Department of Biology, Johns Hopkins University, Baltimore, MD 21218
Received on September 27, 2003; revised on November 7, 2003; accepted on November 10, 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Asn297 / chicken serum IgG / IgG-Fc / monoglucosylated high-mannose-type / N-glycan processing
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We recently found that pigeon serum IgG has unique N-glycan features, such as the presence of a large quantity of highly galactosylated triantennary oligosaccharides as well as monoglucosylated high-mannose-type oligosaccharides (monoGlc-high-Man) (Suzuki et al., 2003), which are not found in mammalian normal serum IgG. MonoGlc-high-Man is probably a characteristic in avian IgG because it had been also found in chicken (Ohta et al., 1991
) and quail (Matsuura et al., 1993
) egg yolk IgGs and chicken serum IgG (Raju et al., 2000
). Except avian IgGs, however, the monoGlc-high-Man is rarely found in secreted mature glycoproteins, and only transiently exists on glycoproteins during folding process in the endoplasmic reticulum (ER). The currently proposed mechanism is that after correct folding of glycoproteins, the monoGlc residue is removed by
-glucosidase II (GII) (Helenius and Aebi, 2001
; Parodi, 2000
). The retained monoGlc-high-Man of avian IgG might have resulted from the steric hindrance imposed by the unique conformational structures of avian IgG. However, the relationship between the protein structure of avian IgG and its N-glycosylation pattern has not been examined. Because chicken IgG is a good source of glycoproteins that contain monoGlc-high-Man and its peptide sequences are known, we believed it is worthwhile investigating how such unique oligosaccharides exist in chicken IgG.
In this study, we first determine the detailed N-glycan structures of chicken serum IgG for the comparison with those of pigeon IgG and then demonstrate the site-specific N-glycosylation on chicken IgG. Our data indicated that high-mannose-type N-glycans are exclusively located on CH3 domains (as we found in pigeon IgG), whereas complex-type N-glycans are present in CH2 domains and Fab regions (most likely variable regions). N-glycosylation patterns, including the oligosaccharide structures, on chicken IgG are more similar to mammalian IgE than to IgG, which is probably due to the structural similarity inherited in molecular evolution from IgY to IgE lineage. By analogy of the 3D structure of human IgE-Fc (containing C2-4 domains) (Wan et al., 2002
), we speculate that protein folding and assembly mechanisms of avian IgG enables retaining monoGlc-high-Man on the secreted proteins.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Elution positions of the remaining eight fractions of neutral oligosaccharides from chicken IgG, n-9, n-10, n-11, n-12, n-14, n-15, n-16, and n-17, were coincidental on ODS and Amide-80 columns with those of human IgG N-glycans F, J, H, L, M, N, O, and P, respectively (Figure 3B, Figure 4B, and Table I). MALDI-TOF MS (Table I) gave good agreement with these results. The structure assignments were also supported by digestion with ß-galactosidase and/or -fucosidase (Figure 4B). After ß-galactosidase digestion, the elution positions of the products on the ODS and Amide-80 columns were shifted as follows: (1) both n-9 and n-11 yielded the same GlcNAc-terminated structure (N-glycan E in Figure 2), which has core
1-6 fucose residues and no bisecting GlcNAc; (2) both n-10 and n-12 yielded the same product (N-glycan I in Figure 2), which has bisecting GlcNAc and no
1-6 core fucose; (3) n-15, n-16, and n-17 all yielded the same product (N-glycan M in Figure 2), which has both core
1-6 fucose and bisecting GlcNAc. After
-fucosidase digestion, n-9, n-11, n-14, n-15, n-16, and n-17 yielded the expected respective fucose-less structures (N-glycans B, D, I, J, K, and L, respectively, as in Figure 2). Before the exoglycosidase-digestion, n-9 (GUODS 13.5; GUAmide 6.3) and n-10 (GUODS 13.5; GUAmide 6.2) exhibit very close GU values. However they are distinguished by m/z values (Table I) as well as by the sensitivity to
-fucosidase. Fractions n-15 (GUODS 19.5; GUAmide 6.5) and n-16 (GUODS 19.6; GUAmide 6.6) also exhibit similar GU values, but
-fucosidase-treated n-15 and n-16 (i.e., N-glycans J and K, respectively) were clearly distinguishable by their elution positions on the ODS column. Thus the structures of n-9, n-10, n-11, n-12, n-14, n-15, n-16, and n-17 were firmly assigned (Table I).
Three fractions of monosialylated PA-oligosaccharides (ms-5, ms-7, and ms-8) and one fraction of disialylated PA-oligosaccharides (ds-7) were isolated, and their structures were analyzed with MALDI-TOF MS and HPLC (Figure 4C). Compositions deduced from the MS data were ms-5, NeuAcHex5HexNAc4dHex-PA; ms-7, NeuAcHex4HexNAc5dHex-PA; ms-8, NeuAcHex5HexNAc5dHex-PA; and ds-7, NeuAc2Hex5HexNAc5dHex-PA. After -sialidase digestion, elution positions of ms-5, ms-7, ms-8, and ds-7 shifted as predicted and were indistinguishable from those of human IgG N-glycans H, O, P, and P, respectively (Figure 4C). Their GUAmide decreased about 0.30.4 after desialylation, suggesting that their sialylation is
2-6 linkage and not
2-3 (Nakagawa et al., 1995
; Takahashi et al., 1995a
,b
; Tomiya and Takahashi, 1998
). The monosialylated branching position on ms-5, ms-7, and ms-8 were determined as follows: when ms-5 and ms-8 were digested with
-fucosidase, followed by ß-galactosidase, and then
-sialidase, the products were identical with N-glycans C and K, respectively (Figures 4 and 5). ß-Galactosidase-treated ms-8 was identical to ms-7. When ms-7 was digested with
-fucosidase and
-sialidase, the products were identical with N-glycan K. These results suggest that monosialylated site in ms-5, ms-7, and ms-8 are on the Man
1-3Man arm. Accordingly, the structures of these monosialylated oligosaccharides were deduced as shown in Table I. Desialylated ds-7 was further digested with
-fucosidase to yield a N-glycan with Gal on both terminals, having bisecting GlcNAc and no core
1-6 fucose (N-glycan L in Figure 2). Therefore, structure of ds-7 was elucidated (Table I).
