N-Glycan structures of pigeon IgG: a major serum glycoprotein containing Gal 1-4Gal termini

Transcription

1 N-Glycan structures of pigeon IgG: a major serum glycoprotein containing Gal 1-4Gal termini Noriko Suzuki*, Kay-Hooi Khoo, Chin-Mei Chen, Hao-Chia Chen#, and Yuan C. Lee* *Department of Biology, The Johns Hopkins University, Baltimore, MD, 21218; Institute of Biological Chemistry, Academia Sinica, Taipei, 115, Taiwan; #Endocrinology and Reproduction Research Branch, NICHD, NIH, Bethesda, MD, 20892; To whom correspondence should be addressed. Tel: (410) Fax: (410) yclee@jhu.edu 1

2 Running Title : N-glycan structures of pigeon serum IgG Key words: pigeon serum IgG, N-glycan, Gal 1-4Gal 1-4Gal 1-4GlcNAc, galabiose, IgY, glycodiversity, monoglucosylated high-mannose-type 2

3 Summary We had previously shown that all major glycoproteins of pigeon egg white (PEW) contain Gal 1-4Gal epitope (Suzuki, N., et al (2001) J. Biol. Chem. 276, ). We now report that (Gal 1-4Gal)-bearing glycoproteins are also present in pigeon serum, lymphocytes, and liver, as probed by western blot with GS-I lectin (specific for terminal -Gal) and anti-p 1 (specific for Gal 1-4Gal 1-4GlcNAc 1--) mab. One of the major glycoproteins from pigeon plasma was identified as IgG (alias IgY), which has Gal 1-4Gal in its heavy chains. HPLC, mass spectrometric (MS), and MS/MS analyses revealed that N-glycans of pigeon serum IgG included i) high-mannose-type (33.3%), ii) disialylated biantennary complex-type (19.2%), and iii) -galactosylated complex-type N-glycans (47.5%). Bi- and tri-antennary oligosaccharides with bisecting GlcNAc and 1-6 Fuc on the Asn-linked GlcNAc were abundant among N-glycans possessing terminal Gal 1-4Gal sequences. Moreover, MS/MS analysis identified Gal 1-4Gal 1-4Gal 1-4GlcNAc branch terminals, which are not found in PEW-glycoproteins. An additonal interesting aspect is that about two thirds of high-mannose-type N-glycans from pigeon IgG were monoglucosylated. Comparison of the N-glycan structures with chicken and quail IgG indicated that the presence of high-mannose-type oligosaccharides may be a characteristic of these avian IgG. 3

4 Pigeon (Columba livia) egg white (PEW) is a rich source of glycoproteins containing Gal 1-4Gal (galabiose). All four major PEW-glycoproteins, ovotransferrin, two ovalbumins, and ovomucoid, contain Gal 1-4Gal (1), and all of their constituent N-glycans were found to possess Gal 1-4Gal at the non-reducing termini of tri, tetra, and penta-antennary structures (2). No sialylation is found on the branch that contains the Gal 1-4Gal sequence. Since biosynthesis and glycosyl modification of major egg white glycoproteins of chicken are carried out in tubular gland cells of oviduct (3), the most likely site of fabricating Gal 1-4Gal linkages on the PEW-glycoproteins would be in the corresponding cells of pigeon oviduct. Gal 1-4Gal in glycoproteins is, however, uncommon in Nature. Mammals (e.g., human, pig, rat, mouse, and hamster) express Gal 1-4Gal mostly on glycolipids (4-8), as in P 1 (Gal 1-4Gal 1-4GlcNAc 1-3Gal 1-4Glc 1-1Cer), P k (Gal 1-4Gal 1-4Glc 1-1Cer) antigens, but rarely on glycoproteins. An exception in mammals is the case of glycoproteins with Gal 1-4Gal in hydatid cyst fluid and membrane infected by tapeworm Echnococcus granulosus (9-11). In animals other than birds and mammals, the only recorded presence of Gal 1-4Gal were both in O-glycosides such as those found in an insect cell line from Mamestra brassicae (12) or egg-jelly mucins from amphibians, such as Xenopus laevis (13), Rana clamitans (14), and Ambystoma mexicanum (15). The presence of Gal 1-4Gal or substances similar to P 1 antigen on glycoproteins is limited even among other birds that have been studied. For example, P 1 antigenic activities is absent in the blood of chicken, gander, turkey, quail, duck, and pheasant (16,17), and no -galactoside is found in the glycans of ovomucoid from chicken, quail, and duck (18-22). On the other hand, P 1 antigenic activities have been found in the blood and/or eggs of pigeon, turtle dove (Streptopelia resoria), budgerigar, and cockatiel (16,23,24). Salivary gland mucin 4

5 glycoproteins of Chinese swiftlets (genus Collocalia) contain O-linked glycans with Gal 1-4Gal sequence (25). Biological significance of Gal 1-4Gal in pigeon and some other avian glycoproteins is still unknown. Whether Gal 1-4Gal is located specifically on PEW-glycoproteins or glycoproteins of other pigeon organs is still unsettled. François-Gérard et al reported the presence of P 1 antigenic activity in pigeon blood (16,17). However, they did not indicate whether the antigenicity was located on glycolipids or glycoproteins, and whether the P 1 antigens were produced by pigeon themselves or derived from foreign bodies. To further understand the characteristics of species-specific oligosaccharides, we investigated the presence of glycoproteins with Gal 1-4Gal in pigeon serum, lymphocyte, and liver. Here we report that one of the major glycoproteins containing Gal 1-4Gal in serum is pigeon IgG, which possesses complex-type N-glycans containing Gal 1-4Gal as well as high-mannose-type oligosaccharides. The high-mannose-type N-glycans are exclusively found on the CH3 domain, and the site-specificity is probably a characteristic in avian IgGs. Unlike IgGs in other animals, the complex-type glycans were mainly tri- as well as bi-antennary structures. Moreover, N-glycans containing both Gal 1-4Gal 1-4Gal 1-4GlcNAc and Gal 1-4Gal 1-4GlcNAc branches were detected by MS/MS analysis, which were not found in PEW-glycoproteins. 5

6 Experimental Procedures Materials Adult (female) pigeons were from a local racing pigeon breeder in Virginia, USA. Pigeon livers were obtained from NASCO (Fort Atkinson, WI). Human serum, alkaline phosphatase-conjugated goat anti-mouse IgM, tributylphosphine, and 4-vinylpyridine were purchased from Sigma (St. Louis, MO). Glycoamidase F 2 (GAF) was from Prozyme Inc. (San Leandro, CA). Trypsin (sequencing grade) was from Worthington Biochem. Co. (Lakewood, NJ). Alkaline-phosphatase conjugated GS-I lectin was purchased from EY Lab., Inc (San Mateo, CA). Anti-P 1 mab (mouse IgM) was from Gamma Biologicals, Inc. (Houston, TX). BCIP/NBT Kit for alkaline phosphatase assay was purchased from Zymed Laboratories Inc. (South San Francisco, CA). Ficoll-Paque TM Plus was from Amersham Pharmacia Biotech (Piscataway, NJ). Dulbecco s phosphate-buffered saline (D-PBS, 10x) was purchased from Life Technologies, Inc. (Grand Island, NY). Chicken IgG and sera from chicken and bovine were from Pel-Freez Biologicals (Rogers, AR). BCA TM Protein Assay reagents were from Pierce (Rockford, IL). Polyvinylidene difluoride (PVDF) membranes for blotting, ultrafiltration membrane (YM 10), and Centricon YM 10 were from Millipore (Bedford, MA). Superdex 200 columns for gel filtration (HR 10/30, 1.0 x 30 cm and HiLoad 26/60, 2.6 x 60 cm) and DEAE-Sepharose Fast Flow column (HiTrap, 1 ml) were from Amersham Pharmacia Biotech (Piscataway, NJ). Shim-Pack CLC-ODS column (6.0 x 150 mm) was from Shimadzu Co. (Kyoto, Japan). TSKgel DEAE-5PW column (7.5 x 75 mm) and TSKgel Amido-80 column (4.6 x 250 mm) were from TosoHaas (Montgomeryville, PA). Carbograph tubes (25 mg) were from Alltech. Buffers and standard procedures TBS contains 50 mm Tris HCl (ph 7.4) and 150 mm 6

7 NaCl. TBST contains 0.1% Tween 20 in TBS. Guanidine HCl (8 M) was made in 0.2 M Tris HCl (ph 8.0). Procedures for SDS-PAGE, GS-I lectin blotting, and N-terminal sequence analyses have been described previously (1). Sequence identity search was performed using NCBI databases. Protein concentrations were measured by the BCA assay (26) using bovine serum albumin (BSA) as a standard. Preparation of extracts from pigeon tissues and cells Blood (ca 5 ml/pigeon) drawn from six pigeons were collected in blood collection tubes (13 x 75 mm) containing ml of 15% K 3 EDTA solution (Becton Dickinson). The lymphocytes were isolated with Ficoll-Paque TM Plus, and washed with D-PBS twice. Pellets of lymphocytes and recovered plasma were stored at 20 C until use. The lymphocytes and a portion of pigeon liver were homogenized in 100 mm sodium cacodylate (ph 7.0) containing 1% Triton X-100 at 4 C. The supernatants separated by centrifugation were used as extracts. Isolation of major glycoproteins from pigeon plasma Diluted pigeon plasma (ca. 6.2 mg of protein/ml) was centrifuged to remove insoluble materials. Supernatant was filtered through a m membrane and injected onto a Superdex 200 column (maximum 0.2 ml for a HR 10/30 column, and 1 ml for a HiLoad 26/60 column) equilibrated with 50 mm sodium phosphate (ph 7.0) containing 150 mm NaCl. The flow rates were 0.25 ml/min for the HR 10/30 column and 2 ml/min for the HiLoad 26/60 column. Proteins in the eluate were monitored by A 280 nm, and individual peaks were collected manually. GS-I-positive fractions were concentrated with ultrafiltration (YM 10 membrane) and desalted by washing with 10 mm Tris HCl (ph 8.0) for further purification by ion exchange chromatography. A column of DEAE-Sepharose Fast Flow 7

