D S S S S K A P P P S L P S P S R L P G P S D T P I L P Q1 10 100 120 121 127 132 138 14011-99

---

*

pep

pep

Figure 3. Digestion at core 1 and core 2. OgpA digestion of human chorionic gonadotropin beta chain (hCGβ). a) The generated glycopeptides from hCGβ confirm glycosylation at S121, S127, S132, S138, and indicate glycosylation at S120 and T140. RP separation was performed on an AdvanceBio Peptide Map C18 column from Agilent and samples were desalted in-line prior to ESI Q-TOF on a Bruker Impact II MS. *Glycan structures according to Valmu et al. (2006)1. b) MS/MS spectra of fragment S121-P131 confirms digestion at the core 2 structure by the presence of HexNAc/HexNAc/Hex as an oxonium ion.

a)

b)