Detailed structural features of glycan chains derived from {alpha}1-acid glycoproteins of several different animals: the presence of hypersialylated, O-acetylated sialic acids but not disialyl residues

Miyako Nakano2, Kazuaki Kakehi1,2, Men-Hwei Tsai3 and Yuan C. Lee4

2 Faculty of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577–8502, Japan; 3 Ambryx Biotechnology Inc., 5603D Foxwood Drive, Oak Park, CA 91377; and 4 Biology Department, Johns Hopkins University, North Charles Street, Baltimore, MD 21218

Received on September 22, 2003; revised on October 26, 2003; accepted on November 14, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
We analyzed carbohydrate chains of human, bovine, sheep, and rat {alpha}1-acid glycoprotein (AGP) and found that carbohydrate chains of AGP of different animals showed quite distinct variations. Human AGP is a highly negatively charged acidic glycoprotein (pKa = 2.6; isoelectic point = 2.7) with a molecular weight of approximately 37,000 when examined by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and contains di-, tri-, and tetraantennary carbohydrate chains. Some of the tri- and tetraantennary carbohydrate chains are substituted with a fucose residue (sialyl Lewis x type structure). In sheep AGP, mono- and disialo-diantennary carbohydrate chains were abundant. Tri- and tetrasialo-triantennary carbohydrate chains were also present as minor oligosaccharides, and some of the sialic acid residues were substituted with N-glycolylneuraminic acid. In rat AGP, very complex mixtures of disialo-carbohydrate chains were observed. Complexity of the disialo-oligosaccharides was due to the presence of N, O-acetylneuraminic acids. Triantennary carbohydrate chains carrying N,O-acetylneuraminic acid were also observed as minor component oligosaccharides. We found some novel carbohydrate chains containing both N-acetylneuraminic acid and N-glycolylneuraminic acid in bovine AGP. Interestingly, triantennary carbohydrate chains were hardly detected in bovine AGP, but diantennary carbohydrate chains with tri- or tetrasialyl residues were abundant. Furthermore the major sialic acid in these carbohydrate chains was N-glycolylneuraminic acid. It should be noted that these sialic acids are attached to multiple sites of the core oligosaccharide and are not present as disialyl groups.

Key words: {alpha}1-acid glycoprotein / HPLC / N-acetylneuraminic acid / N-glycolylneuraminic acid / MALDI-TOF MS


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
{alpha}1-Acid glycoprotein (AGP), a highly heterogeneous glycoprotein, is an acute-phase protein. AGP is produced mainly in the liver (Sarcione et al., 1967Go), but extrahepatic synthesis has also been reported (Sorensson et al., 1999Go). Serum concentrations of AGP in physiological conditions are about 1 g/L in human and increase severalfold during acute-phase reactions, cancer, pregnancy and some other diseases (Fournier et al., 2000Go). Although AGP is an abundant protein, its physiological significance is not fully understood. Involvement of AGP in nonspecific resistance to a Gram-negative infection (Hochepied et al., 2000Go) and stabilization effect on erythrocyte membrane (Matsumoto et al., 2003Go) have been reported.

Human AGP has a molecular mass of 41–43 kDa when examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and contains ~45% carbohydrates (Schmid et al., 1977Go). Carbohydrate chains attach to the protein core as the complex-type of five N-linked glycans (Fournet et al., 1978Go). Various forms of di-, tri-, and tetraantennary carbohydrate chains contribute to the human AGP glycan complexity. Significant heterogeneity of the oligosaccharides also exists due to variation in the linkage of N-acetylneuraminic acid (NeuAc) to galactose (Gal) and due to the presence of fucose (Fuc) residues (Treuheit et al., 1992Go). Micromolar quantities of asialo-carbohydrate chains were isolated from human AGP, and their structures were confirmed using a combination of proton nuclear magnetic resonance and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Stubbs et al., 1997Go).

