Glycosylation of Antibodies:
An Application in Therapy
Joseph H. Musmanno
Biochemistry Comprehensive
Department of Chemistry
The Catholic University of America
Washington, DC
Introduction
Glycoproteins are some of the fundamental building blocks
of
the
mammalian
immune
system.
These
glycoproteins
are
produced by the covalent attachment of carbohydrate groups to a
protein1.
They function in varying capacities within the cell
membrane;
some
of
which
include,
protein
folding,
conferring
stability, and aiding in cell to cell adhesion within the immune
system.
body
Glycoproteins found within the immune system of the
are
at
therapies.
the
root
of
some
diseases
and
also
natural
Recently, glycosylation of antibodies outside of the
body has been explored for therapeutic purposes.
Glycosylation
Glycosylation is an enzyme-directed site-specific process
which
occurs
as
a
post-translational
modification
in
the
endoplasmic reticulum (ER) and in the Golgi complex of the cell.1
The
linkage
linkage
or
of
carbohydrates
N-linkage.
to
O-linked
proteins
occurs
carbohydrates
through
attach
to
Othe
oxygen atom of the side chain of serine or threonine (Figure 1).
The more commonly used method of glycosylation, N-linked, is the
attachment of a sugar to the amide nitrogen atom on the side
chain of asparagine (Figure 1).1
This linkage to amino acids is
imbedded in a conserved sequence of amino acids.
2
Figure 1: N-linked glycosylation of N-acetylglucosamine (GlcNAc)
and O-linked glycosylation of N-acetylgalactosamine (GalNAc).
The glycosidic bond between the carbohydrate (black) and the
amino acids (blue) is seen in red.1
N-linked glycosylation occurs in the ER and continues in
the Golgi complex.
Protein synthesis takes place on ribosomes
on the ER surface.
The proteins are then introduced into the
lumen of the ER for glycosylation.1
Carbohydrates destined for
attachment to the asparagine residue of proteins, are mediated
using dolichol phosphate (Figure 2).
Figure 2: Dolichol phosphate is a lipid located
membrane for attachment to the oligosaccharide.1
in
the
ER
The terminal phosphate group of dolichol phosphate attaches to
the carbohydrate, which then transfers the oligosaccharide to an
asparagine residue of the protein.
3
Glycoproteins once formed in
the lumen of the ER, are transferred to the Golgi complex for
further alterations of the carbohydrate units.1
O-linked glycosylation takes place exclusively in the Golgi
complex.1 The Golgi complex, acting as the sorting center of the
cell,
can
then
direct
the
glycoproteins,
once
modified,
to
lysosomes, secretory granules or the membrane depending on the
amino acid sequence.1
Complex Carbohydrates
Carbohydrates
that
bond
to
their
respective
amino
have two types of structures; high mannose or complex.
these
mannose
structures
and
carbohydrates
two
contains
a
pentasaccharide
N-acetylglucosamine
which
typically
(GlcNAc)
partake
biantennary, taking on a “Y” shape.
in
core
acids
Each of
of
three
residues.1
The
glycosylation
are
Examples of each type of N-
linked oligosaccharide can be seen in Figure 3.
A.
B.
Figure 3: A) High-mannose type of N-linked oligosaccharide and
B) Complex type of N-linked oligosaccharide. The pentasaccharide
core is shaded in gray.1
4
Glycoproteins are key molecules in the immune system, which aid
in immune response.
One relevant issue involving glycosylation,
is the glycosylation of
affinity
of
antibodies
antibody-antigen
in order
to increase the
interactions
for
effective
therapies.2
Antibodies
An antibody is a protein of the immune system which is made
up of four polypeptides, two light chains and two heavy chains.3
Antibodies are also in the shape of a “Y” with the antigen
binding sites at the two ends, named the variable region (Fab),
and the stem, known as the constant region(Fc).
also
homodimers
with
covalent
disulfide-bonding
Antibodies are
at
the
hinge
region (Figure 4).
Figure 4: Antibody structure.3
In order to study these two regions individually, the antibody
can be treated with a protease, which cleaves between the Fc
region and the two identical Fab regions.3
5
Upon the foreign invasion of antigen, the cell, or body,
will
invoke
the
immune response.1
different
production
of
antibodies
against
antigen
as
Antibodies that are produced are specific for
antigens.
