Department of Biomedical Engineering/ND20, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195 USA
Received on July 14, 1999; revised on September 3, 1999; accepted on September 9, 1999.
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Abstract |
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Key words: aggrecan/cartilage/chondroitin sulfate/fine structure/hyaluronan
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Introduction |
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Therefore, an ability to elucidate the composition and fine structure of chondroitin sulfate is a necessary step in the effort to determine the role of specific structures in biologic processes. However, previous methods for analysis of chondroitin sulfate fine structure have required relatively large amounts of starting tissue for analysis, and involved time and labor intensive steps for isolation and purification of tissue proteoglycans, glycosaminoglycans, and their chondroitinase digestion products prior to analysis. Also the analyses themselves have proved time and labor intensive, normally allowing for only one sample to be analyzed at a time, usually by methods such as HPLC chromatography or capillary zone electrophoresis. We have therefore adapted the FACE protocols described in the accompanying article (Calabro et al., 2000) for direct analysis of the internal disaccharide, nonreducing terminal and linkage oligosaccharide structures of chondroitin sulfate in tissues using a predominantly single tube assay system. The sensitivity of the FACE protocols allows for analysis of hyaluronan and chondroitin/dermatan sulfate in as little as 25 µg of wet weight or 12 µg of dry weight of cartilage tissue which makes even topographical analysis of glycosaminoglycan composition in tissues feasible. FACE analyses of the fine structure of chondroitin sulfate chains on purified human aggrecan and in rat chondrosarcoma tissue are compared to previously published results to validate the new methodologies.
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Results |
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Complete FACE analysis of the hyaluronidase and chondroitinase digestion products directly from a proteinase K digest of rat chondrosarcoma tissue
The protocols for complete FACE analysis of the fine structure for hyaluronan and chondroitin sulfate as described in the accompanying article (Calabro et al., 2000) were adapted for direct analysis of tissue digests as described in the Materials and methods. Rat chondrosarcoma tissue was chosen to test these protocols because of the detailed information that already exists about the fine structure of the chondroitin sulfate chains on aggrecan, the predominant proteoglycan in this tissue (Shibata et al., 1992
; Midura et al., 1995
; Plaas et al., 1996
). Figure 2A shows a single gel image containing the complete FACE analysis for this tissue. The image was exposed to over saturate pixel intensities for the major derivatized structures allowing visualization of less abundant derivatized structures. In Figure 2B, the gel image in panel A is repeated with all the bands in each lane from top to bottom referenced by lowercase letters. Lanes 1 through 6 represent the AMAC-derivatized products from six identical aliquots of the proteinase K digest of the tissue which were each processed differently as described below and in Materials and methods.
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Figure 3A shows an image of the same gel as shown in Figure 2, but taken with a shorter exposure time such that all pixels were within a linear 12-bit depth range. This image was analyzed using the Gel-Pro Analyzer program version 3.0 from Media Cybernetics using the "join valley" method of base line correction set at either 1 or 2% slope change. The line plots of integrated optical density versus relative mobility (Rm) generated from this analysis for lanes 1 through 6 are shown in Figure 4, panels A through F, respectively. The peak designations in each line plot correspond to the band designations in Figure 2B. The positions at the top (Rm = 0) and bottom (Rm = 1) of each lane in Figure 2B, which correspond to the same relative mobility in the line plots of Figure 4, are indicated. The values of integrated areas for select peaks in Figure 4 are listed in Table I.
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Since the endogenous glucose and unknown e are present in the original proteinase K tissue digest, their concentrations in the sample aliquots analyzed in lanes 1 through 6 of Figure 2 were initially the same independent of subsequent sample treatment. Therefore, these saccharides could serve as internal standards for differences in samples as a result of processing and gel loading. The initial integrated optical densities obtained from all the peaks in Figure 4 were therefore normalized based on the combined values for the glucose plus unknown e peaks with the value for the SD/ABC sample set at 100%. These normalized values are reported in Table I. Values for the glucose plus unknown e peaks were all within 10% of each other except for the value for the Hg2+ sample, which was 63% of the SD/ABC sample. This is due to selective loss of neutral structures on the Dowex H+ resin used to remove the Hg2+, as compared to the quantitative recovery normally expected for the negatively charged disaccharides and sulfated galNAc structures. When the total fluorescence for all derivatized structures in the SD/ABC lane was compared to the total fluorescence in the Hg2+ lane after adjusting for the observed loss of neutral saccharides, the values are within 2% of each other. Therefore, for the Hg2+ treated sample only, values for uncharged structures (i.e., glucose, unknown e, galNAc, and glcNAc) were normalized based on the relative amounts of glucose plus unknown e (63%), while negatively charged structures (i.e., 4S-galNAc and Di4S) were normalized based on the relative total fluorescence adjusted as described above (2%).
