From the
We earlier reported calcium-dependent, heparin-like L-selectin
ligands in cultured bovine endothelial cells (Norgard-Sumnicht, K. E.,
Varki, N. M., and Varki, A.(1993) Science 261,480-483).
Here we show that these are heparan sulfate proteoglycans (HSPGs)
associated either with the cultured cells or secreted into the medium
and extracellular matrix. Activation of the endothelial cells with
bacterial lipopolysaccharide (LPS) does not markedly alter the amount
or distribution of this material. A major portion of the
glycosaminoglycan (GAG) chains released from these HSPGs by alkaline
Current
understanding of the biosynthesis of heparan sulfate chains indicates
that all glucosamine amino groups must be either N-acetylated
or N-sulfated. However, nitrous acid deamination at pH 4.0
suggests the presence of some unsubstituted amino groups in these
L-selectin-binding GAG chains from endothelial cell HSPGs. This is
confirmed by chemical N-reacetylation and by reactivity with
sulfo-N-hydroxysuccinimide-biotin. These unsubstituted amino
groups are also found on HSPGs from human umbilical vein endothelial
cells, but are not detected in those from Chinese hamster ovary cells.
In both bovine and human endothelial cells, these novel groups are
enriched for in the HS-GAG chains which bind to L-selectin. Despite
this, studies with N-reacetylation and nitrous acid
deamination do not show conclusive evidence for the direct involvement
of the unsubstituted amino groups in L-selectin binding. This may be
because the chemical reactions used to modify the amino groups do not
go to completion. Alternatively, the unsubstituted amino groups may
only be indirectly involved in generating binding, by dictating the
biosynthesis of another critical group. Regardless, these studies shown
that HSPGs from cultured endothelial cells which can bind to L-selectin
are enriched with unsubstituted amino groups on their GAG chains. The
possible biochemical mechanisms for generation of these novel groups
are discussed.
The selectins are a family of glycoproteins responsible for the
initial recognition and binding events in both the normal exit or
``homing'' of leukocytes from the blood stream and in
leukocyte emigration into inflamed
tissues
(1, 2, 3, 4, 5, 6, 7) .
All three members of this family, E-, P-, and L-selectin, contain the
following common domains: an amino-terminal calcium-dependent
carbohydrate recognition domain, an epidermal growth factor-like
domain, variable numbers of complement-regulatory repeat domains, a
transmembrane segment, and a cytoplasmic
domain
(1, 2, 3, 4, 5, 6, 7) .
Each selectin has been shown to recognize carbohydrate ligands on the
opposing cells during various biologically important recognition
processes. Partly because of the therapeutic potential for interrupting
abnormal leukocyte emigration into tissues in pathological situations,
much work has been done to identify the carbohydrate ligands involved
in selectin recognition. Interactions of all three of the selectins can
be blocked by high concentrations of the tetrasaccharide
sialyl-Lewis
We have previously
reported the isolation of heparin-like molecules from calf pulmonary
artery endothelial (CPAE) cells which bind to L-selectin in a
calcium-dependent manner
(24) . In independent work, Nelson
et al.(25) reported that fragments from commercial
heparin as small as tetrasaccharides could block L- and P-selectin
binding to SLe
The remaining pellets from the
saponin and trypsin treatments were extracted with 0.5 ml of ice-cold
PBS with 2% Triton X-100, 1 mM phenylmethylsulfonyl fluoride,
1% (v/v) aprotinin, 10 µg/ml pepstatin, 0.02% NaN
To examine the extracellular matrix material
only, a separate experiment was performed. A P-100 dish of CPAE cells
at passage 10 was labeled with [
Heparin
disaccharides are highly sulfated and contain many
2-O-sulfated iduronic acids, while heparan sulfate
disaccharides contain the less sulfated disaccharide units and
glucuronic acid residues. To explore which type of HS chains were
involved in L-selectin binding, we used a panel of heparin lyases
(I-III) with well-known substrate
specificities
(40, 41, 42, 43, 44, 45) .
The complete abolition of binding by the broad spectrum heparin lyase
II confirms that recognition does indeed involve HS chains and that
with use of heparin lyases, fragments larger than octasaccharides seem
to be required for high affinity binding. Heparin lyase I digestion did
not abolish binding of most of the labeled HS-GAGs, indicating that the
regions involved do not contain the highly sulfated and epimerized
heparin-like sequences recognized by this enzyme. The limited
fragmentation seen indicates that some of these sequences are present
on the flanking ends of large segments which continue to bind to
L-selectin. In contrast, heparin lyase III digestion caused a
substantial loss of recognition. However, it did not completely abolish
binding, and some small fragments were produced which were still able
to bind (see the peak indicated with an asterisk in
Fig. 1
, which contains fragments with
It is
known that the HS-GAG chains can contain many distinct types of
disaccharide units, generated by different patterns of sulfation and/or
epimerization
(46) . The pentasaccharide sequence responsible for
the high affinity binding of heparin to antithrobin III
(66, 67) is a specific sequence which includes an uncommon
3-O-sulfate group. The heparan sulfate sequence which binds to
basic fibroblast growth factor predominantly contains
IdoA(2-O-SO
In 1982, Höök et
al.(72) noted that preparations of purified heparins and
heparan sulfates which were being labeled by
N-[³H]acetylation contained significant
amounts of N-unsubstituted hexosamines. However, they
suggested that these unsubstituted amino groups were either artifacts,
biosynthetic intermediate structures, or products of degradative
enzymes. Since that time, conventional dogma indicates that such amino
groups are transient biosynthetic intermediates that are not retained
in the final product
(26, 27, 28) . We have
presented several lines of evidence that these unsubstituted amino
groups are unlikely to be experimental artifacts. Although the initial
studies with [
Since the amounts of material isolated from
these cultured cells are too small to demonstrate the presence of the
unsubstituted amino groups directly by quantitative chemical or
physical methods, we devised alternate approaches, showing that
chemical N-reacetylation as well as a reaction with
sulfo-NHS-biotin protected the molecules from subsequent nitrous acid
deamination at pH 4.0. In the latter case, the covalent attachment of
biotin residues was also confirmed by demonstrating that a major
fraction of the chains are then able to bind to avidin. For a variety
of reasons, it is difficult to precisely quantitate the number of
unsubstituted amino groups/HS chain. A significant limitation is that
we do not have sufficient amounts of material to ascertain chemically
that the reactions have gone to completion. The sulfo-NHS-biotin
coupling reaction can covalently modify as much as 90% of the
unsubstituted amino groups on the free monosaccharide glucosamine.
Likewise, the N-reacetylation method used is expected to
completely modify unsubstituted glucosamine. However, we cannot rule
out the possibility that these reactions are less efficient when
dealing with a large negatively charged polymer. Furthermore, the
presence of another modification, such as a O-sulfate at the
3-position, could further decrease the efficiency of any one of the
chemical treatments which we have employed. Although precise
quantitation is not possible, we can state some minimal estimates. In
the case of the HUVEC HS-GAGs, at least half of the intact GAG ligands
contain at least one unsubstituted amino group. Furthermore, since
One reason for the
persistent dogma that unsubstituted amino groups should not exist on
natural HS chains is that the N-deacetylation and
N-sulfation reactions that occur during biosynthesis are
catalyzed by the same
enzyme
(49, 50, 51, 52, 53, 54) ,
effectively coupling the two steps. However, much of the structural
work and enzymology studies to date have been done using mast cells,
CHO cells, or intestinal epithelial cells. These cells may or may not
have the identical biosynthetic machinery as endothelial cells. We can
speculate on a few different mechanisms by which these unsubstituted
amino groups might be generated in endothelial cells. First, the
kinetics or the efficiency of the specific enzyme reactions may be
altered in different cell types, i.e. in endothelial cells;
perhaps the N-deacetylase activity may be very high and
efficient, but the N-sulfotransferase activity may be low or
inefficient. This could potentially lead to a disproportionate amount
of glucosamine residues whose amino groups are not substituted.
