From the Chondroitin lyase products of aggrecan and small
proteoglycans from normal and osteoarthritic cartilages were analyzed
for chain internal The function of normal adult joint cartilage resides in its
ability to provide resistance to compressive, tensile, and shear forces
that occur during normal joint motion. The specialized extracellular
matrix consists of a fibrillar collagen network (1), highly organized
and stabilized through inter- and intramolecular cross-links that
restrain the swelling pressure exerted by the high concentration of
negatively charged aggrecan aggregates (2). The metabolic activity of
chondrocytes in adult cartilage is adapted to maintain the composition
of a functional extracellular matrix. The collagen network is
relatively metabolically inert (3), whereas other constituents, such as
aggrecan aggregates, undergo a distinct turnover process (4, 5) in
which catabolic cleavage (6, 7) and removal of molecules from the
extracellular matrix (8, 9) are in balance with synthesis and
deposition of new molecules. However, at the onset and throughout
progression of degenerative joint disease, such as
OA,1 this metabolic balance
is perturbed, and episodes of increased catabolism (10, 11) and
increased synthesis (12-16) of matrix components by the chondrocyte
take place.
It has been proposed that the increased anabolic activity in OA is
related to a change in the differentiated state of the chondrocyte (17)
with expression of pathways that are characteristic of cells in fetal
or postnatal growth cartilages. This hypothesis has been particularly
supported by published reports (18-21) that the GAG composition of
aggrecan synthesized during the early stages of cartilage degeneration
in animal models of OA or accumulated in human OA cartilages was
altered. Changes included decreased keratan sulfate/CS ratio, increased
4-sulfation of CS, and alterations in hydrodynamic size of CS chains.
In addition, the use of monoclonal antibodies to CS epitopes that were
considered abundant on fetal, but not on normal mature adult aggrecan,
indicated that aggrecan from OA cartilages displayed elevated
immunoreactivity compared with normal adult cartilages (22-26).
However, the use of the immunological data to describe CS fine
structures remains problematic, because the structure of the
carbohydrate epitopes recognized by the antibodies has not been
established in many cases. Furthermore, immunoreactivity of CS epitopes
is likely influenced by assay conditions that affect chain presentation
and epitope concentration much as was seen in solid phase immunoassays
with monoclonal antibody 3B3( Despite the large number of reported changes in composition of CS in OA
cartilages as well as of CS in synovial fluids (30-32), methods have
not been available to determine if such disease-related alterations in
structure are randomly distributed within the GAG chain or confined to
specific domains. Because of this limitation it has not been possible
to establish the importance of changes in the process of GAG
biosynthesis as opposed to alterations in matrix catabolism to the
observed changes in GAG composition of tissue and synovial fluid
proteoglycans.
Our previous work on the fine structure of the non-reducing terminal
residues on aggrecan CS (33) showed that such structures are specific
and are a distinguishing feature of aggrecan from human cartilages
before or after skeletal maturity. This was most readily seen for the
GalNAc residues, which represent the terminal on ~90% of CS chains
on aggrecan from all ages. In growth cartilages, they were almost
exclusively 4-monosulfated, whereas in mature cartilages about 50%
were 4,6-disulfated (27). Furthermore, these changes in sulfation of
the termini occurred later in life than the much described alteration
in 4- and 6-monosulfation of the internal repeating disaccharides. This
is clearly in support of separate enzymes for hexosamine sulfation in
the chain interior or terminus. We have now extended such studies to
determine the internal and non-reducing terminal sulfation isomers on
CS and DS from high and low buoyant density PGs purified normal and OA knee cartilages, to delineate potential disease-related alterations in
GAG sulfation.
Chondroitinases ABC (Proteus vulgaris) and ACII
(Arthrobacter aurescens) were from Seikagaku Corp.
