Glycobiology Advance Access originally published online on October 11, 2006
Glycobiology 2007 17(2):157-164; doi:10.1093/glycob/cwl058
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Occurrence of a nonsulfated chondroitin proteoglycan in the dried saliva of Collocalia swiftlets (edible bird's-nest)
Hiroki Nakagawa3,4
3 Department of Biochemistry, Tulane University Health Sciences Center School of Medicine, New Orleans, LA 70112
4 Department of Applied Biological Sciences, Saga University, Saga 840-8502, Japan
5 Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan
6 Laboratory of Proteoglycan Signaling and Therapeutics, Faculty of Advanced Life Science, Hokkaido University, Frontier Research Center for Post-Genomic Science and Technology, Kita 21-jo, Nishi 11-choume, Kita-ku, Sapporo 001-0021, Japan
1 To whom correspondence should be addressed; Tel.: (504)-988-2451; Fax: (504)-988-2739; e-mail: yli1@tulane.edu
Received on June 19, 2006; revised on September 27, 2006; accepted on September 29, 2006
 | Abstract |
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" border="0"> 4GlcUAß1 " border="0"> 3GalNAcß1 " border="0"> )n chains. The average molecular mass of the chondroitin chain was estimated to be 49 kDa by gel filtration. We have isolated a linkage region hexasaccharide, 
HexUA
1 " border="0"> 3GalNAcß1 " border="0"> 4GlcUAß1 " border="0"> 3Galß1 " border="0"> 3Galß1 " border="0"> 4Xyl, from this PG by chondroitinase ABC digestion to show that the GAGs in this PG are also linked to the core protein through the common tetrasaccharide linker, GlcUAß1 " border="0"> 3Galß1 " border="0"> 3Galß1 " border="0"> 4Xyl, found in various PGs. As water was not effective in extracting uronic acid-containing glycoconjugates from the black nest built by black nest swiftlets (A. maximus), we used 4 M guanidium chloride and anion-exchange chromatography in the presence of urea to extract and isolate about 30 mg of a chondroitin PG preparation from 10 g of the desialylated black nest. As the biological significance of chondroitin is still not well understood, bird's nest should become a convenient source for preparing this unique GAG to study its biological functions. Key words: chondroitin / proteoglycan / glycosaminoglycan / bird's nest / saliva
| Introduction |
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; Kjellén and Lindahl 1991; Hardingham and Fosang 1992; Iozzo 1998; Prydz and Dalen 2000; Kolset et al. 2004). Except for hyaluronic acid, all GAGs are sulfated and are linked to a core protein. The nonsulfated GAGs are not usually found in biological materials, except in early stages of biosynthesis or when cultured cells are grown under the condition in which the sulfate donor is deprived (Humphries et al. 1989). It should be noted, however, that nonsulfated chondroitin has been found in the capsular K4 antigen of Escherichia coli O5:K4:H4 (Rodriguez et al. 1988) and in Caenorhabditis elegans (Yamada et al. 1999). Despite their wide distribution, PGs have never been isolated from the saliva of higher animals. Edible birds'-nests are the dried form of the nests made from regurgitated saliva of male Collocalia swiftlets. They can be divided into the white nest and the black nest. The white –nest, built by white nest swiftlets (Aerodramus fuciphagus), is composed of only the dried saliva. In contrast, the black nest, made by black nest swiftlets (A. maximus), contains the birds' feathers together with the saliva (Valli and Summers 1990). Collocalia glycoproteins prepared from birds' nests are known for their high sialic acid content (Howe et al. 1961). While studying the substrate specificity of the two sialidases isolated from Macrobdella leeches (Li et al. 1990), we found that the crude leech sialidase released a disaccharide from Collocalia glycoproteins prepared from both the black and the white birds' nests. By chemical analyses and NMR spectroscopy, the structure of this disaccharide released from the white nest Collocalia glycoprotein was established to be 4,5HexUA1 " border="0"> 3GalNAc, which is identical to that released from chondroitin by chondroitin AC lyase. We reasoned that Collocalia glycoproteins of edible birds'-nests must contain a GAG composed of chondroitin, and that Macrobdella leeches contained a lyase with the specificity similar to that of microbial chondroitin AC lyase (Linhardt et al. 1986). This paper reports the isolation and characterization of a chondroitin PG from edible birds'-nests.
| Results and discussion |
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Characterization of an unknown compound released from white nest Collocalia glycoprotein by crude leech sialidase.
