(Received for publication, December 14, 1994; and in revised form, February 8, 1995)
From the
Chondrocyte cultures derived from the Swarm rat chondrosarcoma
were metabolically labeled with [S]sulfate or
[6-
H]GlcN. Radiolabeled aggrecan was purified
from the cell layer and exhaustively digested with chondroitin ABC
lyase. Digestion products were resolved into disaccharide and
monosaccharide residues using Toyopearl HW40S chromatography. The
separated saccharide pools were reduced with NaBH
and
applied onto a CarboPac PA1 column to resolve all of the internal
disaccharide alditols (unsaturated) from the nonreducing end
disaccharide (saturated) and monosaccharide alditols. Mercuric acetate
treatment was used prior to carbohydrate analysis to identify
unambiguously the saturated from the unsaturated disaccharides. The
chondroitin sulfate (CS) chains from these aggrecan preparations
contained: (a) an internal disaccharide composition of
unsulfated (3-4 per chain), 4-sulfated (
32 per chain),
6-sulfated (
1 per 14 chains), and 4,6-sulfated disaccharides
(
1 per 6 chains) and (b) a nonreducing terminal
composition of 4-sulfated GalNAc (
4 out of every 7 chains),
4,6-disulfated GalNAc (
2 out of every 7 chains), and GlcUA
adjacent to a 4-sulfated GalNAc residue (
1 out of every 7 chains).
Thus, the vast majority of these CS chains terminated with a sulfated
GalNAc residue. The presence of 4,6-disulfated GalNAc at nonreducing
termini is 60-fold more abundant than 4,6-disulfated GalNAc in interior
disaccharides. This observation is consistent with the suggestion that
disulfation of terminal GalNAc residues is involved in chain
termination.
Glycosaminoglycan chain termination is a long-standing issue in
proteoglycan
research(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) .
It has received renewed attention as a result of research suggesting
that changes occur in the sulfation patterns of chondroitin sulfate
(CS) ()chains on proteoglycans synthesized by chondrocytes
in osteoarthritic cartilage (11, 12, 13) as
well as during tissue
development(11, 14, 15, 16) . CS
chains can be terminated by either a GalNAc or a GlcUA, and each of
these terminal residues can be sulfated in various positions. One
approach to this problem has been to isolate and identify the
nonreducing termini of glycosaminoglycans(5, 6) . The
findings of these studies suggest that CS chains are often terminated
with sulfated GalNAc residues. However, it is still not known precisely
how many chains end with a GalNAc versus a GlcUA residue for a
purified proteoglycan species. Furthermore, a quantitative assessment
of the sulfation patterns at the nonreducing termini still needs to be
rigorously addressed.
In this study, a strategy was devised to
quantify the nonreducing termini on a model proteoglycan, aggrecan.
Aggrecan was metabolically labeled with
[S]sulfate and [
H]GlcN
precursors in chondrocyte cultures derived from rat chondrosarcoma
tissue and purified by a series of chromatographic steps. A portion of
the preparation was used to determine the number-average molecular
weight of its CS chain population by monosaccharide analysis to compare
the GalN to Gal (2 residues per chain) contents. Intact aggrecan was
then digested exhaustively using highly purified chondroitin ABC lyase.
This eliminase cleaves the
1
4 bond between GalNAc and
GlcUA residues in CS chains converting the GlcUA residue to a
4,5-unsaturated uronosyl and releasing a GalNAc with a free reducing
group(17, 18) . In limit digests, internal GlcUA
residues yield unsaturated disaccharides. A CS chain with a nonreducing
terminal GlcUA residue will yield one disaccharide with a saturated
GlcUA at its nonreducing end, while a chain with a substituted
nonreducing GalNAc will yield this GalNAc monosaccharide and no
saturated disaccharides(19) . The procedures used in this study
resolves these nonreducing terminal monosaccharides and disaccharides
from the interior unsaturated disaccharides. In addition, mercuric
acetate treatment was employed to convert the unsaturated disaccharides
to their respective substituted GalNAc derivatives by degrading the
unsaturated uronosyl residue and cleaving the glycosidic
bond(20, 21, 22) , while leaving the
saturated disaccharides intact. This procedure unambiguously identifies
a small number of saturated disaccharides in the presence of a large
amount of unsaturated disaccharides. Altogether, this strategy provides
an analysis of the spectrum of nonreducing termini and an estimate of
the number-average molecular weight of the population of CS chains in
the proteoglycan preparation.
