(Received for publication, October 27, 1994; and in revised form, December 16, 1994)
From the
Five major hexasaccharide alditols were isolated from the
carbohydrate-protein linkage region of bovine aorta dermatan sulfate
peptidoglycans after reductive -elimination and subsequent
chondroitinase ABC digestion. These molecules account for at least
55.3% of the total linkage region. Their structures were analyzed by
enzymatic digestion in conjunction with high performance liquid
chromatography, electrospray ionization mass spectrometry, and 500-MHz
one- and two-dimensional
H NMR spectroscopy. Three of these
compounds have the conventional hexasaccharide core;
HexA
1-3GalNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl-ol.
One is nonsulfated, and the other two are monosulfated on C6 or C4 of
the GalNAc residue. They represent at least 6.3, 5.2, and 28.8% of the
total linkage region, respectively. The other two compounds have the
following hitherto unreported hexasaccharide core with an internal
iduronic acid residue in common;
HexA
1-3GalNAc
1-4IdoA
1-3Gal
1-3Gal
1-4Xyl-ol.
One is monosulfated on C4 of the GalNAc, and the other is disulfated on
C4 of the GalNAc and of the galactose residue substituted by the
iduronic acid residue. These two compounds account for 35% of the five
isolated hexasaccharide alditols and at least 4.3 and 10.7% of the
total linkage region, respectively. The latter two structures form a
striking contrast to the currently accepted conception that heparin,
heparan sulfate, and chondroitin/dermatan sulfate share the common
linkage tetrasaccharide core
GlcA
1-3Gal
1-3Gal
1-4Xyl. The biological
significance of the isolated structures is discussed in relation to the
biological functions and the biosynthetic mechanisms of dermatan
sulfate.
Dermatan sulfate is an extracellular matrix component of fibrous
connective tissues and is also present on cell surfaces as a
proteoglycan. Several biological functions of proteodermatan sulfate
are known such as extracellular matrix formation through interaction
with several types of collagen(1, 2, 3) ,
inhibition of the mitogenic activity of transforming growth
factor-(4) , and inhibition of the cell attachment
activity of fibronectin(5) . These functions are attributable
to the core protein. Dermatan sulfate chains also mediate activities
such as anticoagulant activity, self-association activity, and
antiproliferative activity. The anticoagulant activity is expressed by
binding to heparin cofactor II(6) . Self-association via
protein-protein interactions is known, but it also occurs through
carbohydrate-carbohydrate interactions(7) . Dermatan sulfate
chains rich in iduronic acid have been demonstrated to be
antiproliferative and also interact with spermine(8) . However,
the structure-function relationship of dermatan sulfate chains is not
fully understood mainly because of their complicated structure.
Structurally, dermatan sulfate is similar to chondroitin sulfate and
was previously called chondroitin sulfate B(9) . It is
generally accepted that chondroitin sulfate and dermatan sulfate as
well as heparin and heparan sulfate are covalently bound to serine of a
protein core through the common tetrasaccharide structure
GlcA1-3Gal
1-3Gal
1-4Xyl
1- in the
carbohydrate-protein linkage region(10, 11) . Attached
to the linkage region is a long carbohydrate sequence, a so-called
repeating disaccharide region, which is composed of alternately
arranged uronic acid and N-acetylgalactosamine residues. The
uronic acid in the repeating disaccharide units of chondroitin sulfate
is glucuronic acid, whereas that in dermatan sulfate is either iduronic
or glucuronic acid. Thus, dermatan sulfate contains two types of
disaccharide units, -4GlcA
1-3GalNAc
1- and
-4IdoA
1-3GalNAc
1-. (
)The disaccharide
repeats are modified by sulfation. The former can be sulfated at C4 or
C6 of the GalNAc unit, whereas the latter contains almost exclusively
4-sulfated GalNAc units and a minor proportion of IdoA unit which may
be sulfated at C2. Combining sequential arrangements of iduronic acid
residues and sulfate groups results in a wide variety of domain
structures, some of which could have biological activities.
