Site-specific N-glycosylation of chicken serum IgG

Noriko Suzuki1 and Yuan C. Lee

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
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Avian serum immunoglobulin (IgG or IgY) is functionally equivalent to mammalian IgG but has one additional constant region domain (CH2) in its heavy (H) chain. In chicken IgG, each H-chain contains two potential N-glycosylation sites located on CH2 and CH3 domains. To clarify characteristics of N-glycosylation on avian IgG, we analyze N-glycans from chicken serum IgG by derivatization with 2-aminopyridine (PA) and identified by HPLC and MALDI-TOF-MS. There were two types of N-glycans: (1) high-mannose-type oligosaccharides (monoglucosylated 26.8%, others 10.5%) and (2) biantennary complex-type oligosaccharides (neutral, 29.9%; monosialyl, 29.3%; disialyl, 3.7%) on molar basis of total N-glycans. To investigate the site-specific localization of different N-glycans, chicken serum IgG was digested with papain and separated into Fab [containing variable regions (VH + VL) + CH1 + CL] and Fc (containing CH3 + CH4) fragments. Con A stained only Fc (CH3 + CH4) and RCA-I stained only Fab fractions, suggesting that high-mannose-type oligosaccharides were located on Fc (CH3 + CH4) fragments, and variable regions of Fab contains complex-type N-glycans. MS analysis of chicken IgG-glycopeptides revealed that chicken CH3 domain (structurally equivalent to mammalian CH2 domain) contained only high-mannose-type oligosaccharides, whereas chicken CH2 domain contained only complex-type N-glycans. The N-glycosylation pattern on avian IgG is more analogous to that in mammalian IgE than IgG, presumably reflecting the structural similarity to mammalian IgE.

Key words: Asn297 / chicken serum IgG / IgG-Fc / monoglucosylated high-mannose-type / N-glycan processing


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Avian IgG, the predominant serum immunoglobulin in birds, is closely related to both mammalian IgG and IgE, based on their functional and structural properties (Warr et al., 1995Go). In contrast to mammalian IgG, avian IgG contains one additional domain in the constant region of its heavy (H) chains (designated upsilon, {upsilon}), but it lacks functional hinge regions found in mammalian IgG (Figure 1A) (Magor et al., 1992Go; Parvari et al., 1988Go; Warr et al., 1995Go). In short, the CH3 and CH4 domains of chicken/duck IgG resemble the CH2 and CH3 domains of mammalian IgG in structure, respectively, and the equivalent of the CH2 domain in avian IgG is absent in mammalian IgG. Because of its distinct structural difference from mammalian IgG, avian IgG is also called IgY (Leslie and Clem, 1969Go; Warr et al., 1995Go). The IgY-like molecules are also found in reptiles and amphibians (Fellah et al., 1993Go; Warr et al., 1995Go). Structural properties of IgY in birds and amphibians are rather close to mammalian IgE with respect to the number of CH domains as well as the organization of intradomain and interchain disulfide bonds (Figure 1A) (Fellah et al., 1993Go; Parvari et al., 1988Go; Warr et al., 1995Go). It is hypothesized that {gamma} and {varepsilon} genes have been generated from a relatively recent gene-duplication event and that IgY-like molecule was the immediate progenitor both of IgG and IgE (Parvari et al., 1988Go; Warr et al., 1995Go).



