Glycobiology vol. 10 no. 5 pp. 477–486, 2000
© 2000 Oxford University Press
Species-specific variation in glycosylation of IgG: evidence for the species-specific
sialylation and branch-specific galactosylation and importance for engineering
recombinant glycoprotein therapeutics
T.Shantha Raju1, John B.Briggs, Steve M.Borge and
Analytical Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA
Received on August 12, 1999; revised on November 7, 1999; accepted on
December 8, 1999
Immunoglobulins (IgG) are soluble serum glycoproteins in
which the oligosaccharides play significant roles in the
bioactivity and pharmacokinetics. Recombinant immuno-
globulins (rIgG) produced in different host cells by recom-
binant DNA technology are becoming major therapeutic
agents to treat life threatening diseases such as cancer.
Since glycosylation is cell type specific, rIgGs produced in
different host cells contain different patterns of oligosac-
charides which could affect the biological functions. In
order to determine the extent of this variation N-linked
oligosaccharide structures present in the IgGs of different
animal species were characterized. IgGs of human, rhesus,
dog, cow, guinea pig, sheep, goat, horse, rat, mouse, rabbit,
cat, and chicken were treated with peptide-N-glycosidase-F
(PNGase F) and the oligosaccharides analyzed by matrix-
assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF-MS) for neutral and acidic
oligosaccharides, in positive and negative ion modes,
respectively. The data show that for neutral oligosaccha-
rides, the proportions of terminal Gal, core Fuc and/or
bisecting GlcNAc containing oligosaccharides vary from
species to species; for sialylated oligosaccharides in the
negative mode MALDI-TOF-MS show that human and
chicken IgG contain oligosaccharides with N-acetyl-
neuraminicacid (NANA),whereasrhesus,cow, sheep,goat,
horse, and mouse IgGs contain oligosaccharides with N-
glycolylneuraminic acid (NGNA). In contrast, IgGs from
dog, guinea pig, rat, and rabbit contain both NANA and
NGNA. Further, the PNGase F released oligosaccharides
were derivatized with 9-aminopyrene 1,4,6-trisulfonic acid
(APTS) and analyzed by capillary electrophoresis with
laser induced fluorescence detection (CE-LIF). The CE-
LIF results indicate that the proportion of the two isomers
of monogalactosylated, biantennary, complex oligosaccha-
rides vary significantly, suggesting that the branch specifi-
city of β1,4-galactosyltransferase might be different in
different species. These results show that the glycosylation
of IgGs is species-specific, and reveal the necessity for
appropriate cell line selection to express rIgGs for human
therapy. The results of this study are useful for people
working in the transgenic area.
Key words: immunoglobulins/glycoproteins/oligosaccharides/
spectrometry/capillary electrophoresis/laser induced
Immunoglobulins (IgG) are soluble serum glycoproteins
involved in the humoral immune response, binding to antigens
to inactivate them or triggering an inflammatory response
which results in their clearance. On the basis of non-cross-
reacting antigenic determinants in the regions of highly
conserved amino acid sequences in the constant domains of the
heavy chains, five classes of immunoglobulins (IgG, IgA, IgD,
IgE, and IgM) have been distinguished. IgG contains a
conserved N-glycosylation site in the CH2 domain of each
heavy chain of the Fc region (Sutton and Phillips, 1983). The
oligosaccharides present at this site are highly heterogeneous,
and are known to affect the biological, pharmacological and
physicochemical properties of IgGs (Mizuochi et al., 1982;
Fujii et al., 1990). The biological functions affected by the
oligosaccharides include resistance to proteases, binding to
monocyte Fc receptors, interaction with complement compo-
nent C1q, feedback immunosupression of IgG synthesis, and
circulatory half-life in in vivo (Leatherbarrow et al., 1985; Tao
and Morrison, 1989; Walker et al., 1989; Leader et al., 1991).
Changes in N-glycosylation of IgG are associated with the
status in diseases such as rheumatoid arthritis (Parekh et al.,
1985, 1988; Rademachar et al., 1994; Malhotra et al., 1995).
The heterogeneous array of oligosaccharides on nascent
proteins is synthesized in the Golgi compartments, where
glycosyltransferases are localized (Kornfeld and Kornfeld,
1985). These glycosyltransferases are developmentally regu-
lated and differentially expressed (Schachter, 1986; Stanley et
al., 1996). Also, the expression of glycosyltransferases and
protein glycosylation is cell type specific and varies with cell
culture conditions (Stanley, 1984; Patel et al., 1992; Kumpel et
al., 1994; Wright and Morrison, 1998). For example, normal
CHO cells do not express N-acetylglucosaminyltransferase-III
(GlcNAcT-III), the enzyme responsible for the biosynthesis of
bisecting GlcNAc containing oligosaccharides. However, a
mutant cell line isolated by the mutagenesis of parent CHO
cells has been shown to express GlcNAcT-III (Campbell and
Stanley, 1984). Oligosaccharides containing the bisecting
1To whom correspondence should be addressed
T.S.Raju et al.
GlcNAc are found in human and chicken IgG. Hamako et al.