CS tetrasaccharides
recovered from the Toyopearl HW40S column were vacuum-dried,
reconstituted in 100 µl of 0.1 M sodium acetate, pH 7.0,
and then digested with 0.01 unit of chondroitin ABC lyase (1 h at 38
°C). Digestion products (ultrafiltrate) were isolated from the
enzyme (retentate) using Microcon 3 microconcentrators. These digestion
products were reduced using a modified borohydride reduction
procedure(29) . Ultrafiltrates (diluted to 1 mM uronic acid) were adjusted to 50 mM NaBH
in
50 mM sodium acetate, pH 7.0, by adding neutral-pH Nanopure
water and an aliquot of 500 mM NaBH
in 10 mM sodium acetate, pH 7.0 (prepared immediately before adding to the
sample). Reduction of the reducing-end, aldehyde group was achieved by
a 30-min incubation at 38 °C; the unsaturated bond within the
uronosyl residue was not reduced by this procedure(29) .
Following this reaction, the sample was placed on ice, and ice-cold 0.1 M acetic acid was added until all excess borohydride was
exhausted. The reduced disaccharide products were isolated (and
simultaneously desalted) by Toyopearl HW40S chromatography followed by
Speed Vac drying. A portion of this dried sample was treated with 35
mM mercuric acetate, pH 5.0, for 30 min at room temperature to
degrade unsaturated disaccharides (20, 21, 22) . As described
previously(22) , residual mercury was removed from the samples
by Dowex AG 50W-X8 cation exchange before any additional analyses were
performed.
Desalted monosaccharide and disaccharide samples were
reduced with 50 mM NaBH in 50 mM sodium
acetate, pH 7.0, and subsequently retreated with Dowex as described
above. After Speed Vac drying, samples were resuspended with 1 ml of
ice-cold, absolute methanol and dried again to remove residual borate.
Generally, a second cycle of methanol drying was needed to remove all
traces of borate. Samples were then applied to and eluted from a
CarboPac PA1 column, before and after mercuric acetate treatment, as
described above.
A portion of the CS chains was exhaustively digested
with testicular hyaluronidase. The digestion products resolved into one
major and three minor peaks on Toyopearl HW40S (Fig. 1A). The major peak (89% of the S
and 82% of the
H) contains CS tetrasaccharides that eluted
in a position between HA tetra- and hexasaccharides (Fig. 1A, bar). A portion of the
tetrasaccharide peak was digested with chondroitin ABC lyase, reduced
with NaBH
, and reapplied to the HW40S column (Fig. 1B). Two major and three minor peaks were
observed. The first of the two major peaks (49% of the
S
and 45% of the
H) eluted slightly earlier than the CS
disaccharide peak generated by testicular hyaluronidase (A),
while the second (48% of the
S and 44% of the
H) eluted slightly earlier than the saturated HA
disaccharide standard (arrowhead 2, A). These two
major peaks were recovered together as one sample (bar, B). A portion of this sample was reapplied to the HW40S column
without further treatment (Fig. 1C, peaks Iand IIa), while another aliquot was treated
with mercuric acetate as described under ``Experimental
Procedures'' and then reapplied to the HW40S column (Fig. 1D). Peak I was unaltered by mercuric ion
treatment, while peak IIa was cleaved into monosaccharides by the
treatment (peak IIb). These data indicate that peak I consists of
saturated CS disaccharide alditols, while peak IIa consists of
unsaturated CS disaccharide alditols.