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Fig. 1. Structures of Igs and their N-glycosylation sites. (A) Structures of avian IgG, mammalian IgG, and mammalian IgE molecules. Avian (chicken, duck) IgG has five domains in the heavy chain, termed VH (in variable region), CH1, CH2, CH3, and CH4 (in the constant region). The domains of the light chains are termed VL (in the variable region) and CL (in constant region). The potential N-glycosylation sites on avian IgG are shown as hexagons. N-glycosylation on variable regions (gray hexagon) could occur sometimes, depending on the peptide structures. Location of disulfide bonds linking the chains were predicted from analogy to human IgE (Parvari et al., 1988Go; Wan et al., 2002Go), indicated with dotted bold lines. Numbering for chicken IgG H-chain ({upsilon}-chain) is based on the deduced amino acid sequences from cDNA, starting from the first methionine in the leader region (Parvari et al., 1988Go; Reynaud et al., 1989Go). Position of actual N-glycosylated sites on human IgG1 and IgE were shown in hexagons. Interchain disulfide bonds are based on human IgG1 and IgE structures, respectively (Paul, 1999Go; Wan et al., 2002Go), and indicated as bold lines. Numbering for mammalian Ig {gamma}-chain is based on Eu numbering (Edelman et al., 1969Go). Numbering for mammalian Ig {varepsilon}-chain is modified from a reference (Dorrington and Bennich, 1978Go) to comply with an human immunoglobulin {varepsilon}-chain of 547 amino acids. (B) Comparison of amino acid sequences around potential N-glycosylation sites on chicken IgG H-chain and others. Potential N-glycosylation sites were indicated as bold. Residue numbers on Asn were given for chicken IgG H-chain. Homologies of these sequences are not always high, but Trp and Cys residues (indicated with arrows), which are hallmarks of Ig domains, are well conserved each other. Primary accession numbers for Entrez database are; chicken upsilon chain, CAA30161; duck upsilon chain, CAA46322; human gamma chain, CAC20454; human epsilon chain, AAB59424; mouse epsilon chain, EPC_MOUSE; rat epsilon chain, AAA41364; Xenopus upsilon chain, S04845; axolotl upsilon chain, CAA49247.

 
Based on their amino acid sequences, chicken (Parvari et al., 1988Go) and duck (Magor et al., 1992Go) IgGs share two potential N-glycosylation sites, predictable from the consensus sequence (sequon) in constant regions (Figure 1A and 1B). One of them is located in the CH2 (C{upsilon}2) domain, which is absent in mammalian IgG. The other is located in the CH3 (C{upsilon}3) domain, which corresponds to the CH2 (C{gamma}2) domain of mammalian IgG (Asn297, Eu-numbering). Figure 1B shows the sequence alignment for H-chains of avian (chicken, duck) IgG (C{upsilon}-chains), mammalian (human) IgG1 (C{gamma}1-chain), mammalian (human, mouse, rat) IgE (C{varepsilon}-chains), and amphibian (Xenopus, axolotl) IgY (C{upsilon}-chains) around the two sequons on avian IgG H-chains. The sequence alignment indicates that the location of both potential N-glycosylation sites of avian IgGs are also conserved in mammalian IgE (except CH2 in human IgE). In contrast, sequons of amphibian IgY are located at different positions from avian IgG and mammalian IgG/IgE, probably reflecting the evolutional distances between mammals/avians and amphibians.

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., 2003Go), 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., 1991Go) and quail (Matsuura et al., 1993Go) egg yolk IgGs and chicken serum IgG (Raju et al., 2000Go). 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 {alpha}-glucosidase II (GII) (Helenius and Aebi, 2001Go; Parodi, 2000Go). 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 C{varepsilon}2-4 domains) (Wan et al., 2002Go), we speculate that protein folding and assembly mechanisms of avian IgG enables retaining monoGlc-high-Man on the secreted proteins.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Structural analysis of PA-derivatized oligosaccharides from chicken IgG
To investigate structural profile of N-glycans in chicken serum IgG, we utilized a 3D mapping technique (Takahashi et al., 1995bGo), in which 2-aminopyridine (PA)–derivatized N-glycans were chromatographed on high-performance liquid chromatography (HPLC) using (1) anion exchange with a DEAE column, (2) reversed-phase with an octadecylsilica (ODS) column, and then (3) normal phase with an Amide-80 column. The elution positions of each PA-oligosaccharide were recorded as glucose units (GU) (Table I), and their structures were determined based on the elution positions and matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS) data (Table I) by comparing with reference PA-derivatized oligosaccharides (Figure 2).


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Table I. Assignment of the major PA-oligosaccharides from chicken IgG based on HPLC and MS

 


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Fig. 2. Structures of reference oligosaccharides isolated from human IgG and bovine RNase B. All the oligosaccharides were derivatized with AP for HPLC and MALDI-TOF MS analysis. Monosaccharides were denoted by F, fucose; M, mannose; GN, N-acetylglucosamine; G, galactose.

