The anionic character of most known N-linked
oligosaccharides is due to the presence of sialic
acids(1, 2) . However, negative charge in such
molecules can also be due to phosphate esters(3, 4) ,
sulfate
esters(5, 6, 7, 8, 9, 10, 11, 12) ,
or, possibly, uronic acids (13, 14, 15, 16) . Unlike sialylated
chains, the other types of anionic molecules are considered rare, being
reported only in small amounts and/or only on certain proteins. By
metabolic labeling with [
S]sulfate and release
with peptide:N-glycosidase F
(PNGaseF)(
)(17) , we previously identified and
characterized a diverse family of anionic N-linked
oligosaccharides in CPAE cells, a calf pulmonary artery endothelial
cell line(10, 11) . These sugar chains were separated
by size and charge into two general classes. ``Class I'' was
composed of molecules bearing various combinations of primary sulfate
esters and sialic acids, while ``Class II'' molecules carried
sequences susceptible to cleavage by glycosaminoglycan-degrading
enzymes. About half of the negative charge on the sulfated Class I
molecules could be attributed to GlcNAc-6-sulfate units at a position
subterminal to sialic acid and
-galactose. In the case of Class II
molecules, most of the negative charge was susceptible to heparin and
chondroitin lyases, suggesting a novel class of
``N-linked glycosaminoglycans.'' Collectively, all
these sulfated molecules represented
10% of the total
PNGaseF-releasable N-linked oligosaccharides from this cell
line. Further characterization was hindered by their extreme diversity,
as well as by the small quantities of material available (CPAE cells
are a primary cell line with limited growth capacity).
Although the
mammalian lung has many different cell types, about 40% of its mass is
derived from endothelial cells(18, 19) . We reasoned
that the total bovine lung might therefore be a rich source of
molecules similar to those found in CPAE cells. We show here that
PNGaseF treatment of an extract of bovine lung acetone powder releases
anionic N-linked oligosaccharides with some similar
properties. However, there were also many striking differences, both in
the relative quantity of anionic molecules released and in the fact
that the majority of the negative charge is contributed not by sulfate
esters, but by carboxylic acids other than sialic acids. We present
here the identification, fractionation, and partial characterization of
these unusual anionic oligosaccharides. Apart from the novel features
mentioned above, we report several additional properties of the N-linked glycosaminoglycans. This work also emphasizes that
there are very few reported studies of the anionic N-linked
oligosaccharides from intact mammalian tissues. Indeed, the great
majority of previously described anionic N-linked
oligosaccharides originate from a restricted subset of accessible
glycoproteins of the blood plasma and blood cells and of recombinant
proteins derived from cultured cells. The assumption that similar
molecules will predominate in other mammalian tissues is challenged
here.
EXPERIMENTAL PROCEDURES
Materials
Most of the materials used were from Sigma. The following
were from the indicated sources: trifluoroacetic acid, EDC, and NHS,
Pierce; methyl iodide (CH
I) and methylamine
(CH
NH
), Fluka;
[
H]NaBH
, ICN; concanavalin A-agarose
and L-PHA-agarose, E.Y. Laboratories; Arthrobacter ureafaciens sialidase, Calbiochem; heparinase and heparitinase, Seikagaku,
Tokyo. Homogeneous Escherichia coli alkaline phosphatase was a
gift from M. J. Schlesinger, Washington University. Samples of
homogeneous human placental
-hexosaminidase A were generously
provided by Arnold Miller, University of California, San Diego, Don
Mahuran, Hospital for Sick Children, Toronto, and Mario Ratazzi, North
Shore University Hospital, Manhasset, NY. Human
-iduronidase was
the kind gift of Elizabeth Neufeld, University of California, Los
Angeles. Diazomethane in ether was kindly provided by Claudio
Schteingart, UCSD.
Isolation/Extraction of Bovine Lung Acetone Powder
Oligosaccharides
For every 1 g of bovine lung acetone powder (Sigma), 7 ml of
lysis buffer (50 mM Tris-HCl, pH 7.5, 1% SDS, 0.1 M
-mercaptoethanol) was added. This mixture was homogenized
with a Polytron on low speed to solubilize the powder, heated at 37
°C for 30 min, with continued heating overnight at 50 °C. The
sample was then boiled for 10 min and ultracentrifuged at
100,000
g for 40 min. The supernatant was filtered through an
0.8-µm filter with glass wool layered over a pre-filter, and
aliquots (30 ml, representing
5% of the column volume) were
applied to a Sephacryl S-200 column (100
3 cm), eluted with 10
mM Tris-HCl, pH 6.5, 0.2% SDS (fractionation range
250,000-5,000 kDa for globular proteins). The eluent was pumped
to achieve a flow rate of 1 ml/min, and 10-ml fractions were collected.
For large preparations, it was necessary to do multiple runs to
fractionate all of the material. The void area was monitored by A
and pooled, and the proteins precipitated with
9 volumes of ice-cold acetone added slowly to the sample while stirring
at 4 °C. The precipitate was pelleted by centrifugation (2000 rpm
for 15 min), the acetone was decanted, and the pellet was immediately
resuspended in PNGaseF buffer (10 mM Tris-HCl, pH 7.2, 50
mM EDTA, pH 7.5, 0.2% SDS, 1% Nonidet P-40, 20 mM
-mercaptoethanol). The sample was incubated at 37 °C for 30
min with a loosened cap to evaporate excess acetone and split into two
fractions: a control (1% of the sample) and the remainder for PNGaseF
treatment. PNGaseF, prepared in our laboratory as
described(20) , was added at a concentration, which was able to
completely release N-linked oligosaccharides from a similar
amount of fetuin, and incubated overnight at 30 °C under a toluene
atmosphere. After boiling for 10 min, the samples were reapplied to the
S-200 column as above. The released material was included in the column
and detected with the reducing sugar assay (see below). These fractions
were pooled, and SDS was precipitated by adding 1/100 volume of
saturated KCl to the sample followed by refrigeration overnight at 4
°C. The supernatant was collected after spinning at 2000 rpm for 15
min and desalted on either a Sephadex G-25 or a Bio-Gel P-2 column
eluted with H
O.
Reducing Sugar Assay (21, 22
Aliquots of samples were brought up in a total volume of 300
µl of H
O. 700 µl of freshly prepared reagent C was
added, and the mixture was heated at 80 °C for 30 min. After
cooling, the absorbance was read at 560 nm. Reagent C consists of 23 ml
of solution A + 1 ml of solution B + 6 ml of ethanol. To
prepare solution A, 1.5 g of disodium 2,2`-bicinchoninate is dissolved
in 1 liter of water, 71.6 g of anhydrous sodium carbonate is added
while stirring, and the volume is brought up to 1.15 liter final with
dH
O. To prepare solution B, 3.7 g of aspartic acid and 5.0
g of anhydrous Na
CO
are dissolved with shaking
in 100 ml of water, and then mixed with 1.09 g of copper sulfate
dissolved in 40 ml of water.
H Labeling of PNGaseF Released
Bovine Lung Oligosaccharides (23, 24
Purified desalted N-linked oligosaccharides released
by PNGaseF were dried, and 200 µl of 0.2 M sodium borate,
pH 9.8, was added(25) . [
H]Sodium
borohydride (1-5 mCi/oligosaccharides derived from 1 g of bovine
lung acetone powder) were each dissolved in 0.2 M sodium
borate, pH 9.8, mixed in a fume hood, and the reduction was allowed to
proceed for 1-3 h at room temperature. An excess of unlabeled
sodium borohydride (10-fold) over the amount of expected
oligosaccharides was added, and the reduction was continued for 1 h at
room temperature. The reaction was quenched either by acidification
using glacial acetic acid and repeated drying with acidified methanol
or by the addition of a 10-fold molar ratio of acetone over the
non-radiolabeled sodium borohydride which was left at room temperature
for an additional hour. The sample treated by either procedure was
further separated from unreacted radioactivity by direct application to
a disposable 17-ml Bio-Gel P-2 column and desalted in H
O,
collecting only the void volume area.