In human AGP, marked changes in glycoforms are observed during acute-phase reactions. The changes comprise alterations in branching pattern as revealed by reactivity with Concanavalin A and the fucose-binding lectin (Elliott et al., 1997Go). The expression of a sialyl Lewis x{sLex, NeuAc{alpha}2-3Galß1-4(Fuc{alpha}1-3)GlcNAc-} portion in the carbohydrate chains of human AGP molecules has been a challenging target as related to inflammation. De Graaf et al. (1993)Go found a direct relationship between the reactivity of human AGP to the fucose-specific binding lectin (Aleuria auranita) and staining of human AGP by anti-sLex monoclonal antibody under healthy and disease conditions. Thus analysis of sialo- and asialo-oligosaccharides of AGP as well as its glycoforms is important for understanding the biological roles of AGP (Kakehi et al., 2002Go; Sei et al., 2002Go).

Glycosylation is one of the most important posttranslational events for proteins, affecting their functions in health and disease, playing significant roles in various information traffics for intracellular and intercellular biological events. Recently, Fukui et al. (2002)Go proposed a microarray method for the determination of protein–carbohydrate interactions. We also proposed a glycomics approach for interaction analysis between protein and carbohydrate using a set of carbohydrates as library by capillary affinity electrophoresis (Nakajima et al., 2003Go). This method affords simultaneous determination of (1) carbohydrate chains, (2) binding specificity of the constituent carbohydrate chains, and (3) kinetic data, such as the association constant of each carbohydrate. During the course of the studies, we found that a mixture of carbohydrate chains derived from human AGP is one of the best libraries of carbohydrates, because human AGP contains typical di-, tri-, and tetraantennary complex-type N-glycans as already described.

As an extension of the work for establishing glycan libraries for glycomics studies, we have been analyzing carbohydrate chains of many glycoprotein samples obtainable from commercial sources and found that AGP samples from human, bovine, sheep, and rat sera showed distinct variations in carbohydrate chains. Especially bovine AGP contains quite unique and novel sialic acid–containing diantennary oligosaccharides composed of only N-glycolylneuraminic acid.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
MALDI-TOF MS measurement of bovine fetuin and AGP samples from human, bovine, sheep, and rat sera
In the present study we used commercial samples of AGP derived from human, sheep, bovine, and rat. Carbohydrate chains of fetuin from bovine fetal sera have been extensively studied by many research groups (Green et al., 1988Go; Hayase et al., 1992Go; Townsend et al., 1986Go). Therefore, we employed fetuin (Sigma, St. Louis, MO) as a reference glycoprotein. As shown in Figure 1, bovine fetuin showed a broad peak at 48 kDa, and AGP samples showed a broad peak between m/z 30,000 and m/z 40,000. AGP samples from bovine and sheep showed molecular ions at m/z 33,800 and m/z 33,400, respectively. On the other hand, AGP samples from human and rat showed molecular ions at m/z 37,000 and m/z 36,900, respectively.



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Fig. 1. MALDI-TOF MS spectra of bovine fetuin and AGP samples from human, bovine, sheep, and rat sera.

 
It has been shown that human AGP contains multiantennary carbohydrate chains and showed high molecular masses by MALDI-TOF MS. Relatively low molecular masses of bovine and sheep indicate that these proteins have diantennary oligosaccharides as the major carbohydrate chains (see following discussion).

Analysis of carbohydrate chains of fetuin from bovine sera
Carbohydrate chains were released from the peptides with N-glycoamidase F after digestion of the protein with a combination of trypsin and chymotrypsin (Kawasaki et al., 2003Go; Nakano et al., 2003Go), and were labeled with 2-aminobenzoic acid (2AA) and analyzed by high-performance liquid chromatography (HPLC) using an amino column (Figure 2).



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Fig. 2. Analysis of carbohydrate chains of bovine fetuin. 0SA, 1SA, 2SA, 3SA, and 4SA indicate that asialo-, monosialo-, disialo-, trisialo-, and tetrasialo-oligosaccharides are observed in these regions. The numbers and oligosaccharide structures are summarized in Table I and Figure 3. In the box, the region between 85 min and 180 min is enlarged.

 

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Table I. List of molecular masses for sialooligosaccharides found in AGP samples from human, bovine and sheep sera

 


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Fig. 3. List of the structures for sialooligosaccharides found in AGP samples from human, bovine, sheep, and rat sera.