 
Total PA-oligosaccharides from chicken IgG were separated into neutral, mono-, and disialyl oligosaccharides on a DEAE column (Figure 3A). MS analysis revealed that neutral fractions eluted between 5 and 15 min on the ODS column (Figure 3B) were Hex7–11HexNAc2-PA (data not shown), suggesting that these are high-mannose-type oligosaccharides. The three major peaks (n-4, n-5, n-6) were isolated, digested with {alpha}-mannosidase, and further examined by 2D HPLC mapping (Tomiya et al., 1988Go). Fraction n-4 showed the same elution position as Man9GlcNAc2-PA from bovine RNase B (Figure 2), and both moved to the position of Man1GlcNAc2-PA after {alpha}-mannosidase digestion (Figure 4A), suggesting that n-4 has the same structure as Man9GlcNAc2-PA. This was supported by the MS data of n-4 before (m/z 1984.86; Hex9HexNAc2-PA) and after (m/z 687.97; Hex1HexNAc2-PA) {alpha}-mannosidase digestion. On the other hand, neither n-5 (m/z 1984.81; Hex9HexNAc2-PA) nor n-6 (m/z 2147.06; Hex10HexNAc2-PA) could be digested to Hex1HexNAc2-PA under the same conditions used for n-4 (Figure 4A). Mild {alpha}-mannosidase digestion (50 mU/100 pmole PA-N-glycans, 37°C, overnight) of n-6 yielded a major peak on either ODS or Amide-80 column (GUODS 7.1; GUAmide 6.9; m/z 1498.60, Hex6HexNAc2-PA), which was transformed to a peak (GUODS 6.5; GUAmide 5.9; m/z 1336.26, Hex5HexNAc2-PA) on exhaustive digestion with {alpha}-mannosidase (200 mU/100 pmole PA-N-glycans, 37°C, two overnight). This might be a reflection of difficulty of removal of the {alpha}1-6 mannose residue by jack bean {alpha}-mannosidase.



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Fig. 3. HPLC separation of PA-oligosaccharides from chicken serum IgG. (A) Total PA-oligosaccharides from chicken IgG were separated into neutral, mono-, and disialyl oligosaccharides on a DEAE column. (B) Elution profiles of the neutral, mono-, and disialyl PA-oligosaccharides from chicken serum IgG on an ODS column. Structures of human IgG N-glycans (A–P) were shown in Figure 2. Human IgG N-glycans I, J, and K were prepared with {alpha}-fucosidase-digestion of human IgG N-glycans, and their elution positions on an ODS column were indicated. Each peak was collected and analyzed with MS. Fraction numbers indicated for chicken IgG N-glycans correspond to those in Table I.

 


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Fig. 4. Structural analysis of N-glycans from chicken IgG by a 2D mapping technique. (A) Elution position of high-mannose-type PA-oligosaccharides from chicken IgG (n-4, triangle; n-5, square; n-6, open circle) and bovine RNase B (solid circle) on ODS and Amide-80 columns. Structures of bovine RNase B N-glycans (M9 and M5) are shown in Figure 2. Arrows with dot-dot-dashed lines indicate the changes of the coordinates of N-glycans after digestion with {alpha}-mannosidase. (B) Elution positions of neutral complex-type PA-oligosaccharides from chicken (open circle) and human IgG (solid circle). Arrows with dashed lines and with dotted lines indicate the changes of the coordinates of N-glycans after digestion with ß-galactosidase and {alpha}-fucosidase, respectively. Also see Figure 2 for structures. (C) Elution positions of mono-(ms-5, square; ms-7, triangle; ms-8, reversed triangle), and disialylated (ds-7, open circle) PA-oligosaccharides from chicken IgG and human IgG (solid circle). Arrows with solid lines, dashed lines, and dotted lines indicate the changes of the coordinates of N-glycans after digestion with {alpha}-sialidase, ß-galactosidase, and {alpha}-fucosidase, respectively.