It is known that iduronic acid-rich glycosaminoglycans, like dermatan sulfate and heparan sulfate, inhibit fibroblast proliferation and that the antiproliferative activity appears to be related to iduronic acid content(12) . Although the role of IdoA units is important, their distribution along a given dermatan sulfate chain or the relationship between the structure and biological functions is not well understood. A recent structural study of the binding domain to heparin cofactor II is the best known example showing the relationship between the structure and biological functions of dermatan sulfate(13) . The results of this previous study revealed a unique hexasaccharide structure containing a cluster of three IdoA(2-sulfate)-GalNAc(4-sulfate) repeats, which comprises only 5% of the disaccharides present in intact dermatan sulfate.
The biosynthetic mechanisms of such a domain structure of dermatan sulfate remain obscure. Dermatan sulfate is synthesized basically by a mechanism similar to that established for chondroitin sulfate. Chain initiation occurs by xylosylation of the serine residue of a core protein, followed by the addition of 2 galactose residues yielding the Gal-Gal-Xyl trisaccharide sequence which links each polysaccharide chain via a terminal glucuronic acid residue to the core protein. Chain elongation then occurs by alternate addition of GalNAc and GlcA units. Modification reactions take place at the polymer level. Iduronic acid in dermatan sulfate is formed from glucuronic acid by epimerization, most likely after C4 sulfation of the adjacent N-acetylgalactosamine(14) . Epimerization and C4 sulfation of N-acetylgalactosamine are tightly coupled, whereas C2 sulfation of iduronic acid occurs after the epimerization and does not seem to play any role in the formation of iduronic acid.
We have been analyzing the structure of the carbohydrate-protein linkage region of various sulfated glycosaminoglycans to investigate the structure-function relationship and the biosynthetic mechanisms of these glycosaminoglycans(15, 16, 17, 18, 19, 20) . Since the linkage region is first constructed in biosynthesis, possible differences in the structure of the linkage region may influence that of the repeating disaccharide region to be synthesized thereafter. Recently, we found novel 4-sulfated and 6-sulfated galactose units in the linkage region of chondroitin sulfate chains(15, 16, 17, 18, 19) . In this study, we isolated and characterized the linkage region oligosaccharides from bovine aorta dermatan sulfate to examine possible structural variability and differences in this region from other glycosaminoglycans, especially chondroitin sulfate.
The peptidoglycan fraction (100 mg, 1.1 µmol as serine) was
reduced with alkaline NaBH, and a small portion (1 mg) was
treated separately with alkaline NaB[
H]
to prepare tracers by radiolabeling the reducing ends of the
polysaccharides. The nonlabeled (100 mg) and
H-labeled
reduced fractions (1 mg, 6.1
10
cpm) were isolated
by gel filtration on Sephadex G-50, mixed, digested exhaustively by
chondroitinase ABC, and the digest was fractionated by gel filtration
on a Bio-Gel P-2 column. In addition to the major UV-absorbing
disaccharide fraction, the preceding radioactive fractions, presumed to
contain linkage oligosaccharides, were observed and separated into
three subfractions (Fig. 1). Fractions B-1, -2 and -3, which
contained 31, 53, and 16% of the total radioactivity, respectively,
were separately pooled and concentrated. In this study the major
radioactive fraction, B-2, was used for isolation of the linkage
oligosaccharides as described below. Most (
70%) of the
H label in fraction B-1 was associated with presumable
internal oligosaccharides, which were generated probably as a result of
an incomplete depolymerization by chondroitinase ABC, and were not
investigated further. Most (
90%) of the
H label in
fraction B-3 was recovered in a trisaccharide by HPLC, which was
characterized as
HexA
1-3GalNAc(4-sulfate)
1-4[
H]HexA-ol
by enzymatic analyses in conjunction with HPLC, ESI mass spectrometry
and
H NMR spectroscopy. (
)It might have been
produced by alkali-peeling degradation of the linkage region.
Alternatively or in addition it could have been derived from the
reducing termini of free glycosaminoglycan chains in the starting
peptidoglycan preparation, which had been possibly generated by tissue
endoglycosidase as discussed previously for similar sulfated
trisaccharides isolated from various chondroitin/dermatan sulfate
preparations including the one used in this study(21) .
Figure 1:
Gel
filtration of the chondroitinase ABC digests of the H-labeled glycans. The chondroitinase ABC digestion was
carried out as described under ``Experimental Procedures.''
