2Shriners Hospital for Children, 12502 N. Pine Drive, Tampa, FL, 33612, USA; 3Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, FL, USA; and 4The Lerner Research Institute of the Cleveland Clinic Foundation, Euclid Avenue, Cleveland, OH 44195, USA.
Received on February 28, 2001; revised on May 10, 2001; accepted on June 8, 2001.
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Abstract |
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Key words: glycosaminoglycans/keratan sulfate/keratanases/proteoglycans/sulfation
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Introduction |
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A more widespread occurrence of KS-substituted molecules became apparent in the 1980s after the successful production and use of monoclonal antibodies to KS-PGs (Funderburgh et al., 1982, 1987; Caterson et al., 1983
; SundarRaj et al., 1985
; Zanetti et al., 1985
; Keiser and Diamond, 1987
). Furthermore, application of these reagents in immunoassays and immunohistochemical localizations led to the observations that KS substitution of PGs may play a role in the formation and maintenance of structural collagen networks (Quantock et al., 1997
; Scott and Thomlinson, 1998
; Hedlund et al., 1999
; Svensson et al., 2000
) and cell-matrix adhesion processes (Burg and Cole, 1994
; Wendel et al., 1998
; Ota et al., 2000
).
Despite the high sensitivity and relative specificity of such immunoreagents they are of limited use in the quantitation and fine structure analyses of KS chains, due to a common requirement for disulfated KS chain regions (Mehmet et al., 1986), high epitope densities (Seibel et al., 1992
), and possible epitope masking by the fucose and sialic acid substitutions (Thornton et al., 1989b
). Thus an examination of specific aspects of the fine structure and function of KS required the development of analytical procedures for quantitation and complete disaccharide compositional analyses of this glycosaminoglycan.
A range of analytical biochemical methods for KS have been reported; they generally involve depolymerization, either by hydrazinolysis (Shaklee and Conrad, 1986, Brown et al., 1992
) or enzymatic cleavage with specific hydrolases, such as endo-ß-galactosidases from E. freundii (Fukuda, 1981
; Kitamikado, 1984; Kitamikado et al., 1981
; Li et al., 1982
; Scudder et al., 1984
), or from Pseudomonas sp. (designated keratanase) (Fukuda, 1981
; Kitamikado et al., 1981
; Li et al., 1982
) and the endo-ß-N-acetylglucosaminidase from Bacillus sp. (designated keratanase II) (Nakazawa et al., 1989
), followed by separation and quantitation of digestion products by reverse-phase (Yamada et al., 2000) or anion-exchange high-performance liquid chromatography (HPLC) (Brown et al., 1995
; Whitham et al., 1999
). Using these approaches, "compositional fingerprint maps" of KS from tissues, cell cultures, and synovial fluid, can be obtained reproducibly; however, the chromatographic analyses have not been optimized for KS quantitation or disaccharide compositional analyses, and can not evaluate products liberated from unsulfated regions of KS chains. Moreover, despite the wide use of the KS hydrolases for the generation of a variety of structurally distinct oligosaccharide sequences from the chain interior, these enzymes have not been fully described with respect to their capacity for quantitative depolymerization of KS chains of variable size, sulfation and fucosylation.
To address some of these inadequacies in KS analyses, we describe a novel application of fluorophore-assisted carbohydrate electrophoresis (FACE) technology to provide a simple, highly sensitive, and quantitative tool for determination of the range of unsulfated, sulfated, fucosylated, and sialylated KS hydrolase products. We discuss fine structure data obtained by FACE analyses for KS chains substituted on aggrecan from human cartilages of different ages in relation to those previously obtained using HPLC and 1H-nuclear magnetic resonance (NMR) methodological approaches. We also suggest a broader applicability of this procedure for detection and characterization of KS in studies designed to determine whether different GAG fine structures may confer unique biological properties on specific extracellular matrices.