 
Although the final product of {alpha}-mannosidase-digested n-6 had the same mass value as Man5GlcNAc2-PA from bovine RNase B (GUODS 7.3; GUAmide 6.1), it eluted at a distinctly different position on an ODS column (Figure 4A). These results suggest that one of the {alpha}-mannosylated branches on n-6 are blocked at the nonreducing terminus, so that {alpha}-mannosidase could not trim it to Man1GlcNAc2-PA. The blocking is most likely by a glucosylation on the nonreducing terminal of Man{alpha}1-2Man{alpha}1-2Man{alpha}-branch, because the relative elution position of n-6 on both ODS and Amide-80 columns were coincidental with those of Glc1Man9GlcNAc2-PA (Tomiya et al., 1988Go). Fraction n-5, smaller by one hexose than n-6, yielded the same product as n-6 after {alpha}-mannosidase digestion, suggesting that this also has a monoglucosylated branch, but shorter by one {alpha}-mannoside residue than n-6. Because n-5 was eluted earlier than n-6 on the ODS column, one {alpha}1-2-mannoside residue on the Man{alpha}1-3Man{alpha}1-6Manß1-4GlcNAc arm is absent in n-5 (Tomiya et al., 1991Go; Tomiya and Takahashi, 1998Go). Thus the structures of n-4, n-5, and n-6 were deduced as shown in Table I.

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 {alpha}-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 {alpha}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 {alpha}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 {alpha}1-6 fucose and bisecting GlcNAc. After {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-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 0.3–0.4 after desialylation, suggesting that their sialylation is {alpha}2-6 linkage and not {alpha}2-3 (Nakagawa et al., 1995Go; Takahashi et al., 1995aGo,bGo; Tomiya and Takahashi, 1998Go). 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 {alpha}-fucosidase, followed by ß-galactosidase, and then {alpha}-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 {alpha}-fucosidase and {alpha}-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{alpha}1-3Man arm. Accordingly, the structures of these monosialylated oligosaccharides were deduced as shown in Table I. Desialylated ds-7 was further digested with {alpha}-fucosidase to yield a N-glycan with Gal on both terminals, having bisecting GlcNAc and no core {alpha}1-6 fucose (N-glycan L in Figure 2). Therefore, structure of ds-7 was elucidated (Table I).



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Fig. 5. Sequential exoglycosidase digestions for determination of the monosialylated branch of biantennary N-glycans from chicken serum IgG. Products of exoglycosidase-treated ms-5, ms-7, and ms-8 were analyzed with ODS and Amide-80 columns and compared with reference N-glycans (Figure 2). Monosaccharides were denoted by F, fucose; M, mannose; GN, N-acetylglucosamine; G, galactose; NA, N-acetylneuraminic acid.

 
Our HPLC study clearly indicated that chicken serum IgG has high-mannose-type (37.2%) and complex-type (62.8%) oligosaccharides. This ratio is in a range of that of pigeon serum IgG (high-mannose-type, 33.3%; complex-type, 66.7%) (Suzuki et al., 2003Go). The presence of high-mannose-type set avian IgG apart from human (Figure 3B, Figure 2) and other mammalian IgGs that possess complex-type oligosaccharides exclusively. MonoGlc-high-Man (Glc1Man8–9GlcNAc2) was 71.2% of the total high-mannose-type N-glycans. This percent is also in the range of that of pigeon IgG (61.7% of the total high-mannose type).

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, 1965Go; Kubo and Benedict, 1969Go), 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 peaks—fr. 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 sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), 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, 1991Go).



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Fig. 6. Separation of chicken IgG Fab and Fc by DEAE-Sepharose. (A) Elution profile of papain-digested chicken IgG on DEAE-Sepharose. One milligram of papain-digested chicken IgG was loaded onto a DEAE-Sepharose column (1 ml) and eluted by a linear gradient of NaCl in 10 mM Tris–HCl (pH 8.0). (B) Localization of N-glycans in chicken IgG. Lectin- and immunoblottings of chicken IgG Fab and Fc (CH3 + CH4). Fractions from the DEAE-Sepharose (fr. 1 and 3) and affinity column (fr. 2') were heat-denatured with sample buffer containing 3% SDS without reducing, separated by SDS–PAGE (12.5% gel, 1 µg/lane), transferred to polyvinyl difluoride membranes, and stained with CBB, Con A, RCA-I, or anti-chicken IgG-Fc antibodies.