), the crude sialidase prepared from the Macrobdella leech released an unknown compound (UC) with thin layer chromatography (TLC)-mobility faster than that of Neu5Ac and 2,7-anhydro-Neu5Ac from Collocalia glycoproteins prepared from either the black or the white nest (Figure 1). We were intrigued by the fact that the crude leech sialidase released more UC than Neu5Ac and 2,7-anhydro-Neu5Ac combined from the Collocalia glycoprotein prepared from the white nest as shown in Figure 1. We subsequently carried out the isolation of this UC and obtained 1.5 mg of pure UC from 250 mg of white nest Collocalia glycoprotein using the procedure described under the section Methods. On TLC plates, UC did not give a color when sprayed with resorcinol-HCl (Svennerholm 1957) or orcinol-H2SO4 (Winzler 1955), suggesting that this compound was devoid of sialic acid and neutral sugar. In test tubes, UC reacted with the carbazole reagent (Galambos 1967) to give a typical pink color for uronic acids. It also gave a positive Elson–Morgan reaction (Gatt and Berman 1966), suggesting the presence of a hexosamine. The ratio of uronic acid to hexosamine in UC was found to be 1:1. On a TLC plate, UC reacted with iodine vapor to give a brownish color, indicating the possible presence of a double bond. This was also supported by the fact that UC had the UV absorption at 230 nm. When analyzed by gas liquid chromatography (GLC), the hexosamine in the acid hydrolyzate of UC was identified to be galactosamine. The results of chemical analyses suggest that UC contained an unsaturated uronic acid and GalNAc in 1:1 ratio. The structure of UC was finally confirmed by NMR spectroscopy. As shown in supplementary material, the 500-MHz proton NMR chemical shifts of the sugar residues in UC were found to be identical to that of the authentic sodium salt of 4,5HexUA1 " border="0"> 3GalNAc (Yamada et al. 1992).
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Isolation and characterization of a chondroitin PG from the white nest
4,5HexUA1 " border="0"> 3GalNAc from the Collocalia glycoprotein prepared from the white nest by the crude leech sialidase indicated that this source contained a GAG with the structure consistent with that of chondroitin. It also suggested that Macrobdella leeches contained a chondroitinase AC-like lyase capable of de-polymerizing the chondroitin chain. Since the uronic acid-containing glycoconjugates in the white nest were readily soluble in water, we devised a method to use Sepharose CL-6B filtration (Figure 2A), DEAE-Sephacel anion-exchange chromatography (Figure 2B), and Sephadex G-75 filtration (Figure 2C) to isolate approximately 30 mg of chondroitin PG from the water extract prepared from 15 g of the white nest. As shown in Figure 2A, by Sepharose CL-6B gel filtration, sialic acid- and uronic acid-containing glycoconjugates were eluted before the main protein peak. Since PGs with uronic acid-containing GAGs are more negatively charged than sialoglycoconjugates, the anion-exchange chromatography separated the uronic acid-containing PGs from sialoglycoconjugates (Figure 2B). Sephadex G-75 filtration purified the major uronic acid-containing glycoconjugate as shown in Figure 2C. Since chondroitin PG of white nest was highly soluble in water, we chose water as eluent to perform the gel filtration (Figure 2A and C) for the facile isolation of this PG. Under this condition, chondroitin PG was retarded by the Sephadex G-75 column, due to the interaction of the PG with the gel matrix as shown in Figure 2C. Thus, the elution position of the chondroitin PG shown in Figure 2C does not reflect its molecular size. Using this isolation scheme, we were able to isolate approximately 30 mg of chondroitin PG from 15 g of white bird's nest. By Superdex-200 filtration, the average molecular mass of the 2-aminobenzamide (2AB)-derivatized chondroitin GAG chains derived from chondroitin PG was estimated to be 49 kDa, based on the elution position as calculated from a calibration curve obtained with authentic dextrans of various molecular sizes (Figure 3) . The constituent sugars in this PG preparation were determined to be GalNAc (42%), GlcUA (37%), GlcNAc (1.4%), Gal (1.8%) and Xyl (0.6%). It is remarkable that this preparation contained 83% of carbohydrates, of which 79% were GalNAc and GlcUA in equimolar ratio. This preparation was devoid of sialic acid and contained the following amino acids in mole%: Asp, 11.8; Ser, 10.1; Thr, 7.1; Glu, 14.9; Gly, 8.8; Ala, 7.2; Val, 9.0; Ile, 3.5; Leu, 8.7; Tyr, 3.1; Phe, 3.0; His, 3.1; Lys, 4.2; and Arg, 5.6. Upon treatment of this preparation with 0.3 M NaOH at 25 °C for 63 h, 23% of Ser and 19% of Thr were destroyed, suggesting that these two amino acids are linked to a sugar chain through the O-glycosidic linkage. The infrared spectrum of this preparation did not show the absorption at 1230 cm–1 (S = O), 850 cm–1 found in C–O–S of chondroitin 4-sulfate and 820 cm–1 found in C-O-S of chondroitin 6-sulfate (Mathews 1958; Lloyd et al. 1961). We were also not able to detect the presence of sulfate in 0.5 mg of this PG preparation by using the rhodizonate method (Terho and Hartiala 1971). Chondroitin AC lyase liberated 4,5HexUA1 " border="0"> 3GalNAc from this preparation (Figure 4). These results conclusively show that the GAGs in the PG prepared from the white nest are made up of chondroitin ( " border="0"> 4GlcUAß1 " border="0"> 3GalNAcß1 " border="0"> )n chains and that this PG is a chondroitin PG.
