Data are emerging that show roles for CS⁄ DS in a variety of fundamental biological processes including neurite Keywords chondroitin sulfate; dermatan sulfate; glycosaminoglycan; NMR spe
Trang 1chondroitin/dermatan sulfates
1
hexasaccharides
Thomas N Huckerby1, Ian A Nieduszynski1, Marcos Giannopoulos1, Stephen D Weeks1,
Ian H Sadler2and Robert M Lauder1
1 Department of Biological Sciences, Lancaster University, UK
2 Department of Chemistry, University of Edinburgh, UK
There is considerable interest in the detailed molecular
structure [1,2] and function [3] of glycosaminoglycans
(GAGs) including chondroitin and dermatan sulfate
(CS⁄ DS) [4–6] These structurally diverse polymers are
abundant components of extracellular matrices and cell surfaces in humans and other mammals Data are emerging that show roles for CS⁄ DS in a variety of fundamental biological processes including neurite
Keywords
chondroitin sulfate; dermatan sulfate;
glycosaminoglycan; NMR spectroscopy;
sulfation
Correspondence
R M Lauder, Department of Biological
Sciences, IENS, Lancaster University,
Bailrigg, Lancaster LA1 4YQ, UK
Fax: +44 (0)1524 593192
Tel: +44 (0)1524 593561
E-mail: r.lauder@lancaster.ac.uk
(Received 21 July 2005, revised 14
Septem-ber 2005, accepted 7 OctoSeptem-ber 2005)
doi:10.1111/j.1742-4658.2005.05009.x
Chondroitin and dermatan sulfate (CS and DS) chains were isolated from bovine tracheal cartilage and pig intestinal mucosal preparations and frag-mented by enzymatic methods The oligosaccharides studied include a disaccharide and hexasaccharides from chondroitin ABC lyase digestion as well as trisaccharides already present in some commercial preparations In addition, other trisaccharides were generated from tetrasaccharides by chemical removal of nonreducing terminal residues Their structures were examined by high-field 1H and 13C NMR spectroscopy, after reduction using sodium borohydride The main hexasaccharide isolated from pig intestinal mucosal DS was found to be fully 4-O-sulfated and have the
DUA(b1–3)GalNAc4S(b1–4)l-IdoA(a1–3)GalNAc4S(b1–4)l-IdoA(a1–3)GalNAc4S-ol, whereas one from bovine tracheal cartilage CS comprised only 6-O-sulfated residues and had the structure: DUA(b1– 3)GalNAc6S(b1–4)GlcA(b1–3)GalNAc6S(b1–4)GlcA(b1–3)GalNAc6S-ol
No oligosaccharide showed any uronic acid 2-sulfation One novel disac-charide was examined and found to have the structure: GalNAc6S(b1– 4)GlcA-ol The trisaccharides isolated from the CS⁄ DS chains were
found in commercial CS⁄ DS preparations and may derive from endo-genous glucuronidase and other enzymatic activity Chemically generated trisaccharides were confirmed as models of the CS⁄ DS chain caps and included: GalNAc6S(b1–4)GlcA(b1–3)GalNAc4S-ol and GalNAc6S(b1– 4)GlcA(b1–3)GalNAc6S-ol The full assignment of all signals in the NMR spectra are given, and these data permit the further characterization of
CS⁄ DS chains and their nonreducing capping structures
Abbreviations
CS, chondroitin sulfate; DS, dermatan sulfate; GAG, glycosaminoglycan; GalNAc(-ol), 2-deoxy-2-N-acetylamino- D -galactose (-galactitol); GlcA,
D -glucuronic acid; GlcA-ol, D -glucuronic acid alditol; IdoA, a- L -iduronic acid; 6S ⁄ 4S, O-ester sulfate group on C6 ⁄ C4; DUA, 4,5-unsaturated hexuronic acid (4-deoxy-a- L -threo-hex-4-enepyranosyluronic acid); SAX, strong anion-exchange; SEC, size exclusion chromatography.
