Tools and techniques for studying glycosylation and their

applications in health and disease

BIOL2012 21st February 2023

(Protein Glycosylation)

Dr Joel Allen
University of Southampton
 Learning Objectives

• Describe how glycans attached to glycoproteins can

influence the function of the protein, using
antibodies and viral glycoproteins as examples.

• Explain why glycosylation requires specialized

analytical approaches

• Be able to detail some of these methods and what

questions they can be used to answer
 Lecture contents

Lecture 2:

• Why do we want to study glycosylation?

• Why is glycosylation difficult to study?

• Examples of methods that can be used to study glycosylation

 Case study: Antibody glycosylation

• As well as binding to foreign antigens, antibodies recruit other

parts of the immune system to combat pathogens

• These receptors bind in the Fc region, which is glycosylated

• The precise glycan processing state of the Fc can fine tune

which immune effector functions are upregulated/
downregulated.
Case study: Antibody glycosylation
 Glycosylation of IgG Fc fine tunes interactions with the immune system

Deglycosylation abolishes
Fc receptor interactions

De-fucosylated Fc
increases affinity for
FcγRIIIa

De-galactosylated Fc
correlates with
Sialylation exerts anti- pro-inflammatory
inflammatory properties properties
 Afucosylation enhances ADCC by engaging CD16a

Fc Gamma RIIIa PDB ID: 1E4K Stronger Weaker

AKA CD16a

IgG Fc

Crystal structure of soluble human IgG1 Fc

fragment-fc-gamma receptor III complex

Improved antibody dependent cellular

cytotoxicity can lead to better cell killing,
important for cancer treatments Representative SPR sensograms for CD16a N-glycoforms binding IgG1 Fc.
doi: 10.1021/acschembio.8b00342
Antibody drug products developed by selecting desirable glycoforms

More info and examples in:

Dalziel M et al. Glycobiology. 2018 Sep
1;28(9):697-712. Review.
Glycans are found across viral glycoproteins
Glycans shielding protein surfaces

Potential protein
binding antibody
 Glycans shielding protein surfaces

Potential protein
binding antibody

Rare glycan
binding bnAb
 Rare glycan binding antibodies against HIV-1

• The high mutation rate of HIV-1 means that the protein surface
of HIV-1 Env is hyper variable.

• The dense glycan shield inhibits antibody binding

• A subset of patients infected can elicit antibodies that can

neutralize HIV-1 Env and are able to recognize a large number
of circulating strains.

• If these can be elicited by vaccination prior to infection then

they may be able to protect against HIV-1 infection.

• The glycan shield is conserved, and a large number of these

antibodies recognize glycans
 Glycans as antibody epitopes

2G12
VACCINE DESIGN
PGT145

N160
 Glycans as antibody epitopes

• As these antibodies recognize glycans, vaccines will need to

reproduce viral glycan signatures

• As such we need to know where N-linked glycans are attached,

and how they are processed on both the virus and candidate
vaccines aiming to elicit such antibodies
 Summary slide of methods that can be used for structural characterization
GLYCOMICS
Analogous to genomics and proteomics, glycomics represents the systematic methodological
elucidation of the “glycome” (the totality of glycan structures) of a given cell type or organism

Complicated workflows,
“helpful” arrows
 Questions that glycobiologists ask when studying glycoproteins

1)Is a protein glycosylated?

2)How is the glycan processed?

3)Does the protein need the glycan to function correctly?

4)Does specific monosaccharide processing influence the proteins

function?
 Difficulties of studying glycosylation

• Glycan processing is difficult to predict, depends on lots of extrinsic

properties

• Glycans are extremely heterogeneous, many different glycan

compositions can exist at a single site

• Glycans are flexible, and as such are difficult to visualize using other
structural determination methods such as X-ray crystallography and
cryo-electron microscopy

• Monosaccharides can have very similar structures and masses, and it

can be difficult to resolve different isomers and distinct linkages
 Crystallography is not enough to fully identify glycan modifications

Two N-acetylglucosamine
residues

Crystal structure of a glycoprotein Full structure of the attached glycan

zoomed to glycan sites
1)Is a protein glycosylated?

2)How is the glycan processed?

3)Does the protein need the glycan to function correctly?

4)Does specific monosaccharide processing influence the proteins

function?
 Using glycosidase enzymes to remove glycans

• To test whether a protein is

glycosylated, simple experiments can
be performed.

• Peptide N-glycosidase F (PNGaseF) is a

bacterial enzyme that will cleave most
N-linked glycans

• Analysing the protein by SDS PAGE

can show whether or not the protein
contains N-linked glycans.
PNGaseF (1PGS)
PNGaseF can be used to confirm presence of N-linked glycans

Which chain of the IgG contains an

N-linked glycan?

A) Heavy chain

B) Light chain

C) Neither

D) Both

https://international.neb.com/protocols/2014/10/24/rapid-pngase-f-by-sds-page-protocol-p0710
1)Is a protein glycosylated?

2)How is the glycan processed?

3)Does the protein need the glycan to function correctly?