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Structural analysis of the linkage region hexasaccharide prepared from the white bird's nest chondroitin PG
) yields 4,5-unsaturated disaccharide and the linkage region 4,5-unsaturated hexasaccharide–peptide core (Sugahara et al. 1988; Sakaguchi et al. 2001). We found that the rate of degradation of chondroitin by chondroitinase ABC was much slower than that of chondroitin sulfates. Thus, as described under the section Methods, prolonged incubation with chondroitinase ABC was necessary for exhaustive digestion of bird's nest chondroitin PG to prepare the linkage region hexasaccharide. The putative linkage region hexasaccharide prepared from the white nest chondroitin PG was set free from the core protein by LiOH (Sakaguchi et al. 2001), labeled 2AB, and analyzed by anion-exchange high performance liquid chromatography (HPLC) on an amine-bound silica PA-03 column according to Kinoshita and Sugahara (1999). The 2AB-labeled chondroitin PG linkage region hexasaccharide was eluted at the position of the authentic unmodified linkage region hexasaccharide (Figure 5B). A mixture of the authentic 2AB-labeled linkage region hexasaccharides (Sakaguchi et al. 2001) was mixed with the 2AB-labeled chondroitin PG linkage region hexasaccharide fraction and examined by anion-exchange HPLC (Figure 5C). The chondroitin PG linkage region hexasaccharide was co-eluted with the unmodified authentic linkage region hexasaccharide. The 2AB-labeled chondroitin PG linkage region hexasaccharide was digested with chondroitinase AC-II to yield a 2AB-labeled linkage region tetrasaccharide. Upon anion-exchange HPLC analysis, the 2AB-labeled compound in the chondroitinase AC-II digest was eluted in a position of the authentic unmodified linkage region tetrasaccharide (HexUA1 " border="0"> 3Galß1 " border="0"> 3Galß1 " border="0"> 4Xyl-2AB) (data not shown), confirming the structure of the parent linkage region hexasaccharide of the bird's nest chondroitin PG to be HexUA1 " border="0"> 3GalNAcß1 " border="0"> 4GlcUAß1 " border="0"> 3Galß1 " border="0"> 3Galß1 " border="0"> 4Xyl. To further establish the structure of the isolated linkage region hexasaccharide, the delayed extraction (DE) matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to determine its molecular mass. About 30 pmol of the hexasaccharide was mixed with 10 µg of the matrix, 2,5-dihydroxybenzoic acid, and analyzed by MALDI-TOF MS in a positive ion mode using a Voyager DE-RP/Pro (Perspective Biosystems, Framingham, MA) in a linear mode (Sakaguchi et al. 2001). The major molecular masses of the hexasaccharide were calculated to be 1033 and 1055, which corresponded to mono- and di-sodiated forms of the unmodified linkage region hexasaccharide, respectively (Figure 6). By NMR analysis, the proton chemical shifts of the constituents monosaccharides of the linkage region hexasaccharide isolated from bird's nest chondroitin PG were found to be identical to that of the unmodified linkage region hexasaccharide isolated from whale cartilage chondroitin sulfate PG (Figure 7). This analysis clearly established that the structure of the linkage region oligosaccharide isolated from the bird's nest chondroitin PG is HexUA1 " border="0"> 3GalNAcß1 " border="0"> 4GlcUAß1 " border="0"> 3Galß1 " border="0"> 3Galß1 " border="0"> 4Xyl.