(1993) analyzed the asparagine-linked oligosaccharides of
IgGs from 11 different animal species and demonstrated that
the proportion of galactosylated, bisecting GlcNAc containing
and/or core fucosylated oligosaccharides vary among species.
These authors used hydrazinolysis to release oligosaccharides
from the IgGs. Hydrazinolysis causes sialic acids (NGNA and
NANA) and amino sugars to undergo N-deacetylation (in the
case of NGNA, N-deglycolylation), which results in a loss of
information regarding sialylated oligosaccharides (Patel and
Parekh, 1994). Further, the experiments of Hamako et al.
(1993) did not provide information on the diversity of
branching pattern of monogalactosylated oligosaccharides in
IgGs although significant amounts of these oligosaccharides
were observed in IgGs preparations.
Recombinant IgGs (rIgGs), produced by recombinant DNA
technology and/or by transgenic technology, are becoming
major therapeutic agents in the treatment of cancer and other
life threatening diseases. Recently, the FDA (United States
Food and Drug Administration) approved two monoclonal
antibodies, a chimeric monoclonal antibody for treating non-
Hodgkin’s lymphoma and a humanized monoclonal antibody
for treating metastatic breast cancer. These monoclonal anti-
bodies are produced in Chinese hamster ovary cells (CHO).
Different biopharmaceutical companies produce rIgGs in
different host cell lines in which the glycosylation machinery
might be different. In order to understand these issues and also
to obtain information on the nature of sialylated oligosaccha-
rides and the branching pattern of monogalactosylated
oligosaccharides, we undertook a detailed structural study of
N-linked oligosaccharides present in the IgGs of 13 different
animal species. The N-linked oligosaccharides of IgGs from
different animal species were released by PNGase F and
analyzed by MALDI-TOF-MS in the positive ion mode and
negative ion mode, and by CE-LIF after derivatizing with
APTS. In this paper, we show that IgG glycosylation, particu-
larly terminal sialylation and galactosylation of IgGs, is
species-specific suggesting that a careful selection of host cell
lines to produce rIgGs is necessary to avoid potentially immu-
nogenic carbohydrate epitopes.
Initial estimation of neutral sugar content by phenol-sulfuric
acid method (Dubois et al., 1956) showed that the values of
neutral sugars/protein ranged from ∼13 µg/mg for rabbit, goat
and cat IgGs to 32 µg/mg for chicken IgG, with human, cow,
sheep, rhesus, rat, mouse, horse, guinea pig, and dog IgGs
being intermediate (Table I). Determination of NANA and
NGNA content by an RP-HPLC method (Anumula, 1995)
showed that human and chicken IgG contain only NANA,
whereas IgGs from rhesus, cow, sheep, goat, horse, and mouse
Table I. Neutral carbohydrate and sialic acid content of IgGs
The neutral sugars were quantitated by phenol-sulfuric acid method and the
sialic acids were determined by a RP-HPLC method as described by Anumula
(1995). ND, not detectable.
aValues were obtained by assigning 150 kDa as an average molecular weight
IgGs Neutral sugars
Sialic acids (mol/mol of protein)a
Table II. Glycan composition for the quasimolecular ions observed inMALDI-TOF-MS analysis
(M+Na)+m/z Glycan composition
(M-H)–m/z Glycosyl composition
Glycosylation of IgG is species-specific
contain only NGNA. However the IgGs from dog, guinea pig,
rat, and rabbit contain both NANA and NGNA (see Table I),
showing that the distribution of sialic acid residues is different
in IgGs of different animal species.
Variations in neutral oligosaccharides
The N-linked oligosaccharides of IgGs were released by
PNGase F and analyzed by MALDI-TOF-MS in the positive
ion mode using a DHB matrix containing NaCl (Papac et al.,
1998). Consequently, quasimolecular ions of the oligosaccha-
rides (M + Na)+, were observed. The (M + Na)+ions of the
neutral oligosaccharides found in the IgGs and their composi-
tion are provided in Table II. The proposed oligosaccharide
structures for the respective (M + Na)+ions are shown in
Schemes 1 and 2.