8 (HiTrap, 1ml) 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 the sample injection, the column was washed with 10 mm Tris HCl (ph 8.0) for 30 min (flow rate:1 ml/min), and then the concentration of NaCl in the elution was linearly increased up to 0.4 M within 80 min (flow rate: 0.5 ml/min). The major peak was collected and concentrated with a Centricon YM-10. GAF-treatment of glycoproteins Glycoprotein samples (10 g each) in 20 l of 0.4% SDS and 100 mm 2-mercaptoethanol in 10 mm Tris HCl (ph 8.0), were heat-denatured at 90 C for 3 min. After the solutions were cooled to room temperature, 1% (v/v) NP-40, and 1 unit of GAF were added to the solution of glycoproteins. The reaction mixtures were incubated at 37 C for 16 h to complete de-n-glycosylation, and then heated at 100 C for 5 min to inactivate GAF. Preparation of glycopeptides for peptide sequencing To reduce the existing disulfide bonds and to block random reformations of intramolecular disulfide-bonds, 16 mol of tributylphophine and 0.16 mmole of 4-vinylpyridine were added to 1 mg of pigeon IgG in 0.1 M Tris.HCl (ph 8.5) and incubated at room temperature for 2 h under nitrogen. The reaction mixture was dialyzed against water and lyophilized. One third of the reduced and alkylated pigeon IgG was suspended with 40 l of 50 mm NH 4 HCO 3, ph 8.4, and incubated with 12.5 g of trypsin (sequencing grade) at 37 C, overnight. A portion of the supernatant was further treated with GAF (0.5 units/10 l) at 37 C, overnight. The peptide mixtures, before and after the GAF treatment, were analyzed with reversed-phase HPLC on a Shim-pack CLC-ODS column. The mobile phases were (A) 0.05% TFA and (B) 90% CH 3 CN in the 0.05% TFA. Elution (1 ml/min) was conducted by a linear gradient of 0 to 50 % of (B) in (A) developed over 100 min. Each peak 8

9 monitored by A 210 nm was collected and kept at 4 C. A portion of the isolated glycopeptides was treated with GAF, and the released oligosaccharides and the deglycosylated peptide were separated with HPLC. Preparation of oligosaccharides for structural analyses Pigeon IgG (1 mg) was reduced with 180 l of 60 mm dithiothreitol in 8 M guanidine HCl at room temperature for 1 h. For alkylation of thiols, 240 l of 0.18 M iodoacetamide in 8 M guanidine HCl was added, and the mixture was incubated at room temperature for 30 min in the dark. The reaction mixture was dialyzed against water, and lyophilized. The alkylated pigeon IgG was suspended with 200 l of 50 mm NH 4 HCO 3, ph 8.4, digested with 10 g of trypsin at 37 C for 4 h, and followed by addition of 10 g of chymotrypsin (37 C overnight). After inactivating the enzymes at 100 C for 10 min, the digest was lyophilized. Oligosaccharides were released with GAF-treatment in 50 mm NH 4 HCO 3, ph 7.8, at 37 C overnight. After inactivating GAF at 100 C for 5 min, the digest was lyophilized. To remove inorganic cations and peptides, the mixture was loaded onto 1 ml of Dowex 50W 2 (H + form, mesh, Sigma) packed in a 1-ml syringe. The column was washed with 5 ml of water, and the collected effluent was lyophilized. The sample was dissolved with 200 l of water, loaded onto a Carbograph tube (25 mg, Alltech), and washed with 1 ml of water, then eluted with 500 l of 25% CH 3 CN containing 0.05% TFA. Exo-glycosidase digestion The released N-glycan mixtures were digested sequentially with 50 mu of neuraminidase from Arthrobacter ureafaciens (Roche, Indianapolis, IN) in 25 µl of 50 mm sodium acetate buffer, ph 5.0; 0.5 U -galactosidase (from green coffee bean, Calbiochem, San Diego, CA) in 100 µl of 50 mm citrate-phosphate buffer, ph 6.0, followed by 5 9

10 mu of 1-4 galactosidase (from Streptococcus pneumoniae, Calbiochem) in 50 µl of 50 mm sodium acetate buffer, ph 6.0. All digestions were carried out at 37 o C for 24 hr. Each enzyme digestion was desalted by passing through a mixed-bed ion-exchange column packed with a mixture of 1 ml each of Dowex 50W-X8 ( mesh, H + form) and AG 3-X4 ( mesh, free base form, Bio-Rad) resins, prior to taking an aliquot for permethylation and MS analysis. PA-glycans were desalted instead by passing through a Sep-Pak C18 cartridge (Waters), washed with water, and then eluted with 50% methanol in water. Chemical derivatization and GC-MS linkage analysis N-Glycan and PA-derivatized N-glycan samples were permethylated using the NaOH/dimethyl sulfoxide slurry method as described by Dell et al. (27). For GC-MS linkage analysis, partially methylated alditol acetates were prepared from permethylated derivatives by hydrolysis (2 M TFA, 121 o C, 2 h), reduction (10 mg/ml NaBH 4, 25 o C, 2 h), and acetylation (acetic anhydride, 100 o C, 1h). GC-MS was carried out using a Hewlett-Packard Gas Chromatograph 6890 connected to a HP 5973 Mass Selective Detector. Sample was dissolved in hexane prior to splitless injection into a HP-5MS fused silica capillary column (30 m x 0.25 mm I.D., HP). The column head pressure was maintained at around 8.2 psi to give a constant flow rate of 1 ml/min using helium as carrier gas. Initial oven temperature was held at 60 o C for 1 min, increased to 90 o C in 1 min, and then to 290 o C in 25 min. MS analysis for glycans and glycomics For MALDI-TOF MS glycan profiling, the permethylated derivatives in acetonitrile were mixed 1:1 with 2,5-dihydroxybenzoic acid (DHB) matrix (10 mg/ml in acetonitrile), spotted on the target plate, air-dried and recrystallized on-plate with ethanol whenever necessary. Data acquisition was performed manually on a benchtop 10

11 LR system (Mircomass) operated in the reflectron mode. For DHB matrix, the coarse laser energy control was set at high and fine adjusted using the % slider according to sample amount and spectra quality. Laser shots (5 Hz, 10 shots/spectrum) were accumulated until a satisfactory signal to noise ratio was achieved when combined and smoothed. Glycan mass profiling was also performed on a dedicated Q-Tof Ultima MALDI instrument (Micromass) in which case the permethylated samples in acetonitrile were mixed 1:1 with -cyano-4-hydrocinnamic acid matrix (in acetonitrile:0.1% TFA, 99:1 v:v) for spotting onto the target plate. The nitrogen UV laser (337 nm wavelength) was operated at a repetition rate of 10 Hz under full power (300 J/pulse). MS survey data were manually acquired and decision to switch over to CID MS/MS acquisition mode for a particular parent ion was made on-the-fly upon examination of the summed spectra. Argon was used as the collision gas with a collision energy manually adjusted (between 50~200 V) to achieve optimum degree of fragmentation for the parent ions under investigation. Off-line nanoelectrospray (nanoesi) using the borosilicate metal coated glass capillary option was performed on an Q-Tof Ultima API instrument (Micromass) equipped with a nanoflow source, mainly for CID MS/MS analysis of permethylated glycans samples. The capillary voltage was set at about 1.0~1.2 kv. Either protonated and/or sodiated doubly or triply charged parent ions may be selected for MS/MS. A collision energy setting of 20~40 ev was usually sufficient for doubly protonated species but 60~80 ev was needed for singly and doubly sodiated parent ions. Permethylated samples were dissolved in 50% acetonitrile / 0.2% formic acid solvent and 2 µl aliquot was typically loaded into the capillary for nanoesi analysis. Preparation and isolation of PA-derivatized oligosaccharides Lyophilized free 11

12 oligosaccharide fractions (from 2 mg of pigeon IgG) were dissolved in 40 l of 2-aminopyridine solution (1 g of 2-aminopyridine in 580 l conc. HCl, ph 6.8), and heated at 90 C for 15 min with heating block. Freshly prepared NaCNBH 3 solution (7 mg/4 l water) 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 NH 4 HCO 3 ), monitored by a fluorescence (Ex: 300 nm, Em: 360 nm), and lyophilized. The mixture of PA-oligosaccharides was first separated by HPLC with a TSKgel DEAE-5PW column (7.5 x 75 mm) as described previously (28), and the separated neutral, mono-sialyl, and di-sialyl fractions (monitored by fluorescence, Ex: 320 nm, Em: 400 nm) were collected separately and lyophilized. These fractions were individually dissolved in water and separated on a Shim-Pack CLC-ODS column (6.0 x 150 mm) as described previously (1). 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 injection of a sample, the composition of the eluents was changed linearly to A:B = 30:70 in 120 min. Each peak was collected, lyophilized, and analyzed with MALDI-TOF-MS. For determination of N-glycan structures by 2-D mapping with HPLC, the isolated PA-oligosaccharides were analyzed using CLC-ODS and TSKgel Amide-80 columns (4.6 x 250 mm) as described previously (29). Elution position of each of PA-oligosaccharides on CLC-ODS and Amide-80 columns was expressed as Glucose Unit (GU), based on the elution position of iso-malto-oligosaccharide series. Reference PA-oligosaccharides from asialo-human IgG, bovine RNase B, and chicken serum IgG were prepared by the same method. Structures of the reference compounds are shown in Fig. 9. PA-derivatized N-glycans B-1, C-1, D (Fig. 9) were obtained from -fucosidase and N-acetyl- -D-hexosaminidase digested PA-oligosaccharides from human IgG. N-Glycans B-2 12