|
Isolation and lectin-blottings of chicken IgG-Fab and Fc
Chicken serum IgG was known to be cleaved into Fab and Fc fragments by papain digestion (Dreesman and Benedict, 1965; Kubo and Benedict, 1969
), although the exact cleavage sites have not been elucidated yet. To isolate chicken IgG-Fab and Fc fragments, papain-digested chicken IgG was applied to a DEAE-Sepharose column and eluted with a gradient of NaCl to yield three peaksfr. 1, fr. 2, and fr. 3, as shown in Figure 6A. Immunoblottings with anti-chicken IgG-Fc antibody revealed that fr. 1 and fr. 3 were Fab and Fc, respectively (Figure 6B). Fr. 2 is mostly Fab but was slightly contaminated by Fc fragments, which could be removed with an affinity column using anti-chicken IgG-Fc antibody as affinant. The pass-through fraction of the affinity column was designated as fr.2'. Fab was detected as broad bands on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE), perhaps due to peptide heterogeneity of the variable regions (Figure 6B). Fc appeared as a sharp band under the reducing condition (data not shown), but under the nonreducing condition it separated into three bands, all stained with anti-chicken IgG-Fc antibody (Figure 6B, Coomassie brilliant blue [CBB] and anti-Fc antibody staining). The different mobility of the three Fc fragments on the gel is attributable to partial reduction of disulfide bonds and/or multiple or alternative cleavages during papain digestion, as was found in mammalian IgG-Fc (Coligan, 1991
).
|
Concanavalin A (Con A) and anti-chicken IgG-Fc antibody stained only Fc (CH3 + CH4) fractions (fr. 3), but RCA-I stained Fab fractions (fr. 1 and fr. 2' exclusively; Figure 6B). These data strongly suggest the gross difference of glycans between chicken IgG-Fc (CH3 + CH4) and Fab. Based on the lectin specificities, it is most likely that high-mannose-type oligosaccharides are located on the Fc (CH3 + CH4) region, whereas galactosylated glycans are in Fab regions. Oligosaccharides containing ß-galactosides on chicken IgG-Fab were N-glycans (i.e., complex-type), because glycoamidase F (GAF)-treated chicken IgG-Fab could no longer be stained with RCA-I lectin (data not shown). The N-glycosylation on Fab fragments most likely occurred on variable regions, that is, VL and VH domains (Figure 1), as seen in mammalian serum IgG. N-glycosylations on VL and VH occur only when the peptide sequences possess the N-glycosylation signals, and the location of N-glycosylation sites are varied among polyclonal serum IgG. Therefore, N-glycosylation sites on Fab fragments were not determined by isolating glycopeptides originating from VL and VH.
N-glycosylation on chicken IgG-CH3 domain
The known amino acid sequences of chicken Ig upsilon () chains (H-chains) indicate the existence of only one potential N-linked glycosylation site located on the CH3-CH4 domains (Figure 1A) (Parvari et al., 1988
). This was confirmed by GAF treatment and SDSPAGE (data not shown). We also confirmed the presence of N-glycans on the CH3 domain with MALDI-TOF MS. MS of tryptic glycopeptides from the reduced form of chicken IgG-Fc (CH3 + CH4) fragment are as shown in Figure 7, and the [M + H]+ molecular ions are assigned in Table II. Peaks with m/z higher than 5000 were affected by GAF treatment, producing two new peaks of m/z 4036.66 and 4294.34. The larger of these two peaks having additional E and K residues on N-terminal side could have arisen from incomplete trypsin digestion (Table II). The N-glycans on the glycopeptides were assigned to be exclusively high-mannose-type glycans, because the masses of the glycopeptides were completely shifted after treatment with endo-ß-N-acetylglucosaminidase H (Endo H). The intact glycopeptides were assigned as Hex610HexNAc2-peptide (Table II). After
-mannosidase digestion, some of the intact glycopeptides changed to Hex1HexNAc2 peptide, but a large amount of Hex56HexNAc2-peptide was also produced (Figure 7). Concomitant with the decrease in the peaks of Hex6HexNAc2 peptides after exhaustive
-mannosidase digestion, peaks of Hex5HexNAc2 peptides increased. No peaks for Hex24HexNAc2-peptide were detected, even when excess
-mannosidase was used in the digestion. This suggests the presence of Glc-capped oligomannosyl branch. Therefore Hex56HexNAc2 peptides must have been derived from larger monoglucosylated oligosaccharides. Hex1HexNAc2 peptide can be assigned as Manß1-4GlcNAcß1-4GlcNAc-peptide, which can be derived from glycopeptides of high-mannose type without monoglucosylation. These results suggest that both monoglucosylated and nonglucosylated high-mannose-type oligosaccharides were on the same site of CH3 domain.