Analysis of human IgG derived N-linked oligosaccharides
by MALDI-TOF-MS in the positive ion mode afforded (M +
Na)+ions at m/z 1486.3, 1648.4, 1810.5, 1689.4, 1851.5, and
2013.6 (Table III, Figure 1). Since these oligosaccharides were
released by PNGase F, the ions must be due to N-linked
oligosaccharides (Patel and Parekh, 1994). N-Linked oligosac-
charides contain a common core region consisting of three
Man and two GlcNAc residues. The core region of a complex
N-linked oligosaccharide often contains a Fuc (dHex) residue
linked to the reducing terminal GlcNAc residue of the free N-
linked oligosaccharides. Therefore, the additional Hex and
HexNAc residues of each human IgG oligosaccharide beyond
Hex3HexNAc2dHex1sequence found in the fucosylated core
region must be in the oligosaccharide antennae. For example,
in a glycan with composition Hex3HexNAc4dHex1(m/z
1486.3) all but two HexNAc residues can be attributed to resi-
dues found in the fucosylated core sequence. Hence, the two
HexNAc residues would reside in the antennae of an N-linked
oligosaccharide. Since previous studies suggest that HexNAc
residues found in human IgG are GlcNAc residues, the
Hex3HexNAc4dHex1glycan was assigned to structure 9
(Scheme 2). Similarly, the ions at m/z 1648.4 and 1810.5 have
masses consistent with the presence of additional 1 and 2
hexose residues, respectively, beyond those observed in the
oligosaccharide assigned to structure 9. Since biantennary N-
linked oligosaccharides contain 1 or 2 Gal residues, the ions at
m/z1648.4 and 1810.5 were assigned to structures 19 and/or 20
(structures 19 and 20 are the branch isomers of monogalactos-
ylated biantennary structures), and 21 respectively. The (M +
Na)+ion at m/z 1689.4 contains one additional HexNAc
residue compared to the (M + Na)+ion at m/z 1486.3. This ion
might be due to a triantennary structure consisting of three
terminal GlcNAc residues or due to a biantennary oligosaccha-
ride containing a bisecting GlcNAc residue. Mizuochi et al.
(1982) characterized human IgG derived oligosaccharides and
found no evidence for tri- and tetraantennary structures.
Further, these authors reported the presence of a bisecting
GlcNAc residue in some of the oligosaccharides in human IgG
(Mizuochi et al., 1982). Furthermore, if human IgG contained
any triantennary structures, we expect to have observed a (M +
Na)+ion at m/z 2175.4 due to 3 Gal residues. The positive ion
mode MALDI-TOF-MS analysis of human IgG derived
oligosaccharides showed no evidence for such structures.
Scheme 1. Structure of high mannose type N-linked oligosaccharides foundin
Scheme 2. Structureof complextype N-linkedoligosaccharides foundinIgGs.
T.S.Raju et al.
Hence, the (M + Na)+ion at m/z 1689.4 was assigned to the
structure 32 (Scheme 2). Similarly, the (M + Na)+ion at m/z
1851.5 was assigned to structures 33 and/or 34, and the ion at
m/z 2013.6 was assigned to structure 35.
The positive ion mode MALDI-TOF-MS analysis of the
PNGase F released oligosaccharides from chicken IgG gave
(M + Na)+ions at m/z from 1258.1 to 2013.6 (Figure 1, Table
III). The ion at m/z 1258.1 corresponds to the glycosyl compo-
sition of Hex5HexNAc2. From this ion, 3Hex and 2HexNAc
residues could be accounted for the core region of an N-linked
oligosaccharide. The remaining two Hex residues could not be
accounted for by Gal residues because there are no outer core
GlcNAc residues to which Gal residues could be linked. A
high mannose type N-linked oligosaccharide containing 5 Man
and 2 GlcNAc residues could account for this ion (ion at m/z
1258.1). Hence the (M + Na)+ion at m/z 1258.1 was assigned
to structure 1 (see Scheme 1). Similarly the (M + Na)+ions at
m/z 1420.2, 1582.3, 1744.4, 1906.4, and 2068.5 were assigned
to the structures 2A-C, 3A-C, 4A-C, 5 and 6, respectively. In
structures 2A-C, 3A-C, and 4A-C, the symbols A-C are
assigned to the positional isomers which are not distinguish-
able by MS because they produce isobaric ions. Structure 6
was assigned to the (M + Na)+ion at m/z 2068.5 because, in the
biosynthetic pathway of N-linked oligosaccharides, a high
mannosetype N-linked oligosaccharides
maximum 9 Man residues. The additional 1 Hex residue is
interpreted as a Glc residue. According to the biosynthesis of
N-linked oligosaccharides, dolichol linked Glc3Man9GlcNAc2
moiety is transferred en bloc to Asn by oligosaccharyltrans-
ferase (Kornfeld and Kornfeld, 1985). After the initial
oligosaccharide transfer reaction, trimming by glucosidases
and mannosidases takes place. During this trimming reaction,
various intermediates of
Glc2Man9GlcNAc2, Glc1Man9GlcNAc2, Man9GlcNAc2etc. are
produced. Further, Ohta et al. (1991) reported the presence of
a monoglucosylated Man9GlcNAc2structure in chicken IgG
along with an appreciable amount of high mannose type
oligosaccharides. These observations support the structural
assignments to (M + Na)+ions at m/z 1258.1, 1420.2, 1582.3,
1744.4, 1906.4, and 2068.5. The identification of structures 1–
6 suggests that the processing of N-linked oligosaccharides in
chicken IgG is incomplete. The other ions observed from the
positive ion mode MALDI-TOF-MS analysis of chicken IgG
derived N-linked oligosaccharides and their structural assign-
ments are shown in Table III and Figure 1, which suggest that,
in addition to high mannose type structures, complex bianten-
nary structures with or without core Fuc, terminal Gal and
bisecting GlcNAc residues are also present. These data indi-
cate that the N-linked oligosaccharides of chicken IgG are