13 and C-2 were prepared from B-1 and C-1, respectively with -galactosidase digestion. 13

14 Results Detection of -galactoside on pigeon glycoproteins by GS-I lectin and anti-p 1 mab Approximately equal amounts of proteins from pigeon serum, liver, lymphocytes, and egg white were subjected to SDS-PAGE, transferred onto a PVDF membrane, and visualized by staining with Coomassie brilliant blue (CBB) (Fig. 1A, CBB staining) or probed by blotting with GS-I lectin to detect terminal -Gal residues. As shown in Fig. 1A (GS-I staining), some proteins from pigeon serum, liver, and lymphocytes were visualized by this staining as did glycoproteins of PEW. Intensities of the GS-I staining did not coincide with those of the CBB staining, suggesting that proteins carry -Gal residues with varied density. Immunoblotting with anti-p 1 mab (specific for P 1 blood type)(fig. 1A) gave similar staining patterns as shown by the GS-I lectin blotting. Extract from pigeon heart also stained with GS-I and anti-p 1 mab (data not shown). These data indicate that many kinds of glycoproteins in pigeon organs most likely contain terminal Gal 1-4Gal as found in PEW (1). Presence of -Gal residues on proteins was probed in pigeon, chicken, bovine, and human sera. While approximately the same amounts of respective proteins in these sera were stained with CBB, only pigeon serum proteins stained strongly by GS-I (Fig. 1B). Since twice as much proteins were used in this experiment (Fig.1B) as the previous experiment (Fig. 1A), more bands were clearly visualized by the GS-I staining. Bovine is known to produce Gal 1-3Gal (30-32), but its serum was stained with GS-I only faintly. Serum proteins of chicken and human, not known to produce Gal 1-3Gal or other -Gal epitopes, was not stained by GS-I, confirming that these serum proteins have no terminal -galactosyl residues. 14

15 Isolation of pigeon plasma glycoproteins Pigeon plasma proteins (600 g total) were separated by gel filtration using a Superdex 200 HR 10/30, as shown in Fig. 2A. As molecular size markers, chicken IgG (170 kda) and BSA (67 kda) were eluted in separate runs and their positions were marked. Each of the peaks obtained from the Superdex 200 was collected, and 50 l of each fraction was analyzed by SDS-PAGE and GS-I lectin blotting under the reducing conditions (Fig. 2B and C). Proteins in fractions 2 to 8 were positively stained by GS-I, while other fractions (fr. 1, ) were negative to GS-I. The fraction 13 was the largest peak detected by A 280 nm, but no protein/peptide bands were visualized with CBB or GS-I staining. One of the glycoproteins in the fraction 6, which was eluted at the same elution position as chicken IgG, showed the strongest intensity by the GS-I staining. In contrast, major proteins in fraction 8, where BSA is expected to elute, were stained with CBB strongly, but not at all with GS-I. The similar elution profile was detected when 6 mg of pigeon plasma proteins were separated using a semi-preparative Superdex 200 column (2.6 x 60 cm) (data not shown). The fraction 6 from Superdex 200 was further purified with a DEAE-Sepharose column eluting with a gradient of NaCl, as shown in Fig. 2D. The major peak on the anion-exchange column was collected and the purity of the protein was confirmed by SDS-PAGE (Fig. 2D, inset). As in the case of chicken immunoglobulins (33), two distinct bands (67 and 27 kda) were visualized, suggesting it to be IgG. The protein after the purification with gel filtration and anion-exchange columns was estimated to be approximately 5 mg from 1 ml of pigeon serum. This value is within the range of reported concentration of IgG in adult pigeon serum (5.92 mg/ml) (34). Identification of the pigeon serum glycoprotein by partial peptide sequencing 15

16 N-Terminal amino acid sequences of the light (L) and heavy (H) chains were homologous to the corresponding variable regions of immunoglobulins of chicken, duck, and human (Table I). Since avian IgG, IgA, and IgM share the same N-terminal variable domains (35), internal peptide sequencing on the Fc region would be required for the determination of the immunoglobulin classes (36-38). The predominant serum immunoglobulin in birds is IgG, also known as IgY, because of its unique structure. It is functionally equivalent to mammalian IgG, but has one additional constant region domain in its heavy (H) chains (Fig. 3) (33,39). Throughout this paper, we refer to the predominant serum immunoglobulin in birds as serum IgG for convenience. The amino acid sequences of chicken and duck immunoglobulin upsilon chains (H chains) suggest that two potential N-linked glycosylation sites are indeed well conserved between chicken and duck, located in the Fc region (Fig. 3) (36,40). In contrast, positions of potential N-glycosylation sites on Fc regions of avian IgA and IgM are different from those of IgG. Therefore, isolation and peptide-sequencing of N-glycosyl peptides were performed in order to determine the immunoglobulin class based on sequence homology. The C18-HPLC elution profiles of tryptic peptides of pigeon IgG, before and after GAF treatment, indicated that, Peak A shifted its position from 46.4 min to 50.1 min after GAF-treatment (Fig. S1). As shown in Table I, the glycopeptide sequence of the pooled peak A is homologous to that of a CH3 domain of chicken or duck IgG. The sequence alignment showed that conservation of the potential N-glycosylation sites is extended to pigeon, chicken, and duck, but not to chicken IgM and IgA. Based on these sequence data, the isolated protein was identified as pigeon serum IgG. The CH3 domains of chicken and duck IgG resemble the CH2 domain of mammalian IgG (36,39,40). This appears to be the case for pigeon IgG also, because the peptide sequence containing the N-glycosylation site was similar to the corresponding site of the CH2 domains of human (Table I) and other 16

17 mammalian IgG (data not shown). Release of -galactoside-containing oligosaccharides from pigeon IgG with GAF Existence of -galactoside-containing N-glycans in pigeon IgG was probed by SDS-PAGE before and after GAF digestion. Decrease in molecular size after GAF-digestion (analyzed by SDS-PAGE) was apparent in the H chains of pigeon IgG, as was observed in chicken IgG (Fig. S2A). The band of L chains were not shifted by the GAF-treatments. The H chain of pigeon IgG was originally stained with GS-I strongly, but not stained at all after de-n-glycosylated (Fig. S2B). This indicates that -Gal-bearing oligosaccharides on pigeon H chain were completely removed by GAF. The H and L chains of chicken IgG were not stained by GS-I, confirming that they do not possess -galactosyl residues. MS and MS/MS analysis of N-glycans from pigeon IgG The N-glycans released by GAF from pigeon IgG were first profiled by MALDI-TOF MS analysis of the permethylated derivatives. Substantial heterogeneity was evident (Fig. 4A), but the glycans can be conveniently categorized into i) high-mannose-type, ii) disialylated biantennary complex-type, and iii) -galactosylated complex-type structures. i) High-mannose-type N-glycans---The two most abundant peaks in Fig. 4A correspond to Hex 9 HexNAc 2 (m/z 2397) and Hex 10 HexNAc 2 (m/z 2601) which also afforded oxonium type fragment ions at m/z 2097 and 2301, respectively, via cleavage at the chitobiose core. Smaller amounts of Hex 5 HexNAc 2 (m/z 1579) and Hex 8 HexNAc 2 (m/z 2192) were also present. Upon -mannosidase treatment, only these components were digested, giving two major signals at m/z 1579 (Hex 5 HexNAc 2 ) and 1783 (Hex 6 HexNAc 2 ) in addition to a signal corresponding to 17

18 Hex 1 HexNAc 2 (data not shown). The same digestion when applied to high-mannose structures derived from RNase B yielded only the expected product, i.e., Man 1 GlcNAc 2. The data are therefore consistent with the presence of a Glc residue at one of the non-reducing termini of a Man 9 GlcNAc 2 which would block the digestion of the Man residues (later to be shown to be on the 3-arm), allowing a removal of only 4 or 5 Man residues from the 6-arm. The presence of a monoglucosylated Man 9 GlcNAc 2 along with Man 9 GlcNAc 2 as the major glycoforms has been reported for chicken and quail IgG (41-43) and may be a characteristic of avian IgG. Further characterization based on 2D-HPLC mapping of the PA-derivatives confirmed this structure (see later section) and has further resolved Hex 9 HexNAc 2 into Man 9 GlcNAc 2 (n-6, major component) and Glc 1 Man 8 GlcNAc 2 (n-7, minor component), along with Glc 1 Man 9 GlcNAc 2 (n-8) as the predominant high-mannose-type structures. Interestingly, only (±Glc 1 )Man 9 GlcNAc 2 and (±Glc 1 )Man 8 GlcNAc 2 structures were found on the glycopeptide isolated from the CH3 domain (Fig. 4C). ii) Disialylated biantennary N-glycans---The sialylated components were first identified by the m/z values of the molecular ion signals and subsequently confirmed by digestion with neuraminidase (Fig. 4B). A prominent desialylated component afforded a major molecular ion at m/z 2071 corresponding to a biantennary complex-type structure which was absent before desialylation (Fig. 4A). The biantennary branching pattern was established by MS/MS analysis (Fig. S3). In particular, the ion at m/z 1143 from MALDI-MS/MS (Fig. S3B) unambiguously defined the presence of two antenna extending from the trimannosyl core while the ion pair at m/z 432 and 464 from nanoesi-ms/ms (Fig. S3A) indicated an N-acetyllactosamine (LacNAc) unit and not a type 1 chain (Gal 1-3GlcNAc) (27). Removal of two Hex units by 4-galactosidase from Streptococcus pneumoniae resulted in a shift of the corresponding molecular ion signal at 18