|
|
|
Homology modeling of chicken IgG-Fc (CH2 CH4)
Based on the assumption that the 3D structure of chicken IgG is similar to that of human IgE, the 3D structure of chicken IgG-Fc (CH2 CH4) was predicted by homology modeling (Figure 9). The crystal structure of recombinant human IgE-Fc (CH2 CH4) (PDB ID, 1o0v) was utilized as a template. Like the template structure, chicken IgG-Fc (CH2 CH4) was built to form an asymmetric homodimer with highly bent CH2-CH3 junctions. One of the N-glycosylation sites on chicken IgG -chains, Asn407 on the CH3 domain corresponded well to Asn394 on human IgE
-chains (Figure 1A, 1B), and the same orientation as found in the templates was adopted. High-mannose-type oligosaccharides at Asn407 are located in the cave formed by two CH3 + CH4 region chains, and partially buried by the CH2 domains, suggesting that this CH2 domains may confer steric hindrance to access oligosaccharides at Asn407. The model also predicts that without Fab and CH2 regions, two CH3 + CH4 region chains can only form a frame with a cave but cannot confer strict steric hindrance to retain monoglucosylated high-mannose-type oligosaccharides at Asn407 in the frame. If entire chicken IgG molecule formed Y-shaped structure as seen in mammalian IgG (Harris et al., 1997
), terminal glucose residues on Glc1Man89GlcNAc2-oligosaccharides, which occupy wider space than Man5GlcNAc2-, would readily protruded from the (CH3 + CH4) frame and would be accessible to
-glucosidase II in the ER. On the other hand, N-glycosylation sites at Asn308 on CH2 domains are exposed on the surface of the molecule to be easily accessible by processing enzymes. Thus this model supports our experimental results showing the site-specific N-glycans on chicken IgG and can give a rational explanation for the expression mechanism of monoGlc-high-Man on avian IgG.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our previous work for structural analysis of pigeon serum IgG N-glycans by HPLC, MS, and tandem MS have revealed that the prominent N-glycans are triantennary complex type as well as high-mannose-type, both of which are rarely found in mammalian normal serum IgGs (Hamako et al., 1993). When we compared the pigeon IgG N-glycans with those from chicken IgG reported by other groups (Ohta et al., 1991
; Raju et al., 2000
), we realized that confirming the N-glycan structures of chicken serum IgG is necessary by our conventional methods (Suzuki et al., 2001
; Takahashi et al., 2001
) for several reasons. First, although egg yolk IgG, which arises from transport of maternal antibodies by receptor-specific process (Patterson et al., 1962
), has identical biophysical properties with serum IgG (Loeken and Roth, 1983
), it is also reported that egg yolk IgG does not mediate anaphylactic reaction which avian serum IgG does (Faith and Clem, 1973
). In this regard, a possibility that the transport of IgG to egg yolk is restricted by certain carbohydrate structures have not been excluded. Second, Raju et al. (2000)
reported chicken serum IgG N-glycans analyzed by MALDI-TOF MS, but the proposed structures are only based on mass values and deduced by analogy to mammalian IgGs. Although simple MS is a convenient tool for oligosaccharide analysis, it cannot distinguish isoforms, such as bisected biantennary and nonbisected triantennary oligosaccharides. We have demonstrated earlier that N-glycan structures and N-glycosylation pattern of pigeon IgG are quite different from those of mammalian IgGs, so it was necessary to investigate the differences between chicken and pigeon IgG N-glycans carefully. Thus we analyzed the N-glycan structures of chicken serum IgG by HPLC, which can distinguish triantennary and bisected biantennary structures (Tomiya et al., 1988
). Moreover, we also determined the preferred galactosylation and sialylation branches in complex-type as well as isoforms of high-mannose-type oligosaccharides, which have not been reported for chicken serum IgG.
Our results confirmed that structural properties of complex-type N-glycans from chicken serum IgG is more similar to those of human IgG than to those of pigeon serum IgG (Suzuki et al., 2003), because no triantennary or extended branches with ß- and
-galactosylation were detected. This fact points out that structural properties of complex-type N-glycan are somehow conserved between chicken and mammalian IgGs regardless of the different N-glycosylation sites but no longer maintained in pigeon IgG. Both human and chicken IgGs possess biantennary complex-type oligosaccharides with and without core
1-6 Fuc and/or bisecting GlcNAc. Both IgGs have a monogalactosylated branch predominantly on the GlcNAcß1-2Man
1-6Man arm (N-glycans n-9, n-10, and n-15 from chicken IgG; shown in Table I, N-glycans B, F, and N from human IgG; shown in Figures 2 and 3B). Monosialylation in chicken IgG (ms-5, ms-7, ms-8 in Table I) exclusively occurs on the Galß1-4GlcNAcß1-2Man
1-3Man arm, which is also the case in normal human IgG (Takahashi et al., 1995b
). There are, however, some notable difference in structural patterns of complex-type N-glycans between human and chicken IgG. More than half of the complex-type N-glycans from chicken IgG contain bisecting GlcNAc and core
1-6 Fuc and are fully ß-galactosylated (n-17, ms-8, and ds-7 in Table I). Although human IgG also has the same structure, predominant oligosaccharides are core
1-6 fucosylated biantennary N-glycans without bisecting GlcNAc (Figure 3B). The observed difference is consistent with an earlier report (Raju et al., 2000
).
We also confirmed that complex-type N-glycan structures of chicken serum IgG were mostly the same as chicken egg yolk IgG (Ohta et al., 1991) but different from quail egg yolk IgG (Matsuura et al., 1993
) or pigeon serum IgG. In contrast, high-mannose-type N-glycans including monoGlc-high-Man are well conserved among them. Our previous data suggested that pigeon serum IgG-CH3 domain also possess exclusively high-mannose-type (Suzuki et al., 2003
). Because the same site-specific location of high-mannose-type was found even in the distantly removed avian orders such as Galliformes (chicken) and Columbiformes (pigeon), this feature may be widely occurring among avian IgGs.
CH3 domain of avian IgG is equivalent to CH2 domain of mammalian IgG. In mammalian IgG, the N-glycosylation site is located at Asn297 on CH2 domains (Figure 1A), and is well conserved among mammals (Burton, 1987). The N-glycosylation at Asn297 is essential because it influences thermal stability of IgG, recognition by Fc receptors, association with compliment component C1q, and induction of antigen-dependent cellular cytotoxicity (Kobata, 1990
; Mimura et al., 2000
; Radaev and Sun, 2001
). N-glycans at this site are exclusively biantennary complex type. It is reported that recombinant IgG possessing only high-mannose-type oligosaccharides is defective for complement activation (Wright and Morrison, 1994
). Crystal structures of human and mouse IgG studied by X-ray diffraction revealed that their Fc region form a cavity between the two
-chains, and N-glycans on Asn297 can be seen occluding the cavity at the center of the Fc (Harris et al., 1997
). 1Hnuclear magnetic resonance provided evidence that conformational changes in the sugar chains can affect the structure of the Fc (Matsuda et al., 1990
). By analogy of the N-glycans at Asn297 on mammalian IgG, and because of the structural similarity of the complex-type N-glycans of chicken and mammalian IgG, one may assume that the glycans at the corresponding position on chicken IgG (Asn407) are also complex type.