(Rockville, MD). Papain, pepstatin, and aminoethylbenzenesulfonyl
fluoride were obtained from Sigma. Anti-ATEG antiserum to aggrecan
G1 domain was kindly supplied by Dr. John Sandy (Shriners
Hospital, Tampa Unit), and the anti-LEC 7 antiserum to aggrecan G3
domain was supplied by Dr. Kurt Doege (Shriners Hospital, Portland
Unit). Monoclonal antibody 2B6 ascites fluid and rat anti-mouse IgM
were from ICN (Costa Mesa, CA). Nitrocellulose membranes (0.2 µm)
were from Bio-Rad. Nylon N+ membranes, ECL Western blotting
reagent, and Hyperfilm were obtained from Amersham Pharmacia Biotech.
All other chemicals were obtained as described (27, 33).
Cartilage Sampling--
Details of tissue donors and sampling
are given in Fig. 1, A and
B. Cartilage plugs (~5 mm diameter, full depth) were
obtained from the right knees of adult donors from 20 individual sites (Fig. 1A) within 3-18 h postmortem, during bone tissue
harvest. The classification "Normal" was based on the macroscopic
appearance, with no evidence of surface fibrillation, cartilage
erosion, or prior knee injuries. Furthermore, cartilage plugs from
adjacent sites were assayed for collagen II cleavage by immunoassay
described by Hollander et al. (47), and no damage to the
collagen network was detected in the normal
group.2
Shriners Hospital for Children,
Henry Ford Hospital, Bone and Joint Center,
Detroit, Michigan 48202
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
disaccharides and terminal mono- or
disaccharides. Chondroitin and dermatan sulfate chains from arthritic
cartilages were of essentially normal size and internal sulfation but
had significantly altered sulfation of the terminal residues.
Whereas in normal cartilage, ~60% of terminal GalNAc4S
was 4,6-disulfated, it was reduced to ~30% in osteoarthritic
cartilage. This is most likely due to a lower terminal
GalNAc4,6S-disulfotransferase activity and reveals that metabolic
changes in osteoarthritis can affect this distinct sulfation step
during chondroitin and dermatan sulfate synthesis.
GlcA
1,3GalNAc6S-, the mimotope for antibody
3B3(
), was present on ~8 and ~10% of chains from normal and
osteoarthritic cartilages, respectively. 3B3(
) assayed by immunodot
blot was within the normal range for most osteoarthritic samples,
with only 5 of 24 displaying elevated reactivity. This resulted not from a higher content of mimotope, but possibly from other structural changes in the proteoglycan that increase mimotope reactivity. In
summary, chemical determination of sulfation isomers at the non-reducing termini of chondroitin and dermatan sulfate provides a
reliable assay for monitoring proteoglycan metabolism not only during
normal growth of cartilage but also during remodeling of cartilage in
osteoarthritis.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
) (27). Assays with this antibody have
been very widely interpreted as a quantitative measure of the
non-reducing terminal sequence GlcA
1,3GalNAc6S on CS
chains (23-26, 28-30).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Cartilage donor information and tissue
sampling sites for femoral condyles and tibial plateaus. A
illustrates the location of the sampling sites in the normal adult knee
joint. B shows a typical OA specimen after tissue
harvesting. Cartilage erosion was present on the medial femoral
condyles and the tibial plateau, for donors I-III, and these sites
contained insufficient tissue for sampling.
Preparation of Aggrecan and Low Buoyant Density PGs from Human
Knee Cartilages--
For the preparation of purified PGs, cartilages
were pooled as follows: from normal donor V and OA donors I-III, plugs
from the medial femoral condyle, plugs from the lateral femoral
condyle, plugs from the medial tibial plateau, or plugs from the
lateral tibial plateau were pooled separately; from normal donors
I-IV, the plugs from both medial and lateral femoral condyles (FC) or from medial and lateral tibial plateaus were pooled. The tissues were
rinsed with ice-cold phosphate-buffered saline containing protease
inhibitors (5 mM EDTA, 1 mM
aminoethylbenzenesulfonyl fluoride, 1 µg/ml pepstatin, 1 mM N-ethylmaleimide, 10 mM
aminohexanoic acid), minced finely on ice, and then extracted for
48 h at 4 °C in 15 volumes of 4 M guanidine HCl, 50 mM sodium acetate, pH 7.0, 1% (w/v) CHAPS, and the
protease inhibitor mixture described above. The extracts were clarified
by centrifugation at 10,000 × g for 10 min and assayed
for total GAG by dimethylmethylene blue (34). About 84 and 89% of
total GAG was solubilized from normal and OA cartilage samples,
respectively. The extracts were adjusted to a final density of 1.48 g/ml with CsCl and subjected to centrifugation at 38,000 rpm for
48 h in a Beckman 50 Ti rotor. The tubes were divided into four
four equal parts (D1-D4). For both normal and OA preparations, ~87%
of the total GAG was recovered in the D1 fraction, ~6% in each of
the D2 and D3 fractions, and ~1% in the D4 fractions. Each fraction
was dialyzed consecutively against water, 1 M NaCl, and
water and then stored at final GAG concentrations of 2 mg/ml at
20 °C.