more heterogeneous than the N-linked oligosaccharides of
The N-linked oligosaccharides from other IgGs were also
released by PNGase F and analyzed by MALDI-TOF-MS in
the positive ion mode. The observed (M + Na)+ions and their
structural assignments are shown in Table III (see also Figure
1). It is evident that these IgGs also contain complex bianten-
nary structures with or without core Fuc residues and that the
proportion of bisecting GlcNAc containing oligosaccharides
varies among species. For example, ∼67% of sheep IgG
compared to ∼53% of chicken IgG oligosaccharides. However,
the N-linked oligosaccharides of dog, horse and cat IgGs
contain no detectable bisecting GlcNAc residue. Allother IgGs
contain an appreciable proportion of oligosaccharides with
terminal GlcNAc residues which vary from 2% to 40% (see
Table III). Similarly, the variation in the galactosylated
oligosaccharides is also evident (Table III, Figure 1). Rat,
horse, and dog IgGs contain very few galactosylated oligosac-
charides whereas ∼90% of the sheep IgG oligosaccharides are
Variations in acidic oligosaccharides
The PNGase F released oligosaccharides of different IgGs
were also analyzed by MALDI-TOF-MS in the negative ion
mode using THAP as matrix (Papac et al., 1996). The data are
shown in Table IV and Figure 2. The oligosaccharides released
from human IgG gave molecular ions, (M-H)–ions, at m/z
1915.7, 2077.8, 2369.1, 2281.0, and 2572.3 (see Figure 2). The
glycosyl composition of these ions and the corresponding
structural assignments are shown in Tables II and IV. Only
NANA-containing oligosaccharides were detected in human
IgG. The (M-H)–ions corresponding to oligosaccharides with
NGNA (i.e., at m/z values of +16 amu compared to their
NANA counterparts) were not detected in human IgG.
The (M-H)–ions observed for oligosaccharides of other IgGs
and their structural assignments are shown in Table IV. Like
human IgG, chicken IgG also contained oligosaccharides with
only NANA. However, mouse, horse, sheep, rhesus, cow, and
goat IgG-derived oligosaccharides contain only NGNA. In
contrast, the oligosaccharides derived from rat, guinea pig,
rabbit, cat, and dog contain both NANA and NGNA but in
different proportions (see Table IV). These data show that
sialylation of IgG oligosaccharides varies from species to
species. The identification and quantitation of NANA- and
NGNA-containing oligosaccharides of these IgGs by MALDI-
TOF-MS is in good agreement with the data obtained by RP-
HPLC method (Table I). The data in Tables I and IV also
suggest that the amount of sialic acids present in IgGs is signif-
Fig. 1. Positive ion mode MALDI-TOF-MS of PNGase F released N-linked
oligosaccharides of IgGs. The N-linked oligosaccharides of IgGs from the
indicated species were released by PNGase F as described in Materials and
the positive ion mode using a DHB matrix containing NaCl.
Glycosylation of IgG is species-specific
icantly less than the amount of neutral sugars indicating that
the sialylated oligosaccharides are not the major species.
Evidence for branch-specific galactosylation
The oligosaccharides released by PNGase F were labeled with
9-aminopyrene-1,4,6-trisulfonic acid by reductive amination
and analyzed by CE-LIF. Figure 3A shows the electrophero-
gram of human IgG oligosaccharides. In Figure 3A, peaks I
and IV were identified by comparing the migration time of
standard oligosaccharides and assigned to structures 18 and 21,
respectively (data not shown). Peaks II, III, and IV migrated at
the position of peak I after β-galactosidase digestion (see
Figure 3B), and hence the former peaks are galactosylated
biantennary structures with core Fuc residue. Since peak IV
was identified as Structure 21 which contains 2 Gal residues,
peaksII and III must be the two isomersof monogalactosylated
biantennary structures (structures 19 and 20). Jefferis et al.
(1990) reported that human IgG contains two monogalactos-
ylated core fucosylated biantennary structures in which struc-
ture 19 is the predominant species. Based on these
observations, peaks II and III were assigned to structures 19
and 20, respectively. This assignment was independently
confirmed by in vitro galactosylation of structure 18 with β1,4-
galactosyltransferase (β1,4GT) and UDP-Gal (Raju et al.,
unpublished observations) which was in good agreement with
the results described by Paquet et al. (1984).
The electropherogram of APTS-labeled oligosaccharides
derived from cow IgG is shown in Figure 3C in which the
proportion of peaks II and III is different compared to the
proportion observed for human IgG (see Table V). In this case
also, peaks II, III, and IV migrated to the position of peak I
after β-galactosidase treatment (Figure 3D). The data for peaks
II and III (structures 19 and 20, respectively), obtained from
CE-LIF data for other IgGs is summarized in Table V. The data
in Table V show that the proportions of peaks II and III vary,
showing that branch-specific galactosylation of IgGs varies
from species to species.
Table III. Positive ion mode MALDI-TOF-MS analysis of PNGase F released oligosaccharides from IgGs
The N-linked oligosaccharides were released from IgGs by PNGase F and analyzed by MALDI-TOF-MS in the positive ion mode as described in Materials and
methods. The relative proportion (as % of the total) of the quasi-molecular ions, (M+Na)+, was calculated as reported by Papac et al. (1998). The structure of the
oligosaccharides are shown in Schemes 1 and 2.