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Presence of chondroitin PG in the black nest
4,5HexUA1 " border="0"> 3GalNAc from the collocalia glycoprotein prepared from the black nest, suggesting the presence of a GAG with the structure consistent with that of chondroitin in this nest. We found that water was not effective in extracting uronic acid-containing glycoconjugates from the black nest. To prepare PGs from the black nest, we treated the nest with 0.1 M H2SO4 at 100 °C for 1 h to release sialic acids from sialoglycoconjugates followed by using 4 M guanidium chloride (Sajdera and Hascall 1969), and DEAE-cellulose anion-exchange chromatography in the presence of 7 M urea in 0.05 M Tris buffer, pH 6.8 (Antonopoulos et al. 1974), to extract and isolate PGs from the desialylated nest. By this procedure, the amounts of uronic acid-containing glycoconjugates eluted from the DEAE-cellulose column with 0.15 M NaCl and 2 M NaCl in 0.05 M Tris buffer, pH 6.8 containing 7 M urea (Antonopoulos et al. 1974) were 142 mg and 30.7 mg, respectively (Methods). The 0.15-M NaCl eluate was found to contain 25% of carbohydrates. In addition to GlcUA and GalNH2, this preparation also contained GlcNH2, Gal, Man, Xyl, and iduronic acid, and the contents of Gal, GalNH2, and GlcNH2 were higher than that of GlcUA. In view of the heterogeneous nature of the sugar composition, this preparation must contain several species of glycoconjugates. Thus, this preparation was not investigated further. The 2-M NaCl eluate, on the other hand, contained 53% of carbohydrates. The major monosaccharides found in this preparation were GlcUA (20%) and GalNH2 (23%), and the minor monosaccharides found were Gal (4.4%), Man (2.9%), and Xyl (1.3%). By infrared spectroscopy, this preparation was also found to be devoid of sulfate. As shown in Figure 4, chondroitin AC lyase also liberated 4,5HexUA1 " border="0"> 3GalNAc from the PG prepared from the black nest, indicating that this PG also contained chondroitin chains. It should be pointed out that the chondroitin PG from white nest was eluted from the DEAE-Sephacel column with approximately 0.2 M NaCl in water (Figure 2B), whereas the black nest chondroitin PG was eluted from the DEAE-cellulose column by 2 M NaCl in 0.05 M Tris buffer, pH 6.8, containing 7 M urea as described under Methods. This could be due to the difference in the binding avidity of DEAE-Sephacel and DEAE-cellulose under different conditions. Antonopoulos, et al. (1974) used 2 M NaCl in 0.05 M Tris buffer, pH 6.5–6.8 containing 7 M urea to elute PGs from the DEAE-cellulose column. Through the observation of the enzymatic release of 4,5HexUA1 " border="0"> 3GalNAc from the Collocalia glycoprotein, we have uncovered the presence of chondroitin PG in the dried regurgitated saliva of male swiftlets widely known as the edible bird's-nest. We have examined the Collocalia glycoproteins prepared from several commercial bird's nest preparations and found that all of them were susceptible to chondroitin AC lyase to produce 4,5HexUA1 " border="0"> 3GalNAc. The fact that we were able to isolate 30 mg of a highly purified chondroitin PG preparation from 15 g of the white nest indicates that this PG is not a minor constituent of bird's saliva. The water extract prepared from 1 g of white nest contained approximately 35 µmol of uronic acid and 11 µmol of sialic acid. Thus, the uronic acid content in the water extract of the white nest is three times higher than that of sialic acid, indicating that the bird's nest contains more chondroitin PG than sialoglycoconjugates. Most swiftlet nests are collected in caves on cliffs along the seacoast of South East Asia (Valli and Summers 1990). After swiftlets finish making their nests, it may take over a year for them to become commercially available. Thus, it is possible that the chemical compositions of the commercially available birds' nests have undergone partial degradation. There is no doubt that the GAG chains and the core protein of chondroitin PG in these nests have also been partially degraded. It would be interesting to isolate the intact chondroitin PG from the salivary gland of live swiftlets.
