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Volume 272, Number 45, Issue of November 7, 1997 pp. 28227-28231
(Received for publication, February 28, 1997, and in revised form, July 22, 1997)
,
From the Department of Biological Sciences, ABSTRACT The keratan sulfate-containing proteoglycans were
isolated from fourteen pooled human corneas (thirteen from 61- to
86-year-olds, plus one from a 12-year-old). These proteoglycans were
subjected to digestion with the enzyme keratanase II, and the released
oligosaccharides, which included nonreducing termini and repeat region
oligosaccharides but not linkage regions, were reduced with alkaline
borohydride and identified on two separate ion-exchange columns. Both
of the latter had been calibrated with samples, most of which had been derived from bovine corneal keratan sulfate (Tai, G.-H., Huckerby, T. N., and Nieduszynski, I. A. (1996) J. Biol.
Chem. 271, 23535-23546) and all of which had been fully
characterized by NMR spectroscopic analysis.
The capping structures identified in human corneal keratan sulfates
occurred in the relative proportions: NeuAc INTRODUCTION The corneal stroma is a transparent tissue predominantly comprised
of regularly arranged collagen fibrils as well as small proteoglycans
and matrix proteins. The proteoglycans, which are associated with the
collagen fibrils (1), include the keratan sulfate
(KS)1 and the
chondroitin/dermatan sulfate (CS/DS) families. Electron microscopic
studies have demonstrated that several proteoglycan binding sites lie
within the D period of the collagen fibrils in the bovine cornea (2)
with the KSPGs at the a and c step bands and the
CS/DSPGs at the d/e gap zone. Scott (3) has
proposed a model for the structure of the corneal stroma in which
duplexed glycosaminoglycan chains (both double-stranded CS/DS and KS)
bridge collagen fibrils and maintain the precise interfibrillar
distances required for transparency of the tissue.
Molecular biology studies of bovine corneal KS have shown the existence
of several discrete proteoglycans. These have been named lumican (4),
keratocan (5), and osteoglycin (6).
Keratan sulfate was first isolated from bovine corneal stroma by Meyer
et al. (7), who subsequently classified KS types (8).
Corneal KS with an N-linkage between
N-acetylglucosamine and asparagine was called KS-I, and
skeletal KS with an O-linkage from
N-acetylgalactosamine to serine or threonine was designated KS-II. Extensive structural studies of corneal KS showed the linkage region to be of the biantennary complex type in both bovine (9) and
monkey (10) cornea. Other studies examined the distribution of
sulfation along the KS chain in porcine KS (11) and characterized oligosaccharides from the repeat region in bovine corneal KS (12). Recently, a considerable diversity of capping structures such as
Despite the many structural studies there has been little investigation
of human corneal keratan sulfate. Thus, this investigation seeks to
extend our previous work on bovine cornea (13), and it applies
keratanase II fingerprinting methodology (14, 15) to the elucidation of
the capping and repeat region structures in the entire population of
human corneal KSPGs.
EXPERIMENTAL PROCEDURES All materials used in this study were described
previously (13) except that papain (EC 3.4.22.2) was purchased from
Sigma (Poole, Dorset, UK), a Superose 6 column (10 × 300-mm) from
Pharmacia Biotech Inc. (Uppsala, Sweden), a Bio-Gel TSK-30 XL column
(300 × 7.8-mm) from Bio-Rad Laboratories Ltd. (Watford, Herts.,
UK), an analytical Spherisorb S5 SAX column (4.6 × 250-mm) from
Phase Separation Ltd. (Deeside, Clwyd, UK), and an IonPac AS4A SC
column (4-mm) from Dionex (Camberley, Surrey, UK).