Preparation of GAGs from Normal and OA Knee Cartilages-- Cartilage plugs from the individual sampling locations (see Fig. 1) were solubilized with 167 µg of papain in 250 µl of 50 mM sodium acetate, pH 5.5, containing 1 mM cysteine HCl, overnight at 60 °C. The digests were clarified by centrifugation at 10,000 × g for 10 min. Three volumes of 95% ethanol were added to the supernatants, and GAGs were precipitated at 4 °C for 18 h. Precipitates were washed once with 95% ethanol, 5 mM sodium acetate, resuspended in water, and assayed for total GAG with dimethylmethylene blue (34). The GAG content was between 0.10 and 0.15 mg per mg dry weight of tissue for normal specimens and between 0.06 and 0.13 mg per mg dry weight for OA specimens.
Analyses of Chondroitin Lyase Digestion Products--
10-µg
portions of GAG (as dimethylmethylene blue reactivity) of D1 and D2/3
PGs were dissolved in 100 µl of 100 mM sodium acetate, pH
7.3, digested for 18 h at 37 °C with 50 milliunits each
chondroitinase ACII and ABC and reduced prior to analyses by capillary
zone electrophoresis for the quantitation of Di-0S and
Di-HA
(33).
Di-0S was present only in digests from D1 PGs and accounted
for no more than 3% of the total CS disaccharide products.
Di-HA
was in digests from all D2/3 fractions and accounted for 10-35% of
the
disaccharide products.
3B3() Immunodot Blot Assay--
Immunoreactivity of D1 and
D2/3 PGs was assessed essentially as described (27). Briefly, PGs were
diluted in Tris-buffered saline to final concentrations of 0.78-100
µg/ml total GAG. Aliquots (25 µl) of each dilution were adsorbed to
either nitrocellulose or nylon N+ membranes for 1 h at
room temperature, using a 96-well dot blot apparatus (Bio-Rad), and
unbound PGs and buffer were removed by vacuum filtration. Under these
conditions, about 30% of the applied PG remained immobilized. It
should be noted that D2/3 fractions contain other PGs, such as decorin,
biglycan, and versican, which may compete for membrane binding with
aggrecan during immobilization. Decorin, biglycan, or versican were not
quantitated in this study, and published data on the abundance of
decorin, biglycan, or versican relative to aggrecan (i.e.
moles of core protein or GAG) in normal adult or OA cartilages are not
available. Thus no attempt was made to assign the 3B3(
) reactivity to
a single PG species in the D2/3 fraction.
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RESULTS |
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Disaccharide Sulfation Isomers of High and Low Buoyant Density
PGs from Normal and OA Cartilages--
Sulfated
disaccharide
composition of the D1 and D2/3 PGs was examined by fluorescence-based
anion exchange HPLC (Fig. 2). In all D1
digests,
Di-6S was predominant accounting for ~95% of the chain
internal disaccharide in normal samples and ~93% in OA samples (Fig.
2, top panels).