Chondroitin was first isolated from bovine cornea by Davidson and Meyer in 1954 (Davidson and Meyer 1954). Subsequently, this GAG was also found in squid skin in 1964 (Anno et al. 1964) and in the capsular K4 antigen of E. coli O5:K4:H4 in 1988 (Rodriguez et al. 1988). Since then, chondroitin has not been found in other biological materials until the revelation of its presence in C. elegans in 1999 (Yamada et al. 1999). Chondroitin in C. elegans has been shown to be critically involved in cytokinesis of early embryogenesis (Mizuguchi et al. 2003). Although PGs have been isolated from various tissues, they have never been isolated from the saliva of a higher animal. By analyzing the unsaturated disaccharides released by chondroitin AC lyase, rat and human saliva (Iversen et al. 1987; Okazaki et al. 1996) have been indirectly shown to contain chondroitin sulfate. Hyaluronic acid and chondroitin differs only in the hexosamine moiety of the disaccharide repeating units. Hyaluronic acid is usually not linked to a PG molecule. Our results indicate that the chondroitin chain and the core protein in the bird's nest chondroitin PG are also linked through the common tetrasaccharide linker, GlcUAß1 " border="0"> 3Galß1 " border="0"> 3Galß1 " border="0"> 4Xyl, found in various PGs (Rodén and Smith 1966; Hassell et al. 1986; Kjellén and Lindahl 1991; Hardingham and Fosang 1992; Iozzo 1998; Sugahara 1998; Prydz and Dalen 2000; Ueno et al. 2001; Kolset et al. 2004). It is intriguing that the saliva of swiftlets is so rich in chondroitin-containing GAG. Although edible bird's-nest soup has been a culinary delicacy in China since ancient times, the biological importance for the presence of such a high level of chondroitin PG in swiftlet saliva remains to be elucidated. As the biological significance of chondroitin is still not well understood, birds' nests should become a convenient source for preparing this unique PG to study its biological functions.
| Materials and methods |
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Materials
. Crude leech sialidase was prepared according to Li et al. (1990). The following were purchased from commercial sources indicated: 4,5HexUA1 " border="0"> 3GalNAc, DEAE cellulose (fast flow), chondroitin AC lyase, chondroitinase ABC (EC4.2.2.4, from Proteus vulgaris), neuraminidase from Clostridium perfringens (Pronase E, Sigma, St. Louis, MO); precoated silica gel-60 TLC plate (Merck, Darmstadt, Germany). Wakogel C-300 (silica gel; Wako Pure Chemical Industries, Ltd., Osaka, Japan); Sepharose CL-6B, DEAE-Sephacel, Sephadex G-75, Sephadex G-25 (superfine), Superdex-200 (Amersham Pharmacia Biotech Inc., Piscataway, NJ); amine-bound silica PA-03 column (YMC Co., Kyoto, Japan). Methods
Isolation of a UC released from Collocalia glycoprotein
) was dissolved in 5.7 mL of 50 mM sodium acetate buffer, pH 5.5, and incubated with 0.3 mL of crude leech sialidase (Li et al. 1990) at 37 °C for 17 h. The reaction mixture was applied onto a Sephadex G-25 column (2.5 x 100 cm) equilibrated with water. The column was eluted with water at 25 mL/h and 5-mL fractions were collected. A 10-µL aliquot of each fraction was analyzed by TLC to detect mono- and oligo-saccharides, as described under the section Analytical methods. This step separated UC together with Neu5Ac and 2,7-anhydro-Neu5Ac from the Collocalia glycoprotein and other proteins. The fractions containing UC, Neu5Ac, and 2,7-anhydro-Neu5Ac were pooled, lyophilized, redissolved in 0.5 mL of n-butanol/acetic acid/water (2:1:1, v/v/v), and applied onto a Wakogel C-300 silica gel column (1.3 x 20 cm) equilibrated with the same solvent. The column was eluted with the same solvent at 10 mL/h, and 1.1-mL fractions were collected. A 10-µL aliquot of each fraction was analyzed by TLC to locate the positions of UC, Neu5Ac, and 2,7-anhydro-Neu5Ac, as described. Under this condition, UC was eluted first and well separated from Neu5Ac and 2,7-anhydro Neu5Ac. Those fractions containing UC were pooled and evaporated to dryness to obtain approximately 1.5 mg of UC. Isolation of a chondroitin PG from white nest
Determination of the molecular mass of the chondroitin GAG chain in chondroitin PG
). The molecular mass of the 2AB derivative of the free chondroitin chain was analyzed by gel filtration on a Superdex-200 column (0.5 x 30 cm) with 0.2 M ammonium hydrogen carbonate as the eluent at a flow rate of 0.3 mL/min. Preparation of the linkage region hexasaccharide from white bird's nest chondroitin PG