Fourteen human
eyes (thirteen from 61- to 86-year-olds, plus one from a 12 year-old)
were obtained from the Manchester Eye Bank and came from individuals
with no history of ocular disease. The corneas had been removed within
48 h after death and stored in organ culture for periods ranging
from 2 to 3 weeks. The organ culture medium (16) consisted of Eagle's
minimal essential medium with Earles salts and HEPES buffer, 2%
heat-inactivated fetal calf serum, 24 mM sodium
bicarbonate, 2 mM L-glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 0.2 units/ml neomycin. The
corneas were rinsed several times with phosphate-buffered saline to
remove culture medium, and the corneas (with endothelium) were excised
immediately, as described previously (13).
The pooled human corneas (2 g wet weight) were extracted three
times each with 10 ml of 4 M guanidine-HCl containing
0.1 M 6-aminohexanoic acid, 0.01 M EDTA,
0.005 M benzamidine-HCl, and 0.05 M sodium
acetate, pH 5.8, at 4 °C for 24 h. These three extracts were
combined and dialysed thoroughly against 7 M urea, 50 mM Tris-HCl, pH 7.0.
The dialysate was applied to a Q-Sepharose column (15 × 50-mm)
and washed with sufficient 0.15 M NaCl, 7 M
urea, 50 mM Tris-HCl, pH 7.0, to remove the unbound
material. The bound material was eluted with a linear gradient of 0.15 M to 2 M NaCl containing 7 M urea
and 50 mM Tris-HCl, pH 7.0, over 30 min at a flow rate of 1 ml/min. The eluate was monitored (Fig. 1) by absorbance at 280 nm and
assayed for KSPGs by direct enzyme-linked immunosorbent assay (13)
using an anti-KS monoclonal antibody, 5D4. The KSPG-containing material
was recovered by dialysis against water (yield, 11 mg). In a parallel
experiment, 3 g of bovine corneas (wet weight, from 4 eyes) were
extracted in the same way, and 24 mg of bovine proteoglycans were
isolated. These preparations of proteoglycans were directly used for
keratanase II digestion and oligosaccharide fingerprinting.
[View Larger Version of this Image (18K GIF file)]
A proportion of the human proteoglycans (~1.1 mg) and bovine
proteoglycans (~1.2 mg) were further purified by gel permeation chromatography on a Superose 6 column (10 × 300-mm) (see Fig. 2
for human proteoglycan data). The KSPG-containing material (positive to
5D4) was recovered and used for KS chain-sizing experiments.
[View Larger Version of this Image (19K GIF file)]
Aliquots of the purified bovine and
human proteoglycans were treated with chondroitin ABC lyase as
described above. After precipitation with 3 vol of ethanol to remove
CS/DS-oligosaccharides, the KSPGs were recovered and subjected to
papain digestion. 100 µg of KSPGs were dissolved in 100 µl of 50 mM NaH2PO4, pH 7.0, containing 0.2 M NaCl, 50 mM EDTA, and 10 mM
cysteine hydrochloride, and incubated with 20 milliunits papain at
65 °C for 24 h. The digests were dialysed using Spectra/Por
membrane 1 (molecular mass cut-off: 6-8000 kDa protein) to remove
small peptides and chromatographed on a Bio-Gel TSK-30 XL column
(300 × 7.8-mm). The column was eluted with 0.15 M
NaCl and monitored by absorbance at 208 nm (Fig. 3).
[View Larger Version of this Image (18K GIF file)]
Crude preparations of human and bovine proteoglycans
(~10 mg) were dissolved in 1 ml of 10 mM sodium acetate,
pH 6.5, and incubated with 20 milliunits keratanase II. After 30 h
of incubation at 37 °C the digests were heated at 100 °C for 5 min followed by centrifugation at 100,000 × g for
2 h to remove precipitate.
The supernatant was chromatographed on a Bio-Gel P30 column (100 × 10-mm) to separate the oligosaccharides derived by keratanase II
digestion from the intact CS/DSPGs and the core proteins of KSPGs,
which eluted in the void volume (data not shown). After reduction with
NaBH4 the oligosaccharides were chromatographed on an
analytical Spherisorb S5 SAX column (4.6 × 250-mm), eluted first
with 2 mM LiClO4, pH 5.0, for 10 min, followed
by a linear gradient from 2 to 250 mM LiClO4
within 60 min and finally from 250 to 500 mM
LiClO4 within 10 min, at a flow rate of 1 ml/min and
monitored by absorbance at 206 nm. Partial profiles are shown in Fig.