The digest was chromatographed on a column (1.0
115 cm) of
Bio-Gel P-2 with 0.25 M NH
HCO
, 7%
1-propanol as the eluent. The fraction size was 1 ml, and 1-µl
aliquots were used for determination of the radioactivity (
).
Aliquots were also used to measure the absorbance at 232 nm (
).
Fractions B-1, B-2, and B-3 were pooled as indicated. V
is at around fraction 90 (not
shown).
Fraction B-2, which contained 1.26 µmol of oligosaccharides (as
HexA), was fractionated by HPLC (Fig. 2). Of the separated
fractions, fractions 3, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 17 were
labeled with
H, indicating that they probably contained
oligosaccharide alditols derived from the linkage region (Table 2). In this study, the five major fractions 3, 8, 9, 10,
and 14 were further purified by rechromatography, yielding 69, 57, 317,
47, and 131 nmol (as
HexA), respectively, per 100 mg of
peptidoglycans. They represented 6.3, 5.2, 28.8, 4.3, and 11.9% of the
total serine of the starting peptidoglycan preparation, respectively.
Figure 2:
HPLC
separation of the oligosaccharide alditol fraction. The oligosaccharide
alditol fraction (B-2) corresponding to 312 nmol of HexA was
chromatographed on an amine-bound silica column as described under
``Experimental Procedures.'' Elution was performed using a
linear gradient of NaH
PO
as indicated by the dashed line. The elution positions of the authentic
unsaturated chondrodisaccharides are indicated by arrows.
Figure 3:
HPLC analysis of chondroitinase ACII
digests of the isolated sugar alditols. Panel A, elution
positions of the authentic chondrodisaccharides, acetate, and the
authentic tetrasaccharide, HexA-Gal-Gal-Xyl-ol. Panels
B-D, chondroitinase ACII digests of fractions 3, 8, and 9,
respectively. The arrows in panels B-D indicate
the elution positions of fractions 3, 8, and 9 before digestion,
respectively. For the conditions of chondroitinase ACII digestion see
``Experimental Procedures.''
Fraction 14
was partially (10%) degraded by chondroitinase ACII into Di-4S and
the presumed unsaturated tetrasaccharide
HexA
1-3Gal(4-sulfate)
1-3Gal
1-4Xyl-ol
(data not shown), indicating that the structure of this minor component
was most likely
HexA
1-3GalNAc(4-sulfate)
1-4GlcA
1-3Gal(4-sulfate)
1-3Gal
1-4Xyl-ol
reported previously for chondroitin 4-sulfate from whale cartilage (16) . However, the major compound accounting for 90% of this
fraction remained undigested, and fraction 10 was also resistant to
chondroitinase ACII (data not shown), indicating that the structures of
the compound in fraction 10 and of the major component in fraction 14
were clearly different from the hexasaccharide alditols isolated
previously from the linkage region of chondroitin sulfate in terms of
their sensitivity toward chondroitinase ACII. The results suggested
that the internal uronic acid in fractions 10 and 14 was not glucuronic
acid, but more likely iduronic acid.
ESI/MS of underivatized
fractions 10 and 14 were analyzed. Fraction 14 gave doubly charged ions
[M-2H],
[M+Na-3H]
, and
[M+2Na-4H]
at m/z 585, 596, and 607 as well as triply charged ions,
[M-3H]
,
[M+Na-4H]
, and
[M+2Na-5H]
at m/z 390, 397, and 404 (Fig. 4B). The singly charged
ion [M-H]
was also observed at m/z 1,172. All these molecular ions corresponded to
the approximate molecular weight of 1,173 of the compound in this
fraction. Only a doubly charged ion [M-2H]
was observed at m/z 546 for fraction 10 (Fig. 4A), which corresponded to the estimated
molecular weight of 1,094 of the compound in this fraction.
Figure 4: Negative ion mode ESI/MS of fractions 10 and 14. Panel A, fraction 10; panel B, fraction 14.
To
determine the positions of the sulfate groups, aliquots of fractions 10
and 14 were subjected to chondro-4-sulfatase digestion followed by HPLC
analysis. After a 1-h incubation, fraction 10 eluted at position a was
partially degraded, giving rise to a degradation product at position b
which accounted for 30% of fraction 10 used (Fig. 5A).