The
constant
region
of
an
antibody
determines one of the five major classes, IgM, IgG, IgA, IgD,
and IgE.3
These five classes known as immunoglobulin (Ig)3,
designate
the
specificity
in
antigen
binding
and
in
immune
response to bound antigens.
Immunoglobulin G (IgG)4
Immunoglobulin G (IgG) is the most prominent antibody in
humans and it is found in the blood.
sites
for
ligands,
which
activate
glycosylation, are expressed.
effector
functions
glycoforms
and
are
vary
mechanisms
of
The ligands that interact are Fc
receptor types FcγRI, FcγRII, FcγRIII.
effector
On the Fc, interaction
the
essential
The expression of these
efficacy
for
between
glycosylation.
different
The
IgG
structure is consistent with typical immunoglobulin structure.
The
CH2
and
CH3
domains
of
the
Fc
region
on
IgG
are
very
important for receptor binding (Figure 5). The glycosylation of
the CH2 domain through the attachment of oligosaccharides at
asparagine (Asn) 297, is a unique feature of IgG, (Figure 6)
which will become a key player in the immune mechanism.
6
Figure 5: Human
regions are the
chains.4
IgG with the functional regions.
light chains and the orange are
The
the
green
heavy
Figure 6: Interaction of the IgGFc domain with a carbohydrate.
The view is from the hinge region toward the CH3 terminus.2
IgG typically becomes glycosylated by a biantennary complex
which allows for 32 different oligosaccharides and possibly 400
or
more
glycoforms.
The
structure
7
of
the
biantennary
carbohydrates
are
such
that
the
pentasaccharide
invariant, however, external saccharides can vary.
core
is
It has been
shown that the Fc associated sugars from human IgG are complex
biantennary with terminal sialylation and galacosylation.5
The
Fc region of human IgG has N-linked carbohydrates in addition to
the glycosylation site at Asn 297.4
Glycosylation and the Immune System
The immune system aids in the proper folding of proteins
and it controls the assembly of glycoproteins, such as T cell
receptors (TCR), with specific glycoforms.2
Improperly folded
proteins
function
do
not
allow
glycoprotein to occur.
for
the
desired
of
the
Therefore, glycoproteins are required to
engage in a series of interactions with chaperones and enzymes.
An
oligosaccharide
protein in the ER.
precursor,
GlcNAc2Man9Glc3, attaches
to
the
This oligosaccharide is readily processed
into another form, GlcNAc2Man9Glc1, which binds the chaperones
calnexin
(Clx)
and
calreticulin
(Clr).2
Clx
and
Clr
are
essential to access the folding pathway and also in the loading
of antigenic peptides onto the major histocompatibility complex
(MHC).
The MHC plays a large role in recognizing T cells with
TCR in the membrane for attacking foreign antigens.
shows the structure of calnexin and calreticulin.2
8
Figure 7
A
B
Figure 7: Structures of Calnexin (A) and Calreticulin (B).
Glycoproteins which are not folded correctly, or have been left
unfolded, are degraded in the ER in order to avoid improper
functioning.2
then
The proteins which are produced and folded are
presented
to
the
cell
surface
to
respond
to
T
cell
antibodies.
Another role that glycosylation has in the immune system is
T cell recognition of antigen presenting cells (APCs).2
T cells
act in numerous ways in the recognition of antigens in the body.
They are differentiated by the glycoprotein at their surface.
In order for TCRs to recognize APCs, the immunological synapse
must be formed.2
This junction allows for the recognition of
antigenic peptide-loaded MHC by TCRs to generate a response.2
Oligosaccharides on the cell membrane moderate the alignment of
9
opposing cell surfaces as well as restrict the orientation of
cell-adhesion molecules, CD2 and CD48.
Also, they aid in the
transport of MHCs to the center of the junction.2
In Figure 8
the immunological synapse model is seen highlighting the CD2CD48 cell adhesion pair, MHC, and TCR-CD3-CD8 complex.
CD3 and
CD8 act as co-receptors with TCR.
Figure 8: Immunological synapse where T cell recognition of APCs
takes place.
The glycopeptide is in the middle (black and
yellow) which aids in the binding of the complex.2
Glycoproteins have been shown to play a main role in the
immune system through proper folding and antigen recognition.2
By closely examining the variability of glycosylation and its
effect
on
function,
one
can
have
a
better
understanding
health and disease in regards to the immune system.2
10
of
Glycosylation in Vivo Pertinent to Disease
Sialylation6
The addition of sialic acid (SA), sialylation, at Asn 297
on the Fc domain and terminal galactosylation, play a role in
rheumatoid
arthritis
(RA).