HumanRhesus DogCow Guinea
Sheep GoatHorse RatMouseRabbit Cat Chicken
High Mannose type
6.8 2.1 1.028.6
Fig. 2. Negative ion mode MALDI-TOF-MS of PNGase F released N-linked
oligosaccharides of IgGs. The N-linked oligosaccharides were obtained as
described in Figure 1 and analyzed by MALDI-TOF-MS in the negative ion
mode using THAP as a matrix.
T.S.Raju et al.
Serum IgGs are soluble glycoproteins in which the covalently
bound oligosaccharide moieties present in the Fc domain play
significant roles in their bioactivity and serum half life.
Although some glycosylation can occur outside the Fc domain,
this is usually a rare event for IgG (Parekh et al., 1985). The N-
linked oligosaccharides of human IgG have been extensively
characterized. The Fc derived oligosaccharides of human IgG
are mainly complex biantennary type with heterogeneity in
core fucosylation, terminal sialylation, and galactosylation.
Table IV. Negative mode MALDI-TOF-MS analysis of PNGase F released oligosaccharides from IgGs
The N-linked oligosaccharides, released by PNGase F, were analyzed by MALDI-TOF-MS in the negative mode using THAP as a matrix as described in
Materials and methods. The relative proportions (as % of the total) of the observed molecular ions, (M-H)–, were calculated as reported by Papac et al. (1996).
The oligosaccharides structures are shown in Schemes 1 and 2. *The ion at 1728.5 corresponds to an N-linked oligosaccharide with composition
HexNAc3Hex4NANA1(possibly a hybrid oligosaccharide).
HumanRhesus DogCow Guinea
SheepGoat HorseRat Mouse RabbitCat Chicken
Fig. 3. CE-LIF analysis of PNGase F released N-linked oligosaccharides of
human and cow IgGs. The PNGase F released oligosaccharides of human (A)
in the Materials and methods. Identification of peaks I-IV is described in the
Results section. The human (B) and cow (D) IgGs were treated with β-
galactosidase. The oligosaccharides were released by PNGase F, labeled with
APTS and analyzed by CE-LIF as described in Materials and methods.
The PNGase F released oligosaccharides of IgG’s were labeled with APTS
and analyzed by CE-LIF as described in Materials and methods. The Peaks I
and IV (Figure 3) were identified by comparing the elution times of authentic
standard oligosaccharides. The relative proportions (as %) of Peaks II and III
are shown. NI=not identified
Table V. Relative proportions of monogalactosylated complex biantennary
N-linked oligosaccharides present in IgGs
Glycosylation of IgG is species-specific
Heterogeneity of these oligosaccharides also arises due to the
presence or absence of a bisecting GlcNAc (Mizuochi et al.,
1982). However, the structures of oligosaccharides of IgGs
from other animal species have not been studied thoroughly.
Because of their biological importance, and to further our
understanding of thestructure-function relationships, we deter-
mined the structures of N-glycans of IgGs from 13 different
animal species. The results of these studies will enhance our
understanding of recombinant glycoproteins of therapeutic
interest. The IgGs studied are representative of respective
animal species. The IgGs were affinity-purified before analysis
and the oligosaccharides released by PNGase F from the intact
molecules were analyzed. The majority of structural assign-
ments were based on masses (determined by mass spectrom-
compositions of hexose, N-acetylhexosamines, sialic acids,
etc. (see, for example, Table II). This information, together
with the knowledge base of the structures which have been
previously determined (Mizuochi et al., 1982; Nose and
Wigzell, 1983; Leatherbarrow et al., 1985; Fujii et al., 1990),
provided the basis for structural assignments in this work. In
some cases, additional information was derived from the use of
specific enzymes such as glycosidases and glycosyltrans-
ferases, and CE-LIF analysis.
The MALDI-TOF-MS analysis of PNGase F released
oligosaccharides show that IgGs from 13 different animal
species contain a heterogeneous array of biantennary complex
type oligosaccharides. However, there seems to be species-
specific variation in core fucosylation and terminal galactos-
ylation (see Figure 4A,B). For example, the oligosaccharides
derived from human IgG contain mostly core fucosylated
oligosaccharides, whereas the oligosaccharides derived from
rabbit IgG contain mostly nonfucosylated oligosaccharides
(Figure 4A). Similarly, about 90% of sheep IgG oligosaccha-
rides are galactosylated, whereas only ∼10% of rat IgG
oligosaccharides contain galactose residues (Figure 4B). The
MALDI-TOF-MS analyses also show that IgGs from human,
rhesus, cow, guinea pig, sheep, goat, rat, rabbit, and chicken
contain biantennary oligosaccharides with one additional
HexNAc residue (see Tables II and III). Based on the observa-
tions made by Mizuochi et al. (1982) on human IgG derived
oligosaccharides, these were considered as bisecting GlcNAc
containing oligosaccharides. The oligosaccharides from dog,
horse, and mouse did not contain detectable bisecting GlcNAc
residue (Figure 4C). These observations confirm the species-
specific variation in core fucosylation, terminal galactosylation
and the presence of a bisecting GlcNAc residue in IgGs from
different animal species. The presence of oligosaccharides
containing bisecting GlcNAc residues in human, rhesus, cow,
guinea pig, sheep, goat, rat, rabbit, and chicken IgGs suggests
that these animal species express N-acetylglucosaminyltrans-
ferase-III, the enzyme responsible for the biosynthesis of
bisecting GlcNAc containing oligosaccharides (Campbell and
Stanley, 1984). The absence of hybrid and complex tri- and
tetraantennary structures suggests that the glycosylation
observed in this study is consistent with the restriction of Fc
glycosylation to biantennary structures(Parekh et al., 1985). In
all of the species studied here, with the exception of chicken
(which additionally contains high-mannose structures), the
predominant structures are also biantennary complex oligosac-
charides. The data presented here therefore probably describe
only the glycosylation site of the Fc domains.