Trang 2outgrowth [7], disease development [8] and growth
fac-tor binding [9] CS has also been found in
inverte-brates [10–12] including Drosophila melanogaster [13]
and Caenorhabditis elegans [14], where it has been
shown to have fundamental roles in development [15]
CS⁄ DS chains comprise a linkage region, a chain
cap and a repeat region [4,5] The repeat region of CS
is a repeating disaccharide of glucuronic acid (GlcA)
and N-acetylgalactosamine (GalNAc) [-4)GlcA(b1–
3)GalNAc(b1-]n, which may be O-sulfated on the C4
and⁄ or C6 of GalNAc and C2 of GlcA GlcA residues
of CS may be epimerized to iduronic acid (IdoA)
forming the repeating disaccharide
[-4)IdoA(a1–3)Gal-NAc(b1-]n of DS Thus, CS and DS may be found as
pure polymers or a mixed copolymer in which the DS
residues and GalNAc sulfation isoforms may be
located together in large blocks or distributed
through-out the chain These will have very different effects on
molecular interactions and biological function
Both the concentrations and locations of sulfate
ester substituents vary with GAG source [4,16] For
example, in adult human articular cartilage, extensive
6-O-sulfation of GalNAc is observed ( 95%) [4] In
shark cartilage, lower levels of 6-O-sulfation are found
( 70%), with 4-O-sulfation making up the balance
along with 25% 2-sulfation of the uronic acid
resi-dues [4], and in tracheal cartilage lower levels of
6-O-sulfation are found ( 20–40%), with the balance
being mainly GalNAc 4-O-sulfation In D
melanogas-ter, 4-O-sulfation, but not 6-O-sulfation, is observed
whereas in C elegans the chondroitin is unsulfated
[13,14]
Within a tissue, the sulfation profile and levels of
epimerization of GlcA to IdoA change with age For
example, the level of GalNAc 6-O-sulfation reported
above for human articular cartilage applies only to the
adult; at birth this level is close to zero but rises
signi-ficantly during the first 20 years of life [5,17]
Not only does CS⁄ DS structure change with tissue
source and age, but within a single chain there is
vari-ability The chain cap of CS is a GalNAc or GlcA
residue; a 4,6-disulfated GalNAc residue, rare in the
repeat region of human articular cartilage CS,
repre-sents over 50% of the chain caps for a normal adult,
but only 30% at the termini of CS chains from
osteoarthritic cartilage [18,19] Whereas the CS chain
caps may be highly sulfated, the linkage regions, via
which these pendant polymers are attached to a
pro-tein core, have been shown to exhibit low levels of
sulfation relative to residues within the repeat region
[5,6,20], with preferential localization of unsulfated
and 4-O-sulfated GalNAc residues at linkage regions
[5,6,20]
The overall structure of CS chains is thus highly complex, showing significant variation in composition across materials from diverse tissue sources, from tissues of differing ages and also within a single chain Enhanced availability of data to facilitate the characterization of these structures is therefore of value
GAGs are not primary gene products and therefore their analysis cannot rely on genomic approaches; structural analysis requires their isolation followed by
a complex characterization process In our previous work we have used the paradigm of isolation and depolymerization of GAG chains to generate oligosac-charides, the structures of which are determined using NMR spectroscopy [6,16] These oligosaccharides are then integrated into a chromatographic fingerprinting method which can be used for the analysis of biologi-cal samples [4]
Chondroitin lyase enzymes are eliminases which cleave the -3)GalNAc(b1–4)GlcA(b1-⁄ IdoAa(1- bond
in CS⁄ DS in the case of chondroitin ABC lyase (EC 4.2.2.4), whereas chondroitin AC lyases act on CS alone Chondroitin AC and ABC lyases generate disac-charides and tetrasacdisac-charides [4] and have been widely used for the analysis of CS⁄ DS composition These studies have yielded crucial data allowing an under-standing of species, tissue, age and pathology related differences and the estimation of changes in CS⁄ DS abundance and composition However, the reduction
