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)
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{at}tulane.edu
Received on June 19, 2006; revised on September 27, 2006; accepted on September 29, 2006
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Despite their wide occurrence, proteoglycans (PGs) have never been isolated from the saliva of higher animals. We found that the Collocalia glycoproteins isolated from edible birds'-nests (the dried forms of regurgitated saliva of male Collocalia swiftlets) were rich in a PG containing nonsulfated chondroitin glycosaminoglycans (GAGs). We have devised a method to isolate a PG from the water extract of the white nest built by Aerodramus fuciphagus (white nest swiftlets) with a yield of 2-mg PG per gram nest. This PG contained 83% of carbohydrates, of which 79% were GalNAc and GlcUA (D-glucuronic acid) in an equimolar ratio. By using chondroitin AC lyase, the structure of GAGs in this PG was established to be chondroitin (
4GlcUAß1 3GalNAcß1 )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 3GalNAcß1 4GlcUAß1 3Galß1 3Galß1 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 3Galß1 3Galß1 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
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Introduction |
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary dataAcknowledgmentsReferences |
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Proteoglycans (PGs) are ubiquitous constituents of intracellular, pericellular, and extracellular matrices of higher animals. Each PG contains a core protein and covalently linked glycosaminoglycan (GAG) chains. The distribution, structure, and function of PGs have been the subject of intensive studies (Hassell et al. 1986
; 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 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.
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Characterization of an unknown compound released from white nest Collocalia glycoprotein by crude leech sialidase
We found that in addition to Neu5Ac and 2,7-anhydro-Neu5Ac (Li et al. 1990
), 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 3GalNAc (Yamada et al. 1992).
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Isolation and characterization of a chondroitin PG from the white nest
The liberation of
4,5HexUA1 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 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 ( 4GlcUAß1 3GalNAcß1 )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
It has been shown that exhaustive digestion of chondroitin and chondroitin sulfates with chondroitinase ABC (Yamagata et al. 1968
) 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 3Galß1 3Galß1 4Xyl-2AB) (data not shown), confirming the structure of the parent linkage region hexasaccharide of the bird's nest chondroitin PG to be HexUA1 3GalNAcß1 4GlcUAß1 3Galß1 3Galß1 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 3GalNAcß1 4GlcUAß1 3Galß1 3Galß1 4Xyl.
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Presence of chondroitin PG in the black nest
By TLC, the crude leech sialidase also liberated
4,5HexUA1 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 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 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 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 3Galß1 3Galß1 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.
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Materials and methods |
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Materials
The white nest was obtained from a Chinese food market in Yokohama, Japan, and the black nest was purchased from oriental food stores in Boston and San Francisco. Collocalia glycoproteins were prepared from both the white- and the black nests as described by Howe et al. (1961)
. Crude leech sialidase was prepared according to Li et al. (1990). The following were purchased from commercial sources indicated: 4,5HexUA1 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
To isolate sufficient quantities of UC for structural characterization, 250 mg of Collocalia glycoprotein prepared from the white nest (Howe et al. 1961) 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
For the extraction of chondroitin PGs from the white nest, 15 g of the pulverized nest was soaked in 450 mL of water overnight, briefly homogenized with a Polytron homogenizer and centrifuged at 15 000 g for 30 min. The pellet was re-extracted with 450 mL of water and the two extracts were combined and lyophilized to obtain 1.4 g of white powder. This white powder was dissolved in 40 mL of water and 10 mL aliquot of this solution was applied onto a Sepharose CL-6B column (4.0 x 71 cm) equilibrated with water. The column was eluted with water at 35 mL/h and 7.9 mL-fractions were collected. As shown in Figure 2A, the fractions (fractions 39–56) eluted before the main protein peak were found to contain both uronic acid and sialic acid. They were pooled and lyophilized to obtain 240 mg of dried powder. This powder was dissolved in 5 mL of water and applied onto a DEAE-Sephacel column (Cl– -form, 2.8 x 36 cm) equilibrated with water. After washing with water, the column was eluted with a linear NaCl gradient from 0 to 0.3 M (Figure 2B). The uronic acid-containing fractions (fractions 116–126) (Figure 2B) were pooled, dialyzed against water to remove NaCl, and lyophilized to obtain 75 mg of dried powder. This powder was dissolved in 2 mL of water and applied onto a Sephadex G-75 column (1.8 x 74 cm) equilibrated with water. The column was eluted with water at 25 mL/h and 2.8-mL fractions were collected. As shown in Figure 2C, the uronic acid-containing materials were resolved by Sephadex G-75 filtration into one broad and one sharp uronic acid-containing peaks. The sharp peaks (fractions 58–68) were pooled and lyophilized to obtain 7.6 mg of a chondroitin PG preparation.
Determination of the molecular mass of the chondroitin GAG chain in chondroitin PG
To estimate the molecular mass of the chondroitin GAG chain, the chondroitin-PG was treated with 0.5 M LiOH overnight at 4 °C to release the chondroitin chain, and was then labeled 2AB. The excess 2AB reagent was removed by paper chromatography (Kinoshita and Sugahara 1999). 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
To prepare the 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 3GalNAc. The 4,5-unsaturated hexasaccharide–protein core was eluted in fractions 36–48, whereas 4,5HexUA1 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 pulverized black nest (10 g) was soaked in 200 mL of 0.1 M H2SO4 at 4 °C overnight, homogenized with a Polytron homogenizer, and heated at 100 °C for 1 h to remove sialic acids from glycoconjugates. After cooling to room temperature, the mixture was adjusted to pH 7.0 by dropwise addition of 2 M NaOH while stirring. The mixture was exhaustively dialyzed against water and lyophilized to yield 9.5 g of dried asialo bird's nest powder. This powder was then extracted overnight with 280 mL of 4 M guanidium chloride at 4 °C with gentle shaking (Sajdera and Hascall 1969). 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.
Analytical methods
Amino sugars were determined by the Elson and Morgan reaction as modified by Gatt and Berman (1966). 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.
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Supplementary data |
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The supplementary figure shows the 500-MHz 1H NMR spectra of the authentic sodium salt of
4,5HexUA1 3GalNAc and the unknown compound. Supplementary data are available at Glycobiology online (http://glycob.oxfordjournals.org).
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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.).
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2 Present address: Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand
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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.
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TopAbstractIntroductionResults and discussionMaterials and methodsSupplementary dataAcknowledgmentsReferences |
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