Interestingly, Orellana et al.(53) have reported a
4-8-fold higher ratio of N-deacetylase to
N-sulfotransferase activity in the enzyme complex cloned from
a murine mastocytoma cell line as compared to that obtained from rat
liver. As a second possibility, endothelial cells may be expressing
novel tissue-specific enzymes, such as either an independent
N-deacetylase or an N-sulfatase. Enzymes such as
these might be inducible by inflammatory cytokines or triggered to be
expressed in tissue culture, where the cells may perceive themselves to
be in a ``pseudo-inflammatory'' state. Lastly, it is
conceivable that an internal ester migration event (enzyme-dependent or
independent) may be occurring, in which N-sulfate groups
migrate to another position. In this regard, Uchiyama and Nagasawa
(76) have shown that defined chemical conditions can cause a
fairly specific migration from the N-sulfate groups of heparin
to the adjacent 3-hydroxyl groups. Such a migration would cause the
creation of an unsubstituted amino group and perhaps an unusual
O-sulfate group within the heparin chain. Either or both these
modifications due to the migration might be responsible for a high
affinity interaction with the L-selectin molecule.
We have also
shown here that the heparan sulfate GAG chains that bind to L-selectin
are enriched in glucosamine residues whose amino groups are
unsubstituted. These unsubstituted amino groups could potentially be
involved in binding to L-selectin either directly, or indirectly, for
instance, by forming a salt bridge that generates an optimal binding
conformation in an adjacent part of the GAG chain. Attempts were made
to determine if these amino groups were important for binding by
masking them via chemical N-reacetylation. Although some
experiments showed a partial decrease in binding, this was not a
consistent finding. Thus, the presence of these unusual amino groups on
these GAG chains could be serendipitous. However, other possibilities
must be considered. For instance, we have consistently seen that the
chemical N-reacetylation procedure protects most but not all
of the ligand from degradation by nitrous acid at pH 4.0. It is
possible that a small subset of residual groups is sufficient to
maintain binding. Unfortunately, we cannot obtain enough material from
the cells in culture to determine the efficiency of the
N-reacetylation procedure by other direct chemical techniques.
It is also possible that as with anti-thrombin III binding to a highly
specific pentasaccharide sequence in heparin
(66, 67) ,
an unsubstituted amino group is only one of two or more modifications
which direct the high affinity binding to selectins. The unsubstituted
amino group might even serve as a signal for a further modification
during biosynthesis, such as a specific O-sulfation which, in
combination with the unsubstituted amino group, promotes the high
affinity binding to L-selectin. Such a second modification could also
cause a selective difficulty of N-reacetylation if it acts to
sterically inhibit this reaction, e.g. a 3-O-sulfate
residue, immediately adjacent to the unsubstituted amino group. In this
scenario, it is possible that either modification alone could support
binding, but that together they yield the highest affinity. If so, the
amount and ratio of the two groups present on the HS-GAGs obtained from
a given labeling might determine whether subsequent
N-reacetylation could completely destroy binding. In this
regard, it is interesting that commercial heparin (which has very few
if any unsubstituted amino groups) contains relatively few structures
that can bind with high affinity to L-selectin
(24) .
In
contrast to N-reacetylation, nitrous acid degradation at pH
4.0 consistently caused a major loss in the ability of the resulting
fragments to rebind to L-selectin. Of the fragments which still bound
after the nitrous acid degradation, all were rather large in size.
Unfortunately these results are also open to more than one
interpretation. First, the large fragments which are still able to bind
might contain one or more unsubstituted amino groups that escaped
deamination and are responsible for their ability to continue to bind
to L-selectin. Indeed, the fact that the great majority of the
fragments which no longer bind were small in size suggests that these
unsubstituted amino groups may reside at or very near potential
L-selectin-binding sites. However, the coupling of the unsubstituted
amino groups with sulfo-NHS-biotin never caused more than
Regardless of the exact explanation for these results, it appears
likely that these endothelial HS-GAGs carry specific sequences
mediating L-selectin recognition that are not found in high frequency
in commercial heparin preparations. It is interesting to note that
synthetic SLe
While this work was nearing completion, we became
aware of work by others, who have recently used immunohistochemical
approaches to demonstrate the natural occurrence of heparan sulfate
with unmodified amino groups.³(³)
Thus, these
groups may be more common than previously recognized and represent yet
another way in the which the heparin/heparan sulfate family of
glycosaminoglycans can achieve diversity, which in turn can mediate
specific biological functions.
CPAE cells
(passage 10) were grown to subconfluence in 6-well plates. Each well
was labeled with 500 µCi of
Na
We thank Sandra Diaz and Robert Linhardt for helpful
discussions and Andrea Koenig and Gary Sumnicht for their careful
review of the manuscript.
-elimination rebinds to L-selectin in the presence of calcium,
indicating that these saccharides alone can mediate the high affinity
recognition. Heparin lyase digestions indicate that these GAG chains
are enriched in heparan sulfate, not heparin sequences.
(SL(¹)e
-Sia
2-3Gal
1-4(Fuc
1-3)GlcNAc)¹ (8-10). The SLe
epitope has been detected on
some of the specific ligands for the selectins: on the PSGL-1 ligand
for P-selectin (11-14), on the ESL-1 ligand for
E-selectin
(15) , and on the GlyCAM-1 ligand for L-selectin, on
which it appears in a modified form as 6`-sulfated
sialyl-Lewis
(16) . While SLe
and its
isomer SLe
can block selectin-mediated interactions, these
terminal structures are commonly found on many glycoproteins
(17-19), and their apparent affinity for the selectins is quite
poor (3, 6, 10). Thus, these structures appear to be biologically
relevant only when presented in the context of certain intact
glycoprotein ligand structures, and/or perhaps with further
modification, such as
sulfation
(20, 21, 22, 23) . In this
regard, we have suggested that SLe
(and/or other sialylated
or sulfated oligosaccharides) generate high-affinity ligands by forming
unique ``clustered saccharide patches'' on heavily
glycosylated molecules such as mucins (6, 12).
-bovine serum albumin, at concentrations far
lower than those required for SLe
itself. Thus,
heparin-related structures may be another type of high affinity natural
ligand for the selectins. Also, with regard to use as therapeutic
blockers for selectins, these molecules might potentially be more
useful than the sialylated, fucosylated polylactosamines. Here, we
report the initial characterization of these heparin-like ligands from
CPAE cells, aimed at understanding if unusual structure(s) present on
the glycosaminoglycan (GAG) chains might account for their specific
affinity for L-selectin. Our initial approach assumed the accuracy of
current models for biosynthesis of heparin/heparan sulfate (HS) chains,
which indicate that unsubstituted amino groups should not occur on the
glucosamine residues of naturally occurring HS-GAG
chains
(26, 27, 28) . However, we show that these
unusual residues do indeed exist in the L-selectin binding HS-GAGs of
cultured endothelial cells. The involvement of the free amino groups in
binding to L-selectin is also explored.
Materials
Most of the materials used were obtained from Sigma. The
following materials were obtained from the sources indicated:
Arthrobacter ureafaciens neuraminidase (sialidase),
Calbiochem; Proteinase K, Life Technologies, Inc.;
[S]sodium sulfate, ICN; trypsin-treated
L-1-tosyl-amido-2-phenylethyl chloromethyl ketone,
Worthington; diisopropylfluorophosphate, Aldrich; sulfo-NHS-biotin (an
N-hydroxysuccinimide ester of biotin) and immuno pure avidin
coupled to agarose, Pierce; and Centricon-3 Concentrators, Amicon.
Purified heparin lyases (I, II, and III) and
O-sialoglycoprotease were kind gifts from Dr. Robert Linhardt,
University of Iowa, and Dr. Alan Mellors, University of Guelph, Canada,
respectively. Tritium end-labeled heparin octasaccharide standards were
kindly provided by Dr. Magnus Höök, Texas A & M
University. All other chemicals were of reagent grade or better and
were from commercial sources.
Cell Lines
CPAE cells, a calf pulmonary artery endothelial cell line,
was from American Type Culture Collection (CCL 209) and were used at or
before passage 23; HUVEC cells, human umbilical vein endothelial cells,
were from Clonetics, San Diego, CA (CC-2008) and were used within the
first three passages; CHOK1 cells were from ATCC (CCL 9618).
Enzyme Digestions
Heparin lyase digestions were done in 10-20 µl
total volume of 20 mM Tris-HCl, pH 7.2, 1 mM
CaCl using: 50 milliunits of heparin lyase I, 5 milliunits
of heparin lyase II, or 25 milliunits of heparin lyase III/reaction, at
37 °C for 1 h. O-Sialoglycoprotease digestions were done
in a total volume of 10 µl of 20 mM Tris-HCl, pH 7.2, and
2 µl of enzyme, at 37 °C for 1 h (the specific activity of this
enzyme is defined by glycophorin A as a substrate; 1 µl cleaves 5
µg/h).²(²)
Sialidase digestions were done in a
total volume of 10-15 µl of 100 mM sodium acetate pH
5.5 with 5 milliunits of A. ureafaciens neuraminidase
(sialidase), at 37 °C for 1 h. Proteinase K digestions were done
with 0.65 mg of proteinase K in a total volume of 10 µl of 20
mM Tris-HCl, pH 7.2, overnight at 37 °C.