of the polymer to its individual disaccharide units removes any possible sequence data that would allow the reconstruction of biologically important functional motifs In addition, the action of chondroitin lyase enzymes generates a 4,5-unsaturated hexuronic acid (DUA) from the uronic acid of the cleaved bond Thus, the distinction between IdoA and GlcA, epimeri-zation at C5, is lost and it is impossible to distinguish between disaccharides derived from DS and those derived from CS
We have previously reported 1H-NMR data for disaccharides and tetrasaccharides from CS⁄ DS [16], and Sugahara et al [21] have examined, by 1H NMR and MS, a series of chondroitin ABC lyase-resistant fragments derived from CS or DS Several of these were trisaccharides, including DUA(b1–3)GalNAc4, 6diS(b1–4)GlcA, terminated by an unreduced GlcA ring, which could have been derived from polymer chain-reducing termini by a peeling reaction, or, through the action of a tissue endo-b-d-glucuronidase
A series of reduced and unreduced oligosaccharides obtained from DS were previously characterized by1H and 13C NMR [22], including the trisaccharide Gal-NAc4S(b1–4)l-IdoA(a1–3)GalNAc4S-ol
Trang 3More recently, the preparation and structural
char-acterization of unreduced DS oligosaccharides of up to
dodecasaccharide in size has been discussed [23]; a
combination of 1D and 2D NMR together with
elec-trospray MS was employed
The employment of nondestructive analytical
meth-ods in the characterization of GAGs is becoming more
important now that full structural information for
large domains is required as part of the examination
of function for these species Data from other GAG
fragments has already proved valuable in the study of
intact parent polymers; the architecture of keratan
sul-fate chains has been explored in this manner [24,25]
There have already been attempts to examine intact
CS chains isolated from various sources Considerable
difficulties were met when specific assignments of
structural components were sought It is thus
import-ant that polymer characterizations should be facilitated
through the availability of comprehensive parameters
describing the structures of a wide range of
oligosac-charide structures The complete assignments of 1H
and 13C NMR spectra from a series of disaccharides
and tetrasaccharides derived from CS⁄ DS chains have
already been given [16] In this report we present
1H-NMR and some 13C-NMR data for trisaccharides
and hexasaccharides from CS⁄ DS These have the structures shown below:
GalNAc6S(b1–4)GlcA(b1–3)GalNAc4S-ol: CS#604 GalNAc6S(b1–4)GlcA(b1–3)GalNAc6S-ol: CS#606 DUA(b1–3)GalNAc4S(b1–4)GlcA-ol: CS040#
DUA(b1–3)GalNAc6S(b1–4)GlcA-ol: CS060#
GalNAc6S(b1–4)GlcA-ol: CS#60#
DUA(b1–3)GalNAc6S(b1–4)GlcA(b1–3)Gal-NAc6S(b1–4)GlcA(b1–3)GalNAc6S-ol: CS060606 DUA(b1–3)GalNAc4S(b1–4)l-IdoA(a1–3)Gal-NAc4S(b1–4)l-IdoA(a1–3)GalNAc4S-ol: DS040404
Results and Discussion Isolation of oligosaccharides After depolymerization by chondroitin ABC endolyase, oligosaccharides are isolated as disaccharides, trisac-charides, tetrasaccharides and, because of incomplete depolymerization, as hexasaccharides (Fig 1) [4,16] The heterogeneous pools of oligosaccharides generated
in this way were purified by size exclusion chromato-graphy (SEC) to yield individual oligosaccharides [4,16]
In addition, trisaccharides lacking nonreducing ter-minal unsaturated uronic acids have been successfully
Fig 1 Isolation of trisaccharides Insert: SEC of reduced oligosaccharides generated after depolymerization of bovine tracheal CS chains by chondroitin ABC endolyase on a Toyapearl HW40s column (50 cm · 1 cm) eluted in 0.5 M ammonium acetate at 0.4 mLÆmin)1, the eluate was monitored by measuring A232 Disaccharide and tetrasaccharide pools are indicated by 2 and 4, respectively Main chromatogram: after removal of the nonreducing terminal unsaturated uronic acids (Experimental procedures) from the tetrasaccharide mixture (see insert), the crude trisaccharides were purified by SAX chromatography on a Spherisorb S5 column (25 cm · 1 cm) at 2 mLÆmin)1 Bound material was eluted by a linear gradient of 2 m M LiClO4(buffer A) to 250 m M LiClO4(buffer B), pH 5.0, according to the following gradient profile: after a 10-min isocratic phase of 100% buffer A, a gradient of 0–100% buffer B was introduced over 240 min, followed by 10 min of 100% buffer
B The column eluate was monitored online at 206 nm Individual fractions were pooled as indicated.