4. Fractions corresponding to individual peaks, except peaks 7, 8, and
9, which were pooled, were recovered and re-chromatographed using a
calibrated IonPac AS4A SC column (4-mm) as described previously (14).
The chromatograms on IonPac AS4A SC of peaks 3, 4, 7, 8, 9, and 18 are
shown in Fig. 5.
[View Larger Version of this Image (26K GIF file)]
[View Larger Version of this Image (15K GIF file)]
RESULTS AND DISCUSSION The entire population of KSPGs from the
human corneal stroma (plus endothelium) was prepared after dissociative
extraction and initially, one stage of chromatography. This step (Fig.
1) involved ion-exchange chromatography
to separate the proteoglycans from protein contaminants. The
KS-containing proteoglycans from this human preparation elute at a
similar salt concentration to those from the bovine preparation (13)
indicating similar charge densities. The material recovered (Fig. 1,
pooling bar) contained nearly all of the KS and presumably
(see below) substantial amounts of decorin, although the lack of
response with the antibody LF95 (data not shown) indicates that this
antibody does not recognize human decorin. A small peak of 5D4-positive
material is observed prior to the start of the salt gradient, which was
possibly due to column overloading. The gel permeation chromatography
step (Fig. 2) permitted further
purification on the basis of size. The material recovered
(pooling bar) contained all of the KSPGs and decorin but the
procedure had removed protein contaminants such as the tyrosine-rich
acidic matrix protein, TRAMP (17).
The gel permeation chromatographic behavior of
the peptido-KS chains released from the entire population of human
KSPGs was compared with that from the bovine KSPGs (Fig.
3), and they were seen to be of the same
size. In the case of the bovine preparation the chromatogram of free
corneal KS chains released by the enzyme peptide
N-glycosidase F is shown. These chains were estimated (13)
to have a weight average Mr of 14,000 and a
number average Mn of about 10,000. It would,
therefore, seem that these sizes also pertain to the human KS chains,
although, as discussed before (13), the Mr and
Mn values may be slight underestimates because the column was calibrated (18) with linear rather than biantennary oligosaccharides.
The nonreducing terminal and
repeat region oligosaccharides derived from the entire population of
human KSPGs after keratanase II digestion followed by borohydride
reduction were examined on calibrated ion-exchange chromatography
columns. The results (Fig. 4) of the
chromatography on an analytical Spherisorb S5 SAX column for human and
bovine samples are compared and the peaks numbered 1-21. Clearly,
there are several resolution problems, particularly in peaks 7, 8, and
9. (In addition, only minor amounts of fucose-containing peaks had been
observed in the bovine KSPG preparation and hence the SAX column had
not been calibrated for such peaks.) Consequently, every peak from the
SAX column was recovered, and these samples were individually
re-chromatographed on a fully calibrated IonPac AS4A SC column. Those
corresponding to peaks 3, 4, 18, and the composite of peaks 7, 8, and 9 are shown in Fig. 5. Each and every oligosaccharide fragment has been fully characterized previously by NMR
spectroscopic studies and was used in calibrating the elution positions
on both ion-exchange columns. Detailed structural analyses for
fractions 2 and 21 have previously been presented (19), where they were
labeled as fractions R1 and C5. Similar identifications for fractions
1, 3b, 4a, 5, 6, 8, 9, 10, 11, 13, 14, 18a, 18b, and 19 have been
presented (20) where they were labeled as fractions F1, F3, F4, R2, R3,
C1, C2, F5, R4, F6, R5, C3, C4, and R6, respectively. Fractions 3a, 4b,
7a, 7b, 12, 15, 16, 17, 18c, and 20 have also been recognized before
(13), and the identities of the numbered peaks and cross-references to
fraction numbers are given in Table I.