 
One major N-terminal amino acid sequence of the isolated Fc fragment was shown to be SCSPIQL--- (starting from Ser346), located near the initial part of CH3 domain. MALDI-TOF MS revealed that the [M+H]+ values of the whole chicken IgG, Fab, and Fc were 167,940.56, 44,771.61, and 53,925.79, respectively. The value for Fc was close to the theoretical molecular mass of dimerized CH3 + CH4 regions, apparently lacking CH2 domains. Therefore it was designated as Fc (CH3 + CH4). The mass value for the whole chicken IgG molecule were almost the same as previously reported, and the values for Fab were close to those for Fab' prepared by pepsin digestion (Sun et al., 2001Go). The molecular mass value of Fab agrees with theoretical values of {(VL + CL) + (VH + CH1)} but not {(VL + CL) + (VH + CH1 + CH2)}, suggesting that the Fab fragment also lacks CH2 domains. The CH2 domains might have been lost by the papain digestion, presumably by excessive fragmentation.

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 ({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., 1988Go). This was confirmed by GAF treatment and SDS–PAGE (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 Hex6–10HexNAc2-peptide (Table II). After {alpha}-mannosidase digestion, some of the intact glycopeptides changed to Hex1HexNAc2 peptide, but a large amount of Hex5–6HexNAc2-peptide was also produced (Figure 7). Concomitant with the decrease in the peaks of Hex6HexNAc2 peptides after exhaustive {alpha}-mannosidase digestion, peaks of Hex5HexNAc2 peptides increased. No peaks for Hex2–4HexNAc2-peptide were detected, even when excess {alpha}-mannosidase was used in the digestion. This suggests the presence of Glc-capped oligomannosyl branch. Therefore Hex5–6HexNAc2 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.



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Fig. 7. MALDI-TOF MS analysis of the glycopeptides from chicken IgG-Fc (CH3 + CH4). Tryptic digest of chicken IgG-Fc (CH3 + CH4) was analyzed with MALDI-TOF MS before and after digestion with GAF, Endo H, or {alpha}-mannosidase. Assignment of the [M+H]+ molecular ions detected were listed in Table II. Asterisks on the m/z values indicate the peaks of glycopeptides containing two additional amino acid residues (Glu-Lys, 257.29 Da) produced by alternative trypsin digestion. Because all tryptic peptide fragments are <4000 Da, they are not shown in the selected windows.

 

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Table II. Assignment of the [M+H]+ molecular ion signals afforded by glycopeptides from chicken IgGa

 


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Fig. 8. MALDI-TOF MS analysis for glycopeptides from chicken IgG CH2. The isolated chicken IgG CH2 glycopeptide (A) was treated with GAF (B) or sequentially digested with {alpha}-sialidase (C), ß-galactosidase (D), ß-N-acetyl-D-hexosaminidase (E), and {alpha}-fucosidase (F) and analyzed with MALDI-TOF MS. Assignment of the [M+H]+ molecular ions detected were listed in Table II.

 
N-glycosylation on chicken IgG CH2 domain
One of the two potential N-glycosylation sites on chicken IgG CH domains is located on the CH2 domain. To demonstrate the actual glycosylation at this site, the corresponding glycopeptides were isolated. Tryptic peptides of whole chicken IgG were prepared as described in Materials and methods, and the elution profiles on a C18 column before and after GAF treatment were compared. Three of the peaks, designated fr. A, B, and C, shifted their positions after GAF treatment (data not shown). The actual peptide sequence analysis of the pooled fr. A indicated its complete agreement with the predicted peptide sequence with the N-glycosylation site on the CH2 domain (Table II). Likewise, MS and peptide sequencing data indicated that fr. B and fr. C were shorter and longer glycopeptides from the CH3 domain, respectively. The CH2 glycopeptides before and after GAF digestion or sequential exoglycosidase digestions were analyzed with MALDI-TOF MS (Figure 8). Peaks around m/z 2800–3300 (Figure 8A) were eliminated after GAF digestion, resulting in a peak of m/z 1035 (Figure 8B). The m/z value of the GAF-treated glycopeptide corresponded to the expected [M+H]+ molecular ion for de-N-glycosylated CH2 glycopeptides prepared with trypsin digestion (Table II). The signals at about m/z 1700–1800 were not shifted by GAF digestion, suggesting that they are not N-glycosylated peptides. When CH2 glycopeptide was digested with {alpha}-sialidase, one peak, at m/z 3298, disappeared, and the intensity of the m/z 3005 peak increased (Figure 8C). The mass difference indicates that a single NeuAc was removed by {alpha}-sialidase. This agrees with the fact that predominant sialylated N-glycans derived from chicken serum IgG is monosialylated biantennary oligosaccharides as shown in Table I. {alpha}-Sialidase-treated CH2 glycopeptides were further digested sequentially with ß-galactosidase (Figure 8D), ß-N-acetylhexosaminidase (Figure 8E), and {alpha}-fucosidase (Figure 8F), and the [M+H]+ of the product at each step was recorded. Newly produced peaks of glycopeptides were unambiguously assigned as shown in Table II, and the results indicated that N-glycans on the CH2 domain are exclusively complex-type.