 
By comparing the data with those reported by Anumula and Dhume (1998)Go, we could assign major peaks and confirm the structures by MALDI-TOF MS measurement. A list of the structures of carbohydrate chains and their abbreviations are summarized in Figure 3 and Table I. For example, the triantennary oligosaccharide containing three N-acetylneuraminic acid (NeuAc) residues is represented as 3-NNN (peak no. 19 in Table I): The first numeral indicates the branch numbers (triantennary in this case), and NNN indicates the presence of three NeuAc residues. If N-glycolylnueraminic acid is present, "G" instead of "N" is used. In the similar manner, F and Pla indicate the presence of fucose and polylactosamine residues, respectively.



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Fig. 5. Comparison of carbohydrate chains in AGP samples among animals. b, human; c, bovine; d, sheep; e, rat. 0SA, 1SA, 2SA, 3SA, and 4SA indicate that asialo-, monosialo-, disialo-, trisialo-, and tetrasialo-oligosaccharides are observed in these regions. The numbers and oligosaccharide structures are summarized in Figure 3, Table I, and Table II. In the boxes, the region between 70 min and 170 min is enlarged.

 
In the present mode of separation using an amino-bonded polymer-based stationary phase, the carbohydrate chains were eluted according to their charges based on the number of sialic acid residues, and positional isomers of sialic acid residues ({alpha}-2,3 versus {alpha}-2,6) are also resolved. Two earliest eluting groups (2–0 and 3–0; 1 and 2) appearing at ~27 min (Figure 2) were due to diantennary and triantennary carbohydrate chains carrying no sialic acids. The peaks of the second group at around 40 min were mixtures of monosialo tri- and diantennary carbohydrate chains [3-N and 2-N (3 and 4), respectively], and those of the third group at around 52 min were disialo tri- and diantennary carbohydrate chains (3-NN and two 2-NN peaks; 11 and 13). We found two disialo-diantennary carbohydrate chains, apparent isomers in NeuAc{alpha}2-3/6Gal linkages (Green et al., 1988Go), which we could not distinguish by MS technique. The most abundant group of peaks at around 63 min was trisialo-triantennary carbohydrate chains. Two major peaks as well as a small peak (3-NNN; 19) were the positional isomers of {alpha}2-3- and {alpha}2-6-linked NeuAc residues (Green et al., 1988Go). Furthermore, we found another trisialo-triantennary carbohydrate chains (3-NNG; 20) at a later elution position, in which one of the sialic acids is substituted with N-glycolylneuraminic acid (NeuGc). At around 82 min, triantennary chains containing four NeuAc residues (3-NNNN; 30) were found (Townsend et al., 1986Go). Furthermore we found some other minor sialo-carbohydrate chains eluted later. A pentasialo-tetraantennary carbohydrate chain (4-NNNNN; 35) was observed at 99 min. We also found three pentasialo-triantennary carbohydrate chains (3-NNNNN, 3-NNNNG, and 3-NNNGG; 41, 42, and 43) at ~127 min. Interestingly, these carbohydrate chains contained one or two NeuGc residues.

We observed MALDI-TOF mass spectra of these oligosaccharides after separation of each peak. The molecular ions of oligosaccharides which we observed are shown in Table I. MALDI-TOF mass spectra of some unique carbohydrate chains in bovine fetuin that were observed later are shown in Figure 4 (a-series).



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Fig. 4. MALDI-TOF MS spectra of some unique carbohydrate chains found in bovine fetuin and AGP samples from human, bovine and rat. a, b, c, and e mean bovine fetuin, human, bovine, and rat AGP, respectively. The numbers correspond to peak numbers in Table I and Table II.

 

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Table II. List of molecular masses for sialooligosaccharides found in rat AGP

 
A pentasialo-tetraantennary carbohydrate chain (35) at 99 min was confirmed by the molecular ion at m/z 3949. We also found a group of pentasialo-triantennary carbohydrate chains at ~127 min. The most intense peak was that of a triantennary chain having five NeuAc residues (41; m/z 3583). The later eluting peaks (42 and 43) showed the ions at m/z 3599 and m/z 3615, respectively. These molecular ions were 16 and 32 mass units higher than m/z 3583 (3-NNNNN; 41), indicating that these peaks were 3-NNNNG and 3-NNNGG (i.e., triantennary carbohydrate chains substituted with four NeuAc and one NeuGc, and three NeuAc and two NeuGc residues, respectively).