Trang 4prepared from related tetrasaccharides; full 1H-NMR
and some 13C-NMR data for these tetrasaccharides
have been reported elsewhere [16]
Trisaccharides
Two trisaccharides were prepared from the
correspond-ing CS repeat unit tetrasaccharides
DUA(b1–3)Gal-NAc6S(b1–4)GlcA(b1–3)GalNAc4S-ol (CST0604) and
DUA(b1–3)GalNAc6S(b1–4)GlcA(b1–3)GalNAc6S-ol
(CST0606) [16] by chemical removal [26] of the
unsat-urated nonreducing terminal residues (Fig 1) The
1H-NMR data for these species, designated CS#604
and CS#606, are summarized in Table 1 Partial
spectra for the former are shown in Figs 2 and 3,
respectively, and a partial gradient-COSY-45
spec-trum for the latter is given in Fig 4 For both species,
the presence of two anomeric proton sites, together
with two N-acetyl methyl signals and the lack of
responses corresponding to DUA protons all confirm
that these are reduced trisaccharides derived from
endolyase CS#604 and CS#606 are confirmed as
respectively, through examination of the residue
A (galactosaminitol) chemical shifts, which clearly
indicate ester sulfate location and have values closely
similar to those found for the corresponding
tetrasac-charides CST0604 and CST0606 [16] Similarly, the
internal uronic acid residues (ringB) exhibit shift
pat-terns almost identical with those for GlcUA in the
tetramers As would be expected, the nonreducing
ter-minal GalNAc6S protons of both species (ring C)
have chemical shift positions that are perturbed from
those found for the corresponding site in the
DUA-terminated tetrasaccharides That for H3 is displaced
by )0.21 p.p.m., together with movements of )0.185
and )0.12 p.p.m., respectively, for H4 and H2 All
other changes are less than 0.04 p.p.m in magnitude
Two unusual trisaccharides were isolated after
chondroitin lyase digestion of commercial CS
prepara-tions which are related to CS tetrasaccharides,
expected, but lacking the N-acetylgalactosaminitol
reducing terminal moiety These are designated
CS040# and CS060#, respectively, and exhibit the
chemical shift values summarized in Table 2 A
par-tial 600-MHz gradient-COSY-45 NMR spectrum for
CS060# is shown in Fig 5 Comparisons of chemical
shift values for the DUA residues in repeat-unit
tetra-saccharides show only minor perturbations [16] For
CS040#, all (ring C) signals fall within 0.02 p.p.m of
Table 1 Proton chemical shifts for the trisaccharides CS#604 and CS#606 Shift values are from COSY-45 data except for those marked with an asterisk (*).
Residue Proton CS#604 (p.p.m.) CS#606 (p.p.m.)
GalNAc (C) H1 4.558* 4.589*
H2 3.917* 3.930* H3 3.743 3.745 H4 3.988* 4.002* H5 3.967* 3.976* H6 4.24 4.24 H6¢ 4.24 4.24
CH3 2.056* 2.056* GlcA (B) H1 4.628* 4.556*
H2 3.480* 3.451* H3 3.638 3.633* H4 3.77 3.75 H5 3.77 3.75 GalNAc-ol (A) H1 3.70 3.717
H1¢ 3.72 3.784* H2 4.295 4.390* H3 4.257 4.077 H4 4.492* 3.570* H5 4.146* 4.308* H6 3.705 4.07 H6¢ 3.705 4.09
CH 3 2.018* 2.051*
Fig 2 Partial 600-MHz gradient-COSY-45 spectrum for CS#604 at
43 C The spectral width was 1750.7 Hz, and eight acquisitions for each of 1024 increments were sampled into 1024 complex points The array was zero-filled to 2048 · 2048 complex points and trans-formed in each dimension after application of a (sinebell) 2 window function CS#604 has the structure:
GalNAc6S(b1–4)GlcA(b1–3)GalNAc4S-ol.
Trang 5the corresponding (ring D) locations for CST0404; in
the case of CS060#, all are within 0.01 p.p.m of the
shift values seen in either CST0604 or CST0606 with
the sole exception of H3 (+ 0.016 p.p.m relative to
CST0606)
When the internal GalNAc (ring B) data are
exam-ined, they clearly distinguish between sulfation at C4
for CS040# (H4, 4.649 p.p.m.; H6,6¢ at 3.783 and
3.811 p.p.m.) and at C6 for CS060# (H4, 4.173 p.p.m.;
H6,6¢ at 4.205 and 4.238 p.p.m.), but other signal
posi-tions are strongly perturbed compared with their
loca-tions in the related tetrasaccharides There is one
further connected set of protons present for both of
these species This represents a residue consisting of a
nonequivalent methylene group connected to a series
of four further single proton sites The chemical shift
values found for both CS040# and CS060# are quite
similar, the minor perturbations reflecting the different
influences of 4-sulfated and 6-sulfated neighbouring
rings This residue is derived from a GlcA ring, which
has been reductively opened by alkaline borohydride
treatment, forming the corresponding glucuronitol
The methylene proton pair represent the hydrogens at
the reduced C1 site, and the H2–H5 series does not
terminate (as would be the situation for a GalNAc-ol residue) with a further nonequivalent methylene group
at C6
Fig 3 Partial 600-MHz TOCSY spectrum for CS#604 at 43 C The
spectral width was 1750.7 Hz, and eight acquisitions for each of
512 pairs of increments were sampled into 1024 complex points
using a mixing time of 70 ms The array was zero-filled to
2048 · 2048 complex points and transformed in each dimension
after application of a 1.0 Hz exponential window function CS#604
has the structure:
GalNAc6S(b1–4)GlcA(b1–3)GalNAc4S-ol.