In some samples, the high amount of glucose in the sample relative to the amount of hyaluronan and chondroitin/dermatan sulfate requires removal of the glucose prior to processing. Excessive amounts of glucose can deplete the AMAC reagent during derivatization, causing incomplete derivatization of the hyaluronidase and chondroitinase digestion products. Culture medium with 1 g/l of glucose contains ~50 nmol of reducing equivalents in a 10 µl aliquot, the desired upper limit in the derivatization reaction. Further, the fluorescent signal from excessive amounts of derivatized glucose is sufficient to prevent accurate detection of neighboring structures such as DiHA and
Di0S. Glucose (and unknown e) can be selectively removed from proteinase K digested samples after ethanol precipitating the glycosaminoglycans. The small molecular weight glucose partitions into the supernatant fraction while the high molecular weight hyaluronan and chondroitin/dermatan sulfate precipitate. Since most of the protein in the sample has been digested to amino acids and small peptides by the proteinase K (data not shown), the resulting precipitate containing the glycosaminoglycans is normally easily resuspended in the ammonium acetate buffer. In Figure 5A, the results of ethanol precipitation of the proteinase K digested rat chondrosarcoma tissue are shown with the samples in lanes 1 (dAMAC), 3 (SD/ABC), 5 (Hg2+) and 7 (4,6Sase) having been processed the same as the samples in lanes 1, 2, 3, and 5 of Figure 2, respectively, except that no
Di2S standard was added. The samples in lanes 2, 4, 6, and 8 were processed the same as those in lanes 1, 3, 5, and 7, respectively, but after ethanol precipitation as described in Materials and methods to remove selectively glucose and unknown e. Mannose (arrow) was added as an internal standard upon resuspension of the precipitates. For all four precipitated samples, the glucose and unknown e were removed, while the patterns of derivatized hyaluronidase and chondroitinase digestion products were unchanged with recoveries of 94% or better. Note the absence of any
Di2S in any of the samples validating its use as a standard for this tissue.
Identification of nonreducing terminal structures
As seen in Table II, the three nonreducing terminal structures on chondroitin sulfate chains from rat chondrosarcoma tissue as measured by these FACE analyses were 4S-galNAc (51%), galNAc (27%), and Di4S (22%). The galNAc and 4S-galNAc nonreducing terminal structures were measured directly from peaks a and n, respectively, of the SD/ABC (panel B), the 4Sase (panel D), the 6Sase (panel F) and the 4,6Sase (panel E) samples in Figure 4. It is the averages of these four measurements for both galNAc and 4S-galNAc from Table I that are reported as calculated values in Table II. The presence of Di4S as a nonreducing terminal structure was deduced by comparing peak p in the Hg2+ sample (panel C) to those in the dAMAC (panel A), the 4Sase (panel D), and the 4,6Sase (panel E) samples of Figure 4. Peak p in the Hg2+ sample contains a shoulder on the front of the peak compared to peak p in these other three samples. Since the mercuric ion treatment removes any Di4S or
Di6S from this position in the gel, the shoulder potentially contains Di4S, Di6S and/or 4,6S-galNAc. However, all of the shoulder was removed by chondro-4-sulfatase digestion alone (Figure 4D) indicating that it was all Di4S with no Di6S or 4,6S-galNAc. The remaining peak p in the 4Sase and 4,6Sase samples are similar to that in the dAMAC sample, and therefore represent only the contaminant in the AMAC reagent as described above. The calculated value for Di4S as reported in Table II was therefore determined by subtracting the average of the values in Table I for peak p in the dAMAC, 4Sase, and 4,6Sase samples (the value for the AMAC contaminant) from the value in Table I for peak p in the Hg2+ sample. Neither Di0S nor 6S-galNAc was detected as nonreducing terminal structures.