Figure 1:
Isolation
of saturated chondroitin 4-sulfate disaccharide. A, CS chains
digested with testicular hyaluronidase were applied to a Toyopearl
HW40S column. The bar indicates the CS tetrasaccharides
recovered for further analysis. Numbered arrowheads (8, 6,
4, 2, and 1) denote the elution positions for authentic
standards of HA, HA
, HA
, HA
disaccharide, and GlcN, respectively. B, CS tetrasaccharides
from A were digested with chondroitin ABC lyase, reduced with
NaBH
, and eluted on the HW40S column. The bar indicates the disaccharides recovered for further analysis. The arrow denotes the elution position of the CS tetrasaccharide.
These disaccharides were reapplied to the HW40S column in C (designated I and IIa). D, the
disaccharides recovered in B were treated with mercuric
acetate prior to elution on the HW40S column (products are designated I and IIb).
Equal aliquots of the samples
represented in Fig. 1, C and D, were analyzed
on a CarboPac PA1 column (29) in order to identify these
disaccharides. The untreated sample (Fig. 2A) resolved
into two peaks (Iand IIa) of nearly
equal proportions. Peak I eluted in a position (32 min) that was unique
from all known unsaturated disaccharide standards, while peak IIa
eluted in the position of the Di-4S
standard. Analysis
of the mercuric acetate-treated sample (Fig. 2B)
indicated that peak I was resistant to mercuric ion cleavage, while
peak IIa (
Di-4S
) was converted to a new structure
(peak IIb) with a unique elution position (24 min) from all of the
disaccharide standards. Peak IIb is deduced to be N-acetylgalactosaminitol-4-sulfate (4SGalNAc
)
since it is a sulfated monosaccharide derived from
Di-4S
and contains only [
H]galactosaminitol after
acid hydrolysis and hexosamine/hexosaminitol analysis (data not shown).
Figure 2:
Elution position of saturated chondroitin
4-sulfate disaccharide alditol (Di-4S) and 4-sulfated N-acetylgalactosaminitol (4SGalNAc
) on CarboPac
PA1. The column was eluted with a gradient of sodium trifluoroacetate (TFA) in 0.1 M NaOH (indicated by a dashed line in A). A depicts the elution profile for the
intact disaccharides (I and IIa) recovered from HW40S (Fig. 1B). Numbers indicate the elution
positions of the following disaccharide standards: 1,
Di-0S
; 2,
Di-HA
; 3,
Di-4S
; 4,
Di-2S
; 5,
Di-6S
; 6,
Di-2,4S
; 7,
Di-4,6S
; 8,
Di-2,6S
. B shows the elution positions of the
mercuric acetate-treated products (designated I and IIb) derived from disaccharides I and
IIa.
Peak I is deduced to be reduced, saturated chondroitin 4-sulfate
disaccharide (Di-4S) because it (a) is derived
from a chondroitin 4-sulfate tetrasaccharide generated by the hydrolase
testicular hyaluronidase, which leaves a GlcUA residue at the
nonreducing end of all digestion products(34) , (b)
elutes in a disaccharide size range on HW40S after chondroitin lyase
digestion, (c) is resistant to mercuric acetate treatment, and (d) contains only [
H]galactosaminitol
after acid hydrolysis and hexosamine/hexosaminitol analysis (data not
shown). Altogether, these analyses establish the unique elution
positions of Di-4S
and 4SGalNAc
(two potential
nonreducing termini of CS chains) on the CarboPac PA1 column.
Figure 3: Isolation of disaccharides and monosaccharides generated by chondroitin ABC lyase digestion of aggrecan. Labeled aggrecan was exhaustively digested with chondroitin ABC lyase, and its digestion products were applied onto a Toyopearl HW40S column. The monosaccharide peak (1) was well separated from the disaccharide peak (2). The asterisk denotes a trisaccharide peak mentioned in the text. The elution positions of the CS tetrasaccharide (CS tetra) isolated in Fig. 1A and glucosamine (GlcN) are provided as reference standards. The inset depicts the full scale profile.
Figure 4:
Identification of the monosaccharides
generated by chondroitin ABC lyase digestion of aggrecan.
Monosaccharides recovered in Fig. 3(peak 1) were
reduced with NaBH and applied to a CarboPac PA1 column.