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 {upsilon}-chains, Asn407 on the CH3 domain corresponded well to Asn394 on human IgE {varepsilon}-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., 1997Go), terminal glucose residues on Glc1Man8–9GlcNAc2-oligosaccharides, which occupy wider space than Man5GlcNAc2-, would readily protruded from the (CH3 + CH4) frame and would be accessible to {alpha}-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.



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Fig. 9. Ribbon representations of predicted 3D structures of chicken IgG-Fc (CH2 – CH4) by homology modeling. The two chains (A-chain, green and B-chain, orange) are indicated in two orthogonal views. Backbones and side chains of Asn308, Asn407 (light blue), and conserved Cys residues (at 252, 264, 322, 340, 372, 431, 477, and 546, yellow) forming intradomain and interchain disulfide bridges are indicated. Man5GlcNAc2-linking to Asn407 are visualized by superimpose from the template structure (PDB ID: 1o0v). The atom colors for oligosaccharide chains are carbon, white; oxygen, red; nitrogen, blue.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Carbohydrate chains on glycoproteins can have diverse roles, such as mediators of protein foldings, tags for intracellular and extracellular trafficking, protein stabilizer, conferring hydrophilic properties, protectors against proteolytic digestion, ligands of carbohydrate binding receptors, and so on (Varki, 1993Go). Glycans can assume different structures depending on their biological, biochemical, and structural properties of the individual glycoproteins as well as their location in cells and/or in bodies. Such structural and functional complexity of glycans often makes it difficult to understand their biological function immediately. However, comparison of oligosaccharide structures and glycosylation patterns among glycoproteins with similar structures and functions can provide useful information about the structural relationship between the oligosaccharide chains and the core proteins. Mammalian IgG is one of the best studied glycoproteins with regard to their carbohydrate structures and functions, as well as the entire 3D structures (Deisenhofer, 1981Go; Harris et al., 1997Go; Kobata, 1990Go; Matsuda et al., 1990Go; Mimura et al., 2000Go; Radaev and Sun, 2001Go). Indeed, it has been used as a structural model for other classes of mammalian Igs (Burton, 1987Go; Rudd and Dwek, 1997Go). Detailed studies of carbohydrate chains on Igs are, however, mostly limited to those from mammals, and less attention has been paid to other vertebrate immunoglobulins.

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., 1993Go). When we compared the pigeon IgG N-glycans with those from chicken IgG reported by other groups (Ohta et al., 1991Go; Raju et al., 2000Go), we realized that confirming the N-glycan structures of chicken serum IgG is necessary by our conventional methods (Suzuki et al., 2001Go; Takahashi et al., 2001Go) for several reasons. First, although egg yolk IgG, which arises from transport of maternal antibodies by receptor-specific process (Patterson et al., 1962Go), has identical biophysical properties with serum IgG (Loeken and Roth, 1983Go), it is also reported that egg yolk IgG does not mediate anaphylactic reaction which avian serum IgG does (Faith and Clem, 1973Go). 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)Go 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., 1988Go). 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., 2003Go), because no triantennary or extended branches with ß- and {alpha}-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 {alpha}1-6 Fuc and/or bisecting GlcNAc. Both IgGs have a monogalactosylated branch predominantly on the GlcNAcß1-2Man{alpha}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{alpha}1-3Man arm, which is also the case in normal human IgG (Takahashi et al., 1995bGo). 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 {alpha}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 {alpha}1-6 fucosylated biantennary N-glycans without bisecting GlcNAc (Figure 3B). The observed difference is consistent with an earlier report (Raju et al., 2000Go).