Di-4S was low in all samples, and
accounted for ~1 in 20 disaccharides in the normal. This was also
seen in most osteoarthritic samples, and only two were slightly higher
with ~1 in 15 disaccharides 4-sulfated. The disulfated disaccharides,
DiB and
DiD, were detected in all samples
analyzed here (data not shown), but only as minor components (~0.07
and ~1.2%, respectively), and no significant difference was observed between the normal and OA samples. Interestingly,
DiE,
previously shown to be present only in fetal but not postnatal human
aggrecan CS (27), was not detected in any of the normal or OA
samples.
|
Non-reducing Terminal Residues on High and Low Buoyant Density PGs from Normal and OA Cartilages-- The chondroitin lyase products also contained non-reducing termini of the CS and DS chains, as these are quantitively liberated as mono- and saturated disaccharides during the digestion procedure used here. They were also separated and identified by fluorescence anion exchange HPLC (27), before and after mercuric acetate treatment (Fig. 3 and Table I).
|
|
Sulfation Isomer Analyses of Non-reducing Terminal Residues-- The relative contents of the various sulfation isomers in terminal mono- and disaccharides for all D1 and D2/3 PGs from both normal and OA cartilages were determined next from the HPLC analyses (Fig. 4, A and B). As expected (27), both GalNAc4S and GalNAc4,6S were predominant terminal residues for D1 CS from normal cartilages (Fig. 4A, upper panel). GalNAc4,6S was also an abundant terminus on CS and DS from D2/3 PGs in normal cartilages and present on ~55% of chains, compared with GalNAc4S which was on ~37% of chains (Fig. 4A, left-hand panel). Significantly, PGs from OA cartilages revealed striking differences in the abundance of GalNAc4,6S as termini on both D1 and D2/3 PGs (Fig. 4A, right-hand panels). Thus in OA, terminal 4,6-disulfation was present only on ~30% of chains, whereas GalNac4S was increased to about 60% of all chains.
|
3B3() Immmunochemical Analyses of D1 and D2/3 PGs from Normal and
OA Cartilages--
We showed in a recent study, using aggrecan from
normal fetal and adult cartilages, that concentration and presentation
of CS chains containing terminal Di-6S can either enhance or lower 3B3(
) reactivity of PGs immobilized to membranes (27). PGs from
normal and OA cartilages were therefore assayed for 3B3(
) by
quantitative immunodot blotting (see "Experimental Procedures") to
examine if the reported immunochemical distinction between normal and
OA PGs may be due to differential mimotope reactivities. With this dot
blot method, 3B3(
) reactivity was readily detectable in all D1 PG
samples from normal cartilages (Fig. 5,
top panel). Indeed, equivalent reactivities of 100 integrated pixel density units were obtained with ~0.32 and ~0.22
µg of GAG on nitrocellulose or nylon membranes, respectively, in
keeping with our previous results with normal adult human aggrecan
(27). PGs in D2/3 fractions from normal cartilages were also reactive,
and 100 integrated pixel density units were generated with ~0.20 µg
of GAG assayed on nitrocellulose and with ~0.10 µg assayed on nylon
(Fig. 5, bottom panel).
|
|
Non-reducing Terminal GalNAc Sulfation Isomers on GAGs from
Distinct Sites on Normal and OA Knee Joint Cartilages--
The data in
Fig. 4A clearly indicate that the marked decrease of
4,6-disulfation of non-reducing terminal GalNAc residues on CS and DS
could represent a readily quantifiable, specific alteration in the fine
structure of GAGs in cartilages from OA knee joints. We therefore
examined the effect of anatomical site on the proportion of chains with
this terminal structure in normal cartilages. Then we determined if the
decreased content in OA cartilages could be related to any variations
in the gross macroscopic appearance of the OA cartilages. Total tissue
GAGs were prepared from the individual sampling sites (see
"Experimental Procedures" and Fig. 1) from five normal adult joints
and five OA joints. The disaccharide and non-reducing terminal
compositions were essentially identical to those obtained for the D1
PGs prepared from pools of cartilage from the four surfaces of the same
joints (data not shown). No differences in the proportion of the
terminal disaccharides, Di-4S and Di-6S, were found between normal and OA cartilages or between anatomical sites within each group. However, the mean contents of GalNAc4,6S terminating chains in medial tibial, lateral tibial, medial femoral, and lateral femoral joint cartilages from normal donors were 52.1 ± 6.4%, 52.0 ± 8.0%,
50.6 ± 10.2%, and 50.3 ± 9.3%, respectively, indicative
of no marked site-variation in content. Furthermore, this was
significantly reduced to 33.5 ± 14.2%, 28.1 ± 11.9%,
38.4 ± 12.6%, and 31.9 ± 9.8%, respectively, for the
corresponding cartilages from OA patients (Fig.