Analysis of carbohydrate chains from human, bovine, sheep, and rat AGP
Based on the data on the analysis of bovine fetuin, we analyzed carbohydrate chains of AGP samples from human, bovine, sheep and rat sera. The results are shown in Figure 5.

The regions where neutral (0SA), mono- (1SA), di- (2SA), tri- (3SA), and tetra-sialo (4SA) carbohydrate chains were eluted are also indicated in the figure. As reported previously by several groups (Kakehi et al., 2002Go; Sei et al., 2002Go; Stubbs et al., 1997Go), human AGP contains trisialo-carbohydrate chains as the major group, but di- and tetrasialo-carbohydrate chains are also present. Mono- and disialo-carbohydrate chains, observed at ~40 min and 52 min, respectively, were common to all animals. Trisialo- and tetrasialo-carbohydrate chains in human AGP were observed at around 65 min and 75 min, respectively. However, the counterparts from bovine and sheep AGP were obviously observed later. The major group in sheep AGP was disialo-oligosaccharides, but mono- and tri-sialo-oligosaccharides were also found. On the contrary, rat AGP showed much more complex chromatogram indicating that sialic acids of the oligosaccharides were modified with N,O-acetyl groups.

Human AGP contains di-, tri-, and tetraantennary carbohydrate chains, and some of the tri- and tetraantennary carbohydrate chains are substituted with a fucose residue to form the sialyl Lewis x–type structure. We confirmed identities of several peaks in human AGP as well as those also found in fetuin using MALDI-TOF MS measurements (see Figure 4, b-series). In the region (around 40 min) where monosialo-carbohydrate chains were observed, monosialo-diantennary carbohydrate chains (3-N, 2-N, 4-N, 3-NF, and 4-NF; 3, 4, 6, 7, and 8) were detected. Five disialo-carbohydrate chains (4-NN, 4-NNF, 3-NN, 3-NNF, and 2-NN; 9, 10, 11, 12, and 13) were observed at ~52 min. Trisialo-carbohydrate chains were observed at ~63 min. Two trisialo-tri- and -tetraantennary carbohydrate chains containing sialyl Lewis x structure (4-NNNF and 3-NNNF; 17 and 18) were observed, and the structures were confirmed by the molecular ions at m/z 3512 and m/z 3147, respectively. Tetrasialo-carbohydrate chains were observed at ~75 min. Tetrasialo-tetraantennary carbohydrate chain carrying polylactosamine structure (4-NNNNPla; 27) and tetrasialo-tetraantennary carbohydrate chains containing sialyl Lewis x structure (4-NNNNF; 28) were identified by the ions at m/z 4022 and m/z 3803, respectively.

We found that bovine AGP contained surprisingly unique carbohydrate chains that were quite different from those of human AGP. As shown in Figure 5c, abundant peaks derived from disialo-diantennary carbohydrate chains were observed at around 55 min. Diantennary carbohydrate chain (2-NN; 13) with two NeuAc residues was observed first. A carbohydrate chain (2-NG; 14) with both NeuAc and NeuGc residues was more abundantly present and observed slightly later than 13. The largest peak (15) observed last in this region was clearly due to the carbohydrate chain substituted with two NeuGc residues. MALDI-TOF MS spectra of these unique carbohydrate chains are also shown in Figure 4 (c-series). 2-NG and 2-GG (14 and 15; m/z 2360 and m/z 2376, respectively) were easily identified by increase of 16 and 32 mass units to that of 2-NN (13; m/z 2344). Another abundant group of peaks between 70 and 80 min are diantennary carbohydrate chains having trisialo structures (2326). The carbohydrate chain (23) with three NeuAc residues was eluted the earliest, the last peak being that substituted with three NeuGc residues (26). Characteristic molecular ions for 2-NNN (23), 2-NNG (24), 2-NGG (25), and 2-GGG (26) were observed at m/z 2635, m/z 2651, m/z 2667, and m/z 2683, respectively, as expected. Furthermore, we found tetrasialo-diantennary carbohydrate chains (3640) between 95 min and 111 min. These hypersialylated carbohydrate chains were also confirmed by their molecular ions observed by MALDI-TOF MS. Although triantennary carbohydrate chains were also detected in bovine AGP in trace amounts, they also contained tetra- and pentasialyl residues of NeuAc and NeuGc in different ratios. The presence of the triantennary oligosaccharide carrying five NeuGc residues (46) was especially unique. MS data of some unique triantennary carbohydrate chains were also shown in Figure 4 (c-series).