However, we have demonstrated that N-glycosylation pattern of avian IgG is closer to those of mammalian IgE than to mammalian IgG. Although the number and positions of N-glycosylation sites on avian IgG, mammalian IgG, and IgE are varied, one site (Asn297 on CH2 of mammalian IgG, Asn394 on CH3 of IgE, Asn407 on CH3 of avian IgG) is well conserved among them (Figure 1A, 1B). The crystal structure of human IgE-Fc (including C2-4 domains) (Wan et al., 2002
) revealed that N-glycans on Asn394 (see Figure 1A) are buried in a cavity between the two heavy chains, as seen in N-glycans on Asn297 of mammalian IgG. Unlike mammalian IgG, however, N-glycans at Asn394 of IgE is exclusively high-mannose-type oligosaccharides (Baenziger et al., 1974
; Dorrington and Bennich, 1978
), although the precise structures of N-glycans at this position derived from the entire IgE molecule (i.e., not truncated mutants) remain to be elucidated. Other N-glycosylation sites on IgE are complex-type oligosaccharides. The presence of high-mannose-type N-glycans at Asn394 in IgE can be understood from the crystal structure, which shows that the gap between two C
3 domains of the two chains are narrower than those of two C
2 domains and covered with C
2 domains formed by highly bent structure at C
2C
3 junctions (Wan et al., 2002
; Zheng et al., 1992
). In contrast, mammalian IgG has a wider cavity in the Fc region, and its hinge is flexible enough to make the Fc cavity not covered by the Fab regions (Harris et al., 1997
; Zheng et al., 1992
). Enzymes for N-glycan processing apparently can access the N-glycans in C
2 cavity more readily, leading to complex-type structure.
Structural properties of chicken IgG based on the amino acid sequences are known to be close to mammalian IgE in terms of the number of CH domains as well as the organization of intradomain and interchain disulfide bonds (Magor et al., 1992; Parvari et al., 1988
; Warr et al., 1995
) (Figure 1A). In addition, both of them mediate anaphylactic reaction (Faith and Clem, 1973
; Warr et al., 1995
), and it is believed that they are close relatives in molecular evolution (Warr et al., 1995
). Moreover, as we have demonstrated in this study, the site-specific presence of high-mannose-type N-glycan at Asn407 on chicken IgG is also similar to that in human IgE (Asn394). Based on these sequences and with functional similarity in mind, we constructed a 3D structure model of chicken IgG-Fc (CH2 CH4) (Figure 9). The model suggests that unless Fab and CH2 regions conferred steric hindrance against GII, oligosaccharides in the cave of Fc (CH3 + CH4) regions were relatively exposed and could not retain monoglucosylated forms. Although the bent structure of entire chicken IgG with Fab regions should be confirmed precisely by other biochemical and biophysical approaches, such a unique structure is most likely to impose the steric hindrance to N-glycan processing enzymes to retain Glc1Man89GlcNAc2 structures at Asn407.
Glycoproteins bearing monoGlc-high-Man oligosaccharides are usually found in the ER as an early intermediate in the biosynthesis of N-glycans and are supposed to be recognized by lectin-like chaperones, calnexin (CNX) and/or calreticulin (CRT), to aid in the folding process. After the correct folding, the terminal glucose is released by GII, followed by -mannosidase action for further N-glycan processing (Helenius and Aebi, 2001
). Although it is reported that CRT can bind to chicken IgG in vitro (Patil et al., 2000
; Saito et al., 1999
), the role of CNX or CRT in protein folding for IgG in ER is not clear. In contrast, it is reported that partially folded mammalian IgG H-chains form complex with BiP (immunoglobulin heavy-chain binding protein) and some other chaperones, including UDP-Glc:glycoprotein glucosyltransferase, but not with CNX nor CRT (Meunier et al., 2002
).
A currently proposed model for the protein folding and assembly of mammalian IgG (Haas and Wabl, 1983; Lee et al., 1999
) is that VH, CH2, and CH3 domains on mammalian IgG are folded first, then folding of the CH1 domain is accomplished by assembly with light (L) chains. We expected that this two-step protein folding process of mammalian IgG and formation of the IgE-like highly bent structure can account for the presence of monoGlc-high-Man on avian IgG-CH3 domain. If the two-step folding model is also applicable to avian IgG, it follows that VH, CH3, and CH4 domains of the H-chains are folded first, then assembled with L chains to allow the completion of the entire Ig molecule (Figure 10). After the folding of avian CH3 domain, deglucosylation on this domain can only be partially carried out, because avian IgG-CH3 domain possesses both monoglucosylated and nonglucosylated high-mannose-type N-glycans. However, concurrent with but independent of deglucosylation, the H-chains are assembled with L-chains, and the fully folded molecule no longer confers accessibility by GII, so that the IgG-CH3 domain can retain monoglucosylated N-glycans (Figure 10). On the other hand, N-glycosylation sites on the CH2 domain and variable regions are more exposed to allow advanced N-glycan processing, so that they can be possessed to fully ß-galactosylated, bisected, and core fucosylated complex type N-glycans. CH3 domains of chicken IgG contains exclusively high-mannose-type oligosaccharides, whereas CH2 domains only contains complex-type, suggesting that the presence of monoGlc-high-Man N-glycans is mainly due to strict steric hindrance, rather than random, incomplete cleavages by the processing enzymes.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Buffers and standard procedures
Tris-buffered saline (TBS) contains 50 mM TrisHCl (pH 7.4) and 150 mM NaCl. TBST contains 0.1% Tween 20 in TBS. Digestion buffer for papain treatment was 50 mM sodium phosphate (pH 7.0) containing 1 mM EDTA and 10 mM cysteine. Procedures for SDSPAGE, lectin- and immunoblottings, and N-terminal sequence analyses were as described previously (Suzuki et al., 2001). Protein concentrations were measured by the BCA assay (Smith et al., 1985
) using bovine serum albumin as a standard.