Table I.
Oligosaccharides isolated from human and bovine corneal keratan
sulfates
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(2-6)-
>NeuAc(2-3)- >GalNAc(S)
(1-3)-. The other groups of
capping structures which had been identified in bovine corneal keratan
sulfate, i.e. NeuGc(2-3)-, NeuGc(2-6)-,
GlcNAc(S)(1-3)- were absent, although the possibility of the
presence of some Gal(1-3)- structures could not be excluded. In
addition, the human sample showed significantly higher levels of
(1-3)-fucosylated repeat region structures than did the bovine sample, and it is not clear whether this reflects a species or age
dependence as the bovine corneas were from young animals, whereas the
human corneas were predominantly from an older group. The charge
densities and keratan sulfate chain sizes of the human and bovine
keratan sulfate-containing proteoglycans were seen to be similar.
(2-6)- and (2-3)-linked N-acetylneuraminic acids,
(2-6)- and (2-3)-linked N-glycolylneuraminic acids,
(1-3)-linked galactose, as well as (1-3)-linked and 6-sulfated
N-acetylgalactosamine and N-acetylglucosamine
have been shown to be present on bovine corneal KS chains (13), but
neither their functions nor their KSPG distributions are currently
understood.
Fig. 1.
Q-Sepharose ion-exchange chromatography of 4 M guanidine-HCl extract of human corneal stroma. Human
corneal extract was loaded onto a Q-Sepharose column (15 × 50-mm)
and eluted with 0.15 M NaCl, 7 M urea, 50 mM Tris-HCl, pH 7.0 (data not collected). The gradient
started at fraction 20, and the eluate was monitored by absorbance at
280 nm. Fractions (2 ml) were assayed using 5D4 and pooled as shown by
the horizontal bar.
Fig. 2.
Superose 6 chromatography of corneal
KSPG-containing fractions. Aliquots (1 mg) of the crude human
corneal proteoglycans obtained as in Fig. 1 were applied to a Superose
6 HR column (10 × 300-mm) and eluted in 0.15 M NaCl,
7 M urea, 0.05 M Tris-HCl, pH 7.0, at a flow
rate of 0.3 ml/min. Proteoglycans were pooled as shown by the
horizontal bar.
Fig. 3.
Bio-Gel TSK-30 XL chromatography of
peptido-KS derived from bovine and human proteoglycans by papain
digestion. The column (7.8 × 300-mm) was eluted with 0.15 M NaCl at a flow rate of 0.5 ml/min and monitored by
absorbance at 208 nm. The chromatogram of free bovine corneal KS chains
released by the enzyme peptide N-glycosidase F (13) was
plotted to compare with those of peptido-KS. (RI, refractive
index).
Fig. 4.
Spherisorb S5 SAX ion-exchange chromatography
of keratanase II digests of human and bovine corneal
proteoglycans. The reduced oligosaccharides derived from ~10 mg
of either bovine or human proteoglycans were applied to an analytical
Spherisorb S5 SAX column (4.6 × 250-mm) and eluted as described
under "Experimental Procedures" (elution gradient is not shown) and
monitored by absorbance at 206 nm.
Fig. 5.
IonPac AS4A SC anion-exchange
chromatography. Samples corresponding to peaks 3, 4, 18, and the
pool of peaks 7, 8, and 9 in Fig. 4 were chromatographed individually
on an IonPac AS4A SC column (4-mm). The partial chromatograms of each
sample are assembled into a single figure. The column was eluted with 50 mM NaOH for 5 min followed by a linear gradient of 0 to
1.425 M sodium acetate containing 50 mM NaOH
within 48 min. The elution was monitored using a pulsed electrochemical
detector.