From the accumulated data for fetuin, human, and bovine AGPs, we tentatively assigned the major peaks of carbohydrate chains derived from sheep AGP. It is interesting that monosialo-oligosaccharides in sheep AGP were abundantly present. Triantennary carbohydrate chains (19, 20, and 3032) were much less as compared with those of human AGP. Hypersialylated oligosaccharides such as trisialo- and tetrasialo-diantennary oligosaccharides were also observed, and the diantennary-oligosaccharide (2-GG; 15) with only NeuGc was also observed. But tri- and tetrasialo-diantennary oligosaccharides with only NeuGc residue were not observed.

Rat AGP contained a quite complex mixture of oligosaccharides, as shown in Figure 5e. In the present mode of separation using a polymer-based amino column, the separation of oligosaccharides is performed based on a number of structural factors including (1) anionic charge (main factor), (2) the number of antenna, (3) monosaccharide composition, and (4) modification on sialic acid residues (O-substitution, NeuAc, and NeuGc) and the linkage position of sialic acid residues. The largest peak (49) observed at ~50 min showed the molecular ion at 2386 that is 42 mass units higher than the disialo-diantennary oligosaccharide having 2 NeuAc residues (see Figure 4e). This indicates that one of the NeuAc residues is substituted with NeuAc having an O-acetyl group. Hydrolysis of the oligosaccharide mixture (Figure 5e) in 10 mM trifluoracetic acid (TFA) at 80°C for 1 h to remove sialic acids afforded asialo-di- and -triantennary oligosaccharides (1 and 2 respectively in Table I; data not shown). We also observed molecular ions of other peaks as shown in Table II and found that many peaks contained acetyl groups. A few examples are shown in Figure 4 (e-series). These data indicate that the complex chromatogram of rat AGP oligosaccharides was due to sialic acids extensively modified with N- and O-acyl groups, such as acetyl and glycolyl groups. These data were well correlated with our previous data on the analysis of sialic acids in various rat tissues (Morimoto et al., 2001Go).

Analysis of oligosialyl units in bovine AGP
Tri- and tetrasialylated diantennary carbohydrate chains found in bovine AGP have two possible structures. One is the presence of the oligosaccharides which are substituted with extra sialic acids at outer GlcNAc residues as observed for NeuAc{alpha}2-6Galß1-3(NeuAc{alpha}26)GlcNAc branch in fetuin triantennary chains. The other possibility is the oligosialyl structure, and extra sialic acids bind the outermost sialic acid residues to form disialyl residues. Sato et al. (1999)Go reported a method to detect oligosialyl residues released by mild acid hydrolysis using 0.01 M TFA followed by fluorometric labeling with 1,2-diamino-4, 5-methylenedioxybenzene (DMB).

We employed NeuAc{alpha}2-8 NeuAc as model. Although partial acid hydrolysis of NeuAc{alpha}2-8NeuAc was observed during hydrolysis even under mild conditions, the sialic acid dimer was present as the major component even after hydrolysis and derivatization with DMB. The dimer was observed at 9 min using octadecyl silica column according to the procedure reported previously (Morimoto et al., 2001Go). The peak was observed earlier than that of NeuAc (10.5 min). On the contrary, we could not observe any peaks in this region other than NeuAc (10.5 min) and NeuGc (7.7 min) when bovine AGP and oligosaccharide 38 were analyzed after mild hydrolysis (data not shown).