SDS-PAGE
7.5% polyacrylamide gels were poured using a Bio-Rad Mini-Gel
Apparatus as per the manufacturer's instructions. Samples were
boiled in the presence of SDS and 2-mercaptoethanol (reduced), loaded
in a total volume of 40 µl, and gels run at 100 V until the dye
front reached the bottom of the gel. Prestained high molecular weight
standards from Life Technologies, Inc. were included. Following
fixation of the gel in 10% methanol, 10% acetic acid for 30 min and
exposure to En³hance for 1 h as per the manufacturer's
instructions, the gel was dried and exposed to X-MAT Kodak film at
80 °C.
Pulse-Chase Experiments
CPAE cells at passage 10 were grown in 2 6-well
plates to subconfluence (80%). Labeling with
[
S]sodium sulfate was done in sulfate-limited
media (media without cysteine and methionine and a total of 10
uM sodium sulfate as reported
earlier
(24, 29) ). Some wells from each plate were also
stimulated by addition of 1 µg of LPS (Sigma L-6018 from
Escherichia coli 055:B5
-irradiated) at the time of
addition of the
[
S]sulfate
(30, 31) . After an 8-h
pulse, cells to be chased (1 plate) were washed three times with cold
PBS and replenished with
-minimal essential media containing 10%
heat-inactivated fetal calf serum for an overnight chase period. Cells
from each of the four groups (``pulse'' pulsed only; and
``pulse/chase'' pulsed with overnight chase; each with and
without LPS stimulation) were either trypsinized or
saponin-permeabilized.
Trypsin Treatment
Cells were washed three times with
ice-cold PBS, and 0.5 ml of PBS, 0.1% sodium azide containing 0.5 mg of
trypsin-TPCK was added and incubated at room temperature for 15 min.
The trypsin was inactivated by addition of 0.5 µl of 1 M
diisopropylfluorophosphate (in a fume hood). The cells were then
scraped into the buffer and the entire mixture centrifuged at 2000
revolutions/min for 15 min. The supernatant was analyzed as the
trypsin-released fraction, and the remaining pellet was extracted with
Triton-X as described below.
Saponin Treatment
Cells were washed three times
with ice-cold PBS and 0.5 ml of PBS, 0.1% sodium azide containing the
following: 5 mg of Saponin, 30 µg of pepstatin, 6% (v/v) aprotinin,
and 1 mM phenylmethylsulfonyl fluoride was added to each well.
After incubation on ice for 30 min, the cells were scraped into the
buffer, and the entire mixture centrifuged at 2000 revolutions/min for
15 min. The supernatant was analyzed as the saponin-released fraction,
and the remaining pellet was further extracted with Triton X-100 as
described below. Each of the supernatants described above, as well as
the chase media from each group, was analyzed by applying equivalent
amounts of radioactivity (50,000 counts/min) onto an L-selectin
affinity column (described below).
on
ice for 1 h with periodic vortexing. These samples were then
transferred to ultracentrifuge tubes and spun at 13 K in a Ti 50.3
rotor (9500
g) for 1 h. These supernatants
(radioactivity remaining after saponin or trypsin) were taken into
account when calculating the total radioactivity associated with cells,
matrix, and media.
S]sodium sulfate
as described above. After an 8-h pulse, the cells were washed with
ice-cold PBS and the cells scraped from the plate using a rubber
policeman. The plate was inspected to ensure that no cells remained on
the plate and washed a further time with cold PBS. The remaining
extracellular matrix was removed by adding 3 ml of 2% SDS in PBS and
scraped and collected into a 15-ml glass conical tube. The GAG chains
were then released as described below using method 1.
Preparation of Free GAG Chains
Method 1
Free GAG chains were released and
purified using a modification of a previously reported
method
(32) . Briefly, the samples, either before or after
application to a L-selectin column, were digested overnight at 55
°C with 1 mg/ml proteinase K in a buffer containing 50 mM
Tris-HCl, pH 7.5, 1% SDS, 0.1 M 2-mercaptoethanol. The SDS was
precipitated by adding 1/100th volume of saturated KCl and the mixture
left at 4 °C overnight. The potassium SDS precipitate was spun out
at 2000 revolutions/min for 15 min. The supernatant fluid was boiled
for 15 min and DNase (final concentration 3 µg/ml) and MgCl (final concentration 100 mM) was added, and the mixture
incubated at 37 °C overnight. After boiling for 15 min, the mixture
was extracted with 10 volumes of CHCl
/MeOH (2:1). The
aqueous phase which contained the GAG glycopeptides was extensively
dialyzed against water (M
cutoff = 3500)
and the sample lyophilized to dryness. For reductive
-elimination,
22.4 mg of NaBH
was dissolved in 2 ml of 0.4 M
NaOH, and 0.5 ml of this mixture was added to each sample
(33) .
After incubation at room temperature overnight, the reaction was
quenched by acidification with 1 M HCl followed by
neutralization using 1 M NaOH. Excess borates and unreacted
products were then removed by desalting using a Bio-Gel P-2 column
pre-equilibrated and run in 10 mM sodium acetate, pH 5.5. The
free GAG chains from the void volume of this column were then
fractionated on an L-selectin affinity column as described below.
Method 2
A second method of GAG chain preparation
and purification was employed as a check for the possibility of
artifactual production of unsubstituted amino groups and is based on
the protocol described by Bame and Esko
(34) . Briefly, CPAE,
HUVEC, and CHOK1 cells were labeled for 4 days with
[6-³H]GlcNH in complete
-minimal
essential media. Cells were then washed and solubilized with 0.1
M NaOH at room temperature for 15 min. The solubilized cell
mixture was added to the labeling media and the pH adjusted to 7.0.
Proteinase K was added at a final concentration of 0.134 mg/ml to each
sample and incubated at 37 °C overnight. The samples were then
diluted 5-fold with water and the salt concentration lowered to 0.1
M (as checked with a Vapor Pressure Osmometer (5500XR, Wescor)
against calibrated salt solutions). The samples were then applied to
1-ml DEAE-Sephacel columns and washed with 30 ml of 0.1 M
NaCl, 20 mM Tris-HCl, pH 7.4. The GAG peptides were then
eluted with 4 ml of 1 M NaCl, 20 mM Tris-HCl, pH 7.4.
Reductive
-elimination was then done similarly to that described
above. An equivalent volume of a 2
NaOH/NaBH
solution (2
= 44.8 mg NaBH
, 2 ml of
M NaOH) was added to each sample and the pH checked to be at
11.5. The samples were allowed to incubate 24 h at room temperature.
They were then quenched by the addition of glacial acetic acid dropwise
until bubbling stopped and left at room temperature for an additional 2
h for completion of quenching. The samples were then dialyzed
extensively against H
O in dialysis bags with a molecular
weight cutoff of 6,000-8000.
L-selectin Affinity Chromatography
As described previously
(35) , the L-selectin receptor
globulin (LS-Rg) consists of the entire extracellular domain of a human
L-selectin molecule attached to a human IgG Fc
COOH-terminal domain. The construct is expressed in 293 cells and
isolated from the culture media using protein A-Sepharose. Typical
affinity columns were constructed using 0.4-2.7 mg of purified
LS-Rg immobilized by passage over a prepoured column of 2 ml of protein
A-Sepharose. Ligands were bound to the columns in 100 mM NaCl,
20 mM MOPS, pH 7.4, 1 mM CaCl
, 1
mM MgCl
, and 0.02% sodium azide. Bound ligands
were specifically eluted using the above binding buffer in which the
CaCl
and MgCl
were replaced with 5 mM
EDTA. Under all of these conditions, the LS-Rg itself remains attached
to the protein A-Sepharose column.