4,5-unsaturated hexasaccharide–protein core, 110 mg of white nest chondroitin PG was digested under an aseptic condition with 10 units (additional 4 units were added after 3 days) of chondroitinase ABC for 7 days at 37 °C in 3 mL of 0.05 M Tris–HCl buffer, pH 8.0, containing 0.3 mg of bovine serum albumin. After heating the digest in a boiling water bath for 3 min, the digest was applied onto a Sephadex G-25 column (1.8 x 100 cm) equilibrated with 0.15 M ammonium hydrogen carbonate. The column was eluted with the same solution at 6.5 mL/h and 2.8 mL-fractions were collected. Fractions were monitored by UV absorption at 232 nm and 280 nm for the 4,5-unsaturated hexasaccharide–protein core and 10-µL aliquot of each fraction was also examined by TLC for the detection of the released 4,5HexUA1 " border="0"> 3GalNAc. The 4,5-unsaturated hexasaccharide–protein core was eluted in fractions 36–48, whereas 4,5HexUA1 " border="0"> 3GalNAc in fractions 54–60. Fractions 36–48 were pooled and lyophilized to obtain 6.9 mg of hexasaccharide–protein core preparation. To release the putative linkage region hexasaccharide, this preparation was subsequently treated with 1 mL of 0.5 M LiOH at 4 °C for 15 h (Sakaguchi et al. 2001). After neutralization with acetic acid, the reaction mixture was again subjected to Sephadex G-25 gel filtration using the conditions described. Fractions 54–59 were found to contain an oligosaccharide by TLC. These fractions were pooled and lyophilized to obtain 2.6 mg of the putative linkage region hexasaccharide. Isolation of chondroitin PG from black nest
). The mixture was centrifuged at 15,000g for 30 min and the pellet was re-extracted as described. The two extracts were combined, concentrated to 60 mL with an Amicon ultrafiltration cell using a PM 10 membrane, exhaustively dialyzed against 7 M urea in 0.05 M Tris–HCl buffer, pH 6.8, and applied onto a DEAE-cellulose column (Cl– form, 2.5 x 4.2 cm), which had been equilibrated with 7 M urea in the same buffer. After washing with the same buffer containing 7 M urea to remove the unabsorbed materials, the column was eluted at 35 mL/h with 0.15 M NaCl in the same buffer containing 7 M urea followed by 2 M NaCl in the same buffer containing 7 M urea (Antonopoulos et al. 1974). Fractions of 7 mL were collected. The fractions eluted by 0.15 M NaCl and 2 M NaCl were pooled, dialyzed exhaustively against water, and lyophilized. The yields (dried weights) of 0.15-M NaCl and 2-M NaCl eluates were 142 mg and 30.7 mg, respectively. The 0.15-M NaCl eluate was found to contain 25% of carbohydrates. In addition to GlcUA and GalNH2, this preparation also contained GlcNH2, Gal, Man, and Xyl, and also iduronic acid (IdoUA), and the contents of Gal, GalNH2, and GlcNH2 were higher than that of GlcUA. In view of the heterogeneous nature of the sugar composition, this preparation must contain several species of glycoconjugates. Thus, this preparation was not investigated further. The 2-M NaCl eluate, on the other hand, contained 53% of carbohydrates with GalNAc and GlaUA in equimolar ratio. . Uronic acids were determined by the carbazole reaction as modified by Galambos (1967). Quantitative analysis of iduronic acid and glucuronic acid by GLC was performed according to the procedures of Inoue and Miyawaki (1975). Amino sugars and neutral sugars were also determined as their trimethylsilyl derivatives by GLC after methanolysis and reacetylation, as described by Kimura et al. (1994). Sialic acids were released from glycoconjugates by heating the samples with 0.1 M H2SO4 at 80 °C for 1 h and free sialic acids were determined by the modified periodate-thiobarbituric acid method described by Uchida et al. (1977). Monosaccharides and oligosaccharides were also analyzed by TLC using silica gel-60 plates and n-butanol/acetic acid/water (2:1:1, v/v/v) as the developing solvent. The plate was sprayed with the diphenylamine–aniline phosphoric acid reagent and heated at 110 °C for 15–20 min (Anderson et al. 2000). Amino acid composition of protein samples were determined by using a JOEL automatic amino acid analyzer after hydrolyzing the protein samples with 6 M HCl at 110 °C for 24, 48, and 72 h under vacuum. For NMR spectroscopy, the sample (1.5 mg) was repeatedly exchanged with D2O with intermittent lyophilization and dissolved in 0.5 mL of D2O. The 1H NMR spectra of the UC isolated from Collocalia glycoprotein were recorded at 25 °C on a GE Omega PSG 500 NMR spectrometer. The 500-MHz 1H NMR spectra of the hexasaccharide prepared from the bird's nest chondroitin PG was recorded in a Varian VXR-500 spectrometer at a probe temperature of 26 °C. Chemical shifts were measured relative to acetone (
2.225) in D2O. Infrared absorption spectra were recorded using a JOEL FT-IR on potassium bromide disc containing 0.2% of the sample. | Supplementary data |
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The supplementary figure shows the 500-MHz 1H NMR spectra of the authentic sodium salt of 4,5HexUA1 " border="0"> 3GalNAc and the unknown compound. Supplementary data are available at Glycobiology online (http://glycob.oxfordjournals.org).