IgG
can
mediate
anti-
and
pro-
inflammatory activity through interactions of Fc with Fcγ type
receptors.
The distinct properties of IgG Fc are a result of
sialylation of the core carbohydrate structure.
A good example
of a fully processed complex carbohydrate structure observed in
antibodies is shown in Figure 9. The oligosaccharide in Figure 9
is attached to the protein at Asn 297 by the fucosylated GlcNAc
core.
The cleavage at GlcNAc occurs due to the catalytic action
of peptide N-glycosidase F (PNGase F).
This enzyme detaches the
carbohydrate from the protein at the Asn binding site.6
Figure 9: Sialic Acid structure and core carbohydrate structure
observed in antibodies typical in the body. The main backbone
structure is in bold along with the terminal sialic acid and
galactose in red.
PNGase F and neuraminidase are cleavage
enzymes.6
11
Neuraminidase
is
the
enzyme
that
between SA and Gal (Figure 9).
cleaves
glycolytic
bonds
The mass spectroscopic analysis
in Figure 10 detects the mass/charge ratio of the N-glycans of
intravenous gamma globulin (IVIG); specifically, the mass of SA
as IVIG is treated with neuraminidase.
Between the treated and
untreated IVIG, a number of masses for SA are missing.
There is
only a single mass peak for the treated IVIG, as opposed to five
peaks
for
the
untreated
IVIG,
which
demonstrates
the
effectiveness of neuraminidase.
Figure 10: Mass Spectrum of N-glycans of untreated intravenous
gamma globulin (IVIG) and neuraminidase-treated IVIG.
Within
6
the brackets are masses of fragments containing SA.
It has been shown that IgG glycosylation differs within patients
with RA, since Fc galactosylation and sialylation is decreased
when compared with normal patients.
Therefore, a number of IgG
glycoforms have been suggested to contribute to this response.
The analysis of glycoforms by mass spectroscopy to determine the
composition of the carbohydrate was useful to show that minimal
12
SA
residues
existed
in
patients
with
RA.
Antibodies
with
decreased levels of terminal sugars have been suggested to take
on a pathogenic role, thus influencing studies in vivo of SA and
antibody activity.6
The sialylation of a glycoform, 6A6-IgG, reduced the antiinflammatory biological activity which could be restored upon
the
removal
of
the
SA
by
neuraminidase.
Binding
affinity
analysis of sialylated antibodies with their respective FcγRs
also showed the effects of sialylation (Table 1).
glycoforms
that
glycosylation.
were
used
were
IgG
antibodies
with
The four
varying
The binding affinity of each glycoform to the
Fcγ type receptors is best illustrated by the binding constants
in the second to fourth columns of Table 1.
Table 1: FcγR Binding to Antibodies (6A6-IgGs)6
These
binding
constants
physiological effects.
were
in
direct
correlation
with
the
Those glycoforms without SA bound to the
Fcγ type receptors had higher binding constants and inflammation
was enhanced.6
13
Galactosylation7
Galactosylation plays a key role in autoimmune disease by
either
its
presence,
or
lack
thereof,
on
the
CH2
domain.
Increased levels of agalactosyl (G0) IgG isoforms are associated
with
autoimmune
disease.
In
order
to
study
the
role
of
galactosylation in vivo, IgG in the serum of mice was used.
It
was shown that with a reduction in galactosylation of IgG in the
serum,
a
normal
immune
response
to
disease
was
produced.
Sialylation and galactosylation are two forms of glycosylation
in the body that either cause or prevent disease.7
There are a
number of ways in which glycosylation in vitro aids in therapy
for human diseases.
Glycosylation of the Fc Domain for Human Therapy
Oligosaccharides which are present at the N-glycosylation
site
of
the
CH2
domain,
are
known
to
affect
biological
and
pharmacological properties of IgGs.
These changes that occur
might be associated with disease.5
Glycosylation of IgG for
therapeutic
technology.
agents
have
These
been
achieved
particular
using
recombinant
immunoglobulins
recombinant immunoglobulin G (rIgG).
are
known
DNA
as
Glycosylation of IgGs is
“species-specific”, and reveals the necessity for selecting the
proper cell line to express rIgGs for therapy in humans.5
In
a
study
done
by
T.S.
Raju
et
al
in
2000,
N-linked
oligosaccharides in IgGs of thirteen different animal species
14
were studied to find the model for species-specific production
of rIgGs in humans.