Superose 12 FPLC Fractionation
A Pharmacia Superose 12 HR 10/30 FPLC column was used both to
size samples as well as to remove unreacted small products and
reagents. The column was run isocratically in the same buffer used for
the LS-Rg affinity column (see above), allowing for direct application
of any isolated material to the latter. A Pharmacia FPLC system
(P-LKB-Pump P-500; P-LKB-Controller LCC-500 Plus) was used to elute the
column at 0.4 ml/min flow rate with an on-line Pharmacia LKB-Frac 100
fraction collector programmed to collect 1-min fractions after 15 min
from the beginning of the run. Fractions that were to be studied
analytically were collected directly into scintillation vials, 4 ml of
Liquiscint scintillation mixture added, and the radioactivity
determined. The elution profile of this column was highly reproducible
from run to run, as determined by the markers blue dextran (void
volume), [S]sodium sulfate (total volume) and
[³H]heparin octasaccharide standards (partially
included).
Nitrous Acid Degradation
Nitrous acid degradation was performed by the method of
Conrad et al.(36, 37, 38, 39) .
Samples to be treated were either first dialyzed extensively against
water or desalted using a Centricon-3 concentrator unit (spun with
repeated applications of water washes 2-3 2 ml), dried
completely, and chilled on ice. 30 µl of either the chilled nitrous
acid reagent (see below) at pH 1.5 or pH 4.0 was then added, and the
reaction brought to room temperature for 10 min, after which the
solution was neutralized by addition of 5 µl of saturated
Na
CO
, and the final volume adjusted to 200
µl with water. The sample was immediately applied onto the Superose
12 column as described above. For preparation of the pH 1.5 nitrous
acid reagent, 0.5 M H
SO
and 0.5
M Ba(NO
)
were prepared fresh and
cooled separately on ice. Equal volumes were then mixed together and
spun in a microfuge to pellet the BaSO
formed. The
supernatant was aspirated and used immediately. For the pH 4.0 nitrous
acid reagent, 5.5 M NaNO
and 0.5 M
H
SO
were prepared fresh and cooled separately
on ice. Five volumes of NaNO
and 2 volumes of
H
SO
were then mixed on ice and used
immediately. pH values of the nitrous acid reagent were checked before
addition to the sample. For controls, 30 µl of the nitrous acid
solutions actually used were first quenched with 5 µl of saturated
Na
CO
, and the final volume adjusted to 200
µl with water before addition of the samples, which were then
applied immediately to the Superose 12 columns.
N-Reacetylation of Amino Groups
The samples to be N-reacetylated were desalted by
either extensive dialysis against HO or by centrifugation
using a Centricon-3 concentrator unit (spun with repeated applications
of water washes 2-3
2 ml), brought up in a total of 100
µl of H
O, and 12.5 µl of saturated NaHCO
was added followed by 12.5 µl of freshly prepared, ice-cold
5% aqueous acetic anhydride while the mixture was vortexed. At 15-min
intervals, additional aliquots of 12.5 µl of saturated NaHCO
and 12.5 µl of fresh, ice-cold 5% aqueous acetic anhydride
were added for 60-90 min. The sample was then directly injected
onto the Superose 12 FPLC column as described above.
Sulfo-NHS-Biotin Coupling and Purification
Samples to be coupled to sulfo-NHS-biotin were extensively
dialyzed against HO to remove salts. Coupling was performed
in 50 mM sodium borate in volumes ranging from 10 to 27 µl
total. The sample was first brought up in the borate solution and then
added to vials which had 0.1 mg of sulfo-NHS-biotin dried in them.
(Because sulfo-NHS-biotin tends to degrade in aqueous solutions, the
fresh solutions of the compound were quickly aliquoted into individual
Eppendorf tubes, dried on a Savant Speed Vac apparatus, and then stored
refrigerated with desiccation until use). Coupling was allowed to
proceed overnight at 4 °C. Two methods were used to separate
unreacted sulfo-NHS-biotin from the reacted product. Small
oligosaccharides or monosaccharides were spotted directly on Whatman
no. 1 paper and run for 16 h in ethyl acetate/pyridine/acetic
acid/water (5:5:1:3). The paper was then cut into 1-cm strips, eluted
with H
O, and radioactivity determined. Large
oligosaccharides were separated from unreacted sulfo-NHS-biotin by
spinning the unreacted sulfo-NHS-biotin reagent through Centricon-3
concentrator units. The samples were washed with repeated applications
of water (2-3
2 ml).
Avidin Binding of Biotinylated GAG Chains
Samples which had been coupled with sulfo-NHS-biotin and
freed of excess reagents were allowed to interact with immobilized
avidin-agarose beads. 200 µl of avidin-agarose was added to the
sample brought up in 50 µl of sodium borate (50 mM final
borate concentration). After incubation with rotation at 4 °C for 1
h, the sample mixture was then applied to 1-ml pipette tips packed with
a small amount of glass wool. Unbound material was washed through with
3 0.5 ml of 50 mM borate (under slight air pressure).
The run through and wash were counted together as unbound material, and
the beads were counted as bound material.
HSPG L-selectin Ligands Are Present Both as
Cell-associated and Secreted Molecules
We earlier reported the
calcium-dependent intracellular staining of L-selectin ligands in CPAE
cells, which were thought to represent the SO
metabolically labeled material that was subsequently shown to
bind to an L-selectin affinity column
(24) . These molecules are
susceptible to proteases and heparin lyases, indicating that they are
heparan sulfate proteoglycans (HSPGs). We next asked whether all of
these HSPG L-selectin ligands were cell associated, or if any were
associated with the extracellular matrix, and/or secreted into the
media. We were also interested to know if LPS stimulation could induce
increased expression, in a manner similar to the L-selectin ligand
reported by Spertini et al.(30, 31) . In a
series of pulse-chase labeling experiments, we found that the
SO
-labeled L-selectin ligands of CPAE cells
were associated with the intracellular pools (released by saponin),
with the cell surface and extracellular matrix (released by trypsin),
as well as with cell secretions (for an example of the data, see
). Saponin extraction of intracellular pools yielded only a
minority of labeled material capable of binding to L-selectin, while
trypsin treatment yielded a substantially higher amount ().
Since trypsin can both dissociate material from the cell surface as
well as degrade the matrix, we also tried to first scrape the
SO
-labeled cells from the plate, and then
removed the residual matrix with SDS. Indeed, we found that the
SDS-released labeled proteoglycan matrix material carried HS-GAG chains
which bound to L-selectin (data not shown), showing that under these
tissue culture conditions, some of the ligands are deposited into the
extracellular matrix. Addition of LPS did not markedly change the total
amount of material which was able to bind to the L-selectin column in
any fraction, although some increase was seen in the trypsin-released
fraction during the chase period. Also, during the chase period, some
of the labeled ligand shifts from an intracellular location (saponin
releasable) into the chase media (), indicating secretion
of a portion of the ligand. To avoid potential problems with cellular
proteases and detergents, most of the subsequent studies were performed
with the HSPG ligand that was secreted into the culture medium, and
purified by affinity chromatography on an L-selectin column, with EDTA
elution.
Effects of Heparin Lyases on the Structure of the
Glycosaminoglycan Chains, and Their Binding to L-selectin
While
the intact ligand from CPAE cells appears to be an HSPG, 50% of
the free GAG chains released from these molecules by alkaline
-elimination continue to bind to L-selectin affinity
columns
(24) . Further studies were therefore carried out with
these released
SO
-labeled GAG chains. To gain
insight into what type of heparin/heparan sulfate (hereafter referred
to collectively as HS) oligosaccharide units might be interacting with
L-selectin, we digested the CPAE HS-GAG ligands with heparin lyases of
defined specificity and checked to see if any of the resulting
fragments retained their ability to rebind to L-selectin. Following
digestion, the labeled material was separated on an L-selectin affinity
column into a non-binding fraction, and an L-selectin-bound fraction.