Samples were applied either before (A) or after (B)
mercuric acetate treatment. The presence of minor amounts of
Di-0S
and
Di-4S
is the result of
cross-contamination of peak 1 with disaccharides from peak
2.
Figure 5:
Identification of the disaccharides
generated by chondroitin ABC lyase digestion of aggrecan. Disaccharides
recovered in Fig. 3(peak 2) were reduced with
NaBH and applied to a CarboPac PA1 column. Samples were
applied either before (A and C) or after (B and D) mercuric acetate treatment. C and D provide expanded views of the baselines in A and B, respectively. The asterisk in D denotes a
minor peak that might possibly represent a small amount (
0.1%) of
Di-6S
(Anna H. K. Plaas, personal communication). Insets in C and D depict a greatly magnified
view of the baseline between fractions 41 and 56.
SO
, inorganic
sulfate.
After mercuric acetate treatment, one
major and three minor identifiable species were detected during the
CarboPac PA1 run. The major peak eluted in the position of
4SGalNAc and is the degradation product derived from
Di-4S
. An expanded scale in Fig. 5D revealed three minor peaks: (a) GalNAc
which
is derived from
Di-0S
, (b) 4,6SGalNAc
which is derived from
Di-4,6S
, and (c)
Di-4S
. Additionally, the disaccharide peak that eluted in
the position of
Di-6S
was susceptible to mercuric
acetate treatment (Fig. 5D, inset). Thus,
mercuric acetate treatment converted quantitatively all of the
unsaturated disaccharide alditols into their respective, earlier
eluting monosaccharide alditols, thereby exposing the intact
Di-4S
peak.
These data demonstrate that the CS chains
from rat chondrosarcoma aggrecan contain a small amount of Di-4S after chondroitin lyase digestion. The presence of saturated
disaccharides after chondroitin lyase digestion indicates that they are
at the nonreducing ends of CS chains. Table 1summarizes the
proportion of each internal disaccharide and nonreducing terminal unit
accounted for in these analyses as a percentage of the
H
(from GlcN) released from aggrecan by chondroitin ABC lyase. The
nonreducing termini identified in this study account for
3.1% of
this
H activity.
This study has devised a strategy to analyze the nonreducing termini of CS chains. An underlying premise of this strategy is that, when completely degraded by chondroitin lyase, CS chains terminating with a GalNAc residue will yield free, variably sulfated GalNAc monosaccharides, while those terminating with a GlcUA will yield saturated disaccharides of various degrees of sulfation(19) . The strategy involves: (a) exhaustive degradation of the CS chains using highly purified chondroitin ABC lyase which releases nonreducing terminal residues from the internal, repeating disaccharide units; (b) separation of resultant disaccharide and monosaccharide products using high resolution, low molecular weight gel permeation chromatography; and (c) identification of these digestion products using high performance anion exchange chromatography on CarboPac PA1. Additionally, mercuric acetate treatment was used to identify unambiguously any saturated (mercuric ion resistant) from unsaturated (mercuric ion sensitive) disaccharides. Exhaustive chondroitin ABC lyase digestion was critical for this approach since limited digestions revealed trisaccharides that contained a nonreducing terminal GalNAc residue and the adjacent internal disaccharide, thus complicating subsequent structural analyses (see asterisk in Fig. 3).
To achieve validation of this strategy, this study focused on analyzing the saccharide composition of CS chains on aggrecan synthesized by chondrocytes isolated from the Swarm rat chondrosarcoma. This is a well established chondrocyte model system capable of producing a large quantity of aggrecan. Aggrecan synthesized by these cells is relatively easy to purify, and the overall structure of its CS chains is well characterized, with nearly uniform sulfation on C4 of the GalNAc residues(22) . A metabolic labeling approach was chosen because it provides the sensitivity and quantitation required for this analysis and emphasizes a structural analysis of the stable, nonreducing end structures on newly synthesized aggrecan. This strategy is optimal for identifying and quantifying the nonreducing termini of CS chains from an intact proteoglycan.
Table 1and Fig. 6summarize our findings. A novel
observation was made concerning the internal disaccharide composition
of the CS chains from rat chondrosarcoma aggrecan. Minute amounts of
both Di-6S
and
Di-4,6S
were detected
as a result of the high resolution capacity of the CarboPac PA1 column.