QAE Fractionation of Released Oligosaccharides (4, 10, 11
QAE-Sephadex columns (0.8 ml) were washed with 10 column
volumes of 2 mM Tris base. The samples were loaded in 2 mM Tris base and washed with 7
750 µl of 2 mM Tris base. Oligosaccharides were eluted in a batchwise manner with
increasing concentrations of sodium chloride (20, 70, 125, 200, 400,
and 1000 mM NaCl, each in 2 mM Tris base). Each step
consisted of 4 or 5 fractions, with each fraction composed of 2
750 µl additions of buffer.
Enzyme Reactions
A. ureafaciens sialidase digestions were performed
by adjusting the sample to 50 mM sodium acetate, pH 5.5
(final), with 5 milliunits of A. ureafaciens sialidase in a
10-µl reaction volume, and incubating at 37 °C for 2 h.
Alkaline phosphatase digestions were performed by adjusting the sample
to 200 mM Tris-HCl, pH 8.0 (9 µl final volume) with 0.6
milliunit of enzyme, incubating at 37 °C for 30 min. GAG degrading
enzyme digestions were performed using a common buffer, consisting of
10 mM Tris-HCl, pH 7.2, 2.5 mM CaCl
(final) in a 10-µl volume, using 3 µl of each enzyme, and
incubating at 37 °C overnight (15-18 h): heparinase (1
unit/µl), heparitinase (1 unit/µl), chondroitinase AC (1
unit/50 µl), chondroitinase ABC (1 unit/50 µl), or keratanase
(1 unit/50 µl). Human placental
-N-acetylhexosaminidase A incubations were carried out in
100 mM sodium formate, pH 3.5, at 37 °C under a toluene
atmosphere with 0.5 unit of enzyme per 200 µl final reaction
volume. All these enzyme reactions were inactivated by heating the
samples at 100 °C for 10 min.
-Glucuronidase (13 milliunits)
and
-iduronidase (48 milliunits) treatments of oligosaccharides
were carried out in 40 µl of 80 mM sodium citrate, pH 4.6,
overnight under a toluene atmosphere. For some incubations, these
reactions were carried out in the presence of jack bean
-N-acetylhexosaminidase (53 milliunits), bovine
testicular
-galactosidase (2 milliunits), and coffee bean
-galactosidase (5 milliunits). All commercial enzymes were tested
for activity under the conditions used.
Acid Hydrolysis Conditions
To selectively remove sialic acids and cleave phosphodiester
bonds, samples were heated in either 10 mM HCl at 100 °C
for 30 min (4) or 2 M acetic acid at 80 °C for 3
h(26) . Strong acid hydrolysis, needed to break all neutral and
amino sugar glycosidic bonds, was carried out with 2 M trifluoroacetic acid at 100 °C for 4 h (27) .
1,2-Diamino-4,5-methylenedioxybenzene Derivatization and
Analysis (28, 29
1,2-Diamino-4,5-methylenedioxybenzene derivatization and
analysis of sialic acids was done exactly as described previously.
High Voltage Paper Electrophoresis (30
Samples were hydrolyzed with 6 M HCl for 4 h at 100
°C, then spotted on a Whatman No. 3 paper. The entire paper was
moistened with 0.06 M sodium borate, pH 6.5, placed between
two sheets of Mylar, and the ends of the paper were immersed in the
same buffer. Constant voltage was applied at 40 V/cm for 2.5 h with
continuous cooling of the system. Detection was accomplished by cutting
1-cm strips, soaking them in water overnight, and counting in a Beckman
scintillation counter.
HPAEC-PAD Monosaccharide Analysis (27, 31
The samples were strong acid-hydrolyzed as described above,
and monosaccharides were analyzed using a CarboPac PA1 column (250
4 mm; Dionex Corp.) eluted isocratically at 1 ml/min with 18
mM NaOH. The post-column concentration of NaOH was increased
by helium pressure-driven on-line addition of 1 M NaOH at a
rate of 0.3-0.4 ml/min. Detection was accomplished using a PAD-I
cell (Dionex Corp.) with the settings of: E
= 0.15,
E
= 0.7, E
= -0.3, T
= 9, T
= 2, T
= 6,
response time = 1, range = 1, output = 300 nA. The
elution position of each monosaccharide standard was checked
separately, and standard runs were always carried out at the beginning
and end of each set of samples. Occasional inversion in the order of
elution of the pairs Gal/GlcNH
or Xyl/Man were sometimes
noted and seem to be the related to the use of a particular column and
the specific elution conditions used.
Anion Exchange HPLC Analysis of Oligosaccharides
Samples were applied to a TSK-DEAE-2SW (250
4.6 mm)
column from TosoHaas, which was then eluted with the following
gradient: 0-30 min, 0-300 mM NaCl; 30-35
min, hold at 300 mM NaCl; 35-65 min, 300-700
mM NaCl; 65-70 min, hold at 700 mM NaCl;
70-120 min, 700-0 mM NaCl, at a constant flow rate
of 0.6 ml/min. Detection was done with either a Radiomatic
Flow-One-Beta on-line radioactive detector or by collecting fractions
and monitoring radioactivity.
Rhodizonate Assay for Sulfate (32
This procedure was done as described previously. Briefly, the
sample was brought up in 100 µl of H
O. 600 µl of a
barium buffer was added, followed by 300 µl of rhodizonate reagent.
The sample was vortexed, and complex formation was allowed to occur for
10 min at room temperature. The absorbance was then read at 520 nm.
Solvolysis (9, 33
The samples were converted to their pyridinium form by
passage over a Dowex 50 column (hydrogen form). The run-through and
water wash were collected on ice into a 15-ml glass conical tube
containing 4 drops of pyridine and lyophilized to dryness. A 0.2-ml
aliquot of Me
SO containing 10% methanol was added to each
sample and heated at 80 °C for 2 h. The sample was lyophilized,
brought up in 2 mM Tris base, and reapplied to a QAE-Sephadex
column for analysis of loss of negative charge.
Lectin Affinity Chromatography (34
Concanavalin A
A 1-ml column of concanavalin
A-agarose was poured and equilibrated with 10 ml of TBS-NaN
(0.01 M Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM
CaCl
, 1 mM MgCl
, 0.02%
NaN
). The sample was loaded in 750 µl of
TBS-NaN
. Elution of bound material was as follows: 30 ml of
TBS-NaN
, 30 ml of 10 mM
methyl-
-D-glucopyranoside in TBS-NaN
, 30 ml
of 100 mM methyl-
-D-mannopyranoside in
TBS-NaN
, prewarmed to 60 °C.
L-PHA
A 1-ml column of L-PHA-agarose was poured
and equilibrated with 10 ml of phosphate-buffered
saline-NaN
. The samples were loaded and run in
phosphate-buffered saline-NaN
; sample loading volumes were
200 µl. Fractions of 0.4 ml were collected and counted
analytically.
Linkage Analysis by Permethylation (35, 36
The samples were permethylated as described by Hakomori.