Papain digestion of chicken serum IgG
Papain suspension (28 mg/mL) was diluted in digestion buffer to be 1 mg/mL and incubated at 37°C for 10 min for activation. Chicken serum IgG dissolved in digestion buffer (2 mg/ml) was incubated with the activated papain (enzyme:substrate ratio of 1:100) at 37°C for 4 h. The reaction was terminated by adding iodoacetamide (final concentration 30 mM) and incubation at room temperature for 30 min in the dark. The mixture was dialyzed against 10 mM TrisHCl (pH 8.0) for the following anion-exchange chromatography.
Isolation of Fab and Fc fragments of chicken serum IgG
A column of DEAE-Sepharose Fast Flow (HiTrap, 1 mL) was washed with 1 M NaCl in 10 mM TrisHCl (pH 8.0) and equilibrated with 10 mM TrisHCl (pH 8.0). After papain-digested chicken IgG (1 mg) was loaded, the column was washed with 10 mM TrisHCl (pH 8.0) for 20 min (flow rate 1 mL/min), and then concentration of NaCl in the elution was linearly increased up to 0.3 M within 120 min (flow rate 0.5 mL/min). The major peaks detected by A280nm were collected and concentrated with a Centricon YM-10 at 4°C. One of the chicken IgG Fab fractions was incompletely separated from the Fc fraction, and it was further isolated with an affinity column. Avidin-agarose (250 µL) in a 1-mL syringe column was washed with 5 ml water and equilibrated with 5 mL binding buffer (20 mM sodium phosphate, pH 7.4, with 500 mM NaCl). Biotin-conjugated anti-chicken IgG Fc antibodies (100 µg) were loaded onto the column and allowed to stand for 30 min, then the column was washed with 5 mL binding buffer. The sample was loaded into the column and incubated for 30 min; then the column was washed with 2 mL binding buffer. The pass-through fraction was collected and concentrated with a Centricon YM-10. All the affinity purification procedures described were performed at 4°C.
GAF and Endo H treatment of glycoproteins
For GAF digestion, glycoproteins (15 µg) were dissolved with 30 µL 50 mM NaHPO4 (pH 7.5) containing 0.1% SDS and 100 mM 2-mercaptoethanol and heated at 90°C for 3 min for denaturation. After the solution was cooled to room temperature, 1% (v/v) NP-40 was added to the heat-denatured glycoproteins. The mixture was incubated with GAF (40 U/mg substrates) at 37°C for 16 h for complete de-N-glycosylation, and heated at 100°C for 10 min to inactivate GAF. For partial de-N-glycosylation, glycoproteins were incubated with GAF (1 U/mg substrates) for 10 min, 40 min, and 3 h and heated at 100°C for 10 min.
Endo H digestion for glycoproteins was performed similarly, but 50 mM sodium acetate (pH 5.5) was used as the reaction buffer. The glycoproteins before and after the digestions were analyzed with SDSPAGE.
Reduction and alkylation of glycoproteins
The formations of intramolecular disulfide bonds on glycoproteins were reduced and blocked as follows. Glycoproteins (0.2 mg) were dissolved with 120 µL 8 M guanidine-HCl in 0.2 M TrisHCl (pH 8.0) and reduced with 60 µL 0.18 M dithiothreitol in the 8 M guanidine solution at room temperature for 1 h. For thiol alkylation, 240 µL 0.18 M iodoacetamide in the guanidine solution was added to the mixture and incubated at room temperature for 30 min in the dark. The reaction mixture was dialyzed against H2O and lyophilized.
Preparation and isolation of glycopeptides
One-third of the reduced and alkylated glycoproteins were suspended in 50 µL 50 mM NH4HCO3, pH 7.8, and incubated with TPCK-treated trypsin at 37°C overnight. Trypsin was inactivated by heating at 100°C for 5 min. A portion of the reaction mixture was further treated with GAF (1 U/10 µL) at 37°C, overnight. The peptide fragments before and after treatment with GAF were analyzed with reversed-phase HPLC on a Shim-pack CLC-ODS column (6.0 x 150 mm). The mobile phase was (A) 0.05% trifluoroacetic acid (TFA) and (B) 90% CH3CN with 0.05% TFA. Elution (1 ml/min) was conducted by a linear gradient of 050% of (B) in (A) developed over 100 min. Each peak detected by A210nm was collected and kept at 4°C.
Preparation and sepalation of PA-derivatized oligosaccharides for structural analyses
Chicken serum IgG (reductive alkylated, 1 mg) were digested with trypsin and chymotrypsin in 50 mM NH4HCO3, pH 7.8, at 37°C overnight, and the enzymes were inactivated by heating at 100°C for 5 min. Oligosaccharides were released with GAF treatment in 50 mM NH4HCO3, pH 7.8, at 37°C overnight. After inactivating GAF by heating at 100°C for 5 min, the digest was lyophilized. Cations and peptides were removed with Dowex 50W x2 (H+ form, 50100 mesh, 250 µL, Sigma) packed in a 1-mL syringe. The column was washed with 1 mL H2O, and the collected effluent was lyophilized. The sample was reconstituted with 100 µL H2O, loaded onto a Carbograph tube (25 mg, Alltech), and washed with 400 µL H2O, then eluted with 400 µL 50% CH3CN containing 0.05% TFA. For the PA derivatization by reductive amination, lyophilized oligosaccharide fractions were dissolved in 40 µL PA solution (1 g/580 µL in concentrated HCl, pH 6.8), and heated at 90°C for 15 min with heating block. Freshly prepared NaCNBH3 solution (7 mg/4 µL) was added into the reaction mixture, then heated at 90°C for 1 h. PA oligosaccharides were fractionated by gel filtration on a Sephadex G-15 column (1.0 x 40 cm, in 10 mM NH4-HCO3), and the effluent was monitored with a fluorometer (excitation 300 nm, emission 360 nm) and lyophilized.