Fig 4 Partial 600-MHz gradient-COSY-45 spectrum for CS#606 at
43 C The spectral width was 1750.7 Hz, and eight acquisitions for each of 1024 increments were sampled into 1024 complex points The array was zero-filled to 2048 · 2048 complex points and trans-formed in each dimension after application of a (sinebell) 2 window function CS#606 has the structure:
GalNAc6S(b1–4)GlcA(b1–3)GalNAc6S-ol.
Table 2 Proton chemical shifts for the oligosaccharides CS040#, CS060# and CS#60# Shift values are from COSY-45 data except for those marked with an asterisk (*).
Residue Proton
CS040#
(p.p.m.)
CS060#
(p.p.m.)
CS#60# (p.p.m.)
DUA (C) H1 5.297* 5.199 –
H2 3.860 3.805* – H3 3.963* 4.120* – H4 5.971* 5.884* – GalNAc (B) H1 4.835* 4.763* 4.696*
H2 4.092* 4.034* 3.912 H3 4.201* 3.978 3.762 H4 4.649* 4.173* 3.996* H5 3.876 3.968 3.938 H6 3.783 4.205 4.214 H6¢ 3.811 4.238 4.236 CH3 2.113* 2.080* 2.073* GlcA-ol (A) H1 3.644* 3.679* 3.672*
H1¢ 3.724* 3.762* 3.758 H2 3.903 3.922* 3.917 H3 3.845 3.855* 3.848* H4 4.154* 4.092* 4.073* H5 4.247* 4.214 4.201*
Trang 6A novel disaccharide, related to these trisaccharides
has also been characterized, and the chemical shift
data for this species, designated CS#60#, are also
sum-marized in Table 2 In this molecule, the DUA ring is
absent and the presence of GalNAc 6-sulfation is
con-firmed through the presence of H6,6¢ protons at 4.214
and 4.236 p.p.m The GalNAc H4 response is found at
3.996 p.p.m.; this, together with H3 which is displaced
by over )0.2 p.p.m relative to the corresponding
sig-nal in CS060#, confirms that the nonreducing DUA
residue, which would have been attached at C3, is no
longer present
For these novel oligomers, 13C-NMR data are also
available and are summarized in Table 3 The influence
of the sulfation position on the observed signal
posi-tions within rings B and C is very similar to that
observed previously [16] for the corresponding
tetra-saccharides, as is seen in Table 4 where difference data
are presented together with the calculated values
The chondroitin A lyase activity, which acts on
bonds containing 4-sulfated GalNAc residues, is an
order of magnitude greater in chondroitin ABC
endo-lyase than the chondroitin C endo-lyase which acts on bonds
containing 6-sulfated GalNAc residues Thus, during
cleavage, bonds involving a 4-sulfated GalNAc residue
will be cleaved more often, resulting in a predominance
of 4-sulfated GalNAc residues at the reducing terminus
of the resulting oligosaccharides This significantly reduces the number of oligosaccharides actually observed However, it also means that the oligosaccha-ride CS#406, found at the chain cap of articular carti-lage CS [19], is very challenging to produce as the required tetrasaccharide CS0406 is not abundant after chondroitin ABC endolyase digestion [4] Other trisac-charides generated in this study are chain cap model structures; these data will aid identification of chain cap signals in large, or indeed intact, CS⁄ DS chains
In addition, trisaccharides may be isolated from chains that have been partially depolymerized by prior
Fig 5 Partial 600-MHz gradient-COSY-45 spectrum for CS060# at
43 C The spectral width was 1750.7 Hz, and 24 acquisitions for
each of 1024 increments were sampled into 1024 complex points.
The array was zero-filled to 2048 · 2048 complex points and
trans-formed in each dimension after application of a (sinebell) 2 window
function CS060# has the structure:
DUA(b1–3)GalNAc6S(b1–4)GlcA-ol.
Table 3 Carbon chemical shifts for the oligosaccharides CS040#, CS060# and CS#60#.
Residue Carbon
CS040#
(p.p.m.)
CS060#
(p.p.m.)
CS#60# (p.p.m.)