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Results |
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The two prominent products (bands 3 and 8) generated from both KS preparations are the disaccharides galß1,4glcNAc6S and gal6Sß1,4glcNAc6S, respectively. They eluted in the same positions as chondroitin sulfate (CS)-derived disaccharides after fractionation of total digests on Superdex Peptide chromatography (Figure 2, lane c) and were previously reported as the major end products after KII digestion of shark cartilage KS (Nakazawa et al., 1989), bovine (Whitham et al., 1999
) or human corneal KS (Tai et al., 1997
), and bovine (Brown et al., 1994
) or human cartilage KS (Brown et al., 1998
). The human cartilage KS digest contained a product (band 2), not detected in bovine corneal KS that migrated slower than the monosulfated disaccharide (band 3), and in- between the fuc and gal monosaccharide standards (Figure 1A,B). In keeping with previous reports where fucose substitution of glcNAc6S in skeletal KS was detected by 1H-NMR (Tai et al., 1991
) this products was identified as the trisaccharide galß1,4[fuc
1,3]glcNAc6S. It was eliminated when the total KII digests were pretreated with
-fucosidase and this digestion generated an equal amount of fucose and a product that comigrated with the monosulfated disaccharide band 3 (Figure 3A,B).
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Products generated by EB from corneal and skeletal KS were analyzed by FACE (Figure 6A, lanes a and c, respectively). The major product that migrated between the glcNAc and gal6S monosaccharide standards (band 4) was identified as the monosulfated disaccharide glcNAc6Sß1,3gal, as it was recovered in virtually identical yields to the equivalent KII product galß1,4glcNAc6S, band 3 (Table I), it eluted from Superdex Peptide in the disaccharide position (data not shown), and it was also obtained when corneal and skeletal KS were digested with Pseudomonas sp. keratanase (Figure 6A, lanes b and d, respectively), an endo-galactosidase with a strict requirement for a sulfated glcNAc6S adjacent to susceptible galß1,4 linkages (Nakazawa et al., 1989). EB digests of corneal KS included an additional species (band 1), which migrated very slowly on the monosaccharide gel (Figure 6A, lane a). This species was identified as the unsulfated disaccharide glcNAcß1,3gal since it comigrated with such a product prepared by ß-galactosidase digestion of the trisaccharide galß1,4glcNAcß1,3gal (Figure 6B, lane b and c).
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The recovery of all identified hydrolase products and their relative abundance was calculated from the gel images shown in Figures 1 and 6 (see Materials and methods for detail) and data are summarized in Table I. Following a 4-h incubation with 1 mU KII per nmole glcNH2, the proportion of substrate glcNH2 recovered in digestion products was 91% and
79% for corneal or skeletal KS, respectively. Mono- and disulfated disaccharides (bands 3 and 8) constituted
91% of the products, in corneal KS digests, and the remainder were di-and trisulfated tetrasaccharide intermediates. In digests of skeletal KS, the mono- and disulfated disaccharides (bands 3 and 8) and the fucosylated trisaccharide (band 2) were the major products (
80%), nonreducing terminal neuA capped trisaccharides accounted for
10%, and the rest (
10%) was recovered as di- and trisulfated tetrasaccharide intermediates.
Limit digestion of skeletal KS with EB and KII
Skeletal KS was incubated with increasing amounts (0.1, 0.25, and 0.5 mU per nmole glcNH2) of either EB or KII for 4 or 24 h, to optimize quantitate recoveries for all identified hydrolase digestion products. At all EB concentrations and both incubation times, about one-third of the substrate glcNH2 (3 nmol) was recovered as the monosulfated disaccharide product glcNAc6Sß1,4gal and ODS were also produced with the same efficiency under all digestion conditions. There was, however, no evidence for the presence of unsulfated disaccharide products, even with high amounts of enzyme or prolonged incubation times, consistent with previous analyses of human aggrecan KS digests using NMR spectroscopy where unsulfated glcNAc residues were also not detected in the chain interior parts (Brown et al., 1998
).