These were each then sized on an FPLC Superose-12 column which gives
rapid elution and highly reproducible size exclusion profiles. As seen
in Fig. 1, the untreated HS-GAG chains eluted almost exclusively
near the void volume of this column (exclusion limit of 2
10
kDa for globular proteins). Heparin lyase II completely
degraded the ligand, and none of the resulting material was capable of
rebinding to the L-selectin column. As expected from the broad spectrum
of action of this
enzyme
(40, 41, 42, 43, 44, 45) ,
all of the digested material was substantially reduced in size (size
about 1-4 disaccharide units, based on calibration of the column
with known heparin standards). In contrast, heparin lyase I-digested
material still contained some molecules that could rebind, but this
fraction remained very large in size. Notably, many of the fragments
that no longer bound are still relatively large in size (
60% are
>4 disaccharide units in size). This indicates that while the highly
sulfated and epimerized sequences recognized by heparin lyase I are
present in the GAG chains, the regions that mediate L-selectin binding
are unaffected by these treatments. Rather, the heparin lyase I
sensitive regions appear to flank both the L-selectin binding and
non-binding regions. As seen in Fig. 1, heparin lyase III
treatment substantially degraded the ligand, but still produced some
fragments which could rebind to the L-selectin column. Taken together,
the results indicate that although these GAG chains contain both
heparin and heparan sulfate sequences, the majority of the sequences
belong to the heparan sulfate class, and the latter probably include
the binding motifs of the L-selectin ligand. Furthermore, most of the
high affinity ligand surviving after lyase digestions is large (greater
than four disaccharides). The only binding fragments small enough for
further analysis are those derived by heparin lyase III digestion (see
the peak labeled with an asterisk in Fig. 1, panel
F). Unfortunately, these represent a very small fraction (
4%)
of the material subjected to treatment.
Figure 1:
Size analysis of S-labeled
CPAE HS-GAG L-selectin ligands after heparin lyase treatment and
rebinding to an L-selectin column.
[
S]Sulfate-labeled-free GAG chains from CPAE
cells that originally bound to L-selectin were digested with the
individual heparin lyases (heparin lyase I-III) (40,
42-44) and reapplied to an L-selectin (LS) column. The
run through (Unbound) and EDTA-eluted (Bound)
material was then sized using a Superose-12 FPLC column as described
under ``Experimental Procedures.'' The percentages of unbound
and bound label in each case are indicated in the upper right hand
corner of each panel. The arrows mark the V and
V of the column. [³H]Heparin
octasaccharide standards elute at fractions 37-39. The light
line (control) shown in each panel is the elution profile of
undigested material. Panel D is empty because there was no
L-selectin binding material surviving after heparin lyase II digestion.
The peak labeled with an asterisk in panel F represents
4% of the starting material subjected to heparin
lyase III digestion.
Nitrous Acid Deamination Suggests the Presence of
Unsubstituted Amino Groups on the Glycosaminoglycans
To
ascertain whether N-acetyl groups or N-sulfoamino
groups might be involved in L-selectin recognition, we carried out
pH-controlled nitrous acid treatment, which can deaminate certain
glucosamine residues and subsequently cleave the adjacent glycosidic
linkage. Treatment at pH 1.5 or 4.0 are well known to cause cleavage at
N-sulfoamino groups or at unsubstituted amino groups
respectively (N-acetyl groups are not sensitive at either
pH)
(39) . Fig. 2demonstrates the extent of fragmentation
of the SO
-labeled CPAE HS-GAG ligands produced
by each of these treatments. After the pH 1.5 treatment, nearly all of
the sulfate label elutes near the totally included region of the
column. This indicates that almost all of the sulfate label was either
in N-sulfoamino groups or on small fragments (1-2
disaccharide units) produced from the deamination/glycosidic cleavage
of such groups. Furthermore, this fragmentation pattern indicates
either that most of the GAG chains have N-sulfoamino groups
throughout their entire length or that any stretches with primarily
N-acetylated regions do not have any other associated
O-sulfate groups. The latter would not be surprising, based
upon the work of others and current understanding of the biosynthetic
pathway for HS-GAGs
(28, 34, 46) .
Figure 2:
Sizing of S-labeled CPAE
HS-GAG L-selectin ligands after nitrous acid degradation.
[
S]Sulfate-labeled-free GAG chains from CPAE
cells that bound to L-selectin were treated with nitrous acid
(HONO) either at pH 1.5 (upper panel) or at pH 4.0
(lower panel), and then sized on a Superose-12 FPLC column as
in Fig. 1. The light lines show controls for each run.
[³H]Heparin octasaccharide standards elute at
fractions 37-39.
Since
nitrous acid treatment at pH 4.0 will not affect molecules with
N-sulfated or N-acetylated glucosamine residues, this
procedure is usually done after chemical N-deacetylation,
e.g. with hydrazine
(38, 47) . We performed
deamination at pH 4.0 without prior N-deacetylation primarily
as a negative control for the pH 1.5 treatment. Surprisingly, a major
portion of the CPAE HS-GAG chains were cleaved to smaller fragments by
this treatment. This indicated that unlike most reported HS structures,
this ligand might contain a significant amount of unsubstituted amino
groups. As mentioned earlier, the FPLC Superose-12 column used for
sizing gives highly reproducible elution profiles, and the shifts
observed are due to degradation and not to differences between
individual column runs. Previous studies have shown that under the
conditions used for pH 4.0 nitrous acid deamination, small quantities
of sulfoamino groups can be deaminated depending on the amount of
reagent used, the type of acid used to create the nitrosating reagent
(HCl or HSO
), and the length of time of
exposure of the sample with the reagent
(48) . However, if this
was indeed the explanation for all of the fragmentation seen here, one
would have expected to see the release of free
[
S]sulfate following the nitrous acid treatment
at pH 4.0, which was not observed (see Fig. 2).
Unsubstituted Amino Groups Are Not the Result of Growing
Cells in Sulfate-depleted Medium
The
SO
-labeling studies described above were
performed in sulfate-depleted media (concentration 15 µM)
to enhance uptake of the label
(29) . This could theoretically
result in the appearance of unsubstituted amino groups because of an
imbalance in biosynthetic processes of N-deacetylation and
N-sulfation. In practice, this has not been observed in the
past, probably because the two reactions have been shown to be
catalyzed by a single
enzyme
(49, 50, 51, 52, 53, 54) ,
and are therefore coordinately regulated. However, to be certain that
sulfate depletion is not the cause of these findings, CPAE cells were
also labeled with [6-³H]GlcNH
in
complete medium (sulfate concentration 810 µM), and the
HS-GAG ligands for L-selectin isolated and studied as above. Again,
substantial cleavage of the labeled HS-GAG chains by deamination at pH
4.0 was seen (see Fig. 3). The relative difference between the
profiles in Fig. 2(lower panel) and Fig. 3. is
likely to due to the fact that the latter molecules are labeled with
[³H]glucosamine thoughout the entire molecule,
while the former are labeled with [
S]sulfate,
and therefore label only regions of the polymer which are sulfated.
Such regions are known to be non-uniformly distributed in heparan
sulfates
(26, 27, 28) . The present results would
suggest that the free amino groups may be equally common in the
sulfated and non-sulfated regions of the polymer.
Figure 3:
Sizing of
[³H]glucosamine-labeled CPAE HS-GAG L-selectin
ligands after nitrous acid pH 4.0 degradation.
[³H]Glucosamine-labeled released GAG chains from
CPAE cells that bind to L-selectin were treated with nitrous acid, pH
4.0, and sized on a Superose-12 FPLC column as in Figs. 1 and 2. The
light line shows the control run for untreated
ligand.
Human Umbilical Vein Endothelial Cells Also Have HSPG
Ligands Which Bind to L-selectin
The presence of unsubstituted
amino groups could be an unusual anomaly of this particular cell line
(CPAE cells) when grown in culture. We previously reported that both
HUVECs and a second type of bovine endothelial cell (aortic endothelial
cells, ATCC AG08132) produced SO
-labeled
material which bound to L-selectin
(24) . The material from the
latter two cell types appeared very large when analyzed by SDS-PAGE. To
explore if the HUVEC ligand was similar to the CPAE ligand and whether
it too contained unsubstituted amino groups, the media from
[6-³H]GlcNH
-labeled HUVEC cells were
subjected to L-selectin affinity chromatography. When the labeled
material eluted with EDTA was treated with a variety of enzymes and the
products analyzed on SDS-PAGE (Fig. 4), the most obvious change
was produced by heparin lyase digestion, indicating that this material
also carried HS chains. The ability of proteinase K to digest the
material indicates the presence of core protein(s), showing that the
native ligand is an HSPG. Notably, O-sialoglycoprotease, an
enzyme that specifically recognizes mucin-type glycoproteins and
destroys several other previously recognized selectin
ligands
(12, 55, 56, 57) produced only
small changes in the SDS-PAGE profile. Minor shifts were also produced
by sialidase treatment, but these were present also in the pH 5.5
control for this particular enzyme. Thus, the HUVEC ligand is similar
to the one from CPAE cells and very different from the mucin-type
ligands previously described for L- and P-selectin (reviewed in Refs.