4)Does specific monosaccharide processing influence the proteins

function?
 Using glycomics to investigate the structures of glycans

1)Investigate the entire glycome of the protein/ cell surface

2)Investigate the site-specific glycosylation, using the protein

sequence to which the glycan is attached to assign which glycans
are present at a particular glycosylation site
How to study the glycosylation of proteins

Site-specific glycan analysis

Whole glycome analysis

Using UPLC to determine viral glycoprotein glycosylation
Whole glycome analysis by UPLC
Preparing and analysing a sample by UPLC
Preparing andanalysing
Preparing and analysing a sample
a sample by UPLC
by UPLC
Preparing andanalysing
Preparing and analysing a sample
a sample by UPLC
by UPLC

PNGaseF

Asn

Asn

Released N-glycans
Preparing andanalysing
Preparing and analysing a sample
a sample by UPLC
by UPLC

PNGaseF

Asn

Asn

Released N-glycans

Fluorophore
(procainamide)
 Preparing andanalysing
Preparing and analysing a sample
a sample by UPLC
by UPLC

PNGaseF

Asn

Asn

Released N-glycans

Fluorophore
(procainamide)
Waters Acquity UPLC
 How glycans are separated by HILIC UPLC
• HILIC (Hydrophilic Interaction Liquid Chromatography) is a
variant of UPLC (Ultra Performance Liquid
Chromatography)

• It uses a polar stationary phase and a mixed polar/non-

polar mobile phase

• The stationary phase adopts a layer of water that

becomes immobilised

• This allows the hydrophilic analyte to partition into this

layer and react with the stationary phase by means of
hydrogen bonds, ionic bonds and dipole-dipole
interactions
32
 Defining and quantifying the N-glycan compositions

Oligomannose-type Complex-type
glycans glycans

Endoglycosidase Exoglycosidase
Neuraminidase
Galactosidase
N-acetylglucosaminidase
+endoH

Fucosidase

+
 Quantifying oligomannose glycans using endoglycosidase H

Total glycan pool

+endoglycosidaseH
+endoH
 Quantifying oligomannose glycans using endoglycosidase H

Total glycan pool

Complex-type glycans Oligomannose-type glycans

 Why do we want to know this?

HIV virus glycosylation

HIV vaccine glycosylation

Struwe et al. 2018 Cell reports

 Why do we want to know this?

Ensuring vaccine grade material

glycosylation does not differ from that
observed in the laboratory
Applying this method to study antibodies
 What if your protein contains lots of N-linked glycans?

Proteins can contain many different glycosylation sites, a

different method is needed to see which particular sites are
modified
 Preparingsamples
Preparing samples
forfor
sitesite specific
specific analysis
analysis

+6M urea +DTT reducing agent

Unfolded protein Unfolded protein with
no disulphide bonds
Trypsin

+iodoacetamide (IAA)

+DTT reducing agent Unfolded protein with

unpaired capped
Removes excess IAA cysteines (alkylated)

Analysis by liquid
chromatography- mass
spectrometry

Chymotrypsin
 Using this technique to map the glycosylation of the SARS-CoV-2 spike

DOI: 10.1126/science.abb9983
1)Is a protein glycosylated?

2)How is the glycan processed?

3)Does the protein need the glycan to function correctly?

4)Does specific monosaccharide processing influence the proteins

function?
 Point mutations to remove N-linked glycan sites

• To investigate the importance of individual

glycan sites, site-directed mutagenesis can be
used.

• Removing the NxS/T through either amino TRDENDTEEL

aac
acid is sufficient to remove a glycan site
TRDEQDTEEL
caa
• The asparagine is often replaced with
glutamine as the chemistry of the amino acid
is the same vs
WT N234Q

• This was used to study SARS-CoV-2 Measure

infectivity
glycosylation, creating individual point
mutations and then measuring infectivity and
viral production
 Impact of individual point mutations on viral production and infection

• Point mutations at most

glycan sites affected
both viral production
and infectivity

• Likely occurring through

a loss of protein folding
fidelity

• Demonstrates the key

role glycan perform in
viral protein function

DOI: 10.1126/scitranslmed.abm089
1)Is a protein glycosylated?

2)How is the glycan processed?

3)Does the protein need the glycan to function correctly?

4)Does specific monosaccharide processing influence the proteins

function?
 Subtle influence of ACE2 glycan processing on SARS-CoV-2 recognition

• COVID-19 severity varies

from person to person.

• Could the glycosylation of

ACE2 play a role?

• Therapeutic applications,
can affinity be enhanced by
glycan engineering?

Allen et al. 2021 JMB

https://doi.org/10.1016/j.jmb.2020.166762
 Testing glycan dependency of SARS-ACE2 binding

• We wanted to investigate the glycan dependence of

SARS-CoV-2 binding ACE2

• Produce recombinant ACE2 and SARS-CoV-2.

Modify the glycans on ACE2, test the binding

• Therapeutic applications, can affinity be enhanced

by glycan engineering?
 Testing glycan dependency of SARS-ACE2 binding

Protein engineering Expression and purification

WT ACE2 binds to SARS-CoV-2 with nM affinity

KD 80nM
 Removing Fucose does not impact binding

WT Defucosylated
 Removing sialylation modestly increases affinity

WT Desialylated
 Co-transfection with sialyltransferase decreases affinity

WT Sialylated
 Deglycosylation has no major effect

WT Deglycosylated
 Conclusions

• ACE2 binds to SARS-CoV-2 with a high affinity.

• Sialic acids appear to influence the binding, slightly reducing

the affinity. Fucose had no overall effect

• Complete deglycosylation of ACE2 does not remove binding,

suggesting that the glycans are not critical for SARS-CoV-2 to
bind
 Summary

• Glycans are critical post translational modifications for both

structural stability and immune modulation.

• Studying the glycosylation is crucial for understanding how

key biopharmaceuticals perform their function, including
therapeutic antibodies and viral vaccines.

• Glycans can be challenging to study, however bespoke

approaches including enzymatic digests and biophysical
techniques are used to study the glycome.
 Learning Objectives
Summary of the learning objectives of both lectures

Understand the structures of monosaccharides and how they combine to form

glycans.

Understand how glycoproteins attach glycans, with a focus on mammalian/

human cells

Explain why glycans require specialized analytical approaches, and detail some
of these methods

Describe how glycans attached to glycoproteins can influence the function of

the protein, using antibodies and viral glycoproteins as examples.