6).
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DISCUSSION |
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We report here the analyses of the GAG fine structure of CS/DS PGs
from femoral condyles and tibial plateau cartilages from healthy adults
and patients with late stage OA. Our particular interest was in the
separation and quantitation of sulfation isomers of both internal
disaccharides and non-reducing terminal residues of the GAG chains
(27, 33) in order to identify distinct structural alterations of GAGs
in cartilages from OA patients.
Minor increases (1-2%) in 4-sulfation of D1 CS was seen in 2 of 12 OA samples, which had previously been observed using either chemical (20) or immunochemical (26, 42) analyses of CS PGs prepared from OA femoral head cartilages. However, for all other CS and DS PG preparations from the diseased cartilages, the degree of sulfation and the abundance of sulfation isomers in the chain interior was the same as that in healthy adult cartilages (27). Our analyses indicated that the mean size of CS chains on aggrecan in OA cartilages may be slightly smaller than in normal cartilages (Table I). Thus chains from the normal group had on average ~19 repeating disaccharide units, compared with ~16 repeating disaccharide units in the diseased group. This observation is in general agreement with earlier reports describing unchanged (22) or decreased (43) hydrodynamic sizes of GAG chains in OA cartilages as determined by gel filtration chromatography.
Of particular interest were the quantitative chemical data for the
non-reducing terminal sequence GlcA1,3GalNAc6S (Di-6S), and these were present on ~6-8% of chains from PGs from both normal and OA cartilages. This chain terminal structure represents the mimotope recognized by the monoclonal antibody 3B3(
) (27), and all
PGs prepared for this study exhibited easily detectable reactivity in
immunodot blot assays (Fig. 5). These findings are in apparent contrast
to conclusions drawn from reports by others (24, 30) that
3B3(
)-reactive PGs were apparently absent in normal cartilages but
could be demonstrated in OA cartilages. This discrepancy might,
however, in part be explained by observations made here as follows.
First, several PG preparations from OA cartilages displayed a
significantly higher 3B3(
) reactivity than normal PGs (Fig. 5);
however, this was not due to differences in mimotope content (Fig.
4B). On the other hand, the mimotopes were more reactive in
these samples (Table II), and as we had previously shown for
membrane-immobilized PGs, this can be influenced by the sulfation or
length of the chains ending in Di-6S. Second, the threshold for
detection of 3B3(
) in the solid phase assays is very steep (27) and
was also seen in the present study (Fig. 5). With the differences for
individual PGs (Ref. 27, and Table II), it is evident that reactivities
in solid phase immunoassays at low PG concentrations (i.e.
<100 ng of GAG, see Table II) could be easily interpreted as the
presence or absence of mimotope. These data further support our
previous conclusion that 3B3(
) reactivity detected by Western blots
or immunohistochemistry cannot be used alone to quantitate changes in
the fine structure of CS chains during cartilage development (27) or
pathologies (Figs. 4 and 5).
On the other hand, CS and DS PGs in all OA cartilages analyzed here could be distinguished from those in normal adult cartilages by the sulfation of the chain terminal GalNAc residue. PGs from all normal adult human cartilages showed a high content of GalNAc4,6S chain termini (Fig. 5 and Ref. 27), but in OA cartilages, these were 40-50% less, with proportionally more GalNAc4S termini. This sulfation change was seen in both femoral condyle and tibial plateau cartilages, and it was not confined to a particular site on these surfaces, nor was it related to the gross morphological appearance or the degree of erosion of the tissues.
The distinctive compositional change of PGs in OA cartilages could well
be the result of changes in both the catabolic and anabolic pathways of
chondrocytes during the progression of the disease. Thus, during phases
of increased matrix degradation in the OA cartilage, normal adult PGs,
abundantly substituted with chains terminating with GalNAc4,6S, might
be removed from the cartilage into the synovial fluid for clearance. In
this respect it is interesting to note that Hazell et al.