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., 1991Go) but different from quail egg yolk IgG (Matsuura et al., 1993Go) 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., 2003Go). 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, 1987Go). 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, 1990Go; Mimura et al., 2000Go; Radaev and Sun, 2001Go). 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, 1994Go). Crystal structures of human and mouse IgG studied by X-ray diffraction revealed that their Fc region form a cavity between the two {gamma}-chains, and N-glycans on Asn297 can be seen occluding the cavity at the center of the Fc (Harris et al., 1997Go). 1H–nuclear magnetic resonance provided evidence that conformational changes in the sugar chains can affect the structure of the Fc (Matsuda et al., 1990Go). 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 C{varepsilon}2-4 domains) (Wan et al., 2002Go) 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., 1974Go; Dorrington and Bennich, 1978Go), 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{varepsilon}3 domains of the two chains are narrower than those of two C{gamma}2 domains and covered with C{varepsilon}2 domains formed by highly bent structure at C{varepsilon}2–C{varepsilon}3 junctions (Wan et al., 2002Go; Zheng et al., 1992Go). 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., 1997Go; Zheng et al., 1992Go). Enzymes for N-glycan processing apparently can access the N-glycans in C{gamma}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., 1992Go; Parvari et al., 1988Go; Warr et al., 1995Go) (Figure 1A). In addition, both of them mediate anaphylactic reaction (Faith and Clem, 1973Go; Warr et al., 1995Go), and it is believed that they are close relatives in molecular evolution (Warr et al., 1995Go). 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 Glc1Man8–9GlcNAc2 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 {alpha}-mannosidase action for further N-glycan processing (Helenius and Aebi, 2001Go). Although it is reported that CRT can bind to chicken IgG in vitro (Patil et al., 2000Go; Saito et al., 1999Go), 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., 2002Go).

A currently proposed model for the protein folding and assembly of mammalian IgG (Haas and Wabl, 1983Go; Lee et al., 1999Go) 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.



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Fig. 10. Diagram of hypothesis of folding and assembly of avian IgG. In the ER, nascent H-chains of avian IgG possesses Glc3Man9GlcNAc2 on both CH2 and CH3 domains. Partially folded H-chains with Glc1Man9GlcNAc2 can be produced by concerted actions of {alpha}-glucosidase I, II (GI, GII), UDP-Glc:glycoprotein glucosyltransferase (GT) (Parodi, 2000Go), and some ER chaperones. When folding of VH, CH3, and CH4 domains and dimerization of H-chains are proceeded in analogy to mammalian {gamma}-chains (Lee et al., 1999Go), N-glycan processing enzymes such as GII and {alpha}-mannosidase I might be able to partially process Glc1Man9GlcNAc2 on CH3 domain. However, concurrent with but independent on deglucosylation, L-chains are assembled with the H-chains mediated by BiP and other ER chaperones, then the CH3 domains became sterically unaccessible to the processing enzymes after full folding and assembly of the avian IgG molecules. Although timing of the folding of CH2 domain is unknown, folded CH2 domains might confer highly bent structure between Fab and Fc regions in analogy to mammalian IgE (Wan et al., 2002Go). N-glycans on CH2 domains, however, are amenable to processing enzymes and can become complex-type eventually.