After a 14-h incubation, it was completely converted to the compound at
position b (Fig. 5B), corresponding to the authentic
nonsulfated hexasaccharide alditol
HexA
1-3GalNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl-ol.
Thus, the compound in fraction 10 contained one sulfate group on C4 of
either GalNAc or one of the 2 galactose residues. It is known that
chondro-4-sulfatase acts on C4 of both GalNAc and the galactose
substituted by GlcA in the linkage region(16) .
Chondro-4-sulfatase treatment of fraction 14, which eluted at position
c before digestion, resulted in two peaks after a 1-h incubation, one
of which (43%) eluted at position d1 and the other (33%) at position d2 (Fig. 5C). When exhaustively treated, fraction 14 was
completely converted to the compound at position d2, which seemed to
correspond to position b (Fig. 5D). Position d1 was
close to the elution position of fraction 10, indicating that the
compound at position d1 was also a monosulfated hexasaccharide alditol.
Thus, fraction 14 seemed to contain two sulfate groups on C4 of GalNAc
and/or 2 galactose residues. It was noted that only one partially
desulfated product was observed, suggesting that chondro-4-sulfatase
acted sequentially by first removing one then the other sulfate group.
It would appear that this enzyme acted preferentially on one of the two
sulfate groups. Which sulfate group, however, remains to be determined.
The results indicate that fractions 10 and 14 had the common
hexasaccharide core structure
HexA
1-3
GalNAc
1-4HexA1-3Gal
1-3Gal
1-4Xyl-ol,
with one or two sulfate groups on either C4 of GalNAc or galactose
residue(s) underlined.
Figure 5:
HPLC
analysis of the chondro-4-sulfatase digests of the isolated
hexasaccharide alditols. Fractions 10 and 14 were digested with
chondro-4-sulfatase for 1 or 14 h and then chromatographed. Panel
A, fraction 10 after a 1-h digestion; panel B, fraction
10 after a 14-h digestion; panel C, fraction 14 after a 1-h
digestion; panel D, fraction 14 after a 14-h digestion. Black and white arrows indicate the elution positions
of the fractions before and after digestion, respectively. The elution
positions of the authentic chondrodisaccharides and hexasaccharide
alditols derived from the carbohydrate-protein linkage region of whale
cartilage chondroitin 4-sulfate (16) are indicated in panel
A by numbered arrows as follows: 1, Di-0S; 2,
Di-6S; 3,
Di-4S; 4,
Di-diS
; 5,
Di-diS
; 6,
Di-triS; 7,
HexA-GalNAc-GlcA-Gal-Gal-Xyl-ol; 8,
HexA-GalNAc(4-sulfate)-GlcA-Gal-Gal-Xyl-ol; 9,
HexA-GalNAc(4-sulfate)-GlcA-Gal(4-sulfate)-Gal-Xyl-ol.
To identify the internal hexuronic acid
residue, the sensitivity of fractions 10 and 14 toward chondroitinase B
was investigated. Fraction 14 was degraded into two products in
equimolar amounts as detected by absorbance at 232 nm (Fig. 6A), while H radioactivity was found
only in the faster eluting UV-absorbing peak (Fig. 6B),
which corresponded to the position of authentic
HexA
1-3Gal(4-sulfate)
1-3Gal
1-4Xyl-ol.
The nonradiolabeled peak coincided with authentic
Di-4S. Thus, the
compound in fraction 14 most likely had the disulfated hexasaccharide
alditol structure with iduronic acid at the internal position,
HexA
1-3GalNAc(4-sulfate)
1-4IdoA
1-3Gal(4-sulfate)
1-3Gal
1-4Xyl-ol.
In contrast, fraction 10 was resistant to chondroitinase B digestion
(data not shown).
