Analytical Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA
Received on August 12, 1999; revised on November 7, 1999; accepted on December 8, 1999.
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Abstract |
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Key words: immunoglobulins/glycoproteins/oligosaccharides/carbohydrates/sialic acid/glycosyltransferases/mass spectrometry/capillary electrophoresis/laser induced fluorescence detection
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Introduction |
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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 regulated 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 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-Hodgkins lymphoma and a humanized monoclonal antibody for treating metastatic breast cancer. These monoclonal antibodies 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 oligosaccharides 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, particularly 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 immunogenic carbohydrate epitopes.
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Results |
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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 biantennary 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 oligosaccharides contain a bisecting GlcNAc residue compared to ~53% of chicken IgG oligosaccharides. However, the N-linked oligosaccharides of dog, horse and cat IgGs contain no detectable bisecting GlcNAc residue. All other 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 oligosaccharides whereas ~90% of the sheep IgG oligosaccharides are galactosylated.
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.
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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 electropherogram 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 ß-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, peaks II and III must be the two isomers of monogalactosylated biantennary structures (structures 19 and 20). Jefferis et al. (1990) reported that human IgG contains two monogalactosylated core fucosylated biantennary structures in which structure 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 ß1,4-galactosyltransferase (ß1,4GT) and UDP-Gal (Raju et al., unpublished observations) which was in good agreement with the results described by Paquet et al. (1984)
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Discussion |
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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 galactosylation (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 oligosaccharides 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 observations 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-acetylglucosaminyltransferase-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 oligosaccharides. The data presented here therefore probably describe only the glycosylation site of the Fc domains.
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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 variability 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 oligosaccharides 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 chimpanzee 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 microbehost 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 consequences 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-Hodgkins lymphoma and the other to treat metastatic breast cancer overexpressing HER-2 oncogene. Different biopharmaceutical firms use different cell lines and different cell culture conditions to produce the rIgGs. However, different cell lines have different glycosylation machinery. Our data on the glycosylation of IgGs from different species suggest that the glycosylation 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 antibodies produced in mouse cell lines might contain NGNA residues 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 prerequisite 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 transgenic 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 biotechnology, immunology, glycobiology, and others working on glycoprotein therapeutics.
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Materials and methods |
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Analytical methods
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 microscale 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 x 100 µl methanol, 3 x 100 µl water and 1 x 50 µl reduction and carboxymethylation (RCM) buffer (8 M urea containing 360 mM Tris, pH 8.6, and 3.2 mM EDTA). About 2540 µg of glycoprotein was loaded into wells containing 10 µl RCM buffer and the solution was brought to 50 µl by adding additional 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 x 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 carboxymethylation, the wells were washed with water (3 x 300 µl) and the membranes were blocked with 1% aqueous polyvinylpyrrolidone 360 (100 µl) at room temperature for 60 min. The wells were washed with water (3 x 300 µl) to remove blocking agent and incubated with 1.25 U of PNGase-F (Oxford GlycoSciences) 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 evaporative 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-exchange resin (AG50W-X8 resin, H+ form, 100200 mesh, Bio-Rad, Hercules, CA) to remove salt and protein contaminants prior to analysis by mass spectrometry.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)
MALDI-TOF-MS was performed on a Voyager DE Biospectrometry 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 x 103 Torr). 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 (THAP) matrices were used in the analysis of neutral and acidic oligosaccharides, respectively (Papac et al., 1996, 1998).
Analysis of oligosaccharides by CE-LIF
The IgG samples (400500 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 separated 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 fluorescently 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 24 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.
ß-Galactosidase digestion
Human and cow IgGs (~2 mg each) in 100 mM citrate-phosphate buffer, pH 6.4 were treated with ß-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 ß1,4-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 ß1,4-galactosyltransferase (25 mU, human milk or cow, Boehringer Mannheim or Sigma) at 37°C for 04 h. The reaction was stopped by adding ethanol (~1.0 ml). The samples were centrifuged and the centrifugate was dried using a Speed Vac. The oligosaccharides were labeled with APTS and analyzed by CE-LIF as described above.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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