The study focused on the importance of the
terminal sialylation and galactosylation of IgGs.
The initial analysis was to determine the amount of neutral
sugars and sialic acid forms using a phenol-sulfuric acid method
and RP-HPLC method respectively.
Oligosaccharides are released
from IgGs through hydrazinolysis of the glycosidic bonds.5
This
revealed that the distribution of sugars and SA among varying
species is distinct.
Table 2 shows these data and how only
humans and chickens have the SA form N-acetylneuraminic acid
(NANA) while others only contain the N-glycolylneuraminic acid
(NGNA) form but some contain both.5
Table 2: The neutral sugar and SA content of IgGs in 13 animal
species. An average of 150kD was used for the molecular weight
of IgGs in determining the values for SA.5
Following the designation of sugar content, the IgGs of
these species were treated with PNGase F to release the N-linked
15
oligosaccharides from the IgGs.
They were then analyzed through
the use of matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF-MS) for neutral and acidic
oligosaccharides
spectral
shown
analysis
in
Figure
allowed
12A
for
and
a
12B.
This
comparison
mass
between
oligosaccharides of different species. The only oligosaccharides
recovered
were
released
by
PNGase
F
and
therefore
were
determined to be N-linked.5
There were two modes in which the mass spectrometry was
performed,
positive
and
negative.
In
the
positive
mode,
protonated molecular ions are detected and proteins and peptides
are
analyzed.
Negative
mode
identifies
the
deprotonated
molecular ions of oligosaccharides and oligonucleotides.
The
fragments in Figure 12A and 12B represent the PNGase F released
N-linked oligosaccharides of IgGs for each species both in the
positive
ion
mode
and
negative
ion
mode
respectively.
The
oligosaccharides in Figure 12A are shown to be similar in all
species with some variance.
The fragmentation in Figure 12B
represents much more variance between the oligosaccharides from
each
species,
specificity
of
demonstrating
the
oligosaccharides.
importance
of
This
stresses
also
speciesthe
necessity of careful consideration for therapies using rIgGs of
different species for humans.
16
A
B
Figure 12: MALDI-TOF-MS of IgGs in animal species. Determination
of neutral and acidic oligosaccharides based on weight. A)
Positive ion mode in DHB matrix with NaCl B) Negative ion mode
with THAP matrix.5
17
IgGs of varying species contain a wide array of biantennary
complex type oligosaccharides as shown in the core and terminal
galactosylation and sialylation.5
Figure 13 shows the percentage
of glycoforms for each of these species according to variations
of the oligosaccharide complex.
Figure 13: Comparison of the fucosylated core (A) terminal
galactosylation (B) bisecting GlcNAc(C) and NANA (striped bar)
and NGNA (solid bar) (D). All taken from MALDI-TOF-MS analysis.5
It
is
shown
that
the
more
specific
the
glycosylation
cleavage, the fewer glycoforms are present in the species.
18
or
The
most
prominent
difference
lies
within
the
varying
glycoform
percentages of the two forms of SA, NANA and NGNA in Figure 13D.
Some species contain only one form or the other, while some
contain
both.
The
expression
of
these
oligosaccharides
by
glycosylating IgG, has remained consistent with Fc glycosylation
to biantennary carbohydrates, thereby emphasizing the importance
of the Fc domain as site for glycosylation.5
Monoclonal Antibody Technology
Another effective therapy using glycosylation of antibodies
is through monoclonal antibody technology.
There are two main
types of antibodies that can be used for therapies: polyclonal
and monoclonal.
Polyclonal antibodies are comprised of many
different antibodies all binding to different surface features
of the same antigen.
The use of this type of antibody can be
complicated due to heterogeneity.1
Monoclonal antibodies (mAbs)
on the other hand are identical antibodies which recognize one
specific site of the antigen.1
Production of monoclonal antibodies begins with the fusing
of
a
tumor
cell
with
a
mammalian
cell.
Clones
of
single
identical mammalian antibodies is difficult to accomplish since
once
they
are
isolated,
they
die
quickly.
To
increase
the
longevity of the antibodies being produced, they are fused with
specific
tumor
cells.
Tumor
cells
replicate
endlessly,
therefore lengthening the lifespan of antibody-producing cells.1
19
The
fusion
of
hybridoma.8
for
that
these
cells
creates
an
antibody
known
as
a
This hybridoma continually makes antibodies specific
antigen,
antibodies.