19 m/z 2071 (Fig. 5A) to m/z 1661 (Fig. 5B) which confirmed this assignment. Finally, methylation analysis of the desialylated samples clearly demonstrated a disappearance of 6-linked Gal (data not shown) as compared with the GC-MS profile of the sample not treated by neuraminidase (Fig. S4). The molecular ion at m/z 2794 in Fig. 4A could thus be assigned as 2-6 disialylated biantennary complex-type N-glycan which shifted to give a molecular ion of m/z 2071 after neuraminidase digestion (Fig. 4B) and remain resistant to -galactosidase digestion (Fig. 5A). No other sialylated component of high abundance was apparent from the analysis of the total mixtures although monosialylated counterparts of several other complex-type structures could be detected as minor peaks, the detailed analysis of which was made possible after HPLC fractionation (see later). iii) -Galactosylated N-glycans---Consistent with the earlier detection by antibody and lectin, the majority of the non-sialylated complex-type structures were shown to carry terminal -Gal by two criteria. First, the m/z of most of the molecular ions detected shifted to lower values after digestion with -galactosidase (Fig. 5A). Second, methylation analysis indicated a high abundance of terminal Gal and 4-linked Gal (Fig. S4), supporting the presence of Gal 1-4Gal epitope. The mass spectrum (Fig. 4A) indicated a high degree of heterogeneity within the -Gal-containing N-glycans, but their m/z signals could be assigned as having ±(Fuc 1 )Hex 2-5 HexNAc 3 and ±(Fuc 1 )Hex 4-8 HexNAc 4 on the trimannosyl core (Man 3 GlcNAc 2 ). After sequential -galactosidase and 4-galactosidase digestion, the major products observed were (GlcNAc 4 )Man 3 GlcNAc 2 ± core fucosylation (m/z 2153 and 2327) and (GlcNAc 3 )Man 3 GlcNAc 2 ± core fucosylation (m/z 1907 and 2082), as shown in Fig. 5B. MS/MS analysis showed that Fuc was indeed conjugated to the inner GlcNAc of the chitobiose core while all non-reducing terminal HexNAcs occur as single HexNAc linked to the trimannosyl core (data not shown). No 19

20 HexNAc-HexNAc type termini was detected. The other major molecular ion signal at m/z 1661, (GlcNAc 2 )Man 3 GlcNAc 2, corresponded to the digested product of the sialylated biantennary structure whereas the high mannose structures at m/z 2397 and 2601 remain unchanged. When the major molecular ion signals from samples not treated with the exoglycosidase (Fig. 4A) were subjected to MALDI CID-MS/MS analysis, additional unique structural features were unveiled. Fig. 6 shows spectra of MS/MS analysis for the Hex 4-8 HexNAc 4 CF series, where CF denotes fucosylated trimannosyl core. Interpretation of the MS/MS data for the Hex 4-8 HexNAc 4 CF series was summarized in Fig. 7. As expected, the presence of Gal-Gal-GlcNAc terminal epitope was indicated by the sodiated b ion at m/z 690 which could be detected in all of the glycans analyzed. For the larger components, however, another b ion at m/z 894, corresponding to sodiated Hex 3 HexNAc +, was also very prominent. In the case of Hex 8 HexNAc 4 CF, sequential loss of the Hex 2 HexNAc or Hex 3 HexNAc terminal units concomitant with emergence of free OH group(s) yielded the complementary set of y ions (e.g., at m/z 3292, 2420, 1548, 3088, and etc., see Fig. 7E). MS/MS data also indicated that majority of the Hex 8 HexNAc 4 CF components are triantennary carrying a bisecting GlcNAc which is readily lost from the parent ion under the MS/MS conditions employed. Two ions at m/z 1548 (corresponds to the fucosylated trimannosyl core with a bisecting GlcNAc and three free OH groups resulting from the loss of all three antennary sequences, Fig. 7E, left) and m/z 1289 (corresponds to a further loss of the bisecting GlcNAc giving a total of 4 free OH groups, Fig. 7E, right), which were present in all Hex 4-8 HexNAc 4 CF series structures, supported the presence of bisecting GlcNAc. Thus, structure of Hex 8 HexNAc 4 CF is most likely a triantennary bisected and fucosylated trimannosyl core, extended with one Hex-Hex-HexNAc (Hex 2 HexNAc) and two Hex-Hex-Hex-HexNAc (Hex 3 HexNAc) sequences (Fig. 7E). 20

21 Similarly, Hex 7 HexNAc 4 CF was determined to be the triantennary structure with bisected and fucosylated trimannosyl core carrying two Hex 2 HexNAc and one Hex 3 HexNAc sequence (Fig. 7D). Majority of Hex 6 HexNAc 4 CF carries three Hex 2 HexNAc units (Fig. 7C), but the presence of m/z 894, albeit of low abundance, indicated possible isomers with one antennae each of Hex 1 HexNAc, Hex 2 HexNAc, and Hex 3 HexNAc. Likewise Hex 5 HexNAc 4 CF (Fig. 7B) showed the presence of Hex 3 HexNAc (m/z 894) and Hex 1 HexNAc (m/z 486). Hex 4 HexNAc 4 CF showed very little m/z 894, but m/z 282 (terminal HexNAc) was found instead, along with m/z 486 and 690 (Fig. 7A). Similar pattern was found for the Hex 2-5 HexNAc 3 CF series which were predominantly bisected biantennary structures. MS/MS analysis suggested that the main isomer of Hex 4 HexNAc 3 CF contained two Hex 2 HexNAc antenna, while Hex 5 HexNAc 3 CF mostly carries a Hex 2 HexNAc and a Hex 3 HexNAc antennae (data not shown). The predominance of bisected complex-type structures was in harmony with the methylation analysis (Fig. S4). The presence of unsubstituted GlcNAc and a triply substituted (3,4,6-linked) Man supported this conclusion. Only 4-linked GlcNAc was found (indicative of the type 2 chain, Gal 1-4GlcNAc), and the amount of 3-linked GlcNAc residue was insignificant. The Gal residue linked to GlcNAc was susceptible to 4-galactosidase. The linkage analysis data suggest that Hex 2 HexNAc and Hex 3 HexNAc terminal units may be Gal-4Gal-4GlcNAc and Gal-4Gal-4Gal-4GlcNAc, respectively, since no other residue, e.g., branched Gal residue, was found. The results of digestion using - and 4-galactosidases (Fig. 5) suggest that they are most likely to be Gal 1-4Gal 1-4GlcNAc 1- and Gal 1-4Gal 1-4Gal 1-4GlcNAc 1-. These sequences were further demonstrated using fractionated oligosaccharides (see later). Fractionation and structural analysis of PA-derivatized N-glycans from pigeon 21

22 IgG The presence of Gal 3 GlcNAc antennae was unexpected and has resulted in greater heterogeneity, since each glycan as assigned above may comprise smaller amount of isomers with different combination of fully galactosylated to non-galactosylated antenna. This was found to be the case when the glycans from pigeon IgG were derivatized with 2-aminopyridine and fractionated first into neutral, monosialylated and disialylated fractions with a DEAE column (Fig. 8A) and then further fractionated on a ODS column (Fig. 8B), followed by MS and MS/MS analyses of the collected major fractions (Table II, III, S1, and S2). Molar ratios of pigeon IgG N-glycans calculated from the peak areas of PA-oligosaccharides were: neutral 64.8% (high-mannose-type, 33.3%, others, 31.5%), monosialylated 16.0%, and disialylated 19.2%. N-Glycan structures of the three categories were determined as followings. i) PA-derivatized high-mannose-type N-glycans---Mass spectrometric analysis revealed that fractions of neutral N-glycans eluted at around min on the ODS column (n-5 -- n-10) were Hex 5, 8, HexNAc 2 -PA, suggesting that these are high-mannose-type oligosaccharides. Their elution positions on the ODS column also confirm this assignment (29). The fractionated high-mannose-type N-glycans (n-5, n-6, n-7, n-8, and n-9) were further examined by the two-dimensional HPLC mapping (29). The elution position of the PA-N-glycans on both ODS and Amide-80 columns, before and after -mannosidase digestion, were compared with those of the reference compounds derived from bovine RNase B and chicken serum IgG (Fig. 9). Since the elution positions on the two columns as well as mass values for the N-glycans of n-5, n-6, n-7, n-8, and n-9 were indistinguishable with those of Man 8 GlcNAc 2 -PA, Man 9 GlcNAc 2 -PA, Glc 1 Man 8 GlcNAc 2 -PA, Glc 1 Man 9 GlcNAc 2 -PA, and Man 5 GlcNAc 2 -PA, respectively (data not shown), their structures were determined as shown in Table II. ii) PA-derivatized disialylated biantennary N-glycans---The two disialylated N-glycans 22