The mixture of PA-oligosaccharides was separated by HPLC with three different columns as described previously (Nakagawa et al., 1995). In the first stage, the PA-oligosaccharides were separated on a TSKgel DEAE-5PW column (7.5 x 75 mm), and the neutral, monosialyl, and disialyl fractions (monitored by fluorescence, excitation 320 nm, emission 400 nm) were collected separately and lyophilized. In the second stage, neutral, mono-, and disialylated oligosaccharide fractions were individually dissolved in H2O and separated on a Shim-Pack CLC-ODS column (6.0 x 150 mm), monitoring effluent with fluorescence (excitation 320 nm, emission 400 nm). Elution was performed at a flow rate of 1.0 mL/min at 55°C using eluent A (0.005% TFA) and eluent B (0.5 % 1-butanol in eluent A). The column was equilibrated with a mixture of eluents A:B = 90:10 (v/v), and after sample injection, the ratio of the eluents was changed linearly to A:B = 60:40 in 60 min. Each peak was collected, lyophilized, and analyzed with MALDI-TOF MS.
In the third stage, major peaks from the ODS column were dissolved with 50 µL eluent C (CH3CN:3% CH3COOH-trietylamine, pH 7.3, 65:35), and separated on a TSKgel Amide-80 column (4.6 x 250 mm), monitoring effluent with fluorescence, excitation 300 nm, emission 360 nm. Elution position of each PA-oligosaccharides on CLC-ODS and Amide-80 columns was expressed in GUs, based on the elution position of isomaltose series. For the assignment of GUODS, analysis on a CLC-ODS column was performed using 10 mM sodium phosphate, pH 3.8 (Tomiya et al., 1988) instead of 0.005% TFA. Reference PA-derivatized oligosaccharides from asialo-human IgG and bovine RNase B were prepared by the same method. Structures of the reference compounds were shown in Figure 2. PA-derivatized N-glycans A, B, C, D, I, J, K, and L (Figure 2) were obtained from
-fucosidase-digested PA-oligosaccharides from human IgG.
MALDI-TOF MS
MALDI-TOF MS was performed on a Kompact SEQ (Kratos Analytical, Manchester, England), equipped with a 337-nm nitrogen laser and set at 20 kV extraction voltage. Each spectrum was the average of 50 laser shots. Glycoproteins, glycopeptides, and neutral oligosaccharides were analyzed in the linear positive-ion mode, and sialylated oligosaccharides were analyzed in the linear negative-ion mode. SA and ACH were used as matrices in the analysis of glycoproteins and glycopeptides, respectively. These matrices were dissolved to be 10 mg/mL with 50% CH3CN with 0.05% TFA. For neutral and sialylated PA-derivatized oligosaccharides, DHB (10 mg/mL in 5 mM NaCl) and THAP (2 mg/mL in 25% CH3CN with 10 mM dibasic ammonium citrate) were used, respectively (Papac et al., 1998). Samples (0.5 µL) were applied to a target and mixed with matrix (0.5 µL), and allowed to dry under ambient condition (for SA or ACH) or vacuum (for DHB or THAP) at room temperature prior to mass analysis.
Endo- and exoglycosidase digestion for glycopeptides and oligosaccharides
Glycopeptides (about 90 pmol/2.5 µL) were digested with GAF (1 U) in 50 mM NH4HCO3 (pH 7.8) or with Endo H (2.5 µg) in 20 mM sodium acetate (pH 5.6) at 37°C overnight. For exoglycosidase digestion, glycopeptides were dissolved with 20 mM sodium acetate (pH 4.5) to be 40 pmol/µL. Glycopeptides containing high-mannose-type oligosaccharides (90 pmol) were digested with -mannosidase (250 mU/100 pmol glycopeptides) at 37°C overnight. Glycopeptides containing complex-type oligosaccharides (250 pmol) were sequentially digested with
-sialidase (0.63 mU/100 pmol of glycopeptides), ß-galactosidase (0.38 mU), ß-N-acetyl-D-hexosaminidase (1.11 mU), and
-fucosidase (0.5 mU). After the incubation at 37°C overnight and heat-inactivation at 90°C for 5 min at the each step, a portion of the digestion products was analyzed with MALDI-TOF MS. PA-derivatized oligosaccharides were digested with exoglycosidases in the same manner and analyzed with HPLC as described.
Homology modeling
A model of the 3D structure of chicken IgG-Fc (CH2 CH4) (from Ser250 to Gly567) was constructed using the SWISS-MODEL program (Schwede et al., 2003), which is made public by the Swiss Institute of Bioinformatics (University of Basel, Switzerland). The crystal structure of human IgE-Fc (CH2 CH4) (Wan et al., 2002
) (PDB ID: 1o0v) was used as a template for the homology modeling based on their 32% amino acid sequence identity as well as their overall structural similarity. The sequence alignment was generated by T-Coffee (Notredame et al., 2000
), which correctly aligned all Cys and Trp residues (hallmarks of Ig-like domains) conserved between the target and template. The optimize mode for oligomer modeling in the SWISS-MODEL server was chosen for the appropriate modeling. The predicted 3D structure was visualized with DeepView program. Oligosaccharide chains (Man5GlcNAc2-) at Asn 407 on chicken IgG was imposed from the template.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
Abbreviations |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Basu, M., Hakimi, J., Dharm, E., Kondas, J.A., Tsien, W.H., Pilson, R.S., Lin, P., Gilfillan, A., Haring, P., Braswell, E.H., and others. (1993) Purification and characterization of human recombinant IgE-Fc fragments that bind to the human high affinity IgE receptor. J. Biol. Chem., 268, 1311813127.