6, 7).
Figure 4:
SDS-PAGE of L-selectin ligand(s) from
[³H]GlcNH-labeled HUVECs.
[³H]GlcNH
-labeled ligands from the
culture medium of HUVEC cells were purified by affinity chromatography
on an L-selectin column. Bound material was eluted specifically with 5
mM EDTA, and aliquots (8500 counts/min each) were subjected to
the enzyme digestions indicated in the figure and described in detail
under ``Experimental Procedures.'' The enzymes were
inactivated by heating in sample buffer with 2-mercaptoethanol
(reducing conditions) at 100 °C for 10 min and loaded on 7.5%
SDS-PAGE. The gel was processed with En³hance for
fluorography. Controls were incubated at the appropriate pH values (pH
7 for all reactions except the sialidase digestion, which was done at
pH 5.5).
As with the CPAE ligand, a significant fraction (50%) of
the free GAG chains released from the HUVEC ligand by
-elimination
can rebind to L-selectin in a calcium-dependent manner (data not
shown). Again as with the CPAE cells, both
[
S]sulfate- and
[³H]GlcNH
-labeled ligands from HUVECs
were found associated with the cell and/or extracellular matrix, as
well as secreted into the media, and heparin lyase treatments gave
similar profiles (data not shown). Fig. 5illustrates the
fragmentation of [³H]GlcNH
-labeled
HUVEC ligand produced by nitrous acid treatments at pH 1.5 and 4.0.
Treatment at pH 1.5 produces substantial fragmentation, although to a
lesser extent than seen with the
[
S]sulfate-labeled CPAE ligand. Fragmentation by
pH 4.0 nitrous acid treatment was clearly seen (Fig. 5), again to
a somewhat lesser extent than with the CPAE material. (Interestingly,
in some other labelings more unsubstituted amino groups were detected
in the HUVEC ligands, see Fig. 10.) Regardless, these data
indicate that the HSPG ligands from CPAE and HUVECs have generally
similar properties, including the apparent presence of unsubstituted
amino groups.
Figure 5:
Sizing of ³H-labeled HS-GAG
L-selectin ligands from HUVECs after nitrous acid degradation.
[³H]GlcNH-labeled released GAG chains
from HUVEC HSPGs that bound to L-selectin were treated with either
nitrous acid at pH 1.5 (upper panel) or pH 4.0 (lower
panel) and then sized on a Superose-12 FPLC column as in Fig. 2.
The light lines show the profiles obtained with controls for
each treatment. In the case of pH 4.0 treatment, the percentage of
total radioactivity in fraction 28-40 (see bar) was 4%
for the control and 21% for the treated
sample.
Figure 10:
Sizing of total ³H-labeled GAG
glycopeptides from CPAE, HUVEC, or CHO cells before and after nitrous
acid degradation with or without prior N-reacetylation.
[³H]GlcNH-labeled GAG-enriched
glycopeptides from CPAE, HUVEC, and CHO cells were prepared with Method
2 as described under ``Experimental Procedures.'' Aliquots
were treated with nitrous acid at pH 4.0, with or without prior
N-reacetylation, and then sized on a Superose-12 FPLC column
(similar to Figs. 2 and 7). In each profile, the Control trace
is the profile of the material injected on the column without any type
of treatment. To better compare the three different traces, each data
point is presented as percent of total counts/min recovered in the
run.
Unsubstituted Amino Groups Are Not Caused by Chemical
Degradation during
The nitrous acid treatments
presented thus far were performed on free GAG chains released by
-Elimination
-elimination and then repurified on an L-selectin affinity column.
This has the advantage of enriching for the GAG chains capable of
binding to L-selectin. However, because
-elimination is carried
out under alkaline conditions, we considered the possibility that
chemical degradation might account for small amounts of unsubstituted
amino groups seen. We therefore studied the intact proteoglycans from
the culture medium that bind L-selectin without any prior chemical or
enzymatic manipulation. Fig. 6illustrates the fragmentation
profiles of the intact
[³H]GlcNH
-labeled HUVEC HSPG ligand
with heparin lyase treatment or nitrous acid treatment done at pH 4.0.
As expected, the great majority (80%) of the label was digested to
smaller sized fragments with heparin lyase II. Nitrous acid treatment
at pH 4.0 also produced a significant amount of degradation in
comparison to incubation with a pH-adjusted control (about 21% of the
label was found in the included volume, in comparison to 5% in the
control). This provides independent confirmation that the GAG chains
sensitive to pH 4.0 nitrous acid treatment are present in the native
HSPG ligand and are not generated as a chemical artifact during
subsequent processing.
Figure 6:
Effects of digestion with heparin lyase II
or nitrous acid treatment at pH 4.0. on the size of the intact
L-selectin-binding HSPG from ³H-labeled HUVECs. Intact HSPGs
from the culture medium of
[³H]GlcNH-labeled cells were bound to
an L-selectin affinity column and eluted with EDTA. Aliquots were
subjected to heparin lyase II digestion or nitrous acid treatment at pH
4.0, and then sized on a Superose-12 FPLC column. Samples from control
incubations were also sized and are shown in each panel with
the light line. The inset in the lower panel shows the ``released'' region in an expanded scale. With
the nitrous acid treated material, 21% of the label was found in this
region, in comparison to 5% in the control.
Confirmation of the Presence of Unsubstituted Amino
Groups on the HS-GAGs with N-Reacetylation
As discussed
earlier, it is unlikely that the fragmentation seen with the pH 4.0
nitrous acid deamination is due to a small fraction of cleavage at
sulfoamino residues. To further demonstrate the presence of naturally
occurring unsubstituted amino groups, we attempted to protect the
ligands by chemical N-reacetylation of such groups.
N-reacetylation is predicted to prevent deamination and
glycosidic linkage cleavage by blocking the reaction with nitrous acid
at pH 4.0. As seen in Fig. 7, N-reacetylation did indeed
give substantial protection of the HS-GAG ligands of both CPAE and
HUVEC cells from nitrous acid deamination/cleavage at pH 4.0.
Figure 7:
Protection of ³H-labeled
L-selectin HS-GAG ligands from nitrous acid deamination after chemical
N-reacetylation. L-selectin-binding HS-GAG chains from both
HUVEC and CPAE cells were chemically N-reacetylated as
described under ``Experimental Procedures.'' After
N-reacetylation, the ligands were desalted by dialysis,
subjected to deamination/cleavage with nitrous acid at pH 4.0, and
sized using the Superose-12 FPLC system. In each panel, profiles of
ligands subjected to sham N-reacetylation reactions are shown
as a control. Even though the N-reacetylation provided
significant protection from nitrous acid degradation, there is still
some detectable shift compared to the completely non-treated material
(see Fig. 10), indicating that the N-reacetylation is
incomplete.
Confirmation of the Presence of Unsubstituted Amino
Groups on the HS-GAGs by Coupling with Sulfo-NHS-Biotin
As an
independent approach to demonstrate the presence of unsubstituted amino
groups, we reasoned that they should react with sulfo-NHS-biotin (an
N-hydroxysuccinimide ester of biotin), a reagent commonly used
for coupling biotin to the unsubstituted amino groups of proteins (58).
If this reaction occurred with the HS-GAGs, it would not only confirm
the presence of the unsubstituted amino groups, but would also provide
a biotin tag which could be used to retrieve the coupled GAG chain,
using immobilized avidin. Furthermore, by subsequently digesting the
tagged ligand with heparin lyases, it should be possible to estimate
the minimum fraction of disaccharides that carry unsubstituted amino
groups. Since this procedure has not been previously used with
oligosaccharides, we first sought to validate and optimize it by
demonstrating the reaction of sulfo-NHS-biotin with the monosaccharide
glucosamine. After treatment of [6-³H]GlcNH and [
C]N-acetyl-mannosamine
(ManNAc) with sulfo-NHS-biotin under various conditions, we studied the
products by paper chromatography. As shown in Fig. 8, nearly 90%
coupling of the GlcNH
was achieved under optimized
conditions, as evidence by a shift in migration of the molecule. Under
identical conditions there was no reactivity of sulfo-NHS-biotin with
the control ManNAc, which does not have an unsubstituted amino group.
Figure 8:
Paper chromatography of monosaccharides
with and without prior reaction to sulfo-NHS-biotin.