Briefly, to the dried samples, 100 µl of anhydrous Me
SO
was added and the vials were flushed with N
. Dissolution
was accomplished by sonication for 5 min intervals over a period of 4
h. 100 µl of methylsulfinyl carbanion was added, N
flushed into the vials and sonicated every 15 min for 1 h. 100
µl of methyl iodide (CH
I) was added dropwise on ice and
the sample was sonicated every 15 min for 2 h. 400 µl of
methylsulfinyl carbanion was added, and excess of base was ascertained
by testing an aliquot with triphenylmethane. The reaction mixture was
kept overnight at room temperature, and CH
I addition was
repeated as before. An equal volume of chloroform was added to the
sample as well as 10 ml of H
O, and the entire sample was
dialyzed against H
O and lyophilized. The sample was
subjected to acetolysis/hydrolysis as
described(35, 36) . 0.3 ml of 0.5 N
H
SO
in 90% acetic acid was added, and the
samples were heated at 80 °C for 6 h. After cooling, the samples
were neutralized by addition of 0.5 N NaOH and dried with
N
and exposure to P
O
overnight.
Hydrolyzed samples were dissolved in 10 mM NaOH (pH 9.0) and
reduced with NaBD
overnight at room temperature. Excess
NaBD
was destroyed with glacial acetic acid, and the methyl
borates were removed by repeated evaporation with acidified methanol (5
times). Samples were dried over P
O
in a
desiccator. Acetylation was done with acetic anhydride (0.3 ml) at 100
°C for 3 h. After repetitive extraction with toluene, the partially
methylated, partially acetylated alditols were recovered by partition
in CHCl
/H
O. Samples were dissolved in acetone
(10 µl), 1 µl was injected onto a DB-5 (J & W Scientific)
capillary column (25 mm
30 m) with the following temperature
program: start at 50 °C, held for 2 min; at 20 °C/min, from 50
°C to 150 °C, and at 4 °C/min, from 150 °C to 250
°C, and maintaining the final temperature for an additional 5 min.
A Finnigan MAT 4500 gas chromatograph-mass spectrometer with a
computerized data system was used in the EI mode (ionizing potential 70
eV) for detection.
Masking of Carboxylic Acids by Carbodimide Activation and
Coupling to Methylamine
QAE-fractionated
H-oligosaccharides were desalted
by gel filtration using a Bio-Gel P-2 column eluted with water,
desialylated by mild acid treatment with 10 mM HCl for 30 min
at 100 °C, and lyophilized. The samples or
[
H]sialyl-(
2,6)-lactose (standard with
carboxylic acid residues) were dissolved in 5 µl of 50 mM MES buffer, followed by addition of 10 µl of 1 M methylamime. A stock activation solution of EDC/NHS was freshly
prepared by the addition of 100 µl of H
O to an
Eppendorf tube containing 10 mg of EDC and 5 mg of NHS and vortexed. 5
µl of the EDC/NHS solution was immediately added to the samples,
vortexed, and incubated at 37 °C. After 1 h, another 5 µl of
fresh EDC/NHS solution was added and the incubation continued for
another hour. Control samples were treated identically except that the
EDC/NHS solution was replaced with water. The samples were diluted to 4
ml with 2 mM Tris base, loaded onto a QAE-column in 2
2 ml fractions, and step-eluted with sodium chloride as described
above, and radioactivity was monitored.
Methyl Esterification of Carboxylic Acids with
Diazomethane
Samples were converted to their H
form by
passage over a Dowex AG 50W column (H
form),
lyophilized in Reacti-Vials, and 100 µl of dry Me
SO or
methanol was added. The vials were flushed with argon and capped, and
the samples were dissolved by vortexing and cooled on ice.
Approximately 100 µl of cold diazomethane (dissolved in ether) was
transferred into the vials via a cannula and vortexed. This procedure
was repeated twice, until a persistent yellow color was seen. The
samples were then incubated at room temperature for 2 h. Diazomethane
and ether were removed by a stream of argon gas, and the samples were
dried in a Speed Vac evaporator. QAE fractionation was then performed
as described above, but in 2 mM sodium cacodylate, pH 5.8,
instead of Tris base (to avoid base hydrolysis of the methyl esters).
An aliquot of the methyl esterified (neutralized) fraction that ran
through the QAE-column was treated with NaOH (final 20 mM, 1
ml) to regenerate the carboxylic acids. After incubation at room
temperature for 4 h, the base-treated samples were neutralized with
HCl. Controls were similarly treated with 20 mM NaCl. Both
samples were desalted by overnight dialysis with 500 molecular weight
cut-off tubing and then refractionated on QAE-columns.
Methyl Esterification of Carboxylic Acids with Methyl
Iodide in Me
SO or Methanol
Samples were converted to their Na
salt form
by passage over Dowex AG 50W (Na
form) and dried by
vacuum centrifugation. 100-µl of dry Me
SO and 20 µl
of methyl iodide were added to the samples, which were vortexed and
incubated at room temperature for
4 h with occasional vortexing.
QAE fractionation was performed as described above in 2 mM sodium cacodylate, pH 5.8 (instead of Tris base), and monitored
for radioactivity. Control samples were treated similarly, but the
methyl iodide was deactivated by heating in water before addition to
the samples.
Methyl Esterification of Carboxylic Acids by Methyl
Iodide Treatment on a DEAE-column
Carboxylic acid-containing samples were methyl-esterified
with methyl iodide after binding to a DEAE-column as described
previously (37) with the following modifications: (i) the
column and washes were scaled down by a factor of 4 and (ii) the methyl
esterification on the column was carried out somewhat longer and
repeated 4 times. The latter modification from the original protocol
(which was designed for methyl esterification of sialic acids) was
found necessary to adequately neutralize a glucuronic acid standard.
Briefly, the samples in methanol were loaded onto 1-ml DEAE-Sephadex
A-25 columns equilibrated in methanol and washed with dry methanol. The
carboxylic acids were methyl esterified by the addition of 0.4 ml of
Me
SO/CH
I (5:1) which was allowed to flow into
the top of the gel. The flow was then stopped leaving
2 mm of
solvent height above the bed surface, and the column was incubated for
10 min at room temperature. This procedure was repeated 4 times.
Carboxylic acid-containing samples were neutralized by this on-column
treatment and were washed out with methanol. Any remaining negatively
charged glycans were then eluted with 1 M pyridinium acetate,
pH 5.5.
Conversion of Methyl Esters to Methyl Amides by
Methylamine Treatment
Methyl-esterified samples eluted from the DEAE-column as
above were converted to their respective methylamides as described
previously (37) , with minor modifications. Briefly, methanol
and Me
SO were removed from the samples by a stream of
nitrogen gas and lyophilization, respectively. The samples were
redissolved in 200 µl of dry methanol, incubated with 100 µl of
25% methylamine in methanol for 20 min at room temperature, and dried
by vacuum centrifugation.
Regeneration of Charged Carboxylic Acids from Methyl
Esters by Saponification
Saponification of methyl-esterified samples prepared by
methyl iodide treatment on a DEAE-column was performed to regenerate
carboxylic acids and to monitor the extent of conversion to methyl
amides by base resistance. 100 mM NaOH (50 µl) was added
to the samples and incubated for 2 h at 50 °C. The base was then
neutralized by addition of an equal volume of 100 mM HCl. 50
µl of 100 mM NaCl were added to control samples. The
base-treated or control samples were dissolved in 2 ml of methanol,
loaded onto an 0.8-ml DEAE-column, and washed with 4
2 ml
fractions of methanol. Charged glycans were eluted with 4
2 ml
fractions of 1 M pyridine acetate, pH 5.5.