Digestion of skeletal KS with increasing amounts of KII (Figure 7) showed that mono- and disulfated disaccharides, the fucosylated trisaccharide derived from the chain interior, and the two sialylated trisaccharides from the nonreducing termini were readily detectable at all enzyme concentrations after 4 or 24 h incubation. The tetrasaccharides (bands 5 and 7) and other unidentified larger oligosaccharides (designated by *, see also Figure 1B) were detected only in 4-h incubations using lower concentrations (0.1 and 0.25 mU) of enzyme as short-lived intermediates. Calculation of the molar yields of each KII end product (Figure 8A) showed that with 2.55.0 mU of enzyme after 4 or 24 h maximum yields of the fucosylated-monosulfated trisaccharide (band 2), the disulfated disaccharide (band 8) and the neuA-capped terminal trisaccharides (bands 6 and 9) were obtained. In contrast, the recovery of the monosulfated disaccharide (band 3) reached a maximum of 2 nmole after a 4 h, at both enzyme concentrations and then decreased after 22 h, especially when higher amounts of enzyme was used (Figures 7 and 8B). Concurrent with this decreased yield of monosulfated disaccharide, two new products, gal and glcNAc6S (Figure 7) were generated. This suggested the presence of a contaminate exo-galactosidase activity in the KII preparation that cleaves the monosulfated disaccharide galß1,3glcNAc6S. This was also supported by the finding that the monosaccharide products were always recovered in essentially equimolar amounts and their yields corresponded to proportional deficit in recoverable monosulfated disaccharide products.
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Aggrecan KS chain compositional analyses after KII and EB digestions
Portions of purified aggrecan preparations were sequentially digested with KII and EB (2.5 mU for 18 and 4 h, respectively) and products analyzed by FACE (Figure 9). The recovery of substrate glcNH2 as digestion products (including the monosaccharides, gal, and glcNAc6S) was approximately 77, 72, 75, and 92% for aggrecan from the 5-, 13-, 15-, and 68-year-old tissues, respectively, the higher recovery in the older donor consistent with a more extensive KS substitution of aggrecan core bound O-linked oligosaccharides in mature human articular cartilage (Santer et al., 1982).
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Assuming that neuA capping of aggrecan KS chains is almost complete (Dickenson et al., 1991; Thornton et al., 1989a
, 1989b), the molar ratio of neuA to chain internal glNH2 was used to calculate the number-averaged chain length and molecular weights of each KS population (Table III). These computed values showed a gradual but substantial increase in the average length of KS chains with increasing age of cartilage tissue. The chains on 5-year-old aggrecan were composed of about 7 disaccharides; those on the 13- and 15-year-old aggrecan of 810 disaccharides, with the longest chains composed of
14 disaccharides on the 68-year-old sample. Number-averaged molecular weights for these KS chains, computed from the number and composition of internal disaccharides, neuA cap, and the contribution of the oligosaccharide linkage region, ranged from about 4.7 kDa in the 5-year sample to about 9 kDa in the 68-year-old sample. These size ranges are in very close agreement previously reported size ranges of aggrecan KS chains estimated by gel filtration chromatography (Theocharis, 1985; Brown et al., 1998
).
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Discussion |
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The KS hydrolase products identified here (Table I) corresponded to those detected by previous investigations employing size exclusion or ion exchange HPLC separation procedures and/or 1H-NMR spectroscopy. Each disaccharide product from the KS chain interior had a distinct electrophoretic mobility on the monosaccharide gels, and these were in the following ascending order: the EB-generated unsulfated disaccharide (glcNAcß1,3gal), the KII-derived monosulfated disaccharide (galß1,4glcNAc6S), the EB-derived monosulfated disaccharide (glcNAc6Sß1,3gal), and the KII-derived disulfated disaccharide (gal6Sß1,4glcNAc6S). The KII-derived fucosylated trisaccharide (galß1,34[fuc1,3]-glcNAc6S) migrated slower than its unmodified monosulfated disaccharide (Figure 3), confirming fluorescent product separation is determined by both the charge and size characteristics of each saccharide.