23 (ds-6 and ds-8) were analyzed by MALDI-MS before and after permethylation to define their molecular compositions and, by MALDI MS/MS and HPLC for structural assignment (Table III). N-Glycan ds-8 clearly corresponded to the disialylated biantennary structures detected in the MS analysis of the total mixtures for which further MS/MS analysis of the desialylated structures (Fig. S3) have unambiguously established its biantennary structures. In addition, after -neuraminidase digestion, the elution positions of ds-8 on HPLC columns were indistinguishable from human IgG N-glycan D (Fig. S5B), confirming its structure as non-bisected biantennary complex-type without core fucosylation. MS and MS/MS analysis of N-glycans ds-6 (Fig S5A, Table III) led to identification of a unique NeuAc-HexNAc-HexNAc antennae on ds-6 in which the Hex to HexNAc substitution constitutes the sole difference between ds-6 and ds-8. This was supported by the fact that the elution of ds-6 (GU Amide, 6.9) on the Amide-80 column was GU Amide earlier than that of ds-8 (GU Amide, 7.2) (Fig S5B), which is attributable to the difference in unit contribution values between Hex and HexNAc (29,44). Likewise, the GU Amide of both ds-6 and ds-8 was reduced by about GU after desialylation, indicative of their 2-6 but not 2-3 sialylation (45,46). When the desialylated ds-6 was further treated with N-acetyl- -D-hexosaminidase, the elution positions on ODS and Amide-80 columns (GU ODS, 10.3; GU Amide, 5.6) were indistinguishable from the reference N-glycan B-1, but different from C-1 (GU ODS, 7.7; GU Amide, 5.7). This suggests that desialylated ds-6 released two HexNAcs from Man 1-3 branch. Further digestion with -D-galactosidase resulted in a product eluting at the same position as N-glycan B-2 (GU ODS, 9.5; GU Amide, 4.7), but different from C-2 (GU ODS, 7.1; GU Amide, 4.6), thus further confirming that the NeuAc-HexNAc-HexNAc antennae of ds-6 is extending from the Man 1-3 branch. NeuAc-HexNAc-HexNAc unit is most likely corresponding to NeuAc 2-6GalNAc 1-4GlcNAc, 23

24 although no further evidence can be obtained at present due to lack of the sufficient sample quantity. iii) PA-derivatized -galactosylated neutral N-glycans---The ODS-fractionated neutral complex-type structures (eluted over min), identified by MS and MS/MS, are listed in Table S1. Each of the fractions (Fig. 8B) was first screened by MALDI-TOF MS, and the assigned composition was further confirmed by subsequent analysis of the permethylated derivatives. MALDI MS/MS was performed where the sample quantity permits, and the observed fragment ions collectively established the permutation of galactosylated antennae. In general, the fragmentation is similar to the underivatized samples (Fig. 7), except that each of the y fragment ions carrying the reducing terminal HexNAc would have a mass increment of 92 u due to the PA-tag. For clarity, only the characteristic fragment ions essential for structural assignment were listed in Table S1. As illustrated in Fig. S6, among the important features are : 1) Core fucosylation was assigned if a prominent ion at m/z 566 was detected, corresponding to (OH)(Fuc)GlcNAc-PA. This is further supported by b ions resulting from cleavage at the chitobiose core. 2) Sodiated b ions at m/z 894, 690 and/or 486 identified the presence of Hex 3 HexNAc-, Hex 2 HexNAc- and/or Hex 1 HexNAc- antennae, respectively. This was corroborated by sequential loss of the antennary units from the parent ions, giving rise to a series of y ions. The presence of a bisecting GlcNAc was indicated by detection of a facile loss of terminal HexNAc from the parent and other y ions. 3) Multiple cleavages often resulted in a pair of characteristic b ions corresponding to trimannosyl core, the number of free OH groups contained can be used to infer the number of antenna attached originally. 24

25 Conclusions that can be drawn from more detailed MS/MS analysis of the individually purified components are in general consistent with the analyses of the total mixtures (Fig. 4A), except that ODS fractionation of the PA-derivatives separated isomeric series and also led to detection of a more complete range of minor components. -Galactosidase digestion of Hex 7 HexNAc 4 CF-PA from n-37 resulted in a loss of three -Gal residues, and subsequent 4-galactosidase digestion converted it to HexNAc 4 CF-PA (Fig. S7). Together with the MS/MS results (Table S1), the structure of n-37 is most likely (Gal 1-4Gal 1-4Gal 1-GlcNAc)(Gal 1-4Gal 1-GlcNAc) 2 -NCF-PA, where NCF denotes fucosylated trimannosyl core with bisecting GlcNAc. Similarly, sequential / -galactosidase digestions of Hex 6 HexNAc 4 CF-PA from n-38 released two -Gal and four 4-Gal residues and thus implicated a likely structure of (Gal 1-4Gal 1-GlcNAc)(Gal 1-4Gal 1-GlcNAc) 2 -NCF-PA. These results indicated that for n-38, one of the Hex 2 HexNAc antennae was not -Gal capped. The non-reducing termini of pigeon IgG N-glycans may therefore comprise Gal 1-4Gal 1-GlcNAc, Gal 1-4GlcNAc, and GlcNAc in sub-galactosylated structures. vi) PA-derivatized -galactosylated monosialylated N-glycans---Although only a single disialylated biantennary structure was prominent, the presence of other minor sialylated structures were noted from MS analysis of the total mixtures (Fig. 4A). Indeed, after PA-derivatization and fractionation on ODS column, about 60 peaks could be detected for the monosialylated fraction. Select few of the monosialylated components were screened by MALDI-MS before and after permethylation to define their molecular compositions, and, wherever possible, analyzed by MALDI MS/MS for structural assignment (Table S2). MS/MS analysis of the major monosialylated glycans indicated that all sialylated antenna are 25

26 NeuAc-Hex-HexNAc with no additional Gal inserted (Fig. S8, Table S2). However, the other antenna can be Hex 2 HexNAc or Hex 3 HexNAc, likely to be identical to the -galactosylated antenna found in the neutral glycans. It thus appears that the predominant bisected, bi- and triantennary complex-type structures with core 6-fucosylation are highly heterogeneous due primarily to a combination of different capping moieties on the Gal 1-4GlcNAc antennae, i.e., Gal 1-4-, Gal 1-4Gal 1- and NeuAc In the case of Gal 1-4Gal 1-4Gal 1-4GlcNAc termini, an additional -Gal may be added to the LacNAc unit without the concomitant -Gal capping. Although we have not analyzed all of the isomeric structures resolved by HPLC, the general conclusion may apply to the overall glycosylation heterogeneity of pigeon serum IgG. 26

27 Discussion Diversity of carbohydrate structures are manifest in Nature at many different levels, but expression of different oligosaccharide structures among different species is an important subject of inquiry in glycobiology. Highly complex oligosaccharides are speculated to be generated by necessity for survivals among hosts and microbes through symbiotic-commensal-pathogenic continuum (47,48). However, only a few case of species-specific oligosaccharide moiety had been investigated systematically. One of the better known examples is the Gal 1-3Gal epitope, which is widely distributed in all mammals except human, apes, and Old World monkeys. This moiety has been reported to be absent from fish, amphibian, reptile, and bird fibroblasts, but present on both cell surfaces and secreted glycoproteins of the noncatarrhine mammals (31,32,49). N-Glycans possessing Gal 1-3Gal, found in mammalian glycoproteins, have some structural analogy to those possessing Gal 1-4Gal, found in PEW-glycoproteins (1), because both 3-Gal and 4-Gal are located on non-reducing termini of LacNAc. With the example of Gal 1-3Gal in mind, we first examined the presence of Gal 1-4Gal in pigeon serum, lymphocyte and liver, then isolated one of the major serum glycoproteins possessing Gal 1-4Gal, which was identified as immunoglobulin (IgG). Moreover, analysis of oligosaccharide structures of pigeon IgG unveiled several unique glycosylation properties not found in other avian IgGs or in mammalian IgGs. First, little or no -galactosylated oligosaccharides have been detected in normal serum IgG of non-primate mammals, such as dog, cow, sheep, horse, rat, mouse, rabbit, and cat, (42,50), even though these animals have functional -1,3-galactosyltransferases ( -1,3-GalT). A well conserved N-glycosylation site in mammalian IgG is located at Asn297 (Eu-numbering) on CH2 domains 27

28 (51), and oligosaccharide chains can be seen occluding the cavity at the center of the Fc (52). The N-glycosylation at Asn297 with biantennary complex-type oligosaccharides is indispensable, because it influences mammalian IgG in its thermal stability, interaction with Fc receptors, association with C1q, and induction of antigen-dependent cellular cytotoxicity (53-55). It is presumed that N-glycans in the cavity of Fc region cannot possess Gal 1-3Gal termini, due to steric hindrance (56). If this is true, then the highly -galactosylated features of pigeon serum IgG requires a different explanation. The rationalization came from analysis of oligosaccharides in the pigeon IgG-CH3 domain which is structurally equivalent to mammalian IgG-CH2 domain (36,39). The result indicated that no complex-type N-glycans were located in pigeon IgG-CH3 glycopeptides (Fig. 4C). Secondly, about one third of the total N-glycans from pigeon serum IgG was high-mannose-type oligosaccharides, whereas normal mammalian serum IgG contain exclusively biantennary complex-type N-glycans (42). It should be pointed out that 61.7% of the high-mannose-type glycans in pigeon IgG are mono-glucosylated as found in chicken (serum, egg yolk) (41) and quail (egg yolk) IgGs (43). Specifically, we demonstrated that pigeon IgG-CH3 glycopeptides contain only high-mannose-type oligosaccharides. Since the cognate site-specificity was also found in chicken serum IgG (Suzuki, et al, unpublished data), this site-specific N-glycosylation pattern might have resulted from the steric hindrance imposed by the unique conformational structures of avian IgG. The steric hindrance on avian IgG-CH3 may be more severe than that on mammalian IgG-CH2, and allow only limited process on the N-glycans. Since N-glycans located on CH3 domain are exclusively high-mannose-type, complex-type N-glycans were consequently located at other glycosylation sites on avian IgG. 28