Identification of presumptive linkage oligosaccharide (LO) structures
There are several peaks in Figure 4, which do not migrate at known positions. We deduce that three of these peaks (j, k, and l) are derived from the linkage oligosaccharide (LO) region of chondroitin sulfate chains based on the following reasoning. Chondroitinase ABC digestion leaves an unsaturated hexuronic acid adjacent to galNAc (with or without 4-sulfation) at the nonreducing terminus of linkage oligosaccharides from chondroitin sulfate chains from the rat chondrosarcoma (Figure 6, solid inverted triangles) (Shibata et al., 1992). Endo-galactosidase would expose a new reducing end on the linkage oligosaccharides after cleavage at either galactose residue, which could then be derivatized with AMAC (Figure 6, open inverted triangles). Therefore, chondroitinase ABC containing a contaminating endo-galactosidase activity would generate the proposed linkage oligosaccharide derived structures (LOABC) with (4S-) or without (unS-) sulfate as illustrated in Figure 6. Cleavage by endo-galactosidase may be selective for one or the other galactose residue generating two potential structures as indicated in Figure 6. The specific galactose residue cleaved may be affected by steric hindrance of the attached peptide, by sulfation on the galNAc residue or by phosphorylation of the xylose residue as previously reported (Shibata et al., 1992
).
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The quantitation supports the qualitative evaluation described above. The calculated value for the unS-LO in Table II was taken directly from the value in Table I for peak j or the unS-LOHg structure in the Hg2+ sample (Figure 5B, lane 3). The derivatized unS-LOABC structure, which gives rise to peak j after mercuric ion treatment, comigrates with derivatized Di0S in peak i of the SD/ABC sample (Figure 5B, lane 2). This is supported by the fact that the sum of the calculated values in Table II for
DiHA,
Di0S and the unS-LO, and the sum of the values for peaks h (i.e.,
DiHA) and i (i.e.,
Di0S and unS-LO) in Table I of the SD/ABC sample are within 4%. The appearance of peak j, near the position of
Di0S after mercuric ion treatment, might suggest that it was derivatized Di0S, which is not affected by this treatment. However, peak j has shifted position relative to peak i following mercuric ion treatment indicating the presence of a
hexuronic acid residue in the parent compound. Importantly, the sum of the calculated values in Table II for the three identified nonreducing terminal structures is within 3% of the sum of the calculated values for the two proposed linkage oligosaccharide structures. The molar equivalence of these two values is necessary since chondroitin sulfate chains contain one nonreducing terminus and one linkage oligosaccharide per chain. As expected both the linkage oligosaccharides and nonreducing terminal values predict similar number averaged chain lengths for the chondroitin sulfate chains (see below).
The derivatized 4S-LOABC structure which gives rise to peak k (i.e., 4S-LOHg) after mercuric ion treatment and peak l (i.e., 4S-LO4Sase) after chondro-4-sulfatase digestion probably migrates near Di4S in peak q of the SD/ABC sample (Figure 5B, lane 2). In fact, a minor band just ahead of band q can be seen in Figure 2, lanes 2 and 6, which appears as a shoulder on peak q in Figure 4B,F. The large quantity of
Di4S present in the tissue makes it impossible to detect this structure directly in the SD/ABC sample. Therefore, the calculated value for the 4S-LO in Table II was determined by averaging the values in Table I for peak k (the 4S-LOHg structure in the Hg2+ sample) and peaks l (the 4S-LO4Sase structure in both the 4Sase and 4,6Sase samples) (Figure 4CE). The relationship between peaks k and l is inferred from the fact that the value for peak k and the two values for peak l in Table I are all within 5% of their average value as listed in Table II.
Identification of internal disaccharide structures
As seen in Table II, the two internal disaccharides on chondroitin sulfate chains from rat chondrosarcoma tissue as determined by these FACE analyses were
Di4S (93%) and
Di0S (7%). In addition, FACE analysis showed that rat chondrosarcoma tissue contained 5% more internal
disaccharide in the form of
DiHA from hyaluronan. The
Di4S was measured by five independent measurements. The amount of
Di4S was calculated by subtracting the sum of the calculated values in Table II for the Di4S, 4S-LO and AMAC contaminant from the value in Table I for peak q of the SD/ABC and 6Sase samples (see Figure 4B,F, respectively). Peak q in the SD/ABC and 6Sase samples contains predominantly derivatized
Di4S, but also minor amounts of these other three derivatized structures, which migrate at similar positions (see above). Alternatively, the amount of
Di4S was calculated by subtracting the calculated value in Table II for 4S-galNAc as a nonreducing terminal structure from the value for peak n in Table I of the Hg2+ sample (see Figure 4C). Peak n in the Hg2+ sample contains the 4S-galNAc originally present as a nonreducing terminal as well as 4S-galNAc as a result of mercuric ion treatment of
Di4S. Finally, the amount of
Di4S was calculated by subtracting the sum of the calculated values in Table II for the
Di0S,
DiHA, Di4S, and unS-LO structures from the values for the sum of peaks h and i in Table I of the 4Sase and 4,6Sase samples (see Figure 4D,E, respectively). Digestion with chondro-4-sulfatase quantitatively converts
Di4S and Di4S to
Di0S and Di0S, which then both migrate at the position of peak i where the
Di0S and unS-LO in the original chondroitinase digest migrate. Peak h, which contained only
DiHA, was included since it does not completely resolve from peak i in the 4Sase and 4,6Sase samples. These five independent measurements for
Di4S were all within 6% of their average value, which was reported as the calculated value for
Di4S in Table II.