Similarly, the sialic-capped trisaccharides (neuA-2,3galß1,4glcNAc6S, neuA
2,3gal6Sß1,4glcNAc6S) and tetrasaccharides (neuA
2,3galß1,4glcNAc6Sß1,3gal, neuA
2,3-gal6Sß1,4glcNAc6Sß1,3gal) generated from the nonreducing terminus by KII and EB, respectively, had distinct mobilities (Figures 5 and 9) and were thus readily identified and quantitated. Other nonreducing terminal oligosaccharide products such as
2,6neuA,
2,3gal- or ß1,3galNAc-capped oligosaccharides, had been detected previously as minor components in KII digests of human skeletal or bovine corneal KS (Dickenson et al., 1991
; Tai et al., 1992, 1996). These were not identifiable here, even when samples enriched in tri- and tetrasaccharides were analyzed separately (see Figures 4 and 5) and may be explainable if such termination sequences are on subpopulations of KS chains that were enriched as a result of additional steps for KS purification, that were used in other studies (Brown et al., 1996; Huckerby et al., 1998
; Tai et al., 1992, 1996).
When KS was digested with low amounts of KII (e.g., < 0.1 mU of enzyme per/nmole substrate) and for a short time (4 h), several larger oligosaccharide species derived from the chain interior were detected after FACE analyses (Figures 1 and 7B), but their absence following longer incubation times and with higher amounts of enzyme suggests that they are intermediate cleavage products that accumulate only when the endolytic/exolytic depolymerization of the chain interior does not proceeded to completion. Complete digestion of all sulfated and unsulfated regions of KS with KII and EB to identifiable end products (Table I) was therefore considered a prerequisite for quantitative compositional disaccharide analyses of this glycosaminoglycan and can be achieved by increasing enzyme concentrations to 0.250.5 mU per nmol glcNH2. It is of importance to note, however, that under such digestion conditions, the disaccharide galß1,4glcNAc6S is cleaved to its constituent monosaccharide products, gal and glcNAc6S, by a contaminating exoglycosidase in the KII preparations (Figures 8 and 9). Such a finding may explain the discrepancy between our data obtained for the disaccharide compositional analyses (Table III) and previously published reports on changes in gal sulfation of KS on aggrecan from human cartilages of different ages. The FACE analyses performed here included the monosaccharides generated by KII and the data obtained showed only a marginal increase (from 44 to
50%) in gal sulfation in the interior regions of aggrecan KS during tissue maturation and thus did not confirm the substantial increases in this parameter between 4 and 1820 years of age, reported by Brown and colleagues (1998). It is likely that the products from the contaminating galactosidase in the KII were also generated in such studies, but they were not detected during the HPLC separation of digestion products, thus leading to underestimation of the monosulfated disaccharide contents of the samples. It should be noted that a reasonably accurate estimation of the monosulfated disaccharide contents can be obtained after digestion with EB or Pseudomonas keratanase, because these enzymes give good depolymerization efficiencies for monosulfated regions of KS and lack contaminate activities (Figure 6 and Table II). Alternatively, chemical depolymerization of KS using the hydrazinolysis procedure (Shaklee and Conrad, 1986
; Brown et al., 1992
) may be optimized for use in combination with FACE to include cleavage of linkages adjacent to fucose substituted galNAc6S for quantitative depolymerization of fucosylated KS chains, such as those abundant in several cartilages.
Individually purified standards for KS hydrolase digestion products are not commercially available, but the FACE runs can be readily calibrated by inclusion of purified monosaccharide standards (such as shown in Figure 1) and/or hydrolase digestion products obtained from bovine corneal KS obtained from commercial sources. Furthermore, as shown here, the identity of fucosylated and sialylated oligosaccharides can be verified by digestion of hydrolase products by specific exoglycosidases as both the released monosaccharides and the modified oligosaccharides products (Figures 25) are separable and quantifiable on a MonoComposition gels.