RESULTS
Release, Isolation, and Radioactive Labeling of
N-Linked Oligosaccharides from Bovine Lung Acetone Powder
As a
convenient and concentrated source of glycoproteins from bovine lung,
we used a commercially available acetone powder. To specifically
release and purify N-linked oligosaccharides, we used the same
approach (see ``Experimental Procedures'') as previously
employed for N-linked chains from CPAE
cells(10, 11) . Briefly, the acetone powder was
homogenized into a lysis buffer containing SDS and heated to solubilize
glycoproteins. The soluble material was prefractionated on a Sephacryl
S-200 column (run in a buffer containing SDS) to eliminate any small to
medium-sized molecules, e.g. nucleotides, salts, and degraded
cellular products. The material eluting in the void volume of this
column (which includes all large molecules as well as most
glycoproteins that bind SDS and become incorporated into SDS micelles)
was treated with PNGaseF and refractionated on the same Sephacryl S-200
column. The elution profile was monitored using a reducing sugar assay,
which also reacts weakly (about 100-fold less) with proteins. As shown
in Fig. 1, PNGaseF treatment results in the appearance of a new
peak in the partially included area that is not observed in a control
incubation done in the absence of enzyme. The PNGaseF-released material
was pooled as shown in Fig. 1, avoiding any overlap with
fractions where undigested material eluted (as shown in Fig. 1,
this precaution prevents inclusion of some of the largest released
molecules, which were not studied further). Since PNGaseF-released free
oligosaccharides have a reducing end, we next introduced tritium label
into the sugar chains by [
H]NaBH
reduction, as described under ``Experimental Procedures.''
Over the last 5 years, we have made more than 10 such preparations and
characterized each to varying extents. While some batch-to-batch
variation has been noted, the general properties of all of the released
molecules were similar. The following results are representative
examples derived mostly from one of the preparations.
Figure 1:
Release and
isolation of N-linked oligosaccharides from bovine lung.
Bovine lung acetone powder was solubilized in an SDS lysis buffer, and
the extract was fractionated on a Sephacryl S-200 gel filtration column
as described under ``Experimental Procedures,'' collecting
material that eluted in the void volume (as monitored by absorbance at
280 nm, data not shown). This material was pooled, and aliquots were
incubated with (
-
) or without
(
-
) PNGaseF and reapplied to the same S-200
column. Newly created reducing ends were monitored with a reducing
sugar assay as described under ``Experimental Procedures'' (A
). This modified bicinchoninate assay gives a
weak reactivity with proteins and also reacts with
-mercaptoethanol, likely explaining the peaks seen in the void and
totally included areas, respectively. The closed bar indicates
the fractions where the released oligosaccharides were detected and
pooled for further studies. This figure is representative of over 10
such profiles obtained with various
preparations.
A Large Percentage of the N-Linked Oligosaccharides from
Bovine Lung Are Highly Anionic
To purify and partially
fractionate the oligosaccharides by negative charge, we applied the
PNGaseF-released
H-labeled molecules to a QAE-Sephadex
column and eluted them by stepwise increases in sodium chloride
concentration, as described previously(10, 11) . The
salt concentrations chosen for elution were based on prior experience
with anionic N-linked oligosaccharides. In general, N-linked oligosaccharides with a single negative charge are
expected to elute with 20 mM NaCl, those with two charges with
70 mM NaCl, and those with three to four negative charges with
125 mM NaCl (at higher concentrations of salt, the resolution
is less clear, but molecules eluting with 200 mM and 400
mM NaCl are expected to have 4 or more negative charges). As
shown in Fig. 2, about 80% of the radioactivity bound to the
column and eluted with a profile roughly similar to that seen
previously with CPAE oligosaccharides(10) . The CPAE molecules
were labeled with [
S]sulfate, whereas the bovine
lung oligosaccharides had tritium label introduced uniformly in the
reducing end of each molecule. Thus, the ratios of the different
fractions cannot be compared directly. Regardless, there was a
surprisingly high percentage of moderate to highly anionic molecules in
the lung preparation (51% of the negatively charged oligosaccharides
eluting with 200 mM NaCl or higher, representing molecules
expected to have four or more negative charges). Most highly anionic
oligosaccharides reported to date (other than those with polysialic
acid) are of the glycosaminoglycan (GAG) type, e.g. the
heparin/heparan sulfates (HS), chondroitin sulfates (CS), and keratan
sulfates (KS). With the exception of KS, these oligosaccharides are
generally thought to be linked to proteins via O-xylosyl
linkage and therefore should not be released by
PNGaseF(38, 39) . However, in our previous study, a
small fraction (<1%) of the PNGaseF-released oligosaccharides from
CPAE cells were shown to carry GAG chains, either HS or CS. In the
present preparation from bovine lung, about 13% of the anionic
molecules elute with 1 M NaCl, suggesting that a larger
fraction might carry GAG-type structures.
Figure 2:
Fractionation of N-linked
oligosaccharides by negative charge. PNGaseF-released oligosaccharides
were reduced with [
H]NaBH
and
separated from unreacted radioactivity by gel filtration on either a
Sephadex G-25 or Bio-Gel P-2 column, as described under
``Experimental Procedures.'' The labeled oligosaccharides
were applied to a QAE-Sephadex anion exchange column, which was washed
and eluted batchwise by increasing amounts of sodium chloride. This
figure illustrates a profile representative of over 10 such
preparations. The fractions which comprise the Class I and Class II
oligosaccharides are indicated. RT denotes material which ran
through the column and accounts for
20% of total labeled
material.
The Anionic Oligosaccharides from Bovine Lung Fall into
Two Major Classes
Treatment of the oligosaccharide mixture with
sialidases or with GAG-degrading enzymes gave shifts in the profile of
negative charge seen by batch elution on QAE-Sephadex (data not shown).
As an alternate approach to subfractionating these oligosaccharides, we
used a TSK-DEAE-2SW HPLC anion exchange column eluted with a salt
gradient. As seen in Fig. 3, the less charged molecules were
partially susceptible to digestion with A. ureafaciens sialidase (apparently similar to Class I oligosaccharides from
CPAE cells). The highly charged oligosaccharides were resistant to this
treatment, but were completely digested to less negatively charged
fragments by a combination of GAG-degrading enzymes (similar to Class
II oligosaccharides from CPAE cells). Thus, on initial evaluation, the
bovine lung oligosaccharides seem to fall into the same general classes
as the structures found in the CPAE cells.
Figure 3:
Effects of sialidase and GAG-degrading
enzymes on anionic properties of bovine lung N-linked
oligosaccharides. The total mixture of PNGaseF-released
H-labeled oligosaccharides was applied to a TSK-DEAE-2SW
anionic HPLC column and eluted with a gradient of sodium chloride. A shows the profile obtained without any treatment. In B, the mixture was digested with A. ureafaciens sialidase, and in C with a combination of the following
GAG-degrading enzymes: heparinase, heparitinase, chondroitinase ABC,
chondroitinase AC, and keratanase. RT, material running
through the column.
The complete structural
characterization of these oligosaccharides would first require their
separation into distinct species. However, as might be expected for a
whole tissue library, both classes of oligosaccharides are extremely
complex mixtures that have defied many attempts at purification into
defined species. The methods tried included a variety of anionic
exchange systems including QAE-columns with gradient separations, DEAE-
and TSK-DEAE-2SW HPLC columns eluted with either sodium acetate or
sodium chloride gradients, FPLC Mono Q chromatography with either
sodium chloride or sodium sulfate gradients, and a Varian AX-5 HPLC
anion exchange column eluted with phosphate gradients (data not shown).
Gel filtration sizing with Bio-Gel P-4, P-6, or Sephadex G-50 run in
water, Bio-Gel P-60, P-100, and P-300 run in 200 mM Tris-HCl,
pH 6.5, with 0.2% SDS, Bio-Gel P-100 run in 2 M sodium
acetate, and Superose-12 FPLC size fractionation have also been tried.