Figure 6:
HPLC analysis of the chondroitinase B
digest of fraction 14. The isolated fraction 14 was digested with
chondroitinase B as described under ``Experimental
Procedures,'' and the digests were subjected to HPLC on an
amino-bound silica column. Eluates were monitored by absorption at 232
nm (panel A), and H radioactivity (panel
B). The elution positions of the authentic chondrodisaccharides,
tetrasaccharide alditols obtained from whale cartilage chondroitin
4-sulfate(16) , and fraction 14 before digestion are indicated
in panels A and B by numbered arrows: 1,
Di-0S; 2,
Di-6S; 3,
Di-4S; 4,
Di-diS
; 5,
Di-diS
; 6,
Di-triS; 7,
HexA-Gal(4-sulfate)-Gal-Xyl-ol; 8, fraction 14 before
digestion.
The one-dimensional H NMR spectrum of fraction 14
measured at 26 °C is shown in Fig. 7. The inset is
the spectrum recorded at 15 °C to suppress the disturbance by HOD
line. The resonances at
5.962 and 2.115 ppm are characteristic of
the H-4 proton of
HexA (15) and the acetoamide group
protons of GalNAc, respectively. Close inspection of the spectrum
identified the H-4 signal of Xyl-ol at
3.987 ppm. The resonances
between
4.6 and 5.3 ppm are characteristic of anomeric protons,
and those at
4.620, 4.686, 4.736, and 5.265 ppm were identified
as H-1 resonances of Gal-2, GalNAc-5, Gal-3, and
HexA-6,
respectively, by comparison with the spectral data (Table 4) of
the reference compound Fr. D
(
HexA
1-3GalNAc(4-sulfate)
1-4GlcA
1-3Gal(4-sulfate)
1-3Gal
1-4Xylol)
derived from whale cartilage chondroitin 4-sulfate. The signals at
4.620 and 4.736 had a coupling constant J
of
7.5 Hz for the H-1 of
Gal. The signal at
4.686
had a coupling constant J
of
8.5 Hz for the
H-1 of
GalNAc(23) . The signal at
5.147 had a
coupling constant J
of
2.5 Hz, indicating
that it was the H-1 signal of
IdoA-4, since an internal GlcA or
IdoA residue of heparin/heparan sulfate oligosaccharides has been
reported to give an anomeric proton signal at around
4.6 or 5.0
ppm and the different coupling constants J
of
8.0 or 3.0 Hz, respectively(25) . Starting with these anomeric
proton signals, H-2, H-3, and H-4 signals of the corresponding sugar
residues were localized unambiguously with the aid of the COSY spectrum
recorded at 15 °C (not shown) along the cross-section of the
two-dimensional HOHAHA spectrum (Fig. 8). It is worth noting
that a further connection from H-4 of IdoA to the H-5 resonance at
4.742 (26) was clearly observed in the COSY spectrum at
15 °C, and it is indicated along the top horizontal line in the HOHAHA spectrum. The NMR data are summarized in Table 4and were in reasonable agreement with those of the
reference compound Fr. D, except for the data regarding HexA-4. H-1,
H-2, H-3, and H-4 proton signals at
4.686, 4.078, 4.160, and
4.628 are characteristic of 4-sulfated
GalNAc (see the NMR data of
fraction 9 in Table 4). Similarities of the chemical shifts of
H-1, H-3, and H-4 of Gal-3 to those of the reference compound D and the
large downfield shift of H-4 as compared with that of Gal-3 in fraction
3, 8, or 9 support 4-sulfation of this residue. A significant
difference in chemical shift (
0.058) of
GalNAc-5 H-1
was observed between fraction 14 and the reference compound Fr. D,
which was attributed to its linkage to the different HexA isomers.
Thus, the NMR data are consistent with the following structure proposed
above based upon the results of enzyme digestion. The minor component,
disulfated hexasaccharide alditol detected by chondroitinase ACII
digestion in this fraction, was not observed by
H NMR
because of the limited amount.
Figure 7:
Structural reporter group regions of the
500-MHz H NMR spectrum of fraction 14 recorded in
H
O at 26 °C. The inset is the
spectrum recorded at 15 °C. The numbers and letters in the spectra refer to the corresponding residues in the
structures.
Figure 8: Two-dimensional HOHAHA spectrum of fraction 14 recorded at 15 °C.
The NMR data of fraction 10 are presented in Table 4.