We employed another approach to determine whether bovine AGP contains oligosialyl residues or not. Sato et al. (1998)Go also proposed a method to examine the presence of oligosialyl group in glycoproteins using periodate oxidation method. As shown in Figure 6, sialic acids at the nonreducing termini are oxidized with periodate to form a C-7 monosaccharide. On the contrary, the inner sialic acid, of which hydroxyl group of the C8 position is involved in glycosidic linkage to other sialic acid, is not oxidized with periodate. Thus mild hydrolysis of the oligosaccharides affords a C-7 monosaccharide from the outermost sialic acid and intact sialic acids from the inner part.



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Fig. 6. Principle to detect disialyl residues in bovine AGP. Direct acid hydrolysis allows determination of compositions of sialic acids (route a). By hydrolysis after oxidation with periodate, the outermost sialic acids produce C-7 analogs. On the contrary, the inner sialic acids are not oxidized with periodate, and produce intact sialic acids (route b).

 
In this article, we treated a portion of bovine AGP with periodate followed by reduction with sodium borohydride. After hydrolysis of the modified AGP, the constituent sialic acids were analyzed (Figure 6, route b). We also analyzed sialic acids from bovine AGP after direct hydrolysis without periodate oxidation (Figure 6, route a). The results were shown in Figure 7.



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Fig. 7. Detection of disialyl residues in bovine AGP. The dotted line is a chromatogram after direct hydrolysis of bovine AGP. The solid line is a chromatogram after periodate oxidation treatment followed by hydrolysis.

 
Direct hydrolysis of bovine AGP gave two peaks at 7.7 min and 10.5 min due to N-glycolylneuraminic acid and N-acetylneuraminic acid (dotted line in Figure 7). On the contrary, periodate oxidation of bovine AGP showed two products at 8.5 min and 11.5 min (solid line in Figure 7). These two peaks were observed slightly later than those observed after direct hydrolysis of bovine AGP, and identified as C7(Neu5Gc)-DMB and C7(Neu5Ac)-DMB, respectively, by comparison with the data reported by Sato et al. (1998)Go. These results obtained by mild acid hydrolysis and periodate oxidation method clearly indicate that sialic acids in bovine AGP are not present as disialyl residues.


    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Except for human AGP, animal AGPs have received little or no attention with regard to their glycans. We analyzed oligosaccharides of AGP samples from human, bovine, sheep, and rat and compared their patterns after labeling with 2AA (Anumula and Dhume, 1998Go). The fluorescently labeled sialic acid–containing oligosaccharides were superbly resolved based on the number of sialic acids, and isomers of oligosaccharides having sialic acids in different positions were also resolved. In combination with MALDI-TOF MS measurement, we could identify the structures of sialo-oligosaccharides after collection of the peaks, albeit the positions where sialic acids were attached were not determined.

AGP samples from human, bovine, sheep, and rat sera manifested quite different sialo-oligosaccharides patterns. Human AGP contained di-, tri-, and tetraantennary oligosaccharides, and some of the tri- and tetraantennary oligosaccharides included a fucose residue to form sialyl Lewis x structures, as reported previously (Sei et al., 2002Go; Stubbs et al., 1997Go). Rat AGP contained diantennary oligosaccharides as major oligosaccharides. An extremely complex pattern of the chromatogram indicates that rat AGP contains highly acylated oligosaccharides as also shown from MALDI-TOF MS measurement of the major peaks. On the contrary, bovine AGP contained sialo-diantennary oligosaccharides almost exclusively. Furthermore compositions of sialic acids were quite unique, and novel diantennary chains having two NeuGc residues as well as two NeuAc and both NeuAc and NeuGc residues were found abundantly. In addition, we found hypersialylated diantennary oligosaccharides that contained three or four NeuAc and NeuGc residues in various ratios.

We eliminated the possibility of the presence of disialyl linkages by two different approaches using partial acid hydrolysis and periodate oxidation. The results indicate that each sialic acid is attached to different positions (i.e., Gal residues of nonreducing termini and GlcNAc residues of Gal-GlcNAc branches). To the best of our knowledge, there have been no reports on sialyl transferase regulating biosynthesis of such hypersialylated oligosaccharides, and further studies on their biosynthesis will be required.