Burton, D.R. (1987) Structure and function of antibodies. In Calabi, F. and Neuberger, M.S. (Eds.), Molecular genetics of immunoglobulin. Elsevier, Amsterdam, New York, Oxford, pp. 150.
Coligan, J.E. (1991) Current protocols in immunology. Wiley, New York.
Deisenhofer, J. (1981) Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-Å resolution. Biochemistry, 20, 23612370.[ISI][Medline]
Dorrington, K.J. and Bennich, H.H. (1978) Structure-function relationships in human immunoglobulin E. Immunol. Rev., 41, 325.[ISI][Medline]
Dreesman, G. and Benedict, A.A. (1965) Properties of papain-digested chicken 7 S gamma-globulin. J. Immunol., 95, 855866.[ISI][Medline]
Edelman, G.M., Cunningham, B.A., Gall, W.E., Gottlieb, P.D., Rutishauser, U., and Waxdal, M.J. (1969) The covalent structure of an entire gammaG immunoglobulin molecule. Proc. Natl Acad. Sci. USA, 63, 7885.[Abstract]
Faith, R.E. and Clem, L.W. (1973) Passive cutaneous anaphylaxis in the chicken. Biological fractionation of the mediating antibody population. Immunology, 25, 151164.[ISI][Medline]
Fellah, J.S., Kerfourn, F., Wiles, M.V., Schwager, J., and Charlemagne, J. (1993) Phylogeny of immunoglobulin heavy chain isotypes: structure of the constant region of Ambystoma mexicanum upsilon chain deduced from cDNA sequence. Immunogenetics, 38, 311317.[ISI][Medline]
Haas, I.G. and Wabl, M. (1983) Immunoglobulin heavy chain binding protein. Nature, 306, 387389.[ISI][Medline]
Hamako, J., Matsui, T., Ozeki, Y., Mizuochi, T., and Titani, K. (1993) Comparative studies of asparagine-linked sugar chains of immunoglobulin G from eleven mammalian species. Comp. Biochem. Physiol. B, 106, 949954.[CrossRef][ISI][Medline]
Harris, L.J., Larson, S.B., Hasel, K.W., and McPherson, A. (1997) Refined structure of an intact IgG2a monoclonal antibody. Biochemistry, 36, 15811597.[CrossRef][ISI][Medline]
Helenius, A. and Aebi, M. (2001) Intracellular functions of N-linked glycans. Science, 291, 23642369.
Kobata, A. (1990) Function and pathology of the sugar chains of human immunoglobulin G. Glycobiology, 1, 58.[Medline]
Kubo, R.T. and Benedict, A.A. (1969) Comparison of various avian and mammalian IgG immunoglobulins for salt-induced aggregation. J. Immunol., 103, 10221028.[ISI][Medline]
Lee, Y.K., Brewer, J.W., Hellman, R., and Hendershot, L.M. (1999) BiP and immunoglobulin light chain cooperate to control the folding of heavy chain and ensure the fidelity of immunoglobulin assembly. Mol. Biol. Cell, 10, 22092219.
Leslie, G.A. and Clem, L.W. (1969) Phylogeny of immunoglobulin structure and function. 3. Immunoglobulins of the chicken. J. Exp. Med., 130, 13371352.[Medline]
Loeken, M.R. and Roth, T.F. (1983) Analysis of maternal IgG subpopulations which are transported into the chicken oocyte. Immunology, 49, 2128.[ISI][Medline]
Magor, K.E., Warr, G.W., Middleton, D., Wilson, M.R., and Higgins, D.A. (1992) Structural relationship between the two IgY of the duck, Anas platyrhynchos: molecular genetic evidence. J. Immunol., 149, 26272633.
Matsuda, H., Nakamura, S., Ichikawa, Y., Kozai, K., Takano, R., Nose, M., Endo, S., Nishimura, Y., and Arata, Y. (1990) Proton nuclear magnetic resonance studies of the structure of the Fc fragment of human immunoglobulin G1: comparisons of native and recombinant proteins. Mol. Immunol., 27, 571579.[CrossRef][ISI][Medline]
Matsuura, F., Ohta, M., Murakami, K., and Matsuki, Y. (1993) Structures of asparagine linked oligosaccharides of immunoglobulins (IgY) isolated from egg-yolk of Japanese quail. Glycoconj. J., 10, 202213.[ISI][Medline]
Meunier, L., Usherwood, Y.K., Chung, K.T., and Hendershot, L.M. (2002) A subset of chaperones and folding enzymes form multiprotein complexes in endoplasmic reticulum to bind nascent proteins. Mol. Biol. Cell, 13, 44564469.
Mimura, Y., Church, S., Ghirlando, R., Ashton, P.R., Dong, S., Goodall, M., Lund, J., and Jefferis, R. (2000) The influence of glycosylation on the thermal stability and effector function expression of human IgG1-Fc: properties of a series of truncated glycoforms. Mol. Immunol., 37, 697706.[CrossRef][ISI][Medline]
Nakagawa, H., Kawamura, Y., Kato, K., Shimada, I., Arata, Y., and Takahashi, N. (1995) Identification of neutral and sialyl N-linked oligosaccharide structures from human serum glycoproteins using three kinds of high-performance liquid chromatography. Anal. Biochem., 226, 130138.[CrossRef][ISI][Medline]
Notredame, C., Higgins, D.G., and Heringa, J. (2000) T-Coffee: a novel method for fast and accurate multiple sequence alignment. J. Mol. Biol., 302, 205217.[CrossRef][ISI][Medline]
Ohta, M., Hamako, J., Yamamoto, S., Hatta, H., Kim, M., Yamamoto, T., Oka, S., Mizuochi, T., and Matsuura, F. (1991) Structures of asparagine-linked oligosaccharides from hen egg-yolk antibody (IgY). Occurrence of unusual glucosylated oligo-mannose type oligosaccharides in a mature glycoprotein. Glycoconj. J., 8, 400413.[ISI][Medline]
Papac, D.I., Briggs, J.B., Chin, E.T., and Jones, A.J. (1998) A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Glycobiology, 8, 445454.