DUA (C) C1 102.83 104.25 –
C2 71.54 72.60 – C3 67.51 68.89 – C4 109.41 110.11 – C5 147.01 147.62 –
GalNAc (B) C1 102.97 103.70 103.81
C2 55.02 54.12 55.27 C3 78.24 82.50 73.86 C4 79.04 70.51 70.49 C5 77.41 75.60 75.49 C6 63.91 70.36 69.91 CH3 25.27 25.30 25.29
C ¼ O 177.79 178.02 – GlcA-ol (A) C1 65.61 65.46 65.38
C2 74.58 74.78 74.73 C3 73.04 73.16 73.01 C4 82.89 83.16 82.89 C5 76.11 76.59 76.46
Table 4 Carbon chemical shift Dd values between CS040# and CS060#.
Residue Carbon Calculated (p.p.m.) Observed (p.p.m.)
DUA (C) C1 +1.1 +1.4
GalNAc (B) C1 +0.6 +0.7
Trang 7endogenous glucuronidase activity It is noteworthy
that the trisaccharides isolated from tissue sources, i.e
CS040# and CS060#, have only been derived from
commercial samples of CS and not from those
pro-duced in a research laboratory environment, suggesting
that these may not represent major components
in vivo This suggests that commercial samples of CS
contain significant levels of chains that are not intact
and that do not represent an appropriate material for
the study of intact CS chains
Hexasaccharides
Although data from disaccharides, trisaccharides and
tetrasaccharides are valuable for the detailed structural
characterizations of unknown segments derived from
the CS families of polymers, there are many important
features that reside within larger oligosaccharide units
Data are therefore presented in Table 5 giving
compre-hensive 1H-NMR signal assignments for the primary
reduced hexasaccharide repeat units derived from
CS060606 and DS040404 In addition, a partial
600-MHz gradient-COSY-45 NMR spectrum for CS060606
is shown in Fig 6, and that for DS040404 is shown in
Fig 7
Both of the hexasaccharides showed the expected
presence of three N-acetyl methyl singlet resonances
Other regions of the spectra were complex, but the H1
and H4 sites in the DUA nonreducing terminal rings
(F) were readily assignable; 2D correlations to H2
and H3 indicated that for these unsaturated residues
the observed signal positions were closely similar to
the corresponding ringD values in the tetrasaccharides
CST0606 and DST0404 [16] In a similar manner, the
positions of signals corresponding to the GalNAc-ol
(residue A) reducing termini are all found to
corres-pond closely to those of the parallel tetrasaccharides
and those for ring B, residing between a uronic acid
and a GalNAc-ol unit in both the tetrasaccharides
and the hexasaccharides, are likewise little perturbed
The other ring likely to exhibit only small differences
relative to the corresponding tetrasaccharide is E
(C in the smaller oligomer) This is indeed found to
be the case; only for H1 of ring E in CS060606 is
there a relatively strong change, of )0.07 p.p.m.,
relat-ive to H1 of ring C in CST0606 For all of the other
ring E signals in both hexasaccharides the movements
are marginal
The remaining two pairs of residues may now be
assigned; the uronic acids at positionD both comprise
a set of five coupled spins The GalNAc rings located
atC are present as the typical seven-spin systems, and
both are strongly second order, even at 600 MHz, which is typical for these carbohydrate oligomers Rings C and D are located in regions of the hexasac-charides that are beginning to approximate the envi-ronments of polymeric chain segments, therefore the shift values observed are also becoming closer to those observed in macromolecular species These are import-ant data to take forward to the study of intact CS⁄ DS chains
It is becoming clear that a full understanding of the structure-function relationships of the chondroitin⁄ der-matan sulfates will require detailed data on sulfation
Table 5 Proton chemical shifts for the hexasaccharides CS060606, and DS040404 Shift values are from COSY-45 data except for those marked with an asterisk (*).
Residue Proton CS060606 (p.p.m.) DS040404 (p.p.m.)