Anomeric proton resonances of Gal-2, IdoA-4, and HexA-6 were
identified without difficulty in the one-dimensional spectrum at 26
°C by comparison with the spectral data of fraction 14 and the
reference compound D, whereas those of Gal-3 and GalNAc-5 were
identifiable in the one-dimensional spectrum recorded at 15 °C
(data not shown). Although H-1 signals of Gal-3 and GalNAc-5 were close
to each other, they were distinguished based on the slightly different
values of the coupling constants J
as described
above for those of fraction 14. Assignments of most of other resonances
belonging to Gal-2, IdoA-4, and
HexA-6 were made using the COSY
spectrum at 26 °C (data not shown). A cross-peak between H-2 and
H-3 of Gal-2 was not identified in the COSY spectrum because of the
spectral complexity in the corresponding region, but connections from
H-1 to H-2 and from H-4 at
4.205, a structural reporter group of
Gal-2, to H-3 resonances were readily observed. The signal at
4.158 with a coupling constant of 3.0 Hz is characteristic of H-4 of
nonsulfated Gal-3. The chemical shifts of protons belonging to the
component sugar residues except for HexA-4 of this oligosaccharide were
similar to those of the corresponding portion of the hexasaccharide
alditol in fraction 9, whereas the chemical shifts of HexA-4 of
fraction 10 resembled those of
IdoA-4 of fraction 14. A connection
of H-4 to H-5 of this residue was also visible in the COSY spectrum.
The H-1 and H-4 resonances of the
GalNAc-5 at
4.683 and
4.623 were consistent with the 4-sulfation of this residue. Based on
these NMR data, the following monosulfated hexasaccharide alditol
structure was proposed for the compound in fraction 10.
In this study, we isolated five major hexasaccharide alditols from the carbohydrate-protein linkage region of a peptidoglycan preparation of bovine aorta, which is assumed to contain dermatan sulfate chains derived from small proteoglycans such as decorin and biglycan(27, 28, 29) in addition to those from large proteoglycans(30) . Three of these (fractions 3, 8, and 9) were isolated previously from chondroitin 4-sulfate(15, 16) , chondroitin 6-sulfate(18) , and oversulfated chondroitin sulfate rich in 4,6-disulfated GalNAc(20) . However, the other two (fractions 10 and 14), which contain an iduronic acid residue as the innermost uronic acid, have not been reported previously. Since the discovery of the unique common carbohydrate sequence in the linkage region of various sulfated glycosaminoglycans including heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate, it has been accepted that the innermost uronic acid residue immediately adjacent to the linkage trisaccharide sequence of these glycosaminoglycan chains is glucuronic acid(10, 11) . It was demonstrated recently that heparan sulfate shares this tetrasaccharide sequence in the carbohydrate-protein linkage region(25) . In the case of pig skin dermatan sulfate, the first uronic acid residue adjacent to a galactose residue has been demonstrated to be almost exclusively glucuronic acid, although glucuronic acid accounts for only 4-10% of the total uronic acid(31) . No evidence has been obtained for the presence of iduronic acid at this position in such preparations. In contrast, the present study demonstrated iduronic acid at this position in bovine aorta dermatan sulfate, indicating that this structural feature characteristic of dermatan sulfate emerges at the 4th saccharide residue from the attachment site to the core protein. Although how general this structure is among dermatan sulfates of various cells and tissues and how variable it is during development, aging, or pathogenesis remain to be investigated, this structure may have important implications in the expression of biological functions and in the biosynthetic mechanisms of dermatan sulfate. Since a mixed population of proteoglycans from undivided bovine cardiac aorta was used in this study, a next step in these analyses will be to identify the proteoglycan species that manifests the iduronic acid-containing linkage oligosaccharides and to localize such a proteoglycan species in specific tissue areas of aorta.
The dermatan sulfate chain with
iduronic acid closer to the linkage region might be more flexible and
mobile and thus may be able to swing around the core protein due to the
specific conformational properties of iduronic acid. Molecular
mechanics and NMR studies indicated that iduronic acid may be present
in one of the three low energy conformations, C
,
S
, or
C
, or in all these three forms in rapid dynamic
equilibrium. This equilibrium is highly sensitive to sulfation and
carbohydrate sequence as well as to intermolecular factors such as
cation binding(32) . Iduronic acid has never been demonstrated
in the first uronic acid position of the other iduronic acid-containing
glycosaminoglycans heparin or heparan sulfate. Heparan sulfate has a
long nonsulfated stretch of more than eight repeating disaccharide
units, which are assumed to contain only glucuronic
acid(33, 34, 35) , and therefore would be
rather rigid in the proximal portion to the linkage region but plastic
in the distal portion. In heparin, iduronic acid begins appearing
nearer the linkage region than in heparan sulfate, but not in the first
uronic acid position(17, 36) .