Code
Structure
Ha
Bb
Previous
codes
Ref. 19
Ref. 20
Ref. 13
1
Gal
1-4(Fuc1-3)GlcNAc(S)-ol+
+
F1
2
Gal
1-4GlcNAc(S)-ol+
+
R1
2
3a
NeuAc
2-6Gal1-4GlcNAc(S)-ol+
+
6
3b
Gal
1-4GlcNAc(S)1-3Gal1-4(Fuc1-3)GlcNAc(S)-ol+
+
F3
4a
Gal
1-4(Fuc1-3)GlcNAc(S)1-3Gal1-4GlcNAc(S)-ol+
+
F4
4b
Gal
1-3Gal1-4GlcNAc(S)1-3Gal1-4GlcNAc(S)-ol?
+
7
5
Gal
1-4GlcNAc(S)1-3Gal1-4GlcNAc(S)-ol+
+
R2
8
6
Gal(S)
1-4GlcNAc(S)-ol+
+
R3
9
7a
NeuGc
2-6Gal1-4GlcNAc(S)1-3Gal1-4GlcNAc(S)-ol
+
10i
7b
NeuGc
2-3Gal1-4GlcNAc(S)1-3Gal1-4GlcNAc(S)-ol
+
10ii
8
NeuAc
2-6Gal1-4GlcNAc(S)1-3Gal1-4GlcNAc(S)-ol+
+
C1
11
9
NeuAc
2-3Gal1-4GlcNAc(S)1-3Gal1-4GlcNAc(S)-ol+
+
C2
12
10
Gal(S)
1-4GlcNAc(S)1-3Gal1-4(Fuc1-3)GlcNAc(S)-ol+
+
F5
11
Gal(S)
1-4GlcNAc(S)1-3Gal1-4GlcNAc(S)-ol+
+
R4
14
12
Gal
1-3Gal1-4GlcNAc(S)1-3Gal(S)1-4GlcNAc(S)-ol?
+
15
13
Gal
1-4(Fuc1-3)GlcNAc(S)1-3Gal(S)1-4GlcNAc(S)-ol+
F6
14
Gal
1-4GlcNAc(S)1-3Gal(S)1-4GlcNAc(S)-ol+
+
R5
16
15
GalNAc(S)
1-3Gal(S)1-4GlcNAc(S)-ol+
+
17
16
NeuGc
2-3Gal(S)1-4GlcNAc(S)1-3Gal1-4GlcNAc(S)-ol
+
18
17
NeuAc
2-3Gal(S)1-4GlcNAc(S)1-3Gal1-4GlcNAc(S)-ol+
+
19
18a
NeuAc
2-6Gal1-4GlcNAc(S)1-3Gal(S)1-4GlcNAc(S)-ol+
+
C3
21
18b
NeuAc
2-3Gal1-4GlcNAc(S)1-3Gal(S)1-4GlcNAc(S)-ol+
+
C4
18c
GlcNAc(S)
1-3Gal(S)1-4GlcNAc(S)-ol
+
20
19
Gal(S)
1-4GlcNAc(S)1-3Gal(S)1-4GlcNAc(S)-ol+
+
R6
22
20
NeuGc
2-3Gal(S)1-4GlcNAc(S)1-3Gal(S)1-4GlcNAc(S)-ol
+
23
21
NeuAc
2-3Gal(S)1-4GlcNAc(S)1-3Gal(S)1-4GlcNAc(S)-ol+
+
C5
a
Oligosaccharides present (+) or absent ( ) in human
corneal KS.
b
Oligosaccharides present (+) or absent ( ) in bovine
corneal KS; ?, presence uncertain.
These results demonstrate that the human KS chains have a smaller
subset of nonreducing termini than do the bovine ones. The NeuGc(2-3)- (absence of peaks 7b, 16, and 20), NeuGc(2-6)-
(absence of peak 7a), GlcNAc(S)(1-3)- (absence of peak 18c) capping
structures which had been identified in bovine corneal KS were absent.