29 Interestingly, structural analysis of N-glycans revealed that the complex-type N-glycans in IgG were very different between pigeon and chicken. This might be explained that the sites for complex-type N-glycans on these avian IgG are more exposed and accessible by processing enzymes, which may form more branched, longer N-glycans. The presence of triantennary N-glycans is unique in pigeon IgG, as it has not been seen in chicken and quail IgGs (41-43). Thirdly, we found a novel N-glycan structures containing Gal 1-4Gal 1-4Gal 1-4GlcNAc branches on pigeon IgG. Presence of the structures could not be shown by lectin-, and immuno-stainings, because of the coexistence of Gal 1-4Gal 1-4GlcNAc, but MALDI CID-MS/MS analysis identified these two branches. The result was surprising, since N-glycans of major PEW-glycoproteins (2) as well as known N-glycan structures of chicken (41) and quail egg yolk IgG (43) do not contain Gal 1-4Gal sequence at all. The absence of Gal 1-4Gal on PEW-glycoproteins may be because the putative -1,4-galactosyltransferase (Gal: -1,4-GalT) responsible for the synthesis of Gal 1-4Gal is inactive in pigeon oviduct, but is active in IgG-producing cells. Whether the same -1,4-GalT in pigeon mediates -1,4-galactosylation onto both Gal 1-4GlcNAc and Gal 1-4Gal 1-4GlcNAc remains to be established. Only other occurrence of the Gal 1-4Gal 1-4Gal 1-4GlcNAc sequence is in O-linked glycans of salivary gland mucin glycoproteins of Chinese swiftlet (genus Collocalia) (25). While Gal 1-4Gal 1-4Gal 1-4GlcNAc sequence has been found in only two avian species so far, Gal 1-4Gal 1-4GlcNAc sequence is known to occur at either internal or non-reducing terminal position of N-glycans in several fish eggs (57-60). The Gal 1-4Gal 1-4GlcNAc sequence is also found on a glycolipid isolated from ostrich liver (61). Gal 1-4Gal 1-4GlcNAc sequence has not been found in mammals on either glycoproteins nor glycolipids, and high titer of natural antibody against terminal Gal 1-4Gal was detected in human 29

30 sera (61). Thus, the expression of Gal 1-4Gal also appears to have emerged in species-specific fashion. The ability to produce Gal 1-4Gal 1-4Gal 1-4GlcNAc and Gal 1-4Gal 1-4GlcNAc sequence on N-glycans of pigeon IgG also affords greater microheterogeneity. In addition to the variations of ±core -1,6-Fuc, ±bisecting GlcNAc, ± -2,6-sialylation, commonly found in mammalian and chicken IgG, the heterogeneity of N-glycans of pigeon IgG is much higher than those of other IgGs. / -1,4-Galactosylation LacNAc branches seems to compete with -2,6-sialylation in pigeon IgG, since neutral forms are dominant in -galactosylated N-glycans (neutral : monosialyl = 2 : 1). On the other hand, about 20% of disialylated biantennary N-glycans were also detected, and they do not possess core -1,6-Fuc, bisecting GlcNAc, nor -1,4-Gal. One may speculate that some N-glycans on variable region are preferred acceptors for -2,6-sialylatransferase but not for -1,4-GalT. Currently, no clear function has been assigned to Gal / 1-4Gal in animal glycoproteins. The -galactosyl residues are expressed in some species of birds (e.g., pigeon, swiftlet), but absent in chicken even though they belong to the same Class Aves, suggesting that such an epitope is apparently not uniformly required by all animals. Nevertheless, glycoproteins with Gal / 1-4Gal are widely distributed or circulating in the body of pigeon. The enigmatic presence of Gal 1-4Gal, Gal 1-4Gal, Gal 1-4Gal 1-4Gal, may have some important function such as protection against specific carbohydrate binding proteins expressed on infectious microbes. Alternatively, because Gal 1-4Gal, Gal 1-4Gal, or Gal 1-4Gal 1-4Gal on non-reducing termini are strongly antigenic in animals that do not express the antigens by themselves, they may work for species-specific barrier against certain viral infections, as speculated for Gal 1-3Gal (47,62,63). How these Gal / 1-4Gal structures were acquired or lost during the course of 30

31 diversification of birds can be investigated through structural analysis of the genes encoding Gal: -1,4-GalT and -1,4-GalT. Finally, it should be mentioned that biomolecules produced by birds inhabiting close to human habitat influence our lives. For example, pigeon fanciers lung (PFL), one of extrinsic allergic alveolitis, is caused by the repeated inhalation of pigeon droppings and feather bloom (64). The pathogenesis of PFL is related to hypersensitivity reactions to pigeon antigens, one of which had been identified as pigeon intestinal mucin oligosaccharides (65). Although N-glycans on pigeon IgG has not been determined as key antigens for PFL, they deserve future scrutiny. Wild pigeons can be carriers of pathogenic bacteria. Indeed Shiga toxin (Stx)-producing Escherichia coli strains had been detected in faecal samples from some pigeons (66). Since Gal 1-4Gal is known to be a minimum structure recognized by several bacterial adhesins and enterotoxins including Stx (see references in (1)), it may be worthwhile to assess the relation of Gal 1-4Gal production in wild birds and their potentials as carriers of pathogenic bacteria. 31

32 Acknowledgment The authors thank pigeon breeders in Virginia who provided pigeons. This work was supported by National Institutes of Health Research Grant DK09970 (NS, YCL), and Academia Sinica (KHK, CMC). Use of MS instruments in the Core Facilities for Proteomics Research (supported by a National Science Council grant NSC P Y) located at Institute of Biological Chemistry, Academia Sinica, and the techinical assistance of Mr. Pei-Lun Tsai is gratefully acknowledged. 32

33 References 1. Suzuki, N., Khoo, K. H., Chen, H. C., Johnson, J. R., and Lee, Y. C. (2001) J. Biol. Chem. 276, Takahashi, N., Khoo, K. H., Suzuki, N., Johnson, J. R., and Lee, Y. C. (2001) J. Biol. Chem. 276, Burley, R. W., and Vadehra, D. V. (1989) The avian egg : chemistry and biology, Wiley, New York 4. Yang, Z., Bergstrom, J., and Karlsson, K. A. (1994) J. Biol. Chem. 269, Kotani, M., Kawashima, I., Ozawa, H., Ogura, K., Ariga, T., and Tai, T. (1994) Arch. Biochem. Biophys. 310, Nakamura, K., Fujita, R., Ueno, K., and Handa, S. (1984) J. Biochem. (Tokyo) 95, Lyerla, T. A., Gross, S. K., and McCluer, R. H. (1986) J. Cell. Physiol. 129, Gahmberg, C. G., and Hakomori, S. (1975) J. Biol. Chem. 250, Khoo, K. H., Nieto, A., Morris, H. R., and Dell, A. (1997) Mol. Biochem. Parasitol. 86, Russi, S., Siracusano, A., and Vicari, G. (1974) J. Immunol. 112, Cossey, A., Dimichiel, A., and Dunstone, J. (1979) Eur. J. Biochem. 98, Lopez, M., Gazon, M., Juliant, S., Plancke, Y., Leroy, Y., Strecker, G., Cartron, J. P., Bailly, P., Cerutti, M., Verbert, A., and Delannoy, P. (1998) J. Biol. Chem. 273,

34 13. Guerardel, Y., Kol, O., Maes, E., Lefebvre, T., Boilly, B., Davril, M., and Strecker, G. (2000) Biochem. J. 352 Pt 2, Delplace, F., Maes, E., Lemoine, J., and Strecker, G. (2002) Biochem. J. 363, Strecker, G., Wieruszeski, J. M., Michalski, J. C., Alonso, C., Leroy, Y., Boilly, B., and Montreuil, J. (1992) Eur. J. Biochem. 207, Brocteur, J., François-Gérard, C., André, A., Radermecker, M., Bruwier, M., and Salmon, J. (1975) Haematologia 9, François-Gérard, C., Brocteur, J., and André, A. (1980) Vox Sang. 39, Yamashita, K., Kamerling, J. P., and Kobata, A. (1982) J. Biol. Chem. 257, Yamashita, K., Kamerling, J. P., and Kobata, A. (1983) J. Biol. Chem. 258, Hase, S., Okawa, K., and Ikenaka, T. (1982) J. Biochem. 91, Hase, S., Sugimoto, T., Takemoto, H., Ikenaka, T., and Schmid, K. (1986) J. Biochem. 99, Takahashi, N., Matsuda, T., Shikami, K., Shimada, I., Arata, Y., and Nakamura, R. (1993) Glycoconjugate. J. 10, François-Gérard, C., and Brocteur, J. (1980) Blood Transf. Immunohemat. 23, Johnson, J. R., Swanson, J. L., and Neill, M. A. (1992) Infect. Immun. 60, Wieruszeski, J. M., Michalski, J. C., Montreuil, J., Strecker, G., Peter-Katalinic, J., Egge, H., van Halbeek, H., Mutsaers, J. H., and Vliegenthart, J. F. (1987) J. Biol. Chem. 262, 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) Anal. Biochem. 150,

35 27. Dell, A., Reason, A. J., Khoo, K.-H., Panico, M., McDowell, R. A., and Morris, H. R. (1994) in Methods Enzymol. (Lennarz, W. J., and Hart, G. W., eds) Vol. 230, pp , Academic Press, San Diego, New York, Boston, London, Sydney, Tokyo, Toronto 28. Nakagawa, H., Kawamura, Y., Kato, K., Shimada, I., Arata, Y., and Takahashi, N. (1995) Anal. Biochem. 226, Tomiya, N., Awaya, J., Kurono, M., Endo, S., Arata, Y., and Takahashi, N. (1988) Anal. Biochem. 171, Dorland, L., van Halbeek, H., and Vliegenthart, J. F. (1984) Biochem. Biophys. Res. Commun. 122, Galili, U., Clark, M. R., Shohet, S. B., Buehler, J., and Macher, B. A. (1987) Proc. Natl. Acad. Sci. U S A 84, Thall, A., and Galili, U. (1990) Biochemistry 29, Leslie, G. A., and Clem, L. W. (1969) J. Exp. Med. 130, Engberg, R. M., Kaspers, B., Schranner, I., Kösters, J., and Lösch, U. (1992) Avian Pathology 21, Bengten, E., Wilson, M., Miller, N., Clem, L. W., Pilstrom, L., and Warr, G. W. (2000) Curr. Top. Microbiol. Immunol. 248, Parvari, R., Avivi, A., Lentner, F., Ziv, E., Tel-Or, S., Burstein, Y., and Schechter, I. (1988) EMBO J. 7, Dahan, A., Reynaud, C. A., and Weill, J. C. (1983) Nucleic. Acids Res. 11, Mansikka, A. (1992) J. Immunol. 149, Warr, G. W., Magor, K. E., and Higgins, D. A. (1995) Immunol. Today 16,