| Acknowledgments |
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This work was supported by National Institutes of Health Grant NS 09626 (to Y.-T.L.) and by a Grant-in-Aid for Scientific Research-B 16390026, the Human Frontier Science Program, and the Core Research for Evolutional Science and Technology (CREST) Program of the Japan Science and Technology (JST) Agency (to K.S.).
| Footnotes |
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2 Present address: Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand
| Abbreviations |
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2AB, 2-aminobenzamide; DE, delayed extraction; GAG, glycosaminoglycan; GlcUA, D-glucuronic acid; GLC, gas liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; HPLC, high performance liquid chromatography; PG, proteoglycan; TLC, thin-layer chromatography; UC, unknown compound.
| References |
|---|
Anderson K, Li S-C, Li Y-T. (2000) Diphenylamine–aniline–phosphoric acid reagent, a versatile spray reagent for revealing glycoconjugates on thin-layer chromatography plates. Anal Biochem287:337–339.
Anno K, Kawai Y, Seno N. (1964) Isolation of chondroitin from squid skin. Biochim Biophys Acta83:348–349.
Antonopoulos CA, Axelsson I, Heinegård D, Gardell S. (1974) Extraction and purification of proteoglycans from various types of connective tissue. Biochim Biophys Acta 338:108–119.
Davidson EA and Meyer K. (1954) Chondroitin, a new mucopolysaccharide. J Biol Chem211:605–611.
Galambos JT. (1967) The reaction of carbazole with carbohydrates. I. Effect of borate and sulfamate on the carbazole color of sugars. Anal Biochem 19:119–132.
Gatt S and Berman ER. (1966) A rapid procedure for the estimation of amino sugars on a micro scale. Anal Biochem 15:167–171.
Hardingham TE and Fosang AJ. (1992) Proteoglycans: many forms and many functions. FASEB J 6:861–870.
Hassell JR, Kimura JH, Hascall VC. (1986) Proteoglycan core protein families. Annu Rev Biochem 55:539–567.
Howe C, Lee LT, Rose HM. (1961) Collocalia mucoid: a substrate for myxovirus neuraminidase. Arch Biochem Biophys 95:512–520.
Humphries DE, Suguramran G, Silbert JE. (1989) Decreasing sulfation of proteoglycans produced by cultured cells. Methods Enzymol 179:428–434.
Inoue S and Miyawaki M. (1975) Quantitative analysis of iduronic acid and glucuronic acid in sulfated galactosaminoglycuronans by gas chromatography. Anal Biochem 65:164–174.
Iozzo RV. (1998) Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem 67:609–652.
Iversen JM, Keller PJ, Kauffman DL, Robinovitch MR. (1987) The presence of chondroitin sulfate in parotid secretory granules and saliva of the rat. Cell Tissue Res 250:221–226.
Kimura M, Hama Y, Sumi T, Asakawa M, Rao BNN, Horne AP, Li S-C, Li Y-T, Nakagawa H. (1994) Characterization of a deaminated neuraminic acid-containing glycoprotein from the skin mucus of the loach, Misgurnus anguillicaudatus. J Biol Chem 269:32138–32143.
Kinoshita A and Sugahara K. (1999) Microanalysis of glycosaminoglycan-derived oligosaccharides labeled with a fluorophore 2-aminobenzamide by high-performance liquid chromatography: application to disaccharide composition analysis and exosequencing of oligosaccharides. Anal Biochem 269:367–378.
Kjellén L and Lindahl U. (1991) Proteoglycans: structures and interactions. Annu Rev Biochem60:443–475.
Kolset SO, Prydz K, Pejler G. (2004) Intracellular proteoglycans. Biochem J 379:217–227.
Li Y-T, Nakagawa H, Ross SA, Hansson GC, Li S-C. (1990) A novel sialidase which releases 2,7-anhydro-alpha-N-acetylneuraminic acid from sialoglycoconjugates. J Biol Chem265:21629–21633.