 
The reason for the N-glycosylation site in a cavity of Fc region to be conserved among mammalian IgG, IgE, and avian IgG and yet with different N-glycan types remains to be elucidated. Basu et al. (1993)Go had reported that human IgE lacking N-glycosylation at the Asn394 by point mutations tended to self-aggregate, although the loss of N-glycosylation did not influence its binding to Fc{varepsilon} receptors. Therefore the N-glycosylation is probably involved at least in stabilization of the protein structures by conferring suitable hydrophilicity in the cavity of IgE, as well as in IgG. If the hypothesis that IgY evolved to mammalian IgG is correct, mammalian IgG might have gained a hinge region that gives higher segmental flexibility to accommodate a more diverse range of antigens, such as adjusting for cross-linking of epitopes on two large antigens. Accompanying with the changes in protein structures, N-glycans in the cavity of Fc region of mammalian IgG became complex-type but are conserved at the same site (Asn297) to confer the protein stabilization.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Papain (2x crystallized), iodoacetamide, and alkaline phosphatase–conjugated ExtrAvidin were purchased from Sigma (St. Louis, MO). GAF is also known as PNGase F, glycopeptide N-glycosidase F, or N-glycanase. One unit of GAF activity is defined as the amount of enzyme that catalyzes the release of N-linked oligosaccharides from 1 nmol denatured ribonuclease B in 1 min at 37°C, pH 7.5) was from Prozyme (San Leandro, CA), and Endo H from Streptomyces plicatus was a gift from Dr. C. E. Ballou (Berkeley, CA). L-(Tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK)-treated trypsin (3x crystallized) was from Worthington Biochemical (Lakewood, NJ). Alkaline phosphatase–conjugated Con A and RCA-I lectin were purchased from EY Labs (San Mateo, CA). Biotin-conjugated anti-chicken IgG-Fc was from Biotrend Chemicals (Destin, FL). 5-Bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium kit for use with alkaline phosphatase was purchased from Zymed Laboratories (San Francisco, CA). DEAE-Sepharose Fast Flow column (HiTrap, 1 mL) was from Amersham Pharmacia Biotech (Piscataway, NJ). TSKgel DEAE-5PW column (7.5 x 75 mm) and TSKgel Amido-80 column (4.6 x 250 mm) were from TosoHaas (Montgomeryville, PA). Shim-Pack CLC-ODS column (6.0 x 150 mm) was from Shimadzu (Kyoto, Japan). Polyvinylidene difluoride membranes for blotting and Centricon YM 10 were from Millipore (Bedford, MA). Chicken serum IgG was from Pel-Freez Biologicals (Rogers, AR). BCA Protein Assay reagents and immobilized avidin (on 6% cross-linked beaded agarose) were from Pierce (Rockford, IL). Neuraminidase from Arthrobacter ureafaciens was a generous gift from Dr. Tsukada and Dr. Ohta of Kyoto Research Institute (Uji, Japan). Other exoglycosidases used were ß-galactosidase (from jack beans Seikagaku America), ß-N-acetyl-D-hexosaminidase (from jack bean, Sigma), {alpha}-mannosidase (from jack bean, Glyko), and {alpha}-fucosidase (from beef kidney, Roche). The matrices for MALDI-TOF MS, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA), {alpha}-cyano-4-hydroxycinnamic acid (ACH), 2,5-dihydroxybenzoic acid (DHB), and 2',4',6'-trihydroxyacetophenone monohydrate (THAP) were purchased from Aldrich (Milwaukee, WI).

Buffers and standard procedures
Tris-buffered saline (TBS) contains 50 mM Tris–HCl (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 SDS–PAGE, lectin- and immunoblottings, and N-terminal sequence analyses were as described previously (Suzuki et al., 2001Go). Protein concentrations were measured by the BCA assay (Smith et al., 1985Go) 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 Tris–HCl (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 Tris–HCl (pH 8.0) and equilibrated with 10 mM Tris–HCl (pH 8.0). After papain-digested chicken IgG (1 mg) was loaded, the column was washed with 10 mM Tris–HCl (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 SDS–PAGE.

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 Tris–HCl (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 0–50% 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, 50–100 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., 1995Go). 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., 1988Go) 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 {alpha}-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., 1998Go). 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 {alpha}-mannosidase (250 mU/100 pmol glycopeptides) at 37°C overnight. Glycopeptides containing complex-type oligosaccharides (250 pmol) were sequentially digested with {alpha}-sialidase (0.63 mU/100 pmol of glycopeptides), ß-galactosidase (0.38 mU), ß-N-acetyl-D-hexosaminidase (1.11 mU), and {alpha}-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., 2003Go), 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., 2002Go) (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., 2000Go), 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
 
The authors are grateful for Dr. Noboru Tomiya for technical advice concerning oligosaccharide analysis by HPLC and Dr. Hao-Chia Chen for peptide sequencing. This work was supported by NIH Research Grant DK09970.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: nrsuzuki{at}jhu.edu Back


    Abbreviations
 
ACH, {alpha}-cyano-4-hydroxycycinnamic acid; CBB, Coomassie brilliant blue; CNX, calnexin; Con A, concanavalin A; CRT, calreticulin; ER, endoplasmic reticulum; GII, {alpha}-glucosidase II; GAF, glycoamidase F; GU, glucose unit; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; ODS, octadecylsilica; PA, 2-aminopyridine; PBS, phosphate buffered saline; SA, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid); SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TFA, trifluoroacetic acid; THAP, 2',4',6'-trihydroxyaetophenone monohydrate; TOF, time of flight


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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