36 40. Magor, K. E., Warr, G. W., Middleton, D., Wilson, M. R., and Higgins, D. A. (1992) J. Immunol. 149, Ohta, M., Hamako, J., Yamamoto, S., Hatta, H., Kim, M., Yamamoto, T., Oka, S., Mizuochi, T., and Matsuura, F. (1991) Glycoconj. J. 8, Raju, T. S., Briggs, J. B., Borge, S. M., and Jones, A. J. (2000) Glycobiology 10, Matsuura, F., Ohta, M., Murakami, K., and Matsuki, Y. (1993) Glycoconj. J. 10, Tomiya, N., and Takahashi, N. (1998) Anal. Biochem. 264, Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y., and Tomiya, N. (1995) Anal. Biochem. 226, Takahashi, N., Lee, K. B., Nakagawa, H., Tsukamoto, Y., Kawamura, Y., Li, Y. T., and Lee, Y. C. (1995) Anal. Biochem. 230, Gagneux, P., and Varki, A. (1999) Glycobiology 9, Hooper, L. V., and Gordon, J. I. (2001) Glycobiology 11, 1R-10R 49. Galili, U., Shohet, S. B., Kobrin, E., Stults, C. L., and Macher, B. A. (1988) J. Biol. Chem. 263, Hamako, J., Matsui, T., Ozeki, Y., Mizuochi, T., and Titani, K. (1993) Comp. Biochem. Physiol. B 106, Burton, D. R. (1987) in Molecular genetics of immunoglobulin (Calabi, F., and Neuberger, M. S., eds) Vol. 17, pp. 1-50, Elsevier, Amsterdam, New York, Oxford 52. Harris, L. J., Larson, S. B., Hasel, K. W., and McPherson, A. (1997) Biochemistry 36, Kobata, A. (1990) Glycobiology 1,

37 54. Mimura, Y., Church, S., Ghirlando, R., Ashton, P. R., Dong, S., Goodall, M., Lund, J., and Jefferis, R. (2000) Mol. Immunol. 37, Radaev, S., and Sun, P. D. (2001) J. Biol. Chem. 276, Endo, T., Wright, A., Morrison, S. L., and Kobata, A. (1995) Mol. Immunol. 32, Iwasaki, M., Seko, A., Kitajima, K., Inoue, Y., and Inoue, S. (1992) J. Biol. Chem. 267, Taguchi, T., Kitajima, K., Muto, Y., Inoue, S., Khoo, K. H., Morris, H. R., Dell, A., Wallace, R. A., Selman, K., and Inoue, Y. (1995) Glycobiology 5, Taguchi, T., Seko, A., Kitajima, K., Inoue, S., Iwamatsu, T., Khoo, K. H., Morris, H. R., Dell, A., and Inoue, Y. (1993) J. Biol. Chem. 268, Taguchi, T., Seko, A., Kitajima, K., Muto, Y., Inoue, S., Khoo, K. H., Morris, H. R., Dell, A., and Inoue, Y. (1994) J. Biol. Chem. 269, Bouhours, D., Liaigre, J., Naulet, J., Bovin, N. V., and Bouhours, J. F. (2000) Glycobiology 10, Rother, R. P., and Squinto, S. P. (1996) Cell 86, Welsh, R. M., O'Donnell, C. L., Reed, D. J., and Rother, R. P. (1998) J. Virol. 72, Calvert, J. E., Baldwin, C. I., Allen, A., Todd, A., and Bourke, S. J. (1999) Clin. Exp. Allergy. 29, Baldwin, C. I., Todd, A., Bourke, S. J., Allen, A., and Calvert, J. E. (1999) Clin. Exp. Immunol. 117, Schmidt, H., Scheef, J., Morabito, S., Caprioli, A., Wieler, L. H., and Karch, H. (2000) Appl. Environ. Microbiol. 66,

38 67. Reynaud, C. A., Dahan, A., Anquez, V., and Weill, J. C. (1989) Cell 59, 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) Nat. Immunol. 3,

39 Footnotes 1. Abbreviations used are : BCIP, 5-bromo-4-chloro-3-indoyl phosphate; BSA, bovine serum albumin; CBB, Coomassie brilliant blue; CID, collision-induced dissociation; GAF, glycoamidase F; GalT, galactosyltransferase, GC-EI, gas chromatography-electron impact; GU, Glucose Unit; Hex, hexose; HexNAc, N-acetylhexosamine; LacNAc, N-acetyllactosamine; MALDI, matrix-assisted laser desorption/ionization; mab, monoclonal antibody; MS, mass spectrometry; nanoesi, nanoelectrospray; NBT, nitro blue tetrazolium; NeuAc, N-acetylneuraminic acid; ODS, octadecylsilica; PA, 2-aminopyridine; PBS, phosphate buffered saline; PEW, pigeon egg white; PFL, pigeon fanciers lung; Stx, Shiga toxin; TBS, Tris buffered saline; TFA, trifluoroacetic acid; TOF, time of flight. 2. 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 of denatured Ribonuclease B in 1 min at 37 C, ph

40 Figure Legends Figure 1. Detection of -galactosyl residue on pigeon glycoproteins by lectin- and immunoblotting. (A) Expression of Gal 1-4Gal in pigeon. Proteins (5 g each) from pigeon serum, liver, lymphocyte, and egg whites were heat-denatured with sample buffer containing 3% SDS and 5% 2-mercaptoethanol, and separated by SDS-PAGE (12.5%). After electrophoresis, the proteins were transferred to PVDF membranes and stained with CBB, GS-I lectin or anti-p 1 mab. (B) Comparison of the presence of -galactosyl residue on pigeon, chicken, bovine, and human sera glycoproteins from by GS-I lectin blotting. Ten g of proteins from sera were separated by SDS-PAGE, transferred to a PVDF membrane and stained with CBB or GS-I. Figure 2. Isolation of -galactosylated glycoproteins from pigeon plasma. (A) Separation of pigeon plasma proteins by Superdex 200 HR 10/30. Six hundred g of pigeon plasma proteins were loaded onto a Superdex 200 HR 10/30 column (1.0 x 30 cm) and eluted by 50 mm sodium phosphate (ph 7.0) containing 150 mm NaCl. Positions of reference molecules (chicken IgG and BSA) are shown. Individual peaks from the column were collected and analyzed on SDS-PAGE, using CBB staining (B) and GS-I lectin blotting (C). (D) Purification of the major glycoprotein from pigeon plasma with DEAE-Sepharose. Desalted fr. 6 from the Superdex 200 column was applied to a DEAE-Sepharose column (1 ml) and eluted with a linear gradient of NaCl in 10 mm Tris HCl (ph 8.0). The major peak (fr. 3) was collected and analyzed by SDS-PAGE (Inset). Figure 3. Structural diagram of avian IgG. The domains of the light chains are termed VL (in the variable region) and CL (in constant region). Typical avian IgG has five domains in the heavy 40

41 chain, termed VL (in variable region), CH1, CH2, CH3, and CH4 (in the constant region). Position of potential N-glycosylation sites (shown in hexagons) were based on chicken IgG H-chain ( -chain) (36). Numbering for chicken immunoglobulin upsilon chain is based on the deduced amino acid sequences from cdna, starting from the first methionine in the leader reagion (36,38,67). Variable regions do not always have N-glycosylation. Location of inter-chain disulfide bonds were predicted in analogy to those of human IgE (36,68), and indicated as dotted bold lines. Figure 4. MALDI-TOF MS of the permethylated N-glycans from pigeon IgG before (A) and after desialylation by neuraminidase (B). The labeled m/z values correspond to the most abundant isotope peak detected as [M+Na] + molecular ions, except for m/z 2097 and 2301, which are oxonium type fragment ions from Hex 9,10 HexNAc 2 (m/z 2397 and 2601), the two most abundant components. The major non-sialylated complex type structures (resistant to neuraminidase) could be assigned as Hex 4-8 HexNAc 4 CF (m/z 3144, 3349, 3553, 3757 and 3962); Hex 4-8 HexNAc 4 C (m/z 2969, 3174, 3379, 3583 and 3788); and Hex 2-5 HexNAc 3 CF (m/z 2490, 2694, 2898 and 3103), where C and CF denote trimannosyl core and fucosylated trimannosyl core, respectively. The corresponding profile of the N-glycans from the isolated glycopeptides on CH3 domain (C) was devoid of the complex-type structures. The mass region from m/z 2800 to 4000 was magnified to show that no signal was detected. Figure 5. MALDI-TOF MS of the permethylated N-glycans from pigeon IgG after sequential digestion with neuraminidase, -galactosidase (A) and 4-galactosidase (B). As demonstrated by MS/MS analysis (Figs. 6 and 7), most of the non-sialylated complex type structures carried a 41

