WCBP 2012O-linked glycansO-linked glycans

Pauline M RuddDublin-Oxford

Glycobiology groupy gy g p

Conway Institute

From Back – Eoin Cosgrave, Weston Struwe, Oscar Potter, Michael Schomburg, Patrick Jennings, Karina Marino, Jayne Telford, Niaobh McLoughlin, John O’Rourke, Barbara

Keegan, Ciara McManus, Mark Hilliard, Jayesh Kattla, Rebecca Duke, Barbara Adamczyk, R dk F h J WithRadka Fahey, Joanne Withers

Missing from group photograph - Eugene Dempsey, Margaret Doherty, Natalia Artemenko, Tharmala Tharmalingam, Marie Galligan, Giorgio Carta

Carbohydrate – Peptide linkages in Glycoproteins

NtAsn-X-Ser

N-GlycanGelatinase B

N-linked glycans

CXXGG(S/T)CO-fucose

Ser

O

ThrO-GalNAc

O-GalNAc

O-linked glycanSer/Pro/Thr/Ser domain

O fucose

Asn-X-Thr

N-Glycan

C

OGlycan

OOEtN O

PO

O-Inositol

AsnCXSXPC O-glucose

y Glycan O-Inositol

O O

P

OHuman

erythrocyte CD59 EPO

O

O

ON-linked glycan

tPAO OC CO-linked glycanGPI

anchor

tPA

Biosynthesis of O-glycans in Golgi

Specific transferases add initialmonosaccharide (GalNAc)

to Ser/Thr -OH groups accessible

Core I

on fully folded protein

Core II

Core III

Ser/Thr

Core IIIGalNAc

GalCore IV……. XII

Gal

GlcNAc

Sequential additions of monosaccharides by other transferases give branching, elongation and termination

Core 2

Major O-glycan core structures

Core 1 - Gal(β1,3)GalNAc Core 2 - Galβ1,3[GlcNAcβ1,6]GalNAc

Core 3 - GlcNAc(β1,3)GalNAc Core 4 - GlcNAcβ1,3[GlcNAcβ1,6]GalNAc

Symbolic notation- embedded monosaccharide sequence, linkage and anomericity

Linkage positionOpen symbols: hexoseFilled in: HexNAc

Antigenicity

Blood Group

4

86

GlcNAc

pAntigens

A

23

4Gal

GalNAcB

A

2

β li k

Linkage typeFuc (deoxy galactose)

B

β-linkage

α-linkageNeuNAc Ounknown

Functions for O-glycans

O-linked glycansprotect hinge

IgD: a secreted and gmembrane bound

glycoprotein

IgD forms part of the B-Cell Receptor antigen613/11/2003 06:44

IgD forms part of the B-Cell Receptor antigen capture mechanism on lymphocytes

O-linked glycans extend peptide linkers: CD8T cell

140 A80A

MHC ICD8αβ

APC

Lec3.2.8.1 CHO K1

s valuesP values (shape)

(size)1.614.08

1.543.30

Merry, Gilbert, Wormald, Dwek, Classen, Rudd, Davis J. Biol. Chem.

Recognition: sIgA binds pathogenic gut bacteria

Secretory component 7N-links

O-glycosylatedO-glycosylated

hinge regionO glycosylatedhinge region

hinge region

I AJ chainsIgA

sIgAJ-chainI N-link 4 N-links4N-links

Secretory IgA O-glycans 2AB2AB 2AB

2AB2AB

O-glycans2AB

2AB2AB2AB

2AB2AB2AB

2AB2AB

2AB

2AB 2AB2AB

2AB

2AB

2AB2AB

2AB

2AB2AB

2AB2AB2AB2AB

2AB2AB

2AB2AB

2AB2AB

2AB2AB2AB

2AB

2AB2AB 2AB 2AB

2AB2AB

2AB

2AB2AB2AB

Galβ1 3GlcNAc1 2 3 4 5 6 7 8 9 10GU

Galβ1-3GlcNAc

sIgA O-glycans bind bacteria

E.coliE.coli

Actinomyces Naeslundii

Helicobacter pylori

Effect of glycosyltransferase changes O l ion O-glycan processing

tumour cellscontrol cells

Core 2 Truncated

tumour cells

E t d d l

control cells

Core 2

α6ST1

Core 2β6GNT glycanExtended glycan Core 2

β6 GNT

α6ST1

SLex

Elongation of 1,6 GlcNAc arm -

Stop signal forβ3GalT

Premature termination on both arms -SialylTn antigen

expression of Lewis structures

Loss of Golgi organisation: may relate to pH changes and contribute to glycan changes in malignant cells or culture