The Di0S and
DiHA were measured from the galNAc (peak a) and glcNAc (peak g) peaks, respectively, in the mercuric ion treated (Hg2+) sample (Figure 4C). Both these peaks show baseline separation from the peaks adjacent to them. The value for
DiHA in Table II was taken directly from peak g in the Hg2+ sample of Table I. However, the value for
Di0S in Table II was the result of subtracting the calculated value in Table II for galNAc as a nonreducing terminal from the value for peak a in the Hg2+ sample of Table I. The reason that peaks i and h in the SD/ABC sample (Figure 4B) were not used directly to measure the
Di0S and
DiHA was because they contained the derivatized unS-LOABC structure as described above.
The large quantity of Di4S in chondroitin sulfate from rat chondrosarcoma tissue makes it difficult to detect
Di6S directly from the SD/ABC sample (Figure 4B). Chondro-4-sulfatase digestion was used to uncover any minor amounts of
Di6S present (Figure 4D) by shifting the position of the Di4S and
Di4S to that of Di0S and
Di0S, respectively. Only the contaminant from the AMAC reagent in peak p as described above was uncovered indicating that no detectable
Di6S was present. As previously discussed, no
Di2S was detected (see Figure 5A) in the chondrosarcoma tissue. No peaks between a relative mobility of 0.8 and the electrophoresis front (EF) were detected in Figure 4 that would indicate the presence of AMAC-derivatized
Di4,6S or
Di2,6S both of which clearly resolved in this analysis (see Figure 2, lane S13). Finally, although a small peak of fluorescent material is detected at the electrophoresis front, this peak is present in the dAMAC sample (Figure 4A) and is unaffected by any subsequent treatments (Figure 4BF). Therefore, no
Di2,4S, or
Di2,4,6S, which both run at the electrophoresis front were detected.
Determination of number averaged chain size from FACE analysis
The number averaged chain size for the chondroitin sulfate chains from rat chondrosarcoma tissue was determined based on the information in Table II. First, the average number of internal disaccharides per chondroitin sulfate chain was calculated based on the fact that there is only one nonreducing terminus and one linkage oligosaccharide per chain. The total fluorescence for all the internal disaccharides (i.e., Di4S and
Di0S) was divided by either the total fluorescence for all the nonreducing termini (i.e. 4S-galNAc, galNAc, and Di4S) or for all the linkage oligosaccharides (i.e., unS-LO and 4S-LO). Based on this calculation the average number of internal disaccharides per chondroitin sulfate chain was either 30 or 28 depending on whether the nonreducing termini or linkage oligosaccharide values, respectively, were used. An average value of 29 internal disaccharides per chain was then multiplied by an average formula weight for the internal disaccharides of 496 Da, which was calculated from the percent composition and formula weights for the
Di4S and
Di0S listed in Table II. To the resulting calculated number averaged weight of the internal disaccharides was then added the average formula weight of one nonreducing terminus and one linkage oligosaccharide (calculated from the percent composition and molecular weights listed in Table II). These calculations are illustrated in Figure 6, which shows a schematic of the structure of chondroitin sulfate chains from rat chondrosarcoma tissue as determined by FACE analysis. The final estimation of chain size based on the FACE analysis was ~16,000 Da. The weight averaged chain size for these chains, as determined by Superose 6 chromatography, was 24,000 Da (data not shown).