Based on the molar ratio of GalN per 2 Gal residues (
37), the
number of internal and terminal GalNAc residues released from one CS
chain by chondroitin ABC lyase would be
36. The enzyme leaves one
disaccharide, mostly unsulfated(22) , bound on the linkage
oligosaccharide(35, 36) . Excluding this first
disaccharide, the proportions of the internal disaccharides are
32
4-sulfated and 3-4 unsulfated internal disaccharide units per CS
chain, about one 4,6-sulfated disaccharide per 6 CS chains, and
approximately one 6-sulfated disaccharide per 14 CS chains. (
)
Figure 6:
Data summary for nonreducing terminal
structures on CS chains from rat chondrosarcoma aggrecan. Ratios for
nonreducing termini were calculated from data in Table 1as
follows: 1.76/3.03 4/7 for those chains ending with a 4SGalNAc
residue, 0.88/3.03
2/7 for those chains ending with a 4,6SGalNAc
residue, and 0.39/3.03
1/7 for those chains ending with a GlcUA
residue. Arrows indicate the cleavage positions of chondroitin
ABC lyase. Trisaccharides, obtained in small yields, are products of
cleavage of chains terminating in GalNAc at the internal sites
indicated by asterisks. These trisaccharides are digested only
very slowly by chondroitin ABC lyase (R. J. Midura, A. Calabro, M.
Yanagishita, and V. C. Hascall, unpublished
data).
The expected proportion of terminal residues compared
to the total combined internal and terminal units per chain is
calculated to be 1 out of 36 or 2.8%. In close agreement with this
value, the actual proportion of nonreducing termini in these chains is
calculated to be
3.1% of the total chondroitin ABC
lyase-digestible products (Table 1). Three nonreducing termini
were identified:
4 out of 7 CS chains terminated with a 4SGalNAc
residue,
2 out of 7 with a 4,6SGalNAc residue, and
1 out of 7
with a GlcUA residue adjacent to a 4SGalNAc residue (Fig. 6).
Lacking appropriate authentic standards, this study cannot exclude the
possibility that extremely small amounts of 6-sulfated or
4,6-disulfated saturated disaccharides, or 6SGalNAc, were also present
in these aggrecan preparations. These data are consistent with: (i)
studies reporting that a majority of CS chains from embryonic cartilage
terminate with either 4SGalNAc or 4,6SGalNAc residues (5, 6) and (ii) studies suggesting that 4-sulfation of
GalNAc termini on CS chains may stop chain elongation because CS chains
terminating with a 4SGalNAc residue are poor acceptors for subsequent
GlcUA addition in
vitro(1, 2, 3, 4) .
Of
particular interest, the present study argues that there is a 60-fold
greater incidence of 4,6SGalNAc residues at the nonreducing end
position as compared to internal positions. ()Other groups (5, 6) have reported a higher frequency of
4,6-disulfation of GalNAc residues at the nonreducing terminal position
when compared to those in internal positions of CS chains. This
suggests the intriguing possibility that the disulfation of GalNAc is
differentially regulated when this hexosamine resides at the terminus versus in an internal position within the chain. Indeed, some
investigators have identified a 6-O-sulfotransferase activity
which readily adds a sulfate group to the C6 position of 4SGalNAc at
the chain terminus, but not when the 4SGalNAc is in an internal
position within CS chains(7, 8, 37) .
Perhaps, as previously suggested(6, 8, 37) ,
disulfation of GalNAc residues at the nonreducing end is involved in CS
chain termination.
It is not known whether these nonreducing termini represent the actual residues that signal CS chain termination. It is possible that rapid trimming events might occur immediately after chain termination which, in effect, result in the stable, nonreducing end groups reported above. Additionally, the results of this study do not exclude the possibility that other classes of proteoglycans (or aggrecan from other tissue sources, including pathological or developmental conditions) may have a higher proportion of their CS chains terminating with a GlcUA residue or with 6-sulfated residues. Application of the methods described in this paper should help resolve these questions.