All of these methods gave broad peaks, but none gave adequate
separation of the numerous individual species contained within the
mixture. We therefore obtained composite structural information on the
mixtures of the Class I and Class II oligosaccharides obtained by Class
II Sephadex or TSK-DEAE HPLC fractionation, as shown in Fig. 2and Fig. 3, respectively.
The Label Introduced by
NaB[
H]
Is in
N-[
H]Acetylglucosaminitol
Although these
molecules were specifically released from bovine lung acetone powder
using PNGaseF, an enzyme whose specificity is restricted to most known N-linked sugars, we wished to confirm directly that the
H-labeled oligosaccharides had the expected N-[
H]acetylglucosaminitol (GlcNAcitol)
at their reducing end. Strong acid hydrolysis conditions were used to
break all the glycosidic linkages, and the resulting
H-labeled monosaccharides were analyzed by high voltage
paper electrophoresis. As shown in Fig. 4, most of the label
from the Class I fractions migrated with an authentic
[
C]GlcNAcitol internal standard and displayed
similar recovery (55-78%) when compared with authentic
H-labeled N-linked oligosaccharides from bovine
fetuin prepared and analyzed in the same manner (
50% recovery in
the [
H]GlcNAcitol peak, data not shown). The
Class II fraction (Q
) (
)showed a somewhat
lower percentage of recovery of label in
[
H]GlcNAcitol (39%). However, very little
radioactivity co-migrates with either N-acetylgalactosaminitol
or xylitol, the two alditols expected to be derived from the reducing
terminus of common O-linked oligosaccharides. The small
quantities of label migrating in the position of GalNAcitol in some of
the Class II fractions could potentially represent contamination by O-linked glycopeptides which could undergo partial
-elimination during the tritiation/reduction reaction. As shown
below, some unlabeled GalNAc was also found on compositional analysis.
However, these were different fractions from those which contained the
small quantities of labeled GalNAcitol .
Figure 4:
High voltage paper electrophoresis of
reducing end monosaccharides. Each fraction from the QAE-Sephadex
column (see Fig. 2) was mixed with a
[
C]GlcNAcitol internal standard, acid-hydrolyzed
to monosaccharide constituents, spotted on Whatman No. 3 paper, and
subjected to electrophoresis in 0.06 M borate at 40 V/cm for
2.5 h, as described under ``Experimental Procedures.'' One-cm
strips were cut, and radioactivity was determined by liquid
scintillation counting. GalNAcitol and xylitol standards ran at the
positions indicated by the arrows A and B,
respectively (data not shown).
Monosaccharide Compositional Analyses
The
individual QAE-derived fractions were acid-hydrolyzed to their
monosaccharide constituents, and their composition was analyzed with an
HPAEC-PAD system as described under ``Experimental
Procedures.'' Monosaccharides expected in N-linked
oligosaccharides were found in all the fractions, but the quantities of
material in some fractions were inadequate for accurate analyses (data
not shown). Since subsequent studies (presented in Table 4and
discussed below) suggested that most of the negative charge in the
Q
and Q
fractions was due to sialic acids,
while the Q
, Q
, and Q
fractions had other negative charges as well, we grouped together
the fractions as shown in Table 1for further studies of
monosaccharide composition. When normalized to the predicted 3 mannose
residues in the core region of complex-type N-linked
oligosaccharides, a high percentage of both galactose and glucosamine
in many of the pools suggests the presence of polylactosamine repeat
units (Table 1). Since glucosamine residues are also present in
the core of typical N-linked oligosaccharides, all Gal
residues cannot be accounted for by lactosamines. At least some of the
excess is probably accounted for by terminal
-galactose residues,
which appear to cap many of the chains (determined by
-galactosidase treatment, data not shown). The fucose present in
all fractions could represent core or outer chain fucosylation. The
substantial amounts of GalNH
in the Class II molecules is
explained by the content of CS chains (see below), but smaller amounts
are also present in the moderately charged group. The presence of
significant amounts of xylose in the Class II material was unexpected
and is explored further below. Under the elution conditions used,
uronic acids could not be detected.
Both the specificity of the
original PNGaseF release (17) and the demonstration of
[
H]glucosaminitol at the core indicate that both
Class I and Class II molecules consist predominantly of complex-type N-linked oligosaccharides with a chitobiose core and extended
outer chains. Since the two classes of oligosaccharides have otherwise
very distinct properties, their further characterization is presented
separately below.
Linkage Analysis of the Class I
Oligosaccharides
To examine the type of linkages present, the
oligosaccharides were permethylated and analyzed by GC-MS. Class II
molecules proved to be very difficult to dissolve in Me
SO,
even after the addition of 4-methylmorpholine N-oxide, and
gave no useful results. The Class I molecules were grouped together as
in Table 1, ``Group A'' (eluted with 20 or 70 mM NaCl) and ``Group B'' (eluted with 125, 200, or 400
mM NaCl). A summary of the type of linkages found for each
monosaccharide in the Group A oligosaccharide mixture (Q
+ Q
) is presented in Table 2, and the
deduced structural are elements summarized in Fig. 5. These data
indicate that the lesser negatively charged Class I structures contain
many tri- and tetraantennary structures, repeating lactosamine units,
the expected subterminal Gal units (substituted at either positions 3
or 6), and significant amounts of terminal galactose or N-acetylglucosamine residues. Both the excess of terminal Gal
units and the presence of the unusual 6-substituted Man residues were
not pursued further, because of the complexity of the mixture. Group B
molecules were severely undermethylated, again due to poor solubility
in Me
SO. However, the detection of 3,4,6-methylated mannose
suggested the presence of biantennary structures, and lactosamine
repeating units were indicated by the presence of high proportions of
2,4,6-methylated galactose and N,3,6-GlcNAcMe (data not
shown).
Figure 5:
GC-MS analysis of combined Q
and Q
fractions. The QAE-fractions were pooled as
indicated, subjected to permethylation, and partially methylated,
partially acetylated alditol acetates were separated using a fused
silica capillary column, with monitoring by electron impact mass
spectrometry as indicated under ``Experimental Procedures.''
This figure illustrates a composite structure derived from these data
(see text and Table 2for further details). Note that the
structures [3 Gal 1-] and [6 Gal 1] are presumed to
be capped with sialic acids or terminal
-galactose
residues.
Lectin Affinity Chromatography of Class I
Oligosaccharides
As with the Class I material from CPAE cells,
many Class I oligosaccharides from bovine lung ran through a
concanavalin A column (see Table 3for some examples), indicating
that they are complex-type chains(34) . A significant fraction
of these molecules were retarded on an L-PHA affinity column (see Table 3), indicating the presence of the following minimum
structure (34) :

These results further indicate that the Class I oligosaccharides
contain mainly branched complex type oligosaccharides. However, because
many chains had unknown anionic substituents that might unpredictably
affect binding, we did not use other lectin columns for structural
analysis. For the same reason, we also did not study the interactions
of the very highly charged Class II molecules with lectins.