[³H]GlcNH or
[
C]ManNAc were reacted or sham reacted with
sulfo-NHS-biotin as described under ``Experimental
Procedures.'' The samples were spotted on Whatman No. 1 paper and
developed in an ethyl acetate/pyridine/acetic acid/water (5:5:1:3)
solvent system for 16 h at room temperature. The paper was dried, cut
into 1-cm strips, and radioactivity
determined.
Although a coupling efficiency of 90% was possible with
unsubstituted GlcNH
, unsubstituted amino groups within an
intact HS chain might be less susceptible to coupling due to steric
hindrance, charge effects, or salt bridges. However, if
sulfo-NHS-biotin coupling protected the ligand from subsequent
deamination/cleavage by pH 4.0 nitrous acid treatment, then a
reasonably high efficiency of coupling could be assumed. As seen in
Fig. 9
, sulfo-NHS-biotin coupling of the HUVEC HS chains did
indeed provide a substantial, although incomplete protection from
nitrous acid degradation. Interestingly, when HUVEC HS-GAG chains which
had been reacted with sulfo-NHS-biotin were then digested with heparin
lyase, the sulfo-NHS-biotinylation actually seemed to enhance the
degradation by heparin lyase, as detected by a sharpened peak profile,
slightly more included than the uncoupled HS-GAG chains (Fig. 9,
lower panel). This could be because the heparin lyase action
is hindered by the presence of the unsubstituted amino groups, and
restored by their substitution with the biotinyl groups. The intact and
heparin lyase-degraded HS chains were then allowed to interact with
avidin immobilized on agarose beads, and bound and unbound fractions
were determined. As shown in , a substantial fraction of
the intact HS chains from HUVECs were biotinylated by this procedure.
Even among heparin lyase-digested HS fragments, a small but significant
fraction (6.7% as compared with 0.3% in the control) was selectively
retained on the avidin beads. These data confirm that unsubstituted
amino groups are present on a substantial fraction of the HS-GAG
L-selectin ligands. The decrease in the amount of radioactivity binding
to avidin following heparin lyase digestion is not surprising, since
the label is expected to be distributed throughout the polymer, whereas
the amino groups are present only in a limited number of residues. Even
in the HUVEC HS-GAG ligands which have lower levels of unsubstituted
amino groups than the CPAE ligand, we estimate that 1 in 20 to 1 in 50
glucosamine residues have unsubstituted amino groups. Since we cannot
be certain that the reaction with sulfo-NHS-biotin proceeds to
completion, these numbers represent minimum estimates.
Figure 9:
Effects of sulfo-NHS-biotin on the size of
fragments produced by nitrous acid deamination or heparin lyase
digestion of ³H-labeled L-selectin HS-GAG ligand from HUVEC
cells. [³H]GlcNH-labeled L-selectin
binding HS-GAG chains from HUVEC cells were reacted with
sulfo-NHS-biotin as described under ``Experimental
Procedures.'' Portions of the sample (and appropriate sham-treated
controls) were subjected to nitrous acid treatment at pH 4.0 or heparin
lyase II digestion and then sized on a Superose-12 FPLC column, as in
Fig. 2.
Unsubstituted Amino Groups Are Enriched on Endothelial
HS-GAGs as Compared to Those from CHO Cells
The existing
literature on HS-GAGs favors the notion that unsubstituted amino groups
do not occur naturally because of the coordinated enzymatic
N-deacetylation and N-sulfation. The unsubstituted
amino groups described here are present on only a minor fraction of the
glucosamine residues. We therefore carried out a direct comparison of
HS-GAGs from these endothelial cells with those from CHO cells, which
have been extensively studied in the past and have not been found to
contain unsubstituted amino groups in the wild type CHOK1
line
(49) . Following [6-³H]GlcNH labeling under identical conditions, total HS-GAGs from CHO,
CPAE, and HUVEC were isolated by protease digestion and ion-exchange
chromatography, almost exactly as described previously by Bame and
Esko
(34) . To ensure good comparison, total
[6-³H]GlcNH
-labeled material from
cells, extracellular matrix, and medium were pooled together and
digested with proteinase K, a homogeneous protease of broad
specificity. The resulting labeled glycopeptides were desalted and
enriched for GAG chains by step-wise elution from an anion-exchange
column as described previously
(34) . The GAG chains were then
released with
-elimination/NaBH
reduction, and the
resulting material (which would include other types of GAG chains such
as chondroitin sulfate) was treated with nitrous acid at pH 4.0 and
analyzed for any resulting change in size. As shown in Fig. 10,
the total GAG chains from CHO cells showed a very minor (if any) shift
in the profile of elution from the Superose-12 column. In contrast, the
CPAE and HUVEC-derived material showed significant shifts, as before,
which was almost completely protected by N-reacetylation of
the material.
Unsubstituted Amino Groups Are Enriched on the HS-GAGs
That Bind to L-selectin
We wanted to see if the unsubstituted
amino groups which we had detected were associated only with the GAG
chains capable of binding to L-selectin or whether they occurred in all
of the GAG chains isolated from the CPAE cells. To examine this, the
intact [³H]glucosamine-labeled CPAE HSPG fraction
that bound to L-selectin was submitted to -elimination, the
products desalted, and reapplied to the L-selectin affinity column. The
fraction of the radiolabeled material that no longer bound to the
column (50-75%) contains HS-GAG chains, but also may include some
conventional O-linked chains and N-linked
glycopeptides arising from the
-elimination reaction on the intact
HSPG. However, the latter are much smaller in size than the HS-GAG
chains and are discernable on the Superose-12 sizing column as included
material. As shown in Fig. 11, while the L-selectin-bound HS-GAGs
showed extensive cleavage with pH 4.0 nitrous acid treatment,
negligible fragmentation was seen among the non-binding chains. Thus,
the unsubstituted amino groups appear to be highly enriched for in
those HS-GAG chains which bind to L-selectin, suggesting that they may
play a role in recognition.
Figure 11:
Heparan
sulfate L-selectin ligands from endothelial cells are enriched in GAG
chains with unsubstituted amino groups. Intact
[³H]GlcNH-labeled HSPGs from CPAE
endothelial cells that initially bound to an L-selectin (LS)
affinity column and eluted with EDTA were subjected to
-elimination, and the products reapplied to the L-selectin column.
The unbound (upper panel) or bound (lower panel)
fractions were desalted and aliquots treated with nitrous acid
(HONO) at pH 4.0. Treated and untreated samples were studied
by gel filtration on a Superose-12 FPLC as described under in Fig. 1.
Arrows mark the V and V of the column.
[³H]Heparin octasaccharide standards elute at
fractions 37-39.
N-Reacetylation of the HS-GAG Chains Does Not Diminish
Their Ability to Interact with L-selectin
In using chemical
N-reacetylation to demonstrate the presence of unsubstituted
amino groups, we could protect most, but not all, of the ligand from
nitrous acid degradation at pH 4.0 (Fig. 7). To see if these
chemically N-reacetylated ligands maintained their ability to
bind to L-selectin, they were reapplied to an L-selectin affinity
column. Although some experiments showed that a fraction of the ligand
no longer interacted with L-selectin (data not shown), most experiments
gave results such as those shown in the upper panel of
Fig. 12
. Since the majority of the unsubstituted amino groups are
successfully N-reacetylated by this procedure, these data
indicate that all of the free amino groups are not required for high
affinity recognition of HS-GAGs by L-selectin.
Figure 12:
Effects of N-reacetylation and
nitrous acid deamination on the rebinding of HS-GAG ligands to
L-selectin. Samples of
[³H]GlcNH-labeled HS-GAGs from CPAE
cells that had previously bound to L-selectin were treated by
N-reacetylation or nitrous acid (HONO) at pH 4.0, as
described under ``Experimental Procedures.'' After removal of
all salts by dialysis against water and re-equilibration in column
running buffer, the samples were reapplied to an L-selectin affinity
column. The arrow indicates the position of elution with 5
mM EDTA. A control run of untreated sample is shown in the
upper panel.
Nitrous Acid Deamination at pH 4.0 Destroys the Ability
of Some of the HS Chains to Interact with L-selectin
In contrast
to the results with N-reacetylation, nitrous acid treatment of
the HS-GAGs at pH 4.0 (which cleaves at the position of unsubstituted
amino groups) does cause a major portion of the ligand (75%) to
lose binding to L-selectin (see Fig. 12, lower panel).