Altogether, the FACE procedure described here provides a sensitive, rapid separation and quantitation method for the full range of unsulfated, sulfated, fucosylated, and sialylated products generated in sequential incubation of KS with KII and EB. The approach represents an inexpensive and basic laboratory method for routine identification of KS hydrolase products, KS quantitation, and chain fine structure analyses, that can be used as an alternative to the more commonly described applications of 1H-NMR spectroscopy and HPLC.
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Materials and methods |
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Glycosidases
Keratanase II (Bacillus sp.), endo-ß-galactosidase (E. freundii), and keratanase (Pseudomonas sp.) were from Seikagaku (America Inc.). N-acetyl-ß-D-glucosaminidase (D. pneumoniae) and ß-galactosidase (bovine testis) were obtained from Roche Molecular Biochemicals. Neuraminidase type II (V. cholerae) was from Sigma-Aldrich, and almond meal -fucosidase was from Glyko. All enzymes were dissolved in water and stored in small aliquots at 80°C. All reconstituted enzymes showed no notable losses in activity when used within 3 months of preparation.
Saccharide and GAG preparations
Bovine corneal keratan sulfate, para-lacto-N-neo-hexaose (galß1,4glcNAcß1,3galß1,4glcNAcß1,3galß1,4glc), man, fuc, gal, gal6S, glcNAc, galNAc, glcNAc6S, and the 3'-fucosyllactose were from Sigma-Aldrich. 3'- and 6'-Sialyl-N-acetyllactosamine was from V-Labs (Covington, LA). All saccharides were suspended in water and stored in aliquots at 80°C.
Human aggrecan was extracted and purified from normal knee cartilage obtained from a 68-year-old donor as described (West et al., 1999). For the preparation of KS peptides, 100-µg portions of aggrecan (based on S-GAG as determined by the dimethylmethylene blue dye binding assay; Farndale et al., 1986
) were dissolved in 100 µl 50 mM sodium acetate, pH 7.0, and digested with 12.5 µg of proteinase K at 60°C for 18 h. The enzyme was inactivated at 100°C for 10 min and insoluble materials removed by centrifugation at 15,000 x g for 10 min at 4°C. Oligosaccharide peptides, amino acids, and buffer salts were separated from the GAG peptides by centrifugation through MicroCon3 devices at 9000 x g for 15 min at room temperature. The retained GAG peptides were washed with 100 µl water, recentrifuged, and then recovered from the filter in 200 µl of water for storage at 80°C. Using this procedure, recovery of > 95% of the S-GAG from aggrecan was routinely obtained, as determined by the dimethylmethylene blue assay.
Hexosamine composition of GAG substrates
Five micrograms of proteoglycans (as determined by dimethylmethylene blue assay) or 2, 5, 10 nmole of glcNAc and galNAc standards were dispensed into 500-µl polypropylene Eppendorf tubes and dried by speedvac lyophilization. These were resuspended in 100 µl 6 N HCl and heated for 2 h at 100°C. Acid was evaporated in vacuo, and hydrolysates washed twice with 50 µl of water followed by speedvac lyophilization. Reacetylation of hexosamine sugars was carried out essentially as described (Patel and Parekh, 1994), by suspending samples in 50 µl of water, adding 12.5 µl each of 1 M sodium bicarbonate and 5% acetic anhydride (both freshly made in water) and maintaining the mixture at room temperature for 10 min. An additional 12.5 µl of acetic anhydride was added, and the reaction continued for another 20 min at room temperature. Removal of reagent was performed by ion exchange on Dowex H+ by adding the reaction mixture to a 100-µl packed bed volume of washed resin in 0.45-µm Ultrafree MC filtration units, and briefly centrifuged (for 1 min at 5000 x g) to collect the desalted monosaccharides into the filtrate. These were immediately mixed with 2 nmole of glucose, speedvac lyophilized, and fluorotagged for quantitation after FACE separation on MonosaccharideTM composition gels. The yield of fluorotagged glcNAc and galNH2 after the hydrolysis and reacetylation was 65 ± 3% and 58 ± 5%, respectively, relative to the internal glucose standard. Based on this, bovine corneal KS contained 1.45 ± 0.11 nmol glcNH2 per µg of glycosaminoglycan, proteinase generated aggrecan glycosaminoglycan chains contained 1.12 ± 0.18 nmole glcNH2 per µg of sulfated glycosaminoglycan and intact human aggrecan preparation contained between 0.54 and 1.02 nmole of glcNH2 per µg of sulfated glycosaminiglycan.