Linhardt RJ, Galliher PM, Cooney CL. (1986) Polysaccharide lyases. Appl Biochem Biotech12:135–176.
Lloyd AG, Dodgson KS, Price RG, Rose FA. (1961) Infrared studies on sulphate esters. I. Polysaccharide sulphates. Biochim Biophys Acta 46:108–115.
Mathews MB. (1958) Isomeric chondroitin sulphates. Nature 181:421–422.
Mizuguchi S, Uyama T, Kitagawa H, Nomura KH, Dejima K, Gengyo-Ando K, Mitani S, Sugahara K, Nomura K. (2003) Chondroitin proteoglycans are involved in cell division of Caenorhabditis elegans. Nature 423:443–448.
Okazaki J, Kamada A, Gonda Y, Sakaki T, Embery G. (1996) High-performance liquid chromatography analysis of chondroitin sulphate isomers in human whole saliva in a variety of clinical conditions. Oral Dis 2:224–227.
Prydz K and Dalen KT. (2000) Synthesis and sorting of proteoglycans. J Cell Sci 113:193–205.
Rodén L and Smith R. (1966) Structure of the neutral trisaccharide of the chondroitin 4-sulfate-protein linkage region. J Biol Chem 241:5949–5954.
Rodriguez M-L, Jann B, Jann K. (1988) Structure and serological characteristics of the capsular K4 antigen of Escherichia coli O5:K4:H4, a fructose-containing polysaccharide with a chondroitin backbone. Eur J Biochem 177:117–124.
Sajdera SW and Hascall VC. (1969) Proteinpolysaccharide complex from bovine nasal cartilage. J Biol Chem 244:77–87.
Sakaguchi H, Watanabe M, Ueoka C, Sugiyama E, Taketomi T, Yamada S, Sugahara K. (2001) Isolation of reducing oligosaccharide chains from the chondroitin/dermatan sulfate-protein linkage region and preparation of analytical probes by fluorescent labeling with 2-aminobenzamide. J Biochem (Tokyo) 129:107–118.
Sugahara K. (1998) Structural diversity of the glycosaminoglycan–protein linkage region. Available from: http://www.glycoforum.gr.jp/science/word/proteoglycan/PGA06E.html.
Sugahara K, Yamashina I, de Waard P, van Halbeek H, Vliegenthart JFG. (1988) Structural studies on sulfated glycopeptides from the carbohydrate–protein linkage region of chondroitin 4-sulfate proteoglycans of Swarm rat chondrosarcoma. Demonstration of the structure, Gal(4-O-Sulfate)ß1-3Galß1-4Xylß1-O-Ser. J Biol Chem 263:10168–10174.
Svennerholm L. (1957) Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method. Biochim Biophys Acta 24:604–611.
Terho TT and Hartiala K. (1971) Method for determination of the sulfate content of glycosaminoglycans. Anal Biochem 41:471–476.
Uchida Y, Tsukuda Y, Sugimori T. (1977) Distribution of neuraminidase in Arthrobacter and its purification by affinity chromatography. J Biochem (Tokyo) 82:1425–1433.
Ueno M, Yamada S, Zako M, Bernfield M, Sugahara K. (2001) Structural characterization of heparan sulfate and chondroitin sulfate of syndecan-1 purified from normal murine mammary gland epithelial cells. Common phosphorylation of xylose and differential sulfation of galactose in the protein linkage region tetrasaccharide sequence. J Biol Chem 276:29134–29140.
Valli E and Summers D. (1990) Shadow hunters. The nest gatherers of tiger cave. (Eastman Kodak and Thomasson-Grant, Charlottesville (VA)).
Winzler RJ. (1955) Determination of serum glycoproteins. Methods Biochem Anal 2:279–311.
Yamagata T, Saito H, Habuchi O, Suzuki S. (1968) Purification and properties of bacterial chondroitinases and chondrosulfatases. J Biol Chem 243:1523–1535.
Yamada S, van Die I, van den Eijnden DH, Yokota A, Kitagawa H, Sugahara K. (1999) Demonstration of glycosaminoglycans in Caenorhabditis elegans. FEBS Lett 459:327–331.
Yamada S, Yoshida K, Sugiura M, Sugahara K. (1992) One- and two-dimensional H-NMR characterization of two series of sulfated disaccharides prepared from chondroitin sulfate and heparan sulfate/heparin by bacterial eliminase digestion. J Biochem (Tokyo) 112:440–447.
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