When the non-binding and binding material obtained after nitrous acid
treatment are studied by sizing analysis (Fig. 13), the material
which no longer binds has been degraded to smaller-sized fragments,
while that which still binds remains large in size (compare with Figs.
2 and 3, and their discussion above). These data are subject to
different explanations. On the one hand, the survival of some binding
following fragmentation suggests that some or all of the interactions
of the intact molecules is due to regions free of unsubstituted amino
groups. Alternatively, these groups could be critically important for
the majority of the binding, with the remaining binding being explained
either by other types of sequences, or by unsubstituted amino groups
that escaped cleavage and/or N-reacetylation. Because it is
likely that neither treatment (N-reacetylation or nitrous acid
deamination) went to completion, both explanations remain viable.
Figure 13:
Size fractionation of nitrous acid
deamination products following the rebinding of HS-GAG ligands to
L-selectin. After nitrous acid deamination at pH 4.0 (see lower
panel of Fig. 12) HS-GAG products that bound or no longer bound to
L-selectin (LS) were size fractionated on a Superose-12 FPLC
column as described in Fig. 1. The profile obtained with a control
(untreated) sample is shown for comparison.
DISCUSSION
In this study, we have further characterized the heparin-like
ligands for L-selectin reported earlier in CPAE endothelial
cells
(24) . While our report noted that L-selectin staining of
CPAE cells was primarily intracellular in nature
(24) , we have
not further explored the precise intracellular localization here.
However, these ligand(s) are shown to be HSPGs found associated with
the cells and/or extracellular matrix, as well as secreted into the
medium. These observations of secretion and matrix association must of
course be regarded with caution, since cultured cells may have patterns
of expression and/or secretion that are altered relative to their
normal counterparts in vivo. For this reason, it is also
difficult to know what the specific role of this ligand might be in the
normal biology of L-selectin. Since HSPGs are known to be exposed on
the luminal surface of endothelial cells in
vivo(26, 59, 60, 61, 62) ,
it is possible these L-selectin ligands play an initial role in
recognition by L-selectin bearing leucocytes. In this regard, it has
been noted that cytokine-stimulated endothelial cells display an
altered basement membrane structure and a glycosaminoglycan-rich
pericellular matrix
(63, 64) . Alternatively, the
L-selectin binding HS-GAG sequences might be primarily expressed in the
extracellular basement membrane. If so, they may become important only
when endothelial cell junctions become separated in injury, or in
inflammation. Lastly, secreted ligands might act in a negative manner
to inhibit leucocyte adhesion to a normal endothelium. Notably, we
could not induce a major alteration in synthesis or subcellular
distribution of the HS ligand by LPS stimulation of CPAE cells. Thus,
this ligand must be distinct from the inducible L-selectin ligand of
HUVECs reported earlier with either interleukin-1
(30, 31) or interleukin-4
(65) stimulation. Furthermore, the
latter requires new protein synthesis, and recognition is evidently
dependent upon sialic acids
(30, 31) .
4 disaccharide units).
This indicates that while this enzyme was not able to cleave all the
regions which bind L-selectin, it did cleave many regions very near by,
suggesting that the regions which are binding contain heparan sulfate
type sequences. Unfortunately, the amounts of the small fragments that
continue to bind have been too little for more detailed study.
)
1,4-GlcNSO
disaccharides with few 6-O-sulfate groups and it appears
that both the 2-O-sulfate and N-sulfate groups are
essential for the binding activity
(68, 69, 70) .
In contrast, binding of heparan sulfate to hepatocyte growth factor
seems to involve domains with predominantly non-sulfated iduronic
acids, and the highest affinity seems to be most closely associated
with 6-O-sulfated GlcNSO
residues
(71) .
Because HS-GAG-L-selectin binding could be a highly regulated
biological event, we considered the possibility that a unique sequence
and/or unique modification of the HS chains might be present to endow
these ligands with a high affinity. In examining this possibility, we
made the surprising finding that there were a significant amount of
unsubstituted amino groups on glucosamine residues. Given the novelty
of this finding, we chose to focus more attention to it, regardless of
whether or not it was related to L-selectin binding. Interestingly, we
found that the number and ratios of N-sulfo and
N-acetyl groups in the HS ligands from CPAE and HUVEC cells
vary considerably, but both cell types have in common the presence of
these unsubstituted amino groups.
S]sulfate were done using a
sulfate-deficient media, the presence of unsubstituted amino groups is
still noted when labeling with [³H]GlcNH
in a sulfate replete medium. In fact, others who have used
sulfate-deficient media or studied sulfation-negative mutants have not
observed unsubstituted amino groups in HS-GAGs from other cell
types
(27, 34, 49, 73, 74) . In
addition, the chemical technique used to release chains (alkaline
-elimination) is a traditional method that has not been previously
reported to generate unsubstituted amino groups. Regardless, to ensure
that this was not the case in our hands, we also subjected the intact
HSPG (obtained directly from the culture medium by L-selectin binding)
to nitrous acid deamination at pH 4.0; again, evidence was seen for a
small but significant amount of unsubstituted amino groups. Since these
molecules were isolated directly from the culture medium of the cells,
and maintained at neutral pH in an isotonic buffer, they were not
exposed to any harsh chemical conditions that could have artifactually
created the unsubstituted amino groups. Another concern is that under
the conditions used for nitrous acid deamination at pH 4.0, a small
amount of breakdown and release of N-sulfoamino groups might
occur (48). However, when the [
S]sulfate-labeled
HS-GAGs were subjected to this treatment, we did not see a peak of
released free [
S]sulfate. Finally, we have
compared the HS chains from CPAE and HUVEC cells to those from CHO
cells, which have been extensively studied in the
past
(27, 49, 73, 74, 75) .
HS-GAGs were isolated from these three cell types using identical
isolation procedures and were studied for the presence of unsubstituted
amino groups. Again the CPAE and HUVEC GAG chains showed evidence for
unsubstituted amino groups, whereas those from the CHO cells had few,
if any, such groups.
6% of the heparin lyase treated material (fragments between two to
four disaccharides in length) seem to have an unsubstituted amino
group, one can estimate that between 1 in 20 to 1 in 50 of the
disaccharide units can have such groups.
50% of
the ligand to be retained on avidin-agarose. This may indicate that
there are two types of binding of HS-GAGs to L-selectin, one which is
dependent on unsubstituted amino groups and one which is not.
/SLe
molecules whose
N-acetyl-glucosamine residue is replaced with a glucosamine
bind better to L-selectin than the native structures
(10) .
Perhaps one motif recognized by L-selectin is an unique arrangement of
a negatively charged groups near a positively charged group, which
promotes a high affinity interaction. In this regard, it has been
recently reported that Bacteroides fragilis polysaccharides
which induce the formation of intra-abdominal abcesses (collections of
neutrophils that originally bear L-selectin) are characterized by
repeated disaccharide units which contain alternating positively
charged amino groups and neighboring negatively charged
groups
(77) . N-Reacetylation of the unsubstituted amino
groups in these molecules resulted in a dramatic diminution of
neutrophil accumulation
(77) . We are currently exploring the
possibility that such polysaccharides are indeed selectively recognized
by L-selectin.
Table:
Pulse-chase analysis of
[S]sulfate-labeled CPAE cells for expression of
L-selectin ligand and induction by LPS stimulation
SO
. Half of the wells also
received 1 µg/ml LPS at the time of addition of the label. After an
8-h pulse labeling, half of the samples were processed, while fresh
media without label was added to the others for an overnight chase
period. Washed cells from different wells were treated with saponin or
trypsin followed by Triton X-100 extraction of the cell pellet, as
described under ``Experimental Procedures.'' Equal amounts
(50,000 counts/min) of saponin-released, trypsin-released, or secreted
material was loaded onto a L-selectin affinity column, which was washed
and eluted with EDTA to detect calcium-dependent L-selectin ligands.
,
sialyl Lewis-x; HSPG, heparin/heparan sulfate proteoglycan; GAG,
glycosaminoglycan; HS, heparin/heparan sulfate; CPAE, calf pulmonary
artery endothelial cell; CHO, Chinese hamster ovary cell; HUVEC, human
umbilical vein endothelial cell; LPS, lipopolysaccharide; PBS,
phosphate-buffered saline; LS-Rg, L-selectin receptor globulin; MOPS,
3-[N-Morpholino]propanesulfonic acid; PAGE,
polyacrylamide gel electrophoresis; HONO, nitrous acid; FPLC, fast
protein liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.