Enzymatic depolymerization of KS
Bovine corneal KS or aggrecan KS peptides were suspended in 0.1 M ammonium acetate, pH 6.0, at concentrations typically ranging from 512 nmole glcNH2c per 75 µl buffer. Hydrolase digestions were carried out at 37°C using enzyme:substrate concentrations indicated in the figure legends for each experiment, and terminated after 4 or 22 h by heating to 100°C for 10 min. Undigested GAGs and enzyme proteins were precipitated after addition of 900 µl of ice-cold absolute ethanol at 20°C for 2 h and pelleted by centrifugation (15,000 x g, 20 min, at 4°C). The hydrolase products were quantitatively recovered in the supernatants and dried by speedvac lyophilization prior to the fluorotagging procedure described below. In selected experiments, KS hydrolasegenerated oligosaccharides were further digested with exoglycosidases. For this, dried products were resuspended in 50 mM ammonium acetate buffer adjusted to the pH optimum of a given exoglycosidase (as recommended by the supplier). Exoglycosidases were added singly or in combination, and incubations were routinely carried out for 2248 h, after which enzymes were inactivated at 100°C for 10 min, buffer removed by speedvac lyophilization, and the products fluorotagged as described.
Fluorotagging and FACE analyses of sacccharides
Fluorotagging was carried out essentially as described (Jackson, 1994). Briefly, monosaccharide standards (man, 0.25 nmole; fuc, 0.25 nmole; gal, 0.5 nmole; glcNAc 1.5 nmole; gal6S, 3 nmole; glcNAc6S, 6 nmole) and glycosidase products (containing between 1 and 10 nmol reducing sugar) were dried by speedvac lyophilization in 500-µl Eppendorf tubes. They were then mixed with 10 µl 0.1 M 2-amino-acridone (dissolved in glacial acetic acid:dimethylsulfoxide, 3:17, v/v) and incubated for 15 min at room temperature. A 10-µl portion of 1 M sodium cyanoborohydride (freshly prepared in water) was added; samples were mixed again and incubated at 37 °C for 16 h to complete the reductive amination reaction. Forty microliters of diluted glycerol (25% v/v in water) was added to each sample and a portion (6 µl = 10%) was removed immediately for FACE separation on Monosaccharide Composition gels. Electrophoresis was carried out for 6080 min at 4°C, as described in Calabro et al. (2000a).
Gel imaging and quantitation of fluorotagged products
After completion of the electrophoretic run, gel cassettes were removed from the running tank, excess electrophoresis buffer washed off the outside of the glass plates, and cassettes placed on a trans-illuminator light box fitted with a 312-nm light source (Model 33000, from Photodyne, New Berlin, WI). The fluorescent images were displayed using an Eagle Eye II gel documentation system (Stratagene Cloning Systems) and recorded as TIFF files. To display major (150600 pmole) and minor (20100 pmole) bands within the linear range of pixel densities (0 = white, 250 = black), short (30 s) and long (1.2 min) exposures were recorded for each gel. The mean pixel density (= the sum of all pixels per band divided by the number of pixels per band) for each product band was measured using Scion Image Analyses Software. Quantitation of molar yields of enzyme products at each exposure time, was determined from the pixel density per pmole values obtained for the standard monosaccharide bands and accurate product quantitation was achieved between 20 and 400 pmole of product.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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