The
or
tumor
purified
cell,
antibody
thus
creating
attacks
only
monoclonal
the
target
molecule, decreasing the side effects in vivo.8
The
specificity
monoclonal
antibody
of
antibodies
research.8
In
increases
order
to
the
make
value
mAbs
of
more
specific, modification of their glycosylated Fc domain can be
performed.
These antibodies, once glycosylated, can be used
therapeutically
as
well
as
to
diagnose
illnesses
and
detect
“unusual or abnormal substances” within the blood stream.8
Therapies Developed Using in Vitro Glycosylation
Manipulating
the
oligosaccharides
attached
open the door to more effective drugs.
therapeutics
(rMAbs)
antibodies.4
is
now
aimed
to
antibodies
Recombinant antibody
at
the
glycosylation
rMAbs are created by mammalian cell cultures from
Chinese hamster ovary (CHO) cells or other mouse cells.4
are
14
of
rMAbs
Posttranslational
currently
and
modification
more
(PTM)
are
is
being
where
There
studied.
glycosylation
takes place; therefore it is the focus of investigations.4
Some
of
the
therapies
that
have
been
developed
through
glycosylation of the heavy chain regions which are specific for
disease
specific
involve
for
cetuximab
epidermal
and
rituximab.
growth
factor
20
and
Cetuximab
thus
is
is
rMAb
used
for
treatment of colon, head, and neck cancer.
oligosaccharide
directed
is
against
treatment
of
at
the
Asn88.
growth
non-Hodgkin’s
Its main N-linked
Rituximab
is
cancer
cells
of
lymphoma.10
used
This
in
as
therapy
well
therapy
as
a
becomes
effective against malignant cells when the glycoform bears a
bisecting GlcNAc.4
These specificities towards antibodies, along
with therapies, allow for greater potency against disease.
The biopharmaceutical industry manipulates the structure of
antibodies to enhance their efficacy to antigens and create more
helpful therapies.
Occasionally gene knock-outs or gene knock-
ins are made for selected glycosyltransferases.4
Even minute
differences in the glycoform structure have been shown to result
in gross changes.
glycoforms
in
the
Since there are many complex mixtures of
body,
all
of
which
play
a
role
in
vital
effector mechanisms within the body, the possibility to generate
rMAbs for therapeutics is endless.
CONCLUSION
The glycosylation of antibodies and other proteins is the
foundation of many processes within the body.
This has been
shown to be an intricate process involving the asparagine site
on Fc domain of IgG and the variation of the oligosaccharide
composition.
Sialylation
components
in
many
especially
rheumatoid
and
galactosylation
glycosylating
arthritis.
21
processes
The
are
also
involving
development
of
key
disease
therapy
using glycosylation of antibodies is one of the applications in
future biopharmaceuticals.
An important development of therapy
depends on the production of recombinant IgG.
Oligosaccharide
composition of the core of antibodies provides specificity that
is
most
consequential
in
pharmaceutical
applications.
Glycosylation is a natural approach to human health.
modification
of
an
antibody
through
the
The simple
attachment
of
carbohydrates, and their varying structures, is an attractive
mode of therapy.
22
References:
1. Berg, J.; Tymoczko, J.; Stryer, L. Biochemistry. 6th ed. New
York: W.H. Freeman and Company, 2007, pp. 316-319.
2. Rudd, P.; Elliot, T.; Cresswell, P.; Wilson, I.; Dwek, R.
Science 2001, 291, 2370-2376.
3.
University
of
Arizona,
The
Biology
Project
2000.
www.biology.arizona.edu
4. Jefferis, R. Biotechnol. Prog. 2005, 21, 11-16.
5. Raju, T.; Briggs, J.; Borge, S.; Jones, A. Glycobiology 2000,
10, 477-486.
6. Kaneko, Y.; Nimmerjahn, F.; Ravetch, J. Science 2006, 313,
670-673.
7.
Barker,
R.;
Young,
R.;
Leader,
K.;
Elson,
C.
Clin.
Exp.
Immunol. 1999, 117, 449-454.
8. National Health Museum. Monoclonal Antibody Technology 1999,
www.accessexcellence.org/RC/AB/IE/Monoclonal_Antibody.
9. Borman, S. Chemistry and Engineering News 2006, 13-22.
10.
The
Official
Site
for
Rituximab
www.rituximab.com.
23
Information.
2006.