Parodi, A.J. (2000) Protein glucosylation and its role in protein folding. Annu. Rev. Biochem., 69, 6993.[CrossRef][ISI][Medline]
Parvari, R., Avivi, A., Lentner, F., Ziv, E., Tel-Or, S., Burstein, Y., and Schechter, I. (1988) Chicken immunoglobulin -heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus. EMBO J., 7, 739744.[Abstract]
Patil, A.R., Thomas, C.J., and Surolia, A. (2000) Kinetics and the mechanism of interaction of the endoplasmic reticulum chaperone, calreticulin, with monoglucosylated (Glc1Man9GlcNAc2) substrate. J. Biol. Chem., 275, 2434824356.
Patterson, R., Younger, J.S., Weigle, W.O., and Dixon, F. (1962) Antibody production and transfer to egg yolk in chickens. J. Immunol., 89, 272278.[ISI][Medline]
Paul, W.E. (1999) Fundamental immunology. Lippincott-Raven, Philadelphia.
Radaev, S. and Sun, P.D. (2001) Recognition of IgG by Fc. The role of Fc glycosylation and the binding of peptide inhibitors. J. Biol. Chem., 276, 1647816483.
Raju, T.S., Briggs, J.B., Borge, S.M., and Jones, A.J. (2000) Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiology, 10, 477486.
Reynaud, C.A., Dahan, A., Anquez, V., and Weill, J.C. (1989) Somatic hyperconversion diversifies the single Vh gene of the chicken with a high incidence in the D region. Cell, 59, 171183.[ISI][Medline]
Rudd, P.M. and Dwek, R.A. (1997) Glycosylation: heterogeneity and the 3D structure of proteins. Crit. Rev. Biochem. Mol. Biol., 32, 1100.[Abstract]
Saito, Y., Ihara, Y., Leach, M.R., Cohen-Doyle, M.F., and Williams, D.B. (1999) Calreticulin functions in vitro as a molecular chaperone for both glycosylated and non-glycosylated proteins. EMBO J., 18, 67186729.
Schwede, T., Kopp, J., Guex, N., and Peitsch, M.C. (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res., 31, 33813385.
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., and Klenk, D.C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem., 150, 7685.[ISI][Medline]
Sun, S., Mo, W., Ji, Y., and Liu, S. (2001) Preparation and mass spectrometric study of egg yolk antibody (IgY) against rabies virus. Rapid Commun. Mass Spectrom., 15, 708712.[CrossRef][ISI][Medline]
Suzuki, N., Khoo, K.H., Chen, H.C., Johnson, J.R., and Lee, Y.C. (2001) Isolation and characterizaion of major glycoproteins of pigeon egg white: ubiquitous presence of unique N-glycans containing Gal1-4Gal. J. Biol. Chem., 276, 2322123229.
Suzuki, N., Khoo, K.H., Chen, C.M., Chen, H.C., and Lee, Y.C. (2003) N-glycan structures of pegeon IgG: a major serum glycoprotein containing Gal1-4Gal termini. J. Biol. Chem., 278, 4629346306.
Takahashi, N., Lee, K.B., Nakagawa, H., Tsukamoto, Y., Kawamura, Y., Li, Y.T., and Lee, Y.C. (1995a) Enzymatic sialylation of N-linked oligosaccharides using an -(2-3)-specific trans-sialicase from Trypanosoma cruzi: structual identification using a three-dimensional elution mapping technique. Anal. Biochem., 230, 333342.[CrossRef][ISI][Medline]
Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y., and Tomiya, N. (1995b) Three-dimensional elution mapping of pyridylaminated N-linked neutral and sialyl oligosaccharides. Anal. Biochem., 226, 139146.[CrossRef][ISI][Medline]
Takahashi, N., Khoo, K.H., Suzuki, N., Johnson, J.R., and Lee, Y.C. (2001) N-glycan structures from the major glycoproteins of pigeon egg white: predominance of terminal Gal(1-4)Gal. J. Biol. Chem., 276, 2323023239.
Tomiya, N. and Takahashi, N. (1998) Contribution of component monosaccharides to the coordinates of neutral and sialyl pyridylaminated N-glycans on a two-dimensional sugar map. Anal. Biochem., 264, 204210.[CrossRef][ISI][Medline]
Tomiya, N., Awaya, J., Kurono, M., Endo, S., Arata, Y., and Takahashi, N. (1988) Analyses of N-linked oligosaccharides using a two-dimensional mapping technique. Anal. Biochem., 171, 7390.[ISI][Medline]
Tomiya, N., Lee, Y.C., Yoshida, T., Wada, Y., Awaya, J., Kurono, M., and Takahashi, N. (1991) Calculated two-dimensional sugar map of pyridylaminated oligosaccharides: elucidation of the jack bean alpha-mannosidase digestion pathway of Man9GlcNAc2. Anal. Biochem., 193, 90100.[ISI][Medline]
Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology, 3, 97130.[Abstract]
Wan, T., Beavil, R.L., Fabiane, S.M., Beavil, A.J., Sohi, M.K., Keown, M., Young, R.J., Henry, A.J., Owens, R.J., Gould, H.J., and Sutton, B.J. (2002) The crystal structure of IgE Fc reveals an asymmetrically bent conformation. Nat. Immunol., 3, 681686.[CrossRef][ISI][Medline]
Warr, G.W., Magor, K.E., and Higgins, D.A. (1995) IgY: clues to the origins of modern antibodies. Immunol. Today, 16, 392398.[CrossRef][ISI][Medline]
Wright, A. and Morrison, S.L. (1994) Effect of altered CH2-associated carbohydrate structure on the functional properties and in vivo fate of chimeric mouse-human immunoglobulin G1. J. Exp. Med., 180, 10871096.[Abstract]
Zheng, Y., Shopes, B., Holowka, D., and Baird, B. (1992) Dynamic conformations compared for IgE and IgG1 in solution and bound to receptors. Biochemistry, 31, 74467456.[ISI][Medline]