While Sialic Acids and Sulfates Are Present on the Class
I Oligosaccharides, Another Anionic Group Accounts for the Majority of
the Negative Charge
Negative charge on N-linked
oligosaccharides may be due to sialic acids, sulfate esters, phosphate
esters, or, less commonly, uronic acids. Sialidase treatment caused a
decrease in negative charge of many of the Class I molecules, but did
not completely neutralize the majority of them (Table 4). Mild
acid hydrolysis under conditions which selectively remove sialic acids
gave similar results, indicating the absence of sialidase-resistant
sialic acids (data not shown and Table 4). HPLC analysis showed
that the acid-released sialic acids were mainly N-acetylneuraminic acid (Neu5Ac), with small amounts of N-glycolylneuraminic acid (Neu5Gc) (data not shown). We found
no evidence for phosphomonoesters or phoshodiester groups, using
combinations of sialidase, mild acid, and alkaline phosphatase
treatments (data not shown).We previously showed that about
one-third of the
SO
-labeled Class I
oligosaccharides from CPAE cells contained the terminal structure:
Sia
2-(3)6Gal
1-4GlcNAc(6
SO
)
1- (10) . This was demonstrated by release of
S label
after sequential removal of Sia and
-Gal residues, followed by
digestion with human placental
-hexosaminidase A incubated at pH
3.5, which gives specific removal of the exposed terminal
GlcNAc(6SO
)(40) . We subsequently noted that some N-linked structures from CPAE cells also carry terminal
-linked galactose residues(41) . Based on these
observations, we carried out a series of experiments using combinations
of mild acid treatment, jack bean, or bovine testicular
-galactosidases, coffee bean
-galactosidase, and human
placental
-hexosaminidase A (pH 3.5) to look for the presence of
either of the following terminal structures on the bovine lung anionic
oligosaccharides: Sia
2-(3)6
Gal
1-(3)4GlcNAc(6SO
)
1- or Gal
1-3
Gal
1-4 GlcNAc(6SO
)
1-. A small loss of
negative charge was seen with some of these treatments, but the great
majority of the anionic charge remained unchanged (data not shown)
indicating that there are very few subterminal GlcNAc(6SO
)
residues present. Thus, most of the non-sialic acid-dependent negative
charge is different from that found in the CPAE cells or any other
cultured cells analyzed to date.
Chemical analysis (32) or
solvolysis (9, 33) was used to determine total sulfate
groups regardless of location. As expected for GAG-type structures (see
below), the Class II fractions had a sulfate content easily detectable
with the rhodizonate assay: 140 nmol of sulfate per nmol of reducing
end sugar (data not shown). With this assay (3 nmol detection limit),
only trace amounts of sulfate were detected in the Q
fraction, and none was detectable in the lesser-charged
fractions. For the latter, we therefore used solvolysis to remove the
sulfate esters and then reapplied the samples to QAE-columns to check
for loss of negative charge (data not shown). While we did see some
minor shifts with this treatment, the great majority of the negative
charge persisted (data not shown), suggesting that these are not due to
sulfate esters. Thus, unlike Class I oligosaccharides from CPAE cells,
sulfate esters do not account for much of the negative charge.
Much of the Negative Charge in Class I Molecules Is Due
to Carboxylic Acids
The fraction that remains acidic after
removal of sialic acids represents
65% of the anionic
oligosaccharides and
49% of the total oligosaccharides released by
PNGaseF. Since sulfate and phosphate esters do not explain this
negative charge, the most likely candidates were uronic acids. A direct
search for uronic acids by compositional analysis after acid hydrolysis
was not possible because the amounts of material available were
limited, and because uronic acids are notoriously difficult to release
and recover after acid hydrolysis. Furthermore, trace contamination by
Class II molecules (known to contain high proportions of uronic acids
in HS and CS chains, see below) could confound the compositional
analysis of the higher charged subsets of Class I chains. We therefore
sought alternate approaches to look for the presence of carboxylate
groups. First, we chose to specifically neutralize carboxylic acids by
activation with a water-soluble carbodiimide in the presence of NHS.
Any resulting NHS esters were subsequently reacted with an excess of
methylamine, which should give a neutral methylamide.

After establishing general conditions to neutralize 52 ±
10% of the negative charge of the carboxyl groups on a sialyllactose
standard (see ``Experimental Procedures'' for details), we
applied this procedure to the individual Class I fractions after first
treating with mild acid to remove all sialic acids. One example of the
results (from the treatment of the Q
fraction) is shown
in Fig. 6. It can be seen that substantial shifts in negative
charge were obtained as a result of either the complete or partial
neutralization of these oligosaccharide structures. Since the
efficiency of this treatment is incomplete, the results give an
underestimate of the amount of negative charge due to carboxyl groups.
All of the other fractions also showed such partial reduction in
negative charge (data not shown). Thus, the majority of the negative
charge in the Class I oligosaccharides is not due to sialic acids and
may be accounted for by carboxylate substituents (presumably uronic
acids). However, the negative charge on Class I molecules was found to
be completely resistant to the action of either
-glucuronidase or
-iduronidase, with or without the addition of
-galactosidase,
-hexosaminidase, and
-galactosidase (data not shown). Thus,
it is unlikely that the putative carboxylates are part of uronic acid
residues that are present at terminal positions or at subterminal
positions covered by Gal or GlcNAc residues. Alternatively, such
residues might render other adjacent monosaccharides resistant to the
action of the glycosidases used.
Figure 6:
Example of decrease in negative charge
caused by neutralization of carboxyl groups. The individual
QAE-Sephadex-derived fractions were first treated with mild acid to
remove any sialic acids present. The remaining oligosaccharide
structures were then incubated with a mixture of EDC/NHS (closed
circles) or water (open circles) in the presence of
methylamine as described under ``Experimental Procedures.''
Samples were then diluted 160-fold in 2 mM Tris base,
reapplied to a QAE-Sephadex column, and re-eluted batchwise with
increasing sodium chloride concentrations, as in Fig. 2. Shown
is an example of this treatment using the original Q
fraction. Varying degrees of partial neutralization were seen for all
the other QAE-fractions (data not shown).
Reversible Methyl Esterification of the Carboxylate
Groups with Diazomethane or Methyl Iodide
Carboxylate groups can
be methyl-esterified by treatment with diazomethane (reaction proceeds
best on the acid form) or by CH
I (reaction proceeds best on
a salt form).

A mixture of Class I molecules that continued to carry negative
charge after mild acid hydrolysis (desialylation) was subjected to
these treatments, as described under ``Experimental
Procedures.'' In each case, [
C]GlcA or
[
H]Neu5Ac was used as standards containing
carboxylic acid residues. The maximum efficiency of esterification of
the above standards (as detected by loss of binding to a QAE-Sephadex
column, data not shown) were 75% and 94%, respectively (for
diazomethane treatment) and 25% and 41%, respectively (for methyl
iodide treatment). Under these treatment conditions, a substantial loss
of negative charge was also seen in the Class I molecules (as
determined by QAE-Sephadex, see Table 5). To further check for
the presence of methyl esterification, an aliquot of the molecules
neutralized by diazomethane treatment was isolated and subjected to
saponification. This resulted in restoration of the negative charge to
50% of the molecules (data not shown). This is as expected for
methyl esters of carboxylic acids.
Methyl Esterification of Carboxylate Groups with Methyl
Iodide on a DEAE-column and Subsequent Conversion to
Methylamides
The results presented above provide three different
lines of evidence for the presence of carboxylate groups on the Class I
molecules. However, in each case, the neutralization of negative charge
was only partial. Since the carboxylic acid standards also underwent
partial neutralization, it can be extrapolated that the majority of the
non-sialic acid-negative charge is due to these additional carboxylate
residues. However, it is also possible that other unknown groups might
be responsible for the remaining negative charges. To explore this
further, we used a recently described method to obtain near-complete
methyl esterification of sialic acid carboxylate residues on
oligosaccharides which are bound to a DEAE-column, by treatment with
methyl iodide in situ(37) . As described under
``Experimental Procedures,'' this method also has the
advantage that the methyl-esterified molecules will wash off the column
only if they have been completely neutralized. Thus, any molecules that
are eluted from the column upon methyl iodide treatment must have all
of their original negative charge entirely accounted for by carboxylate
groups. When this treatment was optimized for a glucoronic acid
standard and applied to a mixture of desialylated anionic Class I
molecules, 60% of the radioactivity was eluted from the column upon
treatment with methyl iodide, in comparison with elution of 85% of a
GlcA standard (see Fig. 7). Subsequent elution of the Class I
molecules with salt released 37% of the radioactivity, confirming that
most of the non-sialic acid-negative charge was eliminated by methyl
esterification.