Regulation of NeuGc biosynthesis is known in developing pig small intestine (Malykh et al., 2003Go). Sialic acids in bovine fetal and adult tissues were analyzed, and NeuGc was abundantly present in all bovine tissues (Schauer et al., 1991Go). In bovine fetuin, oligosaccharides containing only NeuAc were present almost exclusively as shown in the present data (see Figure 2). We also found that NeuGc was present in various digestive organs of mice and rats in different ratios to NeuAc (Morimoto et al., 2001Go). There are also many reports on adult animals having a higher proportion of NeuGc than young animals. AGP and fetuin are produced in liver. At fetal and newborn stages, fetuin is abundantly present in bovine sera. However, AGP is one of the major acidic proteins at adult stages in mammals, and fetuin is hardly detected. Establishment of the relationship between fetal fetuin and AGP in adult stage will be a challenging target for understanding biological regulation of these proteins and their carbohydrate chains.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Bovine fetuin was obtained from Gibco (Invitrogene, Nihon-bashi, Chuo-ku, Tokyo). AGP samples of human, bovine, sheep, and rat were from Sigma (St. Louis, MO). NeuGc, {alpha}-chymotrypsin, and bicine were also purchased from Sigma. TPCK-treated trypsin was from Worthington (Lakewood, NJ). Sephadex LH-20 was from Amersham Bioscience (Uppsala, Sweden). 2AA and sodium cyanoborohydride for fluorescent labeling of oligosaccharides were from Tokyo Kasei (Chuo-ku, Tokyo). Peptide-N4-(acetyl-ß-D-glucosaminyl)asparagine amidase (N-glycoamidase F, E.C. 3.2.2.18) was from Roche Molecular Biochemicals (Minato-ku, Tokyo). NeuAc was donated by Drs. Tsukada and Ohta (Marukin-Bio, Uji, Kyoto, Japan). Water purified with a Milli-Q purification system (Millipore, Shinagawa-ku, Tokyo) after double distillation of deionized water was used for preparation of the eluent for HPLC. Other reagents were of the highest grade commercially available.

Analysis of carbohydrate chains released from AGP samples with N-glycoamidase F
Carbohydrate chains were released from the protein after digesting with a mixture of trypsin and chymotrypsin as reported recently (Nakano et al., 2003Go). Briefly, a sample of AGP from animal sera (500 µg) was dissolved in 20 mM bicine buffer (pH 8.0, 50 µl) and the solution was kept at 100°C for 10 min. After cooling, trypsin (5 µg) in bicine buffer (5 µl) and chymotrypsin (5 µg) in the same buffer (5 µl) were added to the mixture, and the mixture was incubated at 37°C overnight. After the mixture was kept on a boiling water bath for 10 min, N-glycoamidase F (0.5 U, 1 µl) was added to the mixture and kept at 37°C for 8 h, and the mixture was again kept on a boiling water bath for 10 min.

Carbohydrate chains in the mixture thus obtained were directly labeled with 2AA according to the method reported previously (Anumula and Dhume, 1998Go). To the enzyme reaction mixture, was added a solution (200 µl) of 2AA and sodium cyanoborohydride, freshly prepared by dissolution of both compounds (30 mg each) in methanol (1 ml) containing 4% sodium acetate and 2% boric acid. The mixture was kept at 80°C for 1 h. After cooling, the solution was applied to a column of Sephadex LH-20 (1 x 30 cm) equilibrated with 50% aqueous methanol. Earlier eluting fractions showing fluorescence at 410 nm with irradiating at 335-nm light were collected and evaporated to dryness. The residue was dissolved in water (100 µl), a portion (10 µl) was analyzed by HPLC, and the peaks were collected for MS measurement.