DUA (F) H1 5.193* 5.268*
H2 3.796* 3.843*
H3 4.108* 3.952*
H4 5.885* 5.961*
GalNAc (E) H1 4.593 4.701
H2 4.027 4.066 H3 3.949 4.165*
H4 4.176 4.633 H5 3.996 3.87 H6 4.218 3.80 H6¢ 4.242 3.80 NAc 2.063* 2.123*
UA (D) H1 4.511* 4.886*
H2 3.387* 3.540*
H3 3.587 3.887 H4 3.745 4.115*
H5 3.709 4.728*
GalNAc (C) H1 4.618 4.670
H2 4.031 4.054 H3 3.860 4.024 H4 4.187 4.675 H5 3.984 3.87 H6 4.218 3.80 H6¢ 4.242 3.80 NAc 2.019* 2.082*
UA (B) H1 4.558* 4.975*
H2 3.451* 3.661 H3 3.646* 4.052 H4 3.754 4.132*
H5 3.754 4.634 GalNAc-ol (A) H1 3.718 3.690
H1¢ 3.789 3.722 H2 4.388* 4.260 H3 4.076 4.280 H4 3.573 4.451*
H5 4.300* 4.013*
H6 4.076 3.677 H6¢ 4.085 3.677 NAc 2.054* 2.037*
Trang 8sequence It is not sufficient to gain data on the
average ratio of 4-sulfation to 6-sulfation in a
popula-tion of chains It is likely that many funcpopula-tions will be
associated with domains or capping sequences of
speci-fic structure, and further studies of specispeci-fic
oligosac-charides from CS⁄ DS chains are required to enhance
our understanding of the biological activities of CS
and DS
Experimental Procedures
Materials
A Mono-Q 10⁄ 10 column was from Pharmacia (Uppsala,
Sweden), the Spherisorb S5 SAX column was from Phase
Separations Ltd (Deeside, Clwyd, UK), the Toyapearl
HW-40 s resin was from Anachem (Luton, UK) and the
Bio-Gel P2 resin was from Bio-Rad (Watford, Herts., UK)
Papain was from Sigma Chemical Co (Poole, Dorset, UK),
chondroitin ABC endolyase (protease free; Proteus vulgaris;
EC 4.2.2.4) was from Seikagaku Corp (Tokyo, Japan) via
ICN Biomedicals Ltd (High Wycombe, Bucks., UK), and
lithium perchlorate (ACS grade) and piperazine were from
Aldrich Chemical Co (Gillingham, Dorset, UK) All other
chemicals were of analytical grade
Isolation of CS from cartilage and DS from lung
CS was isolated from fresh articular and tracheal carti-lages as previously described [4,5], and DS was isolated from bovine lung as previously described [22] Briefly, the diced cartilage, or lung, was digested by papain (1 U per
100 mg tissue) in 0.1 m sodium acetate, pH 6.8, with 2.4 mm EDTA and 10 mm cysteine HCl, added just before digestion, for 24 h at 65C The GAGs were pre-cipitated from the soluble fraction by the addition of
4 vol ethanol, and the solution was cooled to 6C and allowed to stand overnight The precipitate was resus-pended in a minimum volume of 50 mm sodium acetate, and the CS, or DS, precipitated by the dropwise addition
of 2 vol ethanol while the solution was stirred The solu-tion was again cooled to 6C and allowed to stand over-night before recovery of the CS, or DS, rich precipitate, which was dialyzed overnight against distilled water and lyophilized
The CS, or DS, chains were released from the attached amino acids by b-elimination with 0.05 m NaOH containing
1 m sodium borohydride at 45C for 48 h [27] The reac-tion was terminated by the careful addireac-tion of 1 m acetic
Fig 6 Partial 600-MHz gradient-COSY-45 spectrum for CS060606
at 43 C The spectral width was 1750.7 Hz, and 24 acquisitions for
each of 1024 increments were sampled into 1024 complex points.
The array was zero-filled to 2048 · 2048 complex points and
Fou-rier transformed in each dimension after application of a 5% offset
(sinebell) 2 window function CS060606 has the structure:
DUA(b1–3)GalNAc6S(b1–4)GlcA(b1–3)GalNAc6S(b1–4)GlcA(b1–3)GalNAc6S-ol.
Fig 7 Partial 600-MHz gradient-COSY-45 spectrum for DS040404
at 43 C The spectral width was 1750.7 Hz, and 56 acquisitions for each of 512 increments were sampled into 1024 complex points The array was zero-filled to 2048 complex points in t 2 and Fourier transformed after application of a 10% offset sinebell window func-tion Data were then extended to 1024 points in t2by forward lin-ear prediction with order 12 before application of a 10% offset sinebell window function, zero filling to 2048 points and Fourier transformation DS040404 has the structure:
DUA(b1–3)GalNAc4S(b1–4) L -IdoA(a1–3)GalNAc4S(b1–4) L -IdoA(a1–3)GalNAc4S-ol.