Figure 7:
Neutralization of carboxyl groups by
methyl iodide treatment on a DEAE-column. Class I molecules were
treated with mild acid to remove sialic acids, and the material
remaining anionic was reisolated on a QAE-Sephadex column by direct
elution with 300 mM NaCl. After desalting, the molecules were
applied to a DEAE-Sephadex A-25 column in methanol and eluted with
CH
I/methanol and then 1 M pyridinium acetate as
described under ``Experimental Procedures.'' The samples
eluted with CH
I (indicated by the asterisks) were
dried and studied further as described in Table 6. As a positive
control, [
C]GlcUA was treated
similarly.
To ensure that the elution from the column was not
due to any nonspecific effect of methyl iodide, we restudied the
behavior of these ``neutralized'' molecules on DEAE-Sephadex.
Indeed, the great majority of these molecules now ran through the
column (data not shown), and binding could be substantially restored by
base treatment (see Table 6, stronger base hydrolysis was not
attempted since the effects of base on the oligosaccharides themselves
could potentially confound the results). To further demonstrate the
presence of methyl ester groups, we converted them to stable methyl
amide groups by treatment with methylamine(37) .

As shown in Table 6, this treatment provided substantial
protection from the base treatment. Appropriate positive and negative
controls gave the expected results (see Table 6and data not
shown). Taken together, these results indicate that the great majority
of the non-sialic acid-negative charge of the Class I molecules is due
to these methyl-esterifiable carboxylate groups. Overall, this as yet
unidentified modification accounts for about half of all the negative
charge on Class I chains.
The Class II Molecules Carry Different Types of GAG
Chains and More Than One Type May Be Present on a Single
Oligosaccharide
Class II molecules were first separated from the
less negatively charged Class I molecules by QAE-Sephadex
chromatography, eluting with 1 M NaCl (see Fig. 2).
Sizing analysis showed these molecules to be generally large, but
heterogeneous in size, ranging from some that are excluded on a Bio-Gel
P-100 column (exclusion limit for globular proteins,
100 kDa), to
some that are partly included on a Bio-Gel P-60 column (exclusion limit
for globular proteins,
60 kDa). However, no distinct peak
separations were obtained by these methods (data not shown). As shown
in Fig. 8, treatment of the mixture with individual
GAG-degrading enzymes produced a shift in the elution profile on a
TSK-DEAE-HPLC anion exchange column, indicating the presence of KS, CS,
and HS chains. However, the products of the individual enzyme
treatments still carried a considerable amount of negative charge (Fig. 8). Totaling the percentage shift produced by the
individual GAG-degrading enzyme treatments gives a value of
140%.
This may be an underestimate of the total loss of charge, since some
highly charged oligosaccharides may not have shifted sufficiently
relative to the control profile to be detected. Regardless, the results
suggested that more than one GAG chain might be associated with each
oligosaccharide reducing end. To explore this possibility, sequential
digestions with the GAG-degrading enzymes were performed. A variety of
such experiments was performed, and one example of the results is shown
in Fig. 9. Digestion of a highly anionic fraction first isolated
from the TSK-DEAE-2SW column with chondroitinase ABC showed a partial
loss of negative charge when reapplied to the same column (Fig. 9, upper panel). Digestion of the resulting peaks
with heparitinase and heparinase gave a shift, and subsequent treatment
with keratanase gave a further loss of negative charge (see example in Fig. 9). The other peaks in each case were also subjected to
sequential digestions and gave similar results, only one example is
shown here. Such experiments suggest that many of these molecules
contain two types of GAG chains, and a small percentage may even
contain all three types of GAG chains. Although these data are
indirect, they suggest the existence of N-linked
oligosaccharides with multiple GAG chains, each perhaps extending from
different antennae of a complex-type N-linked molecule. A less
likely possibility would be the existence of copolymers between the
different types of GAG chains, which has never been reported before in
the literature.
Figure 8:
Susceptibility of Class II molecules to
GAG-degrading enzymes. Class II molecules were isolated by QAE-Sephadex
chromatography (as in Fig. 2) and desalted, and aliquots either
were sham-incubated or treated with the GAG-degrading enzymes as
indicated. After digestion, they were analyzed for loss of negative
charge by reapplication to a TSK-DEAE-2SW HPLC column. The number
shown in the upper left hand corner of each panel indicates the
percent of oligosaccharides which shifted to a lower negative charge in
each case (calculated from the change in the non-overlapping areas of
the profiles from the control and treated
samples).
Figure 9:
Sequential treatments with GAG-degrading
enzymes. A preparative run of undigested Class II molecules was done on
a TSK-DEAE-2SW HPLC column to isolate the most negatively charged
species. Such material was subjected to sequential digestions by
GAG-degrading enzymes, with intermediate refractionation on the same
HPLC column. The initial run is not shown, but see Fig. 7for a
representation of the profile. In each panel, the solid bar indicates the elution position of the peak from the previous run
which was subjected to the enzyme digestion
indicated.
Xylose Present in the Class II Molecules Is Not at the
Reducing End
Most known glycosaminoglycans are linked to the
protein in an O-xylose linkage. Since the molecules studied
here were released with PNGaseF(17) , xylose should not be at
the reducing terminus. This was supported by the results of high
voltage paper electrophoresis following acid hydrolysis (Fig. 4). However, compositional analysis (Table 1)
indicated that xylose was present in the Class II fraction, raising the
possibility of an artifactual contamination with conventional GAG
chains. To rule out the presence of xylose at the reducing end of these
molecules, we analyzed acid hydrolysates of nonreduced or reduced Class
II oligosaccharides by a HPAEC-PAD system under conditions where all
monosaccharide alditols would run through the column, while nonreduced
monosaccharides would be retained and separated. With prior reduction,
only the reducing end monosaccharide would be converted to the alditol
form and therefore would run through the column. As seen in Fig. 10, a xylose standard was completely converted to xylitol
by reduction (consequently running through the column), validating the
method. In contrast, when Class II molecules were reduced before acid
hydrolysis, the xylose peak was retained (Fig. 10), indicating
that the xylose is not at the reducing terminus but somewhere within
the oligosaccharide structure.
Figure 10:
The xylose present in Class II molecules
is not at the reducing terminus. One batch of bovine lung
oligosaccharides released by PNGaseF was subjected to
``blind'' fractionation without introducing a tritium label
into the reducing end, i.e. the reducing end was not converted
to an alditol. The Class II molecules from this preparation were
acid-hydrolyzed and analyzed for monosaccharide composition by the
HPAEC-PAD system (a, fucose; b, galactosamine; c, galactose; d, glucosamine; e, glucose; f, mannose; xyl, xylose). A shows that a
small peak of xylose is detected in nonreduced Class II molecules. B shows that when the Class II molecules are reduced with
NaBH
prior to acid hydrolysis, the xylose peak persists. C and D show profiles of a xylose standard run with (D) and without (C) prior reduction with
NaBH
.