Important considerations
Since tissue glucose concentrations can be measured by the FACE protocols, the tissue was kept on ice after harvesting until the proteinase K digestion step to minimize glucose utilization by the chondrocytes during tissue processing, and therefore to prevent underestimation of tissue glucose concentrations. Only extracellular glucose is measured by this assay since the free reducing group of glucose is normally blocked by phosphorylation upon entering the cell. Unlike with purified proteoglycan or glycosaminoglycan preparations, tissue samples must first be solubilized to release glycosaminoglycan chains regardless of their location within cells, on cell surfaces or in extracellular matrices. This allows for sampling of tissue aliquots which contain the same initial glycosaminoglycan content, and which can therefore be directly compared after the various enzymatic and chemical treatments used in our protocols.
Proteinase K digestion was chosen for solubilizing tissues because of its following properties. Proteinase K is a nonspecific serine protease, so that it is predicted to work equally well on all proteins independent of their amino acid composition. It is stable over a wide pH range (412.5) with optimal activity at a pH of 6.59.5. The activity of proteinase K is increased by denaturing agents, and is stable to metal ions, chelating agents, sulfhydryl reagents, or trypsin/chymotrypsin inhibitors. This provides many options when dovetailing these analyses with already existing protocols. Proteinase K has a temperature optimum of 65°C, with digestion at this elevated temperature facilitating the solubilization of cellular components. Proteinase K is rapidly denatured at temperatures above 65°C, and therefore can be easily inactivated by boiling prior to steps involving other enzymatic treatments. Finally, while autolysis of proteinase K occurs increasingly at alkaline pH, the enzyme is not inactivated by autolysis, thereby minimizing the amount of enzyme required.
There are also some general advantages to protease digestion. Protease digestion removes most of the macromolecular protein, which can prevent complete solubilization of glycosaminoglycans in an ethanol precipitate for those samples, which may require such treatment (see Figure 5A). Protease digestion removes noncovalently bound proteins such as aggrecan and link protein from hyaluronan, which allows for complete digestion of the hyaluronan by hyaluronidase and/or chondroitinase. Protease digestion also releases individual glycosaminoglycan chains from proteoglycan core proteins containing multiple chains (i.e., aggrecan). Finally protease digestion, particularly at 65°C, potentially inactivates endogenous enzymatic activities, which might show specificity for the glycosaminoglycans and complicate the analysis.
The hyaluronidase and chondroitinase digestions can be done on separate sample aliquots to simplify quantitation of hyaluronan. Hyaluronidase SD digestion alone yields only DiHA with minor amounts of
Di0S, and no
Di4S or
Di6S. Presumably, the
Di0S released is from unsulfated regions of two or more disaccharides in the chondroitin sulfate chains. Digestion with chondroitinase ABC alone normally underestimates the amount of hyaluronan. Samples digested with both enzymes are digested first with the hyaluronidase for 1 h prior to chondroitinase digestion, because incomplete hyaluronan digestion was observed when both enzymes were added at the same time. It may be that the
Di0S generated by the chondroitinase inhibits the hyaluronidase activity. Two unknown AMAC-derivatized structures, peaks b and c, which migrate to positions between derivatized galNAc and glucose were observed in all the samples which were hylauronidase and chondroitinase digested (Figures 2 and 4). These minor structures probably arise from the action of contaminating enzyme activities in the hyaluronidase and/or chondroitinase on N- or O-linked oligosaccharides in the proteinase K preparation, since these products are not observed in the ethanol precipitated samples (Figure 5A) and the purified aggrecan samples (Figure 1).
Finally, the derivatized Di2S standard in Figures 2 and 4 contains a minor peak (peak s), which runs slightly ahead of the major
Di2S peak (peak r). This is a break-down product as a result of storage of the standard at 4°C rather than 70°C, and which becomes more prominent with time. A similar break down product running just ahead of the major peak is observed for all of the derivatized
disaccharide standards with storage at 4°C.
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Discussion |
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Compositional analysis of the nonreducing terminal, internal disaccharide and linkage oligosaccharide structures of the chondroitin sulfate chains of aggrecan from rat chondrosarcoma cultures have been made previously (Shibata et al., 1992; Midura et al., 1995
; Plaas et al., 1996
). Some of the methods previously used in these analyses to isolate and purify aggrecan, its chondroitin sulfate chains and their chondroitinase digestion products prior to analysis included dissociative isopycnic density gradient centrifugation (i.e., cesium chloride, guanidine HCl), ultrafiltration (i.e., Centricon 30, Microcon 3), size exclusion chromatography (i.e., Sephadex G-50, Superose 6, Sephacryl S-1000, Toyopearl HW40S, Progel-TSK G2500PWXL) and anion exchange chromatography (i.e., Q-Sepharose, CarboPak PA1, AS4S Ion Pac). We have adapted the FACE protocols as described in the accompanying article (Calabro et al., 2000
) for direct analysis of the fine structure of hyaluronan and chondroitin sulfate in proteinase K digests of tissues with a minimum of sample processing. All sample processing, except mercuric ion treatment, takes place in one tube with the mercuric ion treatment taking place after the hyaluronan and chondroitin sulfate chains have been completely digested to their final digestion products. This single tube approach avoids potential losses of small or undersulfated fractions of chondroitin sulfate chains that are possible when samples are processed by size exclusion (i.e. Sephadex G-50) or ion exchange chromatography (i.e., Q-Sepharose), respectively.