42 bisecting GlcNAc. The triantennary Hex 4-8 HexNAc 4 CF (m/z 3144, 3349, 3553, 3757 and 3962 in Fig 4B) and Hex 6-8 HexNAc 4 C components (m/z 3379, 3583 and 3788 in Fig. 4B) therefore carried a maximum of three terminal -Gal and were trimmed to Hex 2-5 HexNAc 4 CF (m/z 2735, 2939, 3143 and 3348) and Hex 3-5 HexNAc 4 C (m/z 2765, 2969 and 3173) respectively after -galactosidase digestion (A), corresponding to removal of two or three -Gal residues. Likewise the bisected biantennary Hex 2-5 HexNAc 3 CF (m/z 2490, 2694, 2898 and 3103 in Fig. 4B) carried a maximum of two terminal -Gal and could be trimmed to Hex 1-3 HexNAc 3 CF (m/z 2286, 2490 and 2694), corresponding to removal of one or two -Gal residues. The signal at m/z 2735 (Hex 2 HexNAc 4 CF) in (A) could also derive from incomplete digestion by -galactosidase, leaving an -Gal-capped antennae resistant to further -galactosidase digestion (B). The signal at m/z 1711 was contributed by the MALDI matrix used. Figure 6. MALDI CID MS/MS of permethylated -galactosylated triantennary complex-type structures from pigeon IgG. A--E refer to Hex 4-8 HexNAc 4 CF, as labeled on the figure. Assignment of the fragment ions are illustrated by diagrams in Fig. 7. Some fragment ions could not be labeled or assigned, because of complication arising from isomeric structures. Figure 7. Summary of the major -galactosylated triantennary complex-type structures as deduced from MS/MS sequencing data (Fig. 6). (A) Hex 4 HexNAc 4 CF; (B) Hex 5 HexNAc 4 CF; (C) Hex 6 HexNAc 4 CF; (D) Hex 7 HexNAc 4 CF; and (E) Hex 8 HexNAc 4 CF. Full assignment is shown for Hex 8 HexNAc 4 CF (E) where the glycosidic oxygen (-O-) are shown to illustrate the sites of cleavage. The majority of the ions are either b or y ions. The b ions at m/z 690, and 894 showed that both Hex 2 HexNAc + and Hex 3 HexNAc + termini are present. In (A) and (B) the 42

43 presence of incompletely galactosylated termini was also evident from m/z 282 (HexNAc + ) and 486 (Hex 1 HexNAc + ). A combination of multiple cleavages produced the complement of y ions unique to each structure. A facile loss of the bisecting GlcNAc was illustrated in (E) but indicated as further loss from the y ions with an arrow in other structures for simplicity (A-D). All structures from the Hex 4-8 HexNAc 4 CF series gave the common ions at m/z 1548, 1289, 838, 719, 648 and 474 which were shown only in (E). Another common ion at m/z 648 (Fig. 6) is related to m/z 838 (Fig. 6 and 7E left), representing a further loss of the branched terminal Hex (Man) residue. For Hex 4,5 HexNAc 4 CF, the more abundant isomer could be represented by the structures on the right in (B) and (A), but the presence of the isomers shown on the left was evident from the additional ions detected. The location of the different antenna on either 3- or 6-arm of the trimannosyl core was not defined. Symbols used : Triangle, Fuc; Square, HexNAc; Circle, Hex; the darker filled circle at the non-reducing termini is meant to represent -Gal capping although MS data cannot be used to distinguish it from other Hex. Figure 8. Separation of PA-oligosaccharides from pigeon serum IgG by HPLC. (A) Total PA-oligosaccharides from pigeon IgG were separated into neutral, mono-, and disialyl oligosaccharides on the DEAE column. (B) Elution profiles of the neutral, mono-, and disialyl PA-oligosaccharides on the ODS column. Each peak was analyzed with mass spectrometry. Fraction numbers for N-glycans correspond to those in Tables II, III, S1, and S2. Figure 9. Structures of reference oligosaccharides isolated from human IgG, bovine RNase B, and chicken serum IgG. PA-derivatized these N-glycans were utilized for HPLC analysis. 43

44 Figure 10. Summary of the major monosialylated structures as deduced from MS/MS sequencing. (A) NeuAc 1 Hex 3 HexNAc 3 CF-PA from ms-42; (B) NeuAc 1 Hex 4 HexNAc 3 CF-PA from ms-40; (C) NeuAc 1 Hex 5 HexNAc 4 CF-PA from ms-50; and (D) NeuAc 1 Hex 6 HexNAc 4 CF-PA from ms-48. In addition to the key fragment ions illustrated in Fig. S6, key y ions resulting from sequential loss of one or more of the antenna were also illustrated. As in Fig. 7, the facile loss of the bisecting GlcNAc is indicated by an arrow from the primary cleavage ions. The location of the different antenna on either 3 or 6-arm of the trimannosyl core was not defined. Symbols used are as in Fig. 7 and S6. 44

45 Table I Alignment of the amino acid sequences of immunoglobulins of birds and human a Protein Sequence Acc. No. b Identity c (%) light chain (lambda) N-terminal sequence pigeon chicken duck human ALTQPPSVSTTLGGTVQIT ALTQPASVSASLGGTVKIT ALTQPASKSVNPGDTVQIT ILTQPPSVSASLGASVKVT BAB47274 CAA57568 CAA /19 (79) 13/19 (68) 12/19 (63) heavy chain N-terminal sequence pigeon chicken duck human AIELVESGGGLVSPGGSLRL AVTLDESGGGLQTPGGALSL AATLDESGGGLVSPGGSLTL EVQLLESGGGLVQPGGSLRL AAA48846 CAA46322 P /20 (65) 16/20 (80) 15/20 (75) heavy chain (upsilon/gamma) N-glycosylation site pigeon chicken (CH3) duck (CH3) human (CH2) d AQFNGTFTATSEVPVATR EHFNGTYSASSAVPVSTQ EHFNGTFTASSSLAISTQ EQYNSTYRVVSVLTVLHQ CAA30161 CAA46322 CAC /18 (56) 9/18 (50) 5/18 (28) chicken IgM (CH3) e chicken IgA (CH3) e LQSNGLYTVDGVATVCAS RQADGRYTVRSFLRVCAE CAA25762 AAB /18 (28) 5/18 (28) a Sequence identity search was performed using NCBI database. N-Linked glycosylation sites are indicated by bold face. The N-glycosylation site on pigeon IgG was assigned by the presence of Asp occurred after GAF-digestion. b Primary accession numbers for the database. c Identity of the peptide segment (%) were calculated as (number of identical amino acid residues) x 100 / (number of total amino acid residues). d CH2 domains of mammalian IgG is equivalent to CH3 domains of avian IgG. e The positions of indicated sequences correspond to that in CH3 domain of chicken IgG. 45

46 Table II Structures of high-mannose-type PA-oligosaccharides from pigeon IgG based on HPLC and MS analysis Fraction Relative Quantity (%) a GU on ODS GU on Amide m/z b Detected Reference N-Glycans c Deduced Structures d (neutral, high-mannose) n Man8 n Man9 M 1-2M 1 M 1 6 M 1 3 M 1-2M 1-2M 1 M 1-2M 1 M 1-2M 1 6 M 1 3 M 1-2M 1-2M 1 6 M 1-4GN 1-4GN- 3 6 M 1-4GN 1-4GN- 3 n GlcMan8 n GlcMan9 M 1-2M 1 M 1 6 M 1 3 Glc 1-3M 1-2M 1-2M 1 M 1-2M 1 M 1-2M 1 6 M 1 3 Glc 1-3M 1-2M 1-2M 1 6 M 1-4GN 1-4GN- 3 6 M 1-4GN 1-4GN- 3 n Man5 M 1 M 1 6 M 1 3 M 1 6 M 1-4GN 1-4GN- 3 a Relative quantity was calculated based on CLC-ODS elution profiles. b Mass/charge (m/z) of neutral oligosaccharides (non-permethylated) were detected as [M+Na] +. c PA-derivatized reference N-glycans whose elution positions on both CLC-ODS and Amide-80 columns and [M+Na] + coincide with the pigeon IgG PA-oligosaccharides were indicated on the Table. Structures of the reference compounds were shown in Fig. 9. d Monosaccharides were denoted as: M, mannose; Glc, Glucose; GN, N-acetylglucosamine. 46

47 Table III Structures of disialylated PA-oligosaccharides from pigeon IgG based on HPLC and MS analysis Fraction Relative Quantity (%) a GU on ODS GU on Amide Reference N-Glycans b Key Fragment Ion (m/z) c Deduced Structures d (disialyl) ds B-1 + 2HexNAc + 2NeuAc ds D + 2NeuAc 392, 398, 643, 847, 866, 888, 1111 (see Fig.S3) NA 2-6G 1-4GN 1-2M 1 NA 2-6HN 1-?GN 1-2M 1 NA 2-6G 1-4GN 1-2M 1 NA 2-6G 1-4GN 1-2M 1 6 M 1-4GN 1-4GN- 3 6 M 1-4GN 1-4GN- 3 a Relative quantity was calculated based on CLC-ODS elution profiles, as noted in Table II. b PA-derivatized reference N-glycans whose elution positions on both CLC-ODS and Amide-80 columns and [M+Na] + coincide with the pigeon IgG PA-oligosaccharides were indicated on the Table. Structures of the reference compounds were shown in Fig. 9. c Key fragment ions detected in MALDI MS/MS analysis of the selected parents, the production mechanism of which is shown in Fig. S5A. d Monosaccharides were denoted as: M, mannose; GN, N-acetylglucosamine; G, galactose; HN, N-acetylhexosamine; NA, N-acetylneuraminic acid. 47

48 48 Figure 1 Suzuki et al.

49 49 Figure 2A, B, and C Suzuki et al.

50 50 Figure 2D Suzuki et al.

51 51 Figure 3 Suzuki et al.