DISCUSSION
We have demonstrated here that bovine lung contains a
heterogeneous population of unusual N-linked anionic
oligosaccharides. The discovery of these oligosaccharides arose from
our earlier finding that a calf pulmonary artery endothelial cell
(CPAE) synthesized a complex population of anionic N-linked
oligosaccharides which contained both sialic acids and sulfate groups (10, 11) . Hoping to obtain larger amounts of similar
structures for detailed analysis, we studied intact lung in which
40% of the mass is composed of endothelial
cells(18, 19) . Using a commercially available bovine
lung acetone powder and relying on the specificity of the enzyme
PNGaseF(17) , we have found an equally complex array of anionic N-linked oligosaccharides, only some of which are similar to
those from the CPAE cell line. Initial fractionation of these
oligosaccharides on an anion exchange column shows two distinct classes
of structures. Class I chains included the less negatively charged
molecules that were partially susceptible to sialidase. However, in
striking contrast to the CPAE cell molecules, the remaining non-sialic
acid-negative charge in this class of chains was not primarily due to
sulfate esters. Rather, most if not all of the negative charge is due
to carboxylic acids. The more negatively charged Class II
oligosaccharides were similar to those in the CPAE cells, being
sensitive to the GAG-degrading enzymes including heparin lyases,
chondroitin lyases, and keratanases. However, they seemed to be present
in much greater abundance, representing >10% of the total
oligosaccharides released by PNGaseF. This confirms the existence of
the unusual N-linked GAG structures we had previously detected
in CPAE cells. We also show another unusual feature of these molecules,
that multiple GAG chains may be present on a single core structure.
Most structural analyses of oligosaccharides are performed on
purified glycoproteins. The great majority of these have originated
from plasma glycoproteins, blood cell membranes, or from cells grown in
tissue culture. A reasonable assumption is that N-linked
oligosaccharides of intact tissues and organs will be similar, with a
dominance of sialylated complex-type chains. However, this has not been
proven by direct analyses. The present work shows that in a library of
total oligosaccharides from bovine lung, the dominant N-linked
chains are in fact not the expected sialylated ones, but rather a
complex family of unusual structures bearing many unexpected anionic
charges. In this regard, it is worth noting that the only other such
reported attempt at making a library of total N-linked
oligosaccharides from an intact mammalian tissue is the work of Wing et al.(42) , who have created a ``library''
of hydrazine-released N-linked oligosaccharides which also
contain many unexplained anionic structures. It is reasonable to
speculate that the high level expression of these novel molecules in
intact tissues might be the consequence of differentiating stimuli that
are found only in the in vivo situation and perhaps lost or
diminished in in vitro culture. Regardless of the reason, it
may be worthwhile to explore such libraries from other tissues.
A
disadvantage of studying a library of oligosaccharides is the
complexity of the resulting mixture, which can pose major separation
problems to obtain individual structures. In our case, we have tried a
variety of techniques to isolate a single pure species which could be
fully characterized; however, all such approaches have so far resulted
only in dividing the molecules into mixtures of unresolvable
oligosaccharides with similar size or charge. Combinations of
techniques have also been so far unsuccessful in obtaining pure species
for study. We have therefore chosen to group the molecules into several
fractions of similar charge but variable size and gathered preliminary
data on the structures present in these mixtures.
Although all
oligosaccharides studied were specifically released by PNGaseF, we
confirmed their N-linked nature by showing that they contained
the expected N-acetylglucosamine residue as the reducing end
monosaccharide (a small amount of N-acetylgalactosaminitol may
be explained by minor contamination with O-linked
glycopeptides). The Class I oligosaccharides with small numbers of
negative charge contain typical multiantennary N-linked core
structures with sialic acids and no phosphate esters. With the more
highly charged Class I structures, sialic acids contribute only a
minority of the negative charge, and sulfate esters are even less
common. Most if not all of the negative charge seems to be due to
residual carboxylic acids that persist after chemical removal of sialic
acids. We presume that these carboxylic acids are associated with
uronic acids and, as such, present unusual N-linked
structures. There are two prior lines of indirect evidence for the
presence of uronic acids on N-linked oligosaccharides. First,
monoclonal antibodies known to recognize sulfated glucuronosyl groups
on glycolipids are known to cross-react with N-linked
glycoproteins(13, 14, 16) . Secondly,
Kawasaki and colleagues (15) have provided evidence for two
distinct glucuronosyltransferases in brain tissues: one which transfers
to glycolipids, and the other which transfers to N-linked
glycoproteins. While the most obvious explanation for the carboxylates
in these Class I molecules would be the presence of uronic acids, the
residual negative charge on desialylated molecules is completely
resistant to the action of either
-glucuronidase and
-iduronidase, with or without the addition of
-galactosidase,
-hexosaminidase, and
-galactosidase. Thus, it is unlikely
that the putative carboxylates are part of uronic acid residues present
at terminal positions, or at subterminal positions covered by Gal or
GlcNAc residues. Of course, we cannot rule out the possibility that
uronic acid residues are present at more internal positions and/or are
blocking the action of the other glycosidases.
The Class II
oligosaccharides detected in this study seem to be similar to the
corresponding ones previously found in the CPAE cells and thus may be
derived from lung endothelial cells. Although KS chains have been
detected in association with N-linked structures in certain
tissues(43, 44) , HS and CS chains have been thought
to be linked to proteins only via an O-xylosyl linkage. In our
previous study(11) , we showed that the CPAE cells did indeed
contain HS and CS chains releasable by PNGaseF. (
)Here, we
have extended this observation in bovine lung by demonstrating a
reducing end N-acetylglucosamine and raising the possibility
of multiple GAG chains on a single N-linked chain.
Interestingly, although xylose was detected in the compositional
analysis of these oligosaccharides, it is not at the reducing end, but
resides somewhere within the structure. Previous reports have described
the addition of a
1-2-linked xylose to the core
1-4 linked mannose residue in the N-linked
oligosaccharides of horseradish peroxidase(45) , laccase
excreted by sycamore(46) , miraculin from miracle fruit berries (47) , and Drosophila brain(48) . Thus, one
can speculate that this xylose residue might convert the trimannosyl
chitobiose core structure to a substrate for the conventional enzymatic
extension of glycosaminoglycan chains. Another possibility is that the
addition of glucuronosyl residues to the outer antennae of N-linked oligosaccharides might serve to prime GAG chain
biosynthesis. Finally, since the action of PNGaseF requires only the
presence of a chitobiose unit, one can speculate that the core region
of Class II molecules might arise from a biosynthetic pathway distinct
from that of classical N-linked chains. In this regard, it is
interesting to note that earlier work on the biosynthesis of
oligosaccharide dolichols reported the possibility of a chitobiose unit
being transferred from GlcNAc
1-4GlcNAc-P-P-dolichol to
proteins in vitro(49) . Also, we have recently
reported unusual xylosides primed by
4-methylumbelliferyl-
-xyloside in melanoma cells, in which GlcA
residues can be directly linked to Xyl; some of these molecules carry
multiple Xyl residues, suggesting the possibility of branching (50) .
To summarize, we have isolated a heterogeneous and
complex population of anionically charged N-linked
oligosaccharides from bovine lung. These sugar chains can be generally
separated into two groups with unusual characteristics. The majority of
the negative charge from the less negatively charged group (Class I)
arises from carboxylic acids which are not associated with sialic acids
and presumably may be uronic acids. These molecules are clearly
distinct from the sulfated/sialylated ``Class I'' molecules
previously reported in the pulmonary artery endothelial cells. The most
highly charged structures (Class II) contain multiple glycosaminoglycan
chains associated with a single N-linked core and have an
unusual internal xylose residue.
These data also serve to emphasize
the fact that the great majority of previously described N-linked oligosaccharides originate from a restricted subset
of easily accessible glycoproteins of the blood plasma and blood cells
or of recombinant proteins derived from cultured cell lines. The
assumption that similar patterns will occur in other mammalian tissues
is challenged by this study.