FACE analysis of the glycosaminoglycans in the proteinase K digest of rat chondrosarcoma tissue showed that the proportion of DiHA from hyaluronan was 5% of the total of
Di0S and
Di4S from chondroitin sulfate consistent with the stoichiometry of hyaluronan and aggrecan containing complexes normally found in cartilage (Morales and Hascall, 1988
). The internal disaccharide compositions for the chondroitin sulfate chains on rat chondrosarcoma aggrecan as previously measured were
Di0S (510%),
Di4S (9095%),
Di6S (0.20.5%), and
Di4,6S (0.5%) (Shibata et al., 1992
; Midura et al., 1995
; Plaas et al., 1996
). Our result determined by FACE analysis of the chondroitin sulfate chains in the proteinase K digest of rat chondrosarcoma tissue shown in Table II indicate the presence of only
Di0S (7%) and
Di4S (93%) with no
Di6S and
Di4,6S detected. The nonreducing terminal structures for the chondroitin sulfate chains on rat chondrosarcoma aggrecan as previously measured were 4S-galNAc plus 4,6S-galNAc (8587%), and Di4S plus Di6S (1315%) (Midura et al., 1995
; Plaas et al., 1996
). Our results as determined by FACE analysis of the chondroitin sulfate chains in the proteinase K digest of rat chondrosarcoma tissue indicate the presence of 4S-galNAc (51%), galNAc (27%), and Di4S (22%) with no 4,6S-galNAc or Di6S detected (see Table II).
An unexpected consequence of our FACE analysis following chondroitinase digestion was the detection of putative linkage oligosaccharide structures from the chondroitin sulfate chains as a consequence of a proposed contaminating endo-galactosidase activity in the chondroitinase preparation. Results of our FACE analysis of the chondroitin sulfate chains in the proteinase K digest of rat chondrosarcoma tissue strongly suggest the presence of an unsulfated linkage oligosaccharide (unS-LO, 85%) and a 4-sulfated linkage oligosaccharide (4S-LO, 15%). These values are similar to those previously reported by Shibata et al. (1992) of 64% and 36%, respectively. One major difference in our analysis versus previous analyses was that we have measured the bulk mass of aggrecan chondroitin sulfate as synthesized by the tissue in vivo as a subcutaneous tumor while the previous analyses measured only newly synthesized and metabolically labeled ([35S]sulfate and [3H]glucosamine) chondroitin sulfate from primary chondrosarcoma chondrocyte cultures. The difference between the availability of sulfate to the chondrocytes for chondroitin sulfate synthesis under in vivo versus culture conditions may account for the slightly higher level of unsulfated saccharide structures measured for the aggrecan from the tissue versus cultures. This may also explain the absence of any 6-sulfation in the tissue, which may require saturating concentrations of phosphoadenosinephosphosulfate (PAPS) for even the minor amount of 6-sulfation detected in the cultures to be synthesized.
Finally the number averaged chain length estimated by the ratio of the molar amount of the total disaccharides to either the total nonreducing terminal or the total linkage oligosaccharide structures from these FACE analyses was ~16 kDa. This is consistent with previous estimates of chain length for chondroitin sulfate from rat chondrosarcoma aggrecan estimated either by monosaccharide analysis (~18 kDa) or by number averaged chain size calculations using the 2-aminopyridine/AS4S anion exchange chromatography method (~17 kDa) (Midura et al., 1995
; Plaas et al., 1996
). A weight averaged chain size of ~24 kDa (Kd = 0.56) was determined by Superose 6 chromatography for chondroitin sulfate chains from the proteinase K tumor digests used for the FACE analyses described here, and from chondrosarcoma chondrocyte cultures as described previously (Midura et al., 1995
).
