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 protein 1.

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.

Glycoproteins found within the immune system of the body are at

the root of some diseases and also natural therapies. 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 of carbohydrates to proteins occurs through O-linkage

or N-linkage. O-linked carbohydrates attach to the 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 in the ER

membrane for attachment to the oligosaccharide.1

The terminal phosphate group of dolichol phosphate attaches to

the carbohydrate, which then transfers the oligosaccharide to an

asparagine residue of the protein. Glycoproteins once formed in

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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 acids have

two types of structures; high mannose or complex. Each of these

structures contains a pentasaccharide core of three mannose and

two N-acetylglucosamine (GlcNAc) residues.1 The carbohydrates

which typically partake in glycosylation are biantennary, taking

on a Y shape. 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

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Glycoproteins are key molecules in the immune system, which aid

in immune response. One relevant issue involving glycosylation,

is the glycosylation of antibodies in order to increase the

affinity of antibody-antigen 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). Antibodies are

also homodimers with covalent disulfide-bonding 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

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 Upon the foreign invasion of antigen, the cell, or body,

will invoke the production of antibodies against antigen as

immune response.1 Antibodies that are produced are specific for

different 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. On the Fc, interaction

sites for ligands, which activate effector mechanisms of

glycosylation, are expressed. The ligands that interact are Fc

receptor types FcRI, FcRII, FcRIII. The expression of these

effector functions vary the efficacy between different glycoforms

and are essential for glycosylation. 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.

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Figure 5: Human IgG with the functional regions. The green
regions are the light chains and the orange are the heavy
chains.4

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 of the biantennary

carbohydrates are such that the pentasaccharide core is

7
invariant, however, external saccharides can vary. 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 do not allow for the desired function of the

glycoprotein to occur. Therefore, glycoproteins are required to

engage in a series of interactions with chaperones and enzymes.

An oligosaccharide precursor, GlcNAc2Man9Glc3, attaches to the

protein in the ER. 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. Figure 7

shows the structure of calnexin and calreticulin.2

A B

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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 The proteins which are produced and folded are

then 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

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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 CD2-CD48

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 of health

and disease in regards to the immune system.2

Glycosylation in Vivo Pertinent to Disease

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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 Fc 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
Neuraminidase is the enzyme that cleaves glycolytic bonds between

SA and Gal (Figure 9). The mass spectroscopic analysis in Figure

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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 the
brackets are masses of fragments containing SA.6

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

SA residues existed in patients with RA. Antibodies with

decreased levels of terminal sugars have been suggested to take

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on a pathogenic role, thus influencing studies in vivo of SA and

antibody activity.6

The sialylation of a glycoform, 6A6-IgG, reduced the anti-

inflammatory biological activity which could be restored upon the

removal of the SA by neuraminidase. Binding affinity analysis of

sialylated antibodies with their respective FcRs also showed the

effects of sialylation (Table 1). The four glycoforms that were

used were IgG antibodies with varying glycosylation. The binding

affinity of each glycoform to the Fc type receptors is best

illustrated by the binding constants in the second to fourth

columns of Table 1.

Table 1: FcR Binding to Antibodies (6A6-IgGs)6

These binding constants were in direct correlation with the

physiological effects. Those glycoforms without SA bound to the

Fc type receptors had higher binding constants and inflammation

was enhanced.6

Galactosylation7

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 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 agents have been achieved using recombinant DNA

technology. These particular immunoglobulins are known as

recombinant immunoglobulin G (rIgG). 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

were studied to find the model for species-specific production of

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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

oligosaccharides from the IgGs. They were then analyzed through

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the use of matrix-assisted laser desorption/ionization time-of-

flight mass spectrometry (MALDI-TOF-MS) for neutral and acidic

oligosaccharides shown in Figure 12A and 12B. This mass spectral

analysis allowed for a comparison 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,

demonstrating the importance of species-specificity of

oligosaccharides. This also stresses the necessity of careful

consideration for therapies using rIgGs of different species for

humans.

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 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

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 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 or cleavage,

the fewer glycoforms are present in the species. The most

prominent difference lies within the varying glycoform

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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 The

fusion of these cells creates an antibody known as a hybridoma. 8

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This hybridoma continually makes antibodies specific for that

antigen, or tumor cell, thus creating monoclonal antibodies. The

purified antibody attacks only the target molecule, decreasing

the side effects in vivo.8

The specificity of antibodies increases the value of

monoclonal antibody research.8 In order to make mAbs 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 to antibodies

open the door to more effective drugs. Recombinant antibody

therapeutics (rMAbs) is now aimed at the glycosylation of

antibodies.4 rMAbs are created by mammalian cell cultures from

Chinese hamster ovary (CHO) cells or other mouse cells.4 There

are 14 rMAbs currently and more are being studied.

Posttranslational modification (PTM) is where 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 involve cetuximab and rituximab. Cetuximab is rMAb

specific for epidermal growth factor and thus is used for

treatment of colon, head, and neck cancer. Its main N-linked

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oligosaccharide is at Asn88. Rituximab is used in therapy

directed against the growth of cancer cells as well as a

treatment of non-Hodgkins lymphoma.10 This therapy 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. Since there are many complex mixtures of

glycoforms in the 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 and galactosylation are also key

components in many glycosylating processes involving disease

especially rheumatoid arthritis. The development of therapy

using glycosylation of antibodies is one of the applications in

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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. The simple

modification of an antibody through the attachment of

carbohydrates, and their varying structures, is an attractive

mode of therapy.

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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 Information. 2006.
www.rituximab.com.

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