cancerHealthy controll

Golgi apparatus di i d i lldisorganised in cancer cells

cancer associated carbohydrate Ag

High levelsAbsent / low

T, Tn, STn

Golgi pH6: normal pathwaycarbohydrate Ag

expression

High Golgi pH in tumour cells may: Ser/Thr T

• be a result of reduced proton pump activity

• be sub-optimal for glycosyltransferase activity

Ser/Thr

Ser/Thr

Tn

Sialyl Tn

• interfere with ER / Golgi recycling

KelloKumpu et al, FEBS (2002) 516; 217-224

Site occupancy: Overexpression of MUC-1 with increased O-glycosylation aids detachment in breast cancerglycosylation aids detachment in breast cancer

ubiquitous cell surface expression

MUC-1expression

increased sialylation MUC-1

O-glycan site occupancy doubled

ChallengesN il bl f i l f O li k d l• No enzymes available for generic release of O‐linked glycans– O‐glycosidase removes: Gal(β1‐3)GalNAc– But requires exoglycosidases to remove other sugars from core

NeuAc

– But requires exoglycosidases to remove other sugars from core

GalNAcβ1,3 Ser/Thr

NeuAc

Gal

O‐glycosidase

+Sialidase

• Chemical methods available• Hydrazine• Ammonia‐based β‐elimination

Ser/Thr

LC-MS: O-glycan Release ChemistryO-glycan Chemical Release:

O

OR1OH

R ORcX

Reductive β-elimination

R 2O

O O

NHRnH

-OH

AcNH Alditol

OHOR1

OH

R2OOH

NaBH4

Aldose

OOR1

OH

R2O

Fluorescent labelling not

iblH2O

2AcNH

O

OR1OH

RX

HO-

H2O

2O

O HAcNH possible

OHOR1OH

R2OO

H

R 2O

O O

Rc

NHRn

X

AcNH

Further

‘Peeling Product’

R O + H O+OH

OR1OH

-OH2 H

AcNH degradationHR2O- + H2O+

OO

AcNH

Non-reductive β-elimination

Total Area

Optimization of β-elimination release for Bovine Submaxillary Mucin O-glycans

20

β-Elimination Optimization with Bovine Submaxillary Mucin

2 4 6 8

6e+8

Total Area

0 1e+8 2e+8 3e+8

Submaxillary Mucin O glycans

10

12

14

16

18

% P

eele

d

10 12 14 16 18 20 3e+8

4e+8

5e+8

Area

(μV

*sec

)

3e 8 4e+8 5e+8 6e+8

2

4

6

8

15

20

25

30

6065

70

%

Time (

Hours)

0

1e+8

2e+8

10

15

20

25

30

5560

6570

A

Time (

Hours)

T10

4550

55Temperature ( oC)45

50Temperature ( oC)

Total % of Peaks 2-5 Total area unpeeled glycansPeeling: Blue: minimum Recovery: Red maximumPeeling: Blue: minimum Recovery: Red maximum

Tharmala Tharmalingam, Li Liu, Jodie Abrahams, Margaret Doherty, JonathanTharmala Tharmalingam, Li Liu, Jodie Abrahams, Margaret Doherty, Jonathan Bones, Niaobh McLoughlin

E l i f I bili iEvaluation of Immobilisation

Cervical Mucin

Optimised O-glycan releaseCervical Mucin

• Highly reproducible , robust basis for O‐glycan analysis

• Used for Mucins• Bovine Cervical

P i Il d C l• Porcine Ileum and Colon • Chicken mucins

Ammonium hydroxide saturated ith A i b t

3. Evaporate 

Robotic compatible platform for O-glycan release and labelling

Glycoprotein sample

Wash

with Ammonium carbonate

2. Release glycans

ammonia 

4. Salt cleanupCarbon filter to clean up salts

Incubate 65oC for 16 

1. Dehydrated mucins

sample

Waste

Carbon filter to clean up saltshours

Dry5. Formic Acid Treatment1

6. 2AB  fluorescent label

Fluo

rescen

ce 23 4

Compare GU values with 

2AB

7.Elute 2AB‐

Fluo

rescen

ce

10 15 20 25 30 35 40 45 50 55 60 65Time (mins)