Yamada et al. (1997) reported that the galactosylation of
human IgG varies with age and is gender specific. Parekh et al.
(1988) showed that the terminal galactosylation of human IgG
is an indication of disease status in rheumatoid arthritis
patients. Further, Patel et al. (1992) observed that the galacto-
sylation of IgG varies with cell culture conditions. The CE-LIF
data suggest that the galactosylation of IgGs is also branch-
specific (Table V, Figure 3). The MALDI-TOF-MS data
(Table III) suggest that IgGs from different animal species
contain significant amount of monogalactosylated biantennary
structures (for example structures 19 and 20). Using MS tech-
niques it was not possible to distinguish structures 19 and 20.
Hence, we used CE-LIF to determine the proportion of struc-
tures 19 and 20 in IgGs. The data in Figure 3 and Table V
show that the proportion of structures 19 and 20 varies from
species to species. In human and rhesus IgGs the proportion of
structures 19 and 20 is very similar. However, the proportions
observed in cow and mouse IgGs is opposite to each other.
These data (Table V ) suggest that the tendency for one branch
to be galactosylated over another branch is species-specific.
The branch and species-specific galactosylation of IgGs
implies species-specific variation in the activity of β1,4GT.
The enzyme, β1,4GT is a constitutively expressed, trans-Golgi
resident, type II membrane-bound glycoprotein that catalyzes
the transfer of Gal to GlcNAc from UDP-Gal (Beyer and Hill,
1968). β1,4GT enzymatic activity is widely distributed in the
vertebrate kingdom, in both mammals and nonmammals,
including avians and amphibians (Shaper et al., 1997).
Recently, Lo et al. (Lo et al., 1998) reported the presence of a
family of β1,4GT genes. Our in vitro galactosylation of agalac-
tosylated biantennary complex oligosaccharide with commer-
cially available human and cow β1,4GT suggest that the
enzyme preferentially adds Gal to GlcNAc linked to α1,3-Man
Fig. 4. Comparison of core fucosylated (A), terminal galactosylated (B),
bisecting GlcNAc containing (C), and NANA and NGNA (D) containing
oligosaccharides of IgGs. The PNGase F released N-linked oligosaccharides
of IgGs from 13 different animal species were analyzed by MALDI-TOF-MS
in the positive and negative ion mode, as described in Materials and methods.
Therelativeproportionsofthe quasi-molecular or (M-H)-ionswere calculated
as described by Papac et al. The distribution of core Fuc, terminal Gal,
bisecting GlcNAc, and NANA and NGNA residues containing
oligosaccharides is shown.
T.S.Raju et al.
arm (Raju et al., unpublished observations). These observa-
tions also suggest that the activity of β1,4GT is species- and
Sialic acid determinations indicate that the IgGs from
different animal species contain an appreciable amount of
sialylated oligosaccharides (see Table I). Data in Tables I and
IV and Figure 2 provide additional information on the varia-
bility of NANA and NGNA content from species to species
and the structures in which they are found. For example,
human and chicken IgG-derived acidic oligosaccharides
contain exclusively NANA (Figure 4D), whereas sheep, goat,
rhesus, cow, horse, and mouse derived acidic oligosaccharides
contain only NGNA. Interestingly, the acidic oligosaccharides
derived from dog, guinea pig, rat, rabbit, and cat IgGs contain
both NANA and NGNA residues in different proportions,
confirming significant variations in the sialylated oligosaccha-
rides of IgGs. Muchmore et al. (Muchmore et al., 1998)
reported that NGNA is essentially undetectable on human
plasma proteins and erythrocytes, but is a major component in
chimpanzee, bonobo, gorilla, and orangutan. In our study also,
NGNA is essentially undetectable in human IgG (see Table I
and VI). Biosynthetically, NGNA arises from the action of a
hydroxylase that converts the nucleotide donor sugar, CMP-
NANA to CMP-NGNA (Chou et al., 1998). The activity of
CMP-NANA hydroxylase is reported to be present in chim-
panzee cells but not in human cells (Varki, 1992; Chou et al.,
1998; Muchmore et al., 1998). As major terminal structures on
cell surfaces,sialic acids are involved in intercellular cross-talk
and microbe–host recognition. The level of NGNA is known to
positively or negatively affect several of these endogenous and
exogenous interactions (Varki, 1993, 1994, 1997, 1998; Varki
and Marth, 1995). Hence there are potential functional conse-
quences of this structural change which affect the cell surface
functions (Muchmore et al., 1998).