HPLC of the fluorescent labeled carbohydrate chains
HPLC was performed with a Jasco apparatus equipped with two PU-980 pumps and a Jasco FP-920 fluorescence detector. Separation was done at 50°C with a polymer-based Asahi Shodex NH2P-50 4E column (Showa Denko, Tokyo; 4.6 x 250 mm) using a linear gradient formed by 2% acetic acid in acetonitrile (solvent A) and 5% acetic acid in water containing 3% triethylamine (solvent B). The column was initially equilibrated and eluted with 70% solvent A for 2 min, at which point solvent B was increased to 95% over 80 min and kept at this composition for further 100 min. The flow rate was 1.0 ml/min throughout the analysis. Detection was performed by fluorometry with {lambda}ex = 350 nm and {lambda}em = 425 nm.

MALDI-TOF MS
MALDI-TOF mass spectra of AGP samples and the fluorescent labeled oligosaccharides were measured on a Voyager DE-PRO apparatus (PE Biosystems, Framingham, MA). A nitrogen laser was used to irradiate samples at 337 nm, and an average of 50 shots was taken. The instrument was operated in linear mode using positive polarity for proteins and negative polarity for oligosaccharides, respectively, at an accelerating voltage of 20 kV. Samples (~10 pmol, 0.5 µl each) were applied to a polished stainless steel target, to which was added a solution (0.5 µl) of 2,5-dihydroxybenzoic acid in a mixture of methanol–water (1:1). The mixture was dried in atmosphere by keeping it at room temperature for several minutes.

Detection of oligosialyl units
Bovine AGP or a fluorescent labeled oligosaccharide collected by HPLC was dried and hydrolyzed in 0.01 N TFA (20 µl) at 50°C for 1 h to release disialyl unit, and the solution was directly derivatized with DMB (Dojin) (Morimoto et al., 2001Go; Sato et al., 1999Go). In brief, to the hydrolyzed solution was added 100 µl of 7 mM DMB solution containing 5.0 mM TFA, 1 M 2-mercaptoetanol and 18 mM sodium hydrosulfite. The mixture was incubated at 50°C for 2 h. In the similar manner, Neu5Ac, Neu5Gc, and Neu5Ac{alpha}2-8Neu5Ac (donated by Drs. Tsukada and Ohta of Marukin-Bio, Kyoto) were derivatized with DMB and used as the standard samples. A portion of the reaction mixture was analyzed on an octadecyl silica column (YMC-Pack ODS-A, 4.6 mm ID, 150 mm length, YMC Co., Kyoto, Japan) using a Shimadzu SLC10A HPLC apparatus with a Jasco FP-920 fluorometer at {lambda}em 448 nm and {lambda}ex 373 nm. The elution was performed in isocratic mode using a mixture of methanol-acetonitrile-water (14:2:84, v/v) at a flow rate of 0.9 ml/min at 40°C. At this condition, NeuAc, NeuGc, and Neu5Ac{alpha}2-8Neu5Ac were observed at 10.5 min, 7.7 min, and 9.0 min, respectively.

Another approach to detect disialyl residues was performed according to the method reported by Sato et al. Briefly, an aqueous solution (10 µl) of bovine AGP (200 µg) was mixed with 50 mM sodium metaperiodata in 50 mM actate buffer (pH 5.0, 10 µl), and the mixture was kept at room temperature for 10 min in the dark. After addition of an aqueous solution of 10% ethyleglycol (10 µl) followed by incubation of the mixture for further 15 min at room temperature. 1 M sodium borohydride in saturated sodium bicarbonate solution (10 µl) was added to the mixture and kept at room temperature for 15 min. To remove the reagents, the reaction mixture was diluted with 300 µl water and filtered through an ultrafiltration tube (molecular cutoff 10,000; Millipore). After washing the retentate with water three times, the retentate was collected with 10 mM TFA (40 µl) and was kept for 1 h at 80°C to release sialic acids by hydrolysis. A portion (20 µl) of the mixture was derivatized with DMB and analyzed by HPLC in the same manner as described.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: k_kakehi{at}phar.kindai.ac.jp


    Abbreviations
 
2AA, 2-aminobenzoic acid; AGP, {alpha}1-acid glycoprotein; DMB, 1,2-diamino-4,5-methylenedioxybenzene; HPLC, high-performance liquid chromatography; MALDI-TOF MS, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry; TFA, trifluoroacetic acid


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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