In the biosynthesis of dermatan sulfate chains, C5 epimerization of glucuronic acid to iduronic acid is considered to be critical(37) . Once one iduronic acid is formed, the C5 epimerase seems to continue to make more iduronic acid thereafter, indicating that the formation of the very first iduronic acid is a key step. Although the selection mechanism of such a target glucuronic acid is unknown, the iduronic acid found at the most proximal position to the linkage region might have been one of the first. Transfer of N-acetylgalactosamine to a dermatan sulfate oligosaccharide with iduronic acid at the nonreducing terminus has been observed(38) , which is in contrast to the biosynthesis of heparin where transfer of N-acetylglucosamine residues occurs only to glucuronic acid and not to iduronic acid residues(39) . Thus, the first epimerization reaction of glucuronic acid to iduronic acid may trigger dermatan sulfate synthesis rather than heparan sulfate synthesis. However, it does not seem to be a prerequisite for the selection of dermatan sulfate over chondroitin sulfate since the innermost uronic acid residue in the linkage region of pig skin dermatan sulfate has been reported to be almost exclusively glucuronic acid(31) . The iduronic acid adjacent to a galactose residue also supports the notion that the C5 uronosyl epimerase for dermatan sulfate is different from that for heparin or heparan sulfate. It has been suggested that microsomes from cultured fibroblasts contain two different uronosyl epimerases, one for biosynthesis of heparan sulfate and the other for that of dermatan sulfate, and that they have different cofactor and pH requirements(40, 41) . Our results indicate that the latter enzyme can recognize the glucuronic acid residue attached to the adjacent galactose unit and may trigger the epimerization reactions.
Both major and minor compounds in fraction 14 contain another novel 4-sulfated galactose structure, which was first demonstrated in chondroitin 4-sulfate of rat chondrosarcoma (15) and afterwards in that of whale cartilage(16) . It has been suggested that epimerization of glucuronic acid to iduronic acid is enhanced by concomitant C4 sulfation of the N-acetylgalactosamine residue(14, 42) . It would be of interest to investigate whether 4-sulfation of the galactose residue accelerates epimerization of the adjacent glucuronic acid residue.
The results of the present study indicate that there are at least five different subclasses of dermatan sulfate chains with respect to the structure of the linkage region. It is likely that different chains have different patterns of modification. It remains to be determined whether biologically active domain structures such as the binding domain to heparin cofactor II is found on a specific subclass chain. The presence of iduronic acid in the vicinity of the linkage region raises the possibility that biologically active domain structures that require iduronic acid may well be found near the linkage region along the dermatan sulfate chains and presented to the corresponding ligands. Another interesting structural feature is 6-sulfation of the first GalNAc residue found in fraction 8. It should be noted that GalNAc-6-sulfate, which is a minor component of dermatan sulfate, can be found in the closest vicinity of the linkage region.
This study
revealed the unexpected substrate specificity of chondroitinase B using
the iduronic acid-containing linkage fragments as substrates. Previous
studies indicated that the enzyme produced hexa- or larger
oligosaccharides attached to the linkage glycopeptides (29) .
However, in this study it was shown to degrade the major disulfated
hexasaccharide alditol in fraction 14 into a disaccharide and a linkage
tetrasaccharide alditol. In contrast, it was inert to the monosulfated
counterpart in fraction 10. The enzyme seems to recognize not only the
iduronic acid but also the 4-sulfate group on the neighboring
galactose. The detailed structural requirements remain to be determined
for this enzyme which is potentially useful for structural studies of
dermatan sulfate, especially for identification of the highly specific
structure
GalNAc(4-sulfate)1-4IdoA
1-3Gal(4-sulfate) in the
linkage region of dermatan sulfate.