(30) detected an increase in 3B3()-reactive PG fragments in human
synovial fluid after traumatic knee injuries, by competitive
enzyme-linked immunosorbent assay. Based on our findings that
3B3(
)-reactive CS is abundant in the normal adult cartilage matrix,
the immunoassay might have detected release of degraded aggrecan that
is resident in the healthy adult articular cartilage, rather than
release of degraded aggrecan synthesized in response to the injury.
Future work aimed at quantitation of CS chains with GalNAc4,6S in
synovial fluids of such a patient group, as an alternative measure for
release of normal resident aggrecan, should yield additional
information on the origin of PG fragments released from cartilages
after joint trauma.
The terminal GalNAc 4,6-disulfation reaction occurs as a final step in
CS/DS biosynthesis, is carried out by a distinct 6-sulfotransferase with specificity for GalNAc4S termini, and is modulated independently of the chain interior 6-sulfation (27, 44). Alterations in the GAG
biosynthesis pathways in OA cartilages may lower their capacity for
4,6-disulfation of the non-reducing termini, thus resulting in the
synthesis and deposition of CS/DS with predominantly monosulfated
termini. It should be noted that terminal 4,6-disulfation of aggrecan
CS is also low or absent in normal fetal and postnatal growth human
knee cartilages (27). However, the change in terminal GalNAc sulfation
in OA cannot simply be explained by the often quoted suggestion
(18-21) that chondroytes in human OA cartilages synthesize aggrecan
substituted with CS typical of fetal or growth chondrocytes. CS chains
in fetal and postnatal growth cartilages are typically large
(containing ~40 disaccharide repeats) and have a high content
(~50%) of 4-sulfated internal disaccharides (27). If the proportion
of normal adult aggrecan CS which remains in the OA cartilages,
estimated from the abundance of chains with GalNAc4,6S termini, ranged
from 50 to 80% of the total, then 20-50% of chains present on
aggrecan would have been synthesized during the progression of the
disease. If this newly synthesized CS was in the fetal or growth
structure, both the Di-4S content and the average chain lengths
would be expected to be significantly increased in OA (by 10-20%),
but this was not detected in the current analyses (Fig. 2 and Table I).
However, based on chain size and both internal and non-reducing
terminal sulfation patterns, it is tempting to speculate that newly
synthesized CS in OA cartilages may be similar to that in cartilages
taken from adolescents (15-17 years of age), just before reaching
skeletal maturity (27).
In conclusion, the data described here provide for the first time quantitative chemical evidence to support the contention that metabolic changes in osteoarthritic cartilages may affect distinct sulfation steps in chondroitin and dermatan sulfate synthesis. Furthermore, the detection and quantitation of such alterations from assays of sulfation isomers of non-reducing terminal GalNAc residues, using the methods described here, should provide a powerful new approach to monitoring proteoglycan metabolism during cartilage remodeling in osteoarthritis (46).
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ACKNOWLEDGEMENTS |
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We thank J. Boyer and V. Thompson (Shriners Hospital, Tampa Unit) for performing the Western blot analyses. We are grateful to Dr. W. E. Kilgore and the Operating Room Staff of Morton Plant Hospital, St. Petersburg, FL, as well as the Organ Procurement Agency of Michigan for their invaluable cooperation in the collection of the cartilage specimens.
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FOOTNOTES |
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* This work was supported by a grant from the Shriners of North America (to A. H. K. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Cell Biology Laboratory, Shriners Hospital for Children, 12502 North Pine Dr., Tampa, FL 33612. Tel.: 813-972-2250; Fax: 813-975-7127; E-mail: annaplaas{at}delphi.com.
1
The abbreviations used are: OA, osteoarthritis;
PG, proteoglycan; GAG, glycosaminoglycan; CS, chondroitin sulfate;
HPLC, high performance liquid chromatography; DS, dermatan sulfate;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; the structures of the unsaturated disaccharides Di0S
Di-4S,
Di-6S,
DiB,
DiD and
DiE
are given (33); GalNAc4S, N-acetylgalactosamine 4-sulfate;
GalNAc4,6S, N-acetylgalactosamine 4,6-disulfate.
2 F. Nelson and A. R. Poole, unpublished observations.
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REFERENCES |
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