In summary, we have developed a fast, simple, and sensitive method for detection and quantitation of the hyaluronidase and chondroitinase digestion products of hyaluronan and chondroitin/dermatan sulfate chains using 2-aminoacridone (AMAC) derivatization and fluorophore-assisted carbohydrate electrophoresis (FACE). The FACE protocols described here and in the accompanying article (Calabro et al., 2000) have several important features that make them useful for a wide variety of applications. The amount of the fluorescence from the AMAC is independent of the chemistry of the saccharide to which it is attached. This means that the fluorescence is a direct measure of the molar amount of a derivatized structure, which is a powerful tool in elucidating the nature of unexpected chemistries such as the linkage oligosaccharides identified in these analyses. The sensitive nature of the assay (in the nanogram/picomole range) allows for detection of hyaluronan and chondroitin sulfate from small tissue samples. The hyaluronan and chondroitin sulfate digestion products visualized in each lane of the FACE gel in Figure 2 is from 25 µg of rat chondrosarcoma tissue based on wet weight. Assuming the tissue is 95% water, that amounts to 12 µg of tissue by dry weight. This level of sensitivity when combined with the protocols described in this paper for detection of hyaluronan and chondroitin sulfate directly from a proteinase K tissue digest brings experiments such as topographic analysis of glycosaminoglycans in tissues like cartilage into the realistic and manageable range, especially considering the single tube nature of the assay and the ability to process and image a number of samples simultaneously. As shown in Figure 2 complete characterization of a sample can be accomplished with a single gel in less than a week using the combination of direct fluorescent derivatization, hyaluronidase and chondroitinase digestion, mercuric ion treatment, and sulfatase digestions.
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Materials and methods |
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Preparation of purified aggrecan samples for fluorescent derivatization
Aggrecan samples from fetal and 68-year-old human cartilage were prepared by guanidine HCl extraction and cesium chloride gradient centrifugation as previously described (Plaas et al., 1996). The aggrecan preparations were generous gifts of Dr. Anna Plaas (Shriners Hospital for Crippled Children, Tampa, FL). Aggrecan preparations (in ultrapure water) were processed for derivatization as follows. Three identical aliquots from each preparation were frozen on dry ice, and lyophilized until dry on a vacuum concentrator. Each aliquot contained no more than 50 nmol of potential disaccharide reducing equivalents. Each aliquot was resuspended in 100 µl of 0.0005% phenol red, 100 mM ammonium acetate, pH 7.0. One aliquot was immediately frozen on dry ice, and lyophilized until dry on a vacuum concentrator prior to derivatization (dAMAC). The remaining two aliquots were digested with chondroitinase ABC (100 mU/ml) for 3 h at 37°C, and then frozen on dry ice, and lyophilized until dry on a vacuum concentrator. One chondroitinase digest was derivatized directly (ABC). The other chondroitinase digest was treated with mercuric ion prior to derivatization (Hg2+) as described in the accompanying article (Calabro et al., 2000
).
Preparation of proteinase K digest of Swarm rat chondrosarcoma tissue
The Swarm rat chondrosarcoma tissue was propagated in rats as described previously (Calabro and Hascall, 1994). Briefly, one tumor-laden rat was sacrificed by CO2 asphyxiation. The tumor and surrounding facia were excised, and immediately placed on ice. The tumor was quickly pressed through a wire mesh sieve to mince the cartilage tissue and to remove the surrounding facia. The minced cartilage tissue (~3 g) was immediately transferred to a preweighed tube on ice. A 100 mg aliquot of this cartilage tissue was transferred to a 1.5 ml screw cap tube on ice. The tissue was assumed to have a density equal to water (1 mg/µl) per its high water content, and the tissue sample was diluted to a final volume of 900 µl containing 0.0005% phenol red, 100 mM ammonium acetate, pH 7.0. A 50 µl aliquot of a fresh solution of proteinase K (2.5 mg/ml) in 0.0005% phenol red, 100 mM ammonium acetate, pH 7.0 was added, and the tissue digested for 2 h at 60°C with mixing every 30 min. A second 50 µl aliquot of a fresh solution of proteinase K was added, and the tissue digested for an additional 2 h at 60°C with mixing every 30 min. The sample was then boiled for 10 min to inactivate the proteinase K, and centrifuged at 10,000 x g for 15 min at room temperature to pellet any undigested material. An enzyme/buffer sham sample containing only the buffer was also digested with proteinase K as described above. A 200 µl aliquot of the proteinase K digested tissue was eluted on a Superose 6 column in 0.5 M ammonium acetate, pH 7, to obtain an estimate of the weight averaged chain size of the chondroitin sulfate chains.