Glycobase 

10. Structure Assignment

glycans 8.NPHPLC profile of glycan pool Transfer to SPE 

plate

Time (mins)

cence

Dry, redissolve in set volume

9. Exoglycosidase digestions

Fluo

resc

Fluo

rescen

ce

Time (mins)

autoGU  

Exoglycosidase Digestions: O-glycans Bovine cervical mucins

Preliminary assignments for bovine cervical mucinO-glycans - HPLC

Unicarb‐DB: Glycan LC‐MS Database

Glycan Structure

LC-MS DetailsLC MS Details

ReferencesBiological Information

Composition

Sequence Information

Possible Structural Isomershttp://unicarb-db.comp

Hayes C., Campbell, M., Struwe W., Karlsson, N., Rudd, PM et al. Bioinformatics, 2011

Giorgio CartaJohn O’Rourke

LC-MS: O-glycan Sample Prep WorkflowO-glycoprotein Purification

Glycan Release - Reductive β-elimination: 50 mM NaOH and 0.50 M NaBH4 at 50°C for 16 h

g y p

B t R l

Peptide Removal and Desalting:DOWEX AG-50 cation exchange resin on the top of a C18 Zip-tip (Millipore).

(resin was washed with 1 mL MeOH, primed with 1M HCl and equilibrated with 1 mL H2O)

Borate Removal: 5 washes of 60 μL 1% acetic acid in MeOH

glycan samples were reconstituted in 10 μL H2O prior to LC-MS analysis

LC MS A l iLC-MS AnalysisAnalysis of glycan alditols performed in negative mode using an ion trap mass spectrometer with an Agilent

1100 HPLC binary pump and HTC-PAL autosampler.HPLC analytical columns consisted of 5 μm porous graphatized carbon in a fused silica capillary (20 cm x 180

μm i d ; o d 375 μm) The solvent flow rate was between 8 12 μL per minuteμm i.d.; o.d. 375 μm). The solvent flow rate was between 8-12 μL per minute.

Structural Annotationglycan analysis was performed with the aid of the Unicarb-DB glycan database

(http://www unicarb db com)(http://www.unicarb-db.com)

Weston Struwe

Mucin O-glycans from Chicken Intestines

Analysis of chicken intestinal mucin O‐glycans:

C j j i i l b i• C. jejuni is a commensal bacteria in chickens

• Chicken mucin attenuated bacterial binding & is attributed tobacterial binding & is attributed to mucin glycosylation (Alemka et al.)

• Large intestine inhibited C. jejunibinding and internalization betterbinding and internalization better than small Intestine and caecum

• Goal: To Identify glycosylation differences in the chickendifferences in the chicken intestinal tract as it pertains to C. jejuni colonization

Chicken Mucins are Highly Sulphated and Sialylated OH

315 OH

OH

OH

NHAcOO

NHAcOH

O

OH

O

OH

OH

SO4-

OH

241

301315

OH

OH

444241 444 OH

OH

OH

NHAcOO

NHAcOH

O

O

OH

OH

OH

OH

SO4-

282

505

Mucin O-glycans are extended core III and core IV

• Large Intestine contains N-glycolylneuranimic acid

Mucin O glycans are extended core III and core IV• Large Intestine contains more glycan diversity• More charged structures in the LI

g g y y

• 18 structures present in Large Intestine

Sulphation is present throughout

8 st uctu es p ese t a ge test e• 13 structures present in Small Intestine• 13 structures present in Caecum

http://www.extension.org/pages/31093/campylobacter-jejuni

FDA Approved Glycoprotein Biomarkers

PROTEIN/MARKER CANCER TYPE

α-fetoprotein (AFP) gastric, liver, testicular

CA 125 (MUC16) gastric, lung, ovarian

CA 15.3 (MUC1) breast

CA 19 9 l t l t i li tiCA 19.9 colorectal, gastric, liver, pancreatic

Carcinoembryonic antigen (CEA) breast, colorectal, gastric, lung, pancreatic, thyroid

Complement factor H protein bladderComplement factor H protein bladder

Human chorionic gonadotropin-β testicularKallikrein 3 (prostate specific antigen, PSA) prostate

O-glycosylated therapeutucs

Follicle Stimulating Hormone)Prolactin endometrial carcinoma

Thyroglobulin thyroid

ErbB2 Her2/neu breast

Follicle Stimulating HormoneLuteinising HormoneVon Willebrand factor

Th id ti l ti hErbB2, Her2/neu breastThyroid stimulating hormone

Glycan analysis is complexThe question determines the technological approach

Marino Rudd et al Nature Chemical Biology 2010

Instrument Acquisitions Agilent TLAOscar Potter

Marino, Rudd et al Nature Chemical Biology 2010

Oscar Potter

Beckmann CEGiorgio Carta Glycobase

Key Personnel

Jonathan Bones

Mark Hilliard

StefanMittermayer

WestonStruwe

Margaret Docherty

Michael Schomberg

Andras Guttman group