However, NeuAc(2-6)- (peaks 3a, 8, and 18a), NeuAc(2-3)- (peaks
9, 17, 18b, and 21) and GalNAc(S)(1-3)- (peak 15), in decreasing
order of abundance, were detected in the human KS chains. Observation of minor peaks at 4b and 12 also suggested the presence of some Gal(1-3)- structures in the human KS. The repeat region
oligosaccharides derived from the human sample are similar to those
from the bovine but show a higher proportion of fucosylation.
Comparison of these results on human KSPGs with those from bovine cornea show that the proteoglycans have similar charge densities and KS chain sizes. The similarity of KS chain size between human and bovine KSPGs is particularly interesting in the light of the double-stranded glycosaminoglycan model (3), which has been proposed for controlling the regular collagen interfibrillar spacing. Indeed, the collagen fibril surface-to-surface spacing (21) in human (31 nm, but decreases with increasing age) and bovine cornea (25.4 nm) are similar. However, great care must be taken in interpreting our results, because in both our bovine and human studies the KS chain preparation from the entire population of KSPGs has been examined, but not that from a single collagen fibril-associated proteoglycan such as lumican.
Comparison of the carbohydrate structures present in the human and
bovine KS chains show differences both in levels of fucosylation and in
nonreducing termini (capping structures). In our previous study of
bovine corneal KS only low levels of (1-3)-fucose (perhaps 1/10
chains) were observed. However, the bovine cornea were from young
(15-month-old to 3-year-old) animals. By contrast, this study of human
KS detects 2-4 times higher (1-3)-fucose levels (see Figs. 4 and
5), but the human samples predominantly derive from older individuals
(61- to 86-year-olds). Previous studies on bovine and human articular
cartilage KS have shown that fucose levels increase with
age,2 and thus it is probable
that the fucose levels noted in this study relate more to age than
species. The capping structures from human corneal KS do not contain
NeuGc(2-3)- or NeuGc(2-6)-, which are generally absent from human
adult tissues (23) nor was the GlcNAc(S)(1-3)- cap found.
Surprisingly, because humans do not normally have a functional
(1-3)-galactosyltransferase (22), a low level of Gal(1-3)- caps
was detected and this was not believed to be a contaminant. When
identified in humans the Gal(1-3)- structure is normally believed to
be associated with autoimmune conditions. The relative abundance of the
three major caps detected in the human corneal KS were NeuAc(2-6)-
>NeuAc(2-3)- >GalNAc(S)(1-3)- (although these data are not
fully quantitative), and these relative proportions are similar to
those observed in the bovine sample. It is interesting to note the
differences here with the capping proportions in articular cartilage KS
where for both bovine and human samples the content of NeuAc(2-3)-
>NeuAc(2-6)-, and there are age-related changes in NeuAc(2-6)-
content.2 A further understanding of the significance of
the diverse capping structures in corneal KS will require a closer
description of how they are distributed between antennae and specific
KSPGs.
In an as yet unreported part of these studies, we have made considerable but unsuccessful efforts to fractionate the bovine KSPGs on a preparative scale to permit characterization of the individual KS chains present. We believe that the best future chance of achieving this goal will depend upon antibody-based KSPG fractionation followed by high sensitivity keratanase II fingerprinting using a fluorescence-based method. Such a methodology should permit the full characterization of lumican and keratocan KS chains and may establish whether any relationship exists between specific chain capping and chain length, as the control of KS chain length in the cornea would seem to be a requirement for any precise collagen fibril spacer mechanism (3).
FOOTNOTES
To whom correspondence and requests for reprints should be
addressed: Dept. of Biological Sciences, Inst. of Environmental and
Natural Sciences, Lancaster University, Bailrigg, Lancaster LA1 4YQ,
UK. Tel.: 44-1524-593191; Fax: 44-1524-843854; E-mail: I.Nieduszynski{at}lancaster.ac.uk.
ACKNOWLEDGEMENTS
We thank Dr. G. M. Brown for helpful discussions and H. Morris and R. Berry for technical assistance.
REFERENCES
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