Processing of proteinase K digested tissue for fluorescent derivatization
One 100 µl aliquot of the supernatant from the proteinase K digested tissue sample was diluted to 1 ml with 0.0005% phenol red, 100 mM ammonium acetate, pH 7.0. Six 100 µl aliquots of the diluted sample were transferred to new microfuge tubes. One aliquot remained untreated (dAMAC). The remaining five aliquots were digested for 1 h at 37°C with 100 mU/ml of hyaluronidase SD followed by 3 h at 37°C with the addition of 100 mU/ml of chondroitinase ABC. One digested aliquot received no further treatment (SD/ABC). Three digested aliquots were further incubated during the last 2 h of the chondroitinase ABC digestion with 100 mU/ml each of chondro-4-sulfatase alone (4Sase), chondro-6-sulfatase alone (6Sase) or the two sulfatases together (4,6Sase). Finally, one digested aliquot was further treated with mercuric ion (Hg2+) as described in the accompanying article (Calabro et al., 2000). To each of the six processed aliquots from the diluted tissue sample, 1 ml of -20°C absolute ethanol was added, the samples mixed, and stored a minimum of 2 h at -20°C. The samples were centrifuged at 10,000 x g for 15 min at 4°C to pellet any macromolecular material, and the supernatants containing the hyaluronidase and chondroitinase digestion products were transferred to new microfuge tubes. Both the supernatant and pellet fractions were dried on a vacuum concentrator prior to derivatization with 2-aminoacridone (AMAC) as described in the accompanying article (Calabro et al., 2000
). The pellet fractions contained less than 1% of the total derivatized digestion products (data not shown). After derivatization an aliquot of each supernatant sample was mixed 1:1 with one AMAC-derivatized
Di2S standard solution containing 16, 31, 62.5, 125, 250, or 500 pmol of derivatized
Di2S per 5 µl. The
Di2S standards were calibrated by FACE analysis using AMAC-derivatized glucose standards. Samples were run with (Figures 2, 3) and without (Figure 5A)
Di2S standards on FACE gels as described below. Enzyme/buffer shams for all six treatments were prepared similarly for derivatization.
Preparation of ethanol precipitated proteinase K digested tissue for fluorescent derivatization
One 100 µl aliquot of the supernatant from the proteinase K digested tissue sample was transferred to a new microfuge tube. One milliliter of -20°C absolute ethanol was added, the sample mixed, and stored a minimum of 2 h at -20°C. The sample was centrifuged at 10,000 x g for 15 min at 4°C to pellet any macromolecular material including the hyaluronan and chondroitin sulfate chains, and the supernatant containing the small molecular weight molecules such as glucose and salts were aspirated to waste. The pellet was washed with 1 ml of -20°C absolute ethanol by inverting, and centrifuged at 10,000 x g for 15 min at 4°C. The supernatant was aspirated to waste, and the precipitate dried for 5 min on a vacuum concentrator. The precipitate was resuspended in 1 ml of 20 nmol/ml mannose, 0.0005% phenol red, 100 mM ammonium acetate, pH 7. The concentration of mannose was determined by FACE analysis using AMAC-derivatized glucose standards, and it served as an internal standard in place of the endogenous glucose removed by the ethanol precipitation. Six 100 µl aliquots of the precipitated tissue sample were transferred to new microfuge tubes, and then processed as described above for the direct tissue samples (i.e., dAMAC, SD/ABC, Hg2+, 4Sase, 6Sase, and 4,6Sase).
Gel electrophoresis, gel imaging, and data analysis
Samples were run on MONO® composition gels with MONO® gel running buffer as described in the accompanying article (Calabro et al., 2000). The gels were illuminated with UV light (365 nm) from an Ultra Lum Transilluminator, and imaged with a Quantix cooled CCD camera from Roper Scientific/Photometrics as described in the accompanying article (Calabro et al., 2000
). Digital images for each gel were taken at two exposures. One exposure over saturated pixel intensity for the major derivatized structures in order to allow visualization of less abundant derivatized structures as seen in Figures 1, 2, and 5. The second exposure had all pixels within a linear 12-bit depth range, and was used for quantitation as seen in Figure 3A. The images were analyzed using the Gel-Pro Analyzer program version 3.0 from Media Cybernetics. Base line was determined using the "join valleys" method set at either 1 or 2%.
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References |
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