rIgGs are being developed by many biopharmaceutical
companies as therapeutic agents to treat human diseases. Two
rIgGs, produced in CHO cells were approved by regulatory
agencies around the world, one to treat non-Hodgkin’s
lymphoma and the other to treat metastatic breast cancer over-
expressing HER-2 oncogene. Different biopharmaceutical
firms use different cell lines and different cell culture condi-
tions to produce the rIgGs. However, different cell lines have
different glycosylation machinery. Our data on the glycosyla-
tion of IgGs from different species suggest that the glycosyla-
tion varies from species to species. This in turn suggests that
the glycosylation of rIgGs produced in different cell lines
might be significantly different. For example, monoclonal anti-
bodies produced in mouse cell lines might contain NGNA resi-
dues which are potentially immunogenic to humans (Cho et
al., 1996; Noguchi, 1995). The data obtained by the analysis of
oligosaccharides of IgGs from 13 different animal species
clearly suggests that a careful selection of cell line is a prereq-
uisite to produce rIgGs for human therapy. The data is also
helpful to understand the effect of glycosylation on protein
therapeutics produced by transgenic technology. There is a
growing interest to express protein therapeutics using trans-
genic animals such as goat, cow, sheep, etc. The data presented
here clearly suggest that IgGs of goat, cow, and sheep have
different glycosylation pattern which might influence their
biological and pharmacological functions. Further, Cabanes-
Macheteau et al. expressed a mouse IgG in transgenic tobacco
plants and shown that they contain unusual carbohydrates
which might be immunogenic to humans (Cabanes-Macheteau
et al., 1999). Such studies clearly show the importance of our
results on species-specific glycosylation in the areas of
working on glycoprotein therapeutics.
Materials and methods
Human, rhesus, dog, cow, guinea pig, sheep, goat, horse, rat,
mouse, rabbit, cat, and chicken IgGs were obtained from Sigma
Chemical Co. (St. Louis, MO) and purified on protein A/G
columns before use. PNGase-F and human β1,4-galacto-
syltransferase were obtained from Boehringer Mannheim
(Germany) or from Oxford GlycoSciences (London, UK). The
cow β1,4-galactosyltransferase and polyvinylpyrrolidone were
from Sigma Chemical Co. Immobilized Protein A and Protein G
cartridges were obtained from Pharmacia and used as per the
manufacturers protocol. The matrices 2′,4′,6′-trihydroxyace-
tophenone monohydrate and 2,5-Dihydroxybenzoic acid were
purchased from Aldrich (Milwaukee, WI). 9-Aminopyrene-
1,4,6-trisulfonic acid was purchased from Beckman (Jackson,
Neutral hexoses were quantitated by phenol-sulfuric acid assay
(Dubois et al., 1956), and the sialic acids were measured by
RP-HPLC (Anumula, 1995).
Release of N-linked oligosaccharides by PNGase-F
The N-linked oligosaccharides from IgGs (at least two
different batches of IgGs from each species were used in the
study) released by PNGase-F using a high-throughput micros-
cale method as described by Papac et al. (1998). The PVDF
membrane wells of a MultiScreen-IP plate (pore size 0.45 µm,
Millipore) were preconditioned by washing with 1 × 100 µl
methanol, 3 × 100 µl water and 1 × 50 µl reduction and
carboxymethylation (RCM) buffer (8 M urea containing
360 mM Tris, pH 8.6, and 3.2 mM EDTA). About 25–40 µg of
glycoprotein was loaded into wells containing 10 µl RCM
buffer and the solution was brought to 50 µl by adding addi-
tional RCM buffer. Reduction of protein was performed in the
presence of 50 µl of 0.1 M dithiothreitol in RCM buffer for 1 h
at 37°C followed by washing with water (3 × 300 µl).
Carboxymethylation was accomplished by adding 50 µl of 0.1
M iodoacetic acid in RCM buffer and incubating at room
temperature for 30 min in the dark. Following carboxymethyl-
ation, the wells were washed with water (3 × 300 µl) and the
membranes were blocked with 1% aqueous polyvinylpyrro-
lidone 360 (100 µl) at room temperature for 60 min. The wells
were washed with water (3 × 300 µl) to remove blocking agent
and incubated with 1.25 U of PNGase-F (Oxford Glyco-
Sciences) in 50 µl of 10 mM Tris-acetate buffer (pH 8.3) at
37°C for 3 h in plates covered with Parafilm to prevent evapo-
rative loss of the digestion buffer. The solution containing
released oligosaccharide, buffer and enzyme was transferred to
an Eppendorf tube and treated with 150 mM acetic acid for 3 h
at room temperature. The solution containing the released
oligosaccharides was passed through a 0.6 ml of cation-
Glycosylation of IgG is species-specific
exchange resin (AG50W-X8 resin, H+form, 100–200 mesh,
Bio-Rad, Hercules, CA) to remove salt and protein contami-
nants prior to analysis by mass spectrometry.