Trang 9acid, and the solution dialyzed extensively against distilled
water and lyophilized
Purification of CS/DS on Mono-Q
CS⁄ DS was separated from any remaining non-GAG
material using a Mono-Q (10⁄ 10) column (1 cm · 10 cm)
in a gradient of 2–500 mm LiClO4⁄ 10 mm piperazine, pH
5, at a flow rate of 1 mLÆmin)1 The elution of bound
chains was monitored online by measuring A232
Depolymerization of GAGs
Aliquots, 1–10 mg, of CS or DS chains were depolymerized
with 1 U per 100 mg chondroitin ABC endolyase at
20 mgÆmL)1 in 0.1 m Tris⁄ HCl, pH 8, at 37 C for 15 h
The enzyme was inactivated by heating at 100C for
1 min, and the oligosaccharides generated were reduced by
the addition of 25 mm NaBH4
Isolation of oligosaccharides
Reduced oligosaccharides were subjected to SEC on a
Toyapearl HW40s column (50 cm· 1 cm) eluted in 0.5 m
ammonium acetate at 0.4 mLÆmin)1, the eluate being
mon-itored by measuring A232 Disaccharides, trisaccharides,
tetrasaccharides and hexasaccharides were separately
pooled as previously described [4], subjected to repeated
lyophilization, and then stored at)20 C
The individual oligosaccharides were purified, from the
CS trisaccharide, tetrasaccharide and hexasaccharide pools
recovered after HW40 SEC, by strong anion-exchange
(SAX) chromatography as previously described [6,16,20] In
each case a 10-mg aliquot of oligosaccharide was
resus-pended in 500 lL 2 mm LiClO4, pH 5.0, and
chromato-graphed on a Spherisorb S5 column (25 cm· 1 cm) at
2 mLÆmin)1 Bound material was eluted by a linear gradient
of 2 mm LiClO4 (buffer A) to 250 mm LiClO4 (buffer B),
pH 5.0, according to the following gradient profile: after a
10-min isocratic phase of 100% buffer A, a gradient of 0–
100% buffer B was introduced over 240 min, followed by
10 min of 100% buffer B The column eluate was
monit-ored online at 232 nm or, in the case of trisaccharides, at
206 nm Individual fractions were pooled, desalted by SEC
on a column of Bio-Rad P2 resin (1· 12 cm) running at
0.4 mLÆmin)1and 50C, and then lyophilized
Trisaccharide preparation by chemical removal of
unsaturated chain termini from tetrasaccharides
For removal of the nonreducing terminal unsaturated uronic
acids [26], an aliquot of reduced tetrasaccharide mixture was
incubated at room temperature with 500 lL 35 mm mercuric
acetate, pH 5, prepared as previously described [26] After
1 h, excess reagent was removed by mixing with 2 mL Dowex AG-50X (H+ form) which had previously been washed with 5 mL 5% HCl followed by 50 mL distilled water The oligosaccharides were separated from the resin
by centrifugation through a 0.45-lm nylon filter, and the resin was subsequently washed with 2 mL distilled water fol-lowed by 500 lL 1 m NH4HCO3and the sample lyophilized The crude trisaccharides were purified by SAX chromatogra-phy on a Spherisorb S5 column as described above, and the purified trisaccharides were desalted by SEC on Bio-Gel P2
as described above and then lyophilized
NMR spectroscopy
Samples were dissolved in 0.5 mL 99.8%2H2O, buffered to
pH 7 with phosphate (10 mm) and referenced with sodium 3-trimethyl[2H4]propionate as internal standard After micro-filtration through 0.45-lm nylon filters, samples were lyophi-lized using a rotary concentrator and exchanged several times with 0.5 mL 99.8%2H2O and then once with 99.96%
2H2O before final dissolution in 0.7 mL 99.96%2H2O Preliminary 1H-NMR spectra and all 13C-NMR spectra were obtained at 400 MHz (100 MHz for 13C) on a JEOL GSX400 spectrometer fitted with a 5 mm probe For 1D
13
C-NMR spectra, 50 000–250 000 acquisitions were per-formed, using 60 pulses at 1 s intervals High-field 1D and 2D correlation (gradient-COSY-45 and TOCSY) 1H-NMR spectra were determined at 600 MHz on a Varian Unity INOVA spectrometer fitted with a 5 mm triple nucleus probe capable of field-gradient experiments All spectra were determined at 43C, and1H and 13C chemical shifts are quoted relative to internal sodium 3-trimethyl-silyl[2H4]propionate at 0.0 p.p.m Experimental details for 2D spectra are given in the legends to the Figures The
C⁄ H-correlation 13
C-NMR spectrum was obtained using similar conditions to those described previously by Huc-kerby et al [28–30]
Spectra were reprocessed for presentation using the soft-ware packages Gifa V4.2 [31], obtained from Dr M.-A Del-suc (University of Montpellier, France), and nmrPipe [32]
Acknowledgements
We thank the Arthritis Research Campaign (arc) (grant N0528) for support, and the Engineering and Physical Sciences Research Council are acknowledged for provision of 600-MHz NMR facilities at the Uni-versity of Edinburgh
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