MALDI-TOF-MS was performed on a Voyager DE Biospec-
trometry Work Station (Perseptive Biosystems, Framingham,
MA) equipped with delayed extraction. A nitrogen laser was
used to irradiate samples with ultraviolet light (337 nm), and an
average of 240 scans was taken. The instrument was operated in
linear configuration (1.2 m flight path), and an acceleration
voltage of 20 kV was used to propel ions down the flight tube
after a 60 ns delay. Samples (0.5 µl) were applied to a polished
stainless steel target to which 0.3 µl of matrix was added and
dried under vacuum (50 × 10–3Torr). Oligosaccharide standards
were used to achieve a two-point external calibration for mass
assignment of ions (Papac et al., 1996). 2,5-Dihydroxybenzoic
acid (DHB)and 2,4,6-trihydroxyacetophenone
matriceswere used in the analysisof neutraland acidicoligosac-
charides, respectively (Papac et al., 1996, 1998).
Analysis of oligosaccharides by CE-LIF
The IgG samples (400–500 µg, at least two different batches of
IgGs from each species were used in the study) were treated
with PNGase-F (50 U/mg) in 20 mM phosphate buffer (pH 8.2,
200 µl) containing 50 mM EDTA and 0.02% (w/v) sodium
azide at 37°C for 24 h. Released oligosaccharides were sepa-
rated from protein and enzyme by ethanol precipitation and/or
by heat denaturation. The supernatant was evaporated to
dryness using a Savant Speed Vac. The samples were fluores-
cently labeled by adding 15 µl of a 19 mM solution of 9-
aminopyrene-1,4,6-trisulfonic acid (APTS, Beckman) in 15%
acetic acid, and 5 µl of 1 M sodium cyanoborohydride in
tetrahydrofuran. The labeling reaction was carried out for two
h at 55°C, followed by ∼25-fold dilution with water prior to
capillary electrophoretic (CE) analysis. CE analysis of the
labeled oligosaccharides was performed on a P/ACE 5000 CE
system (Beckman) with reversed polarity, using a 50-µm
internal diameter coated capillary and 20 cm effective length
(eCAP, N-CHO coated capillary, Beckman). The samples
were introduced by pressure injection at 0.5 psi for 2–4 s, and
the separation was carried out at a constant voltage of 20 kV.
The temperature of the capillary was maintained at 20°C. The
separations were monitored on-column with a Beckman laser-
induced fluorescence detection system using a 3 mW argon ion
laser with an excitation wavelength of 488 nm and emission
bandpass filter at 520 ± 10 nm.
Human and cow IgGs (∼2 mg each) in 100 mM citrate-phos-
phate buffer, pH 6.4 were treated with β-D-galactosidase
(Diplococcus pneumoniae, 40 mU/ mg protein, Boehringer
Mannheim) at 37°C for 24 h. The antibodies were purified
using a HiTrap Protein A cartridge (Pharmacia) as described
by the manufacturer. The modified antibodies were treated
with PNGase F to release the oligosaccharides which were
analyzed by CE-LIF as described above.
In vitro galactosylation with β1,4-galactosyltransferase
Oligosaccharide samples (20 µg) in 50 mM sodium cacodylate
buffer, pH 6.7 (in a final volume of 100 µl) were treated with
UDP-Gal (5 µmol, Sigma Chemical Co.) and β1,4-galactosyl-
transferase (25 mU, human milk or cow, Boehringer
Mannheim or Sigma) at 37°C for 0–4 h. The reaction was
stopped by adding ethanol (∼1.0 ml). The samples were centri-
fuged and the centrifugate was dried using a Speed Vac. The
oligosaccharides were labeled with APTS and analyzed by CE-
LIF as described above.
We thank Drs. John O’Connor and Pamela Stanley (Albert
Einstein College of Medicine, New York) for helpful discus-
sions. We greatly acknowledge Mr. Michael Wilks for sialic
acids analysis by RP-HPLC.
CHO cells,Chinesehamster ovary cells;IgG,immunoglobulinG;
rIgG, recombinant IgG; MALDI-TOF-MS, matrix-assisted laser/
desorption ionization time-of-flight mass spectrometry; ESI-MS,
electrospray ionization-mass spectrometry; RP-HPLC, reverse
phase high-performance liquid chromatography; HPAEC-PAD,
high-performance anion exchange chromatography with pulsed
amperometric detection; CE-LIF, capillary electrophoresis with
laser induced fluorescence detection; RCM, reduction and
carboxymethylation; NANA, N-acetyl neuraminic acid; NGNA,
N-glycolyl neuraminic acid: APTS, 9-aminopyrene 1,4,6-trisul-
fonic acid; PNGase F, Peptide-N-glycosidase-F; GlcNAc, N-
acetyl D-glucosamine; Gal, D-galactose; Fuc, L-fucose; β1,4GT,
β1,4-galactosyltransferase; GlcNAcT-III, N-acetylglucosaminyl-
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