(Received for publication, June 13, 1995; and in revised form, July 14, 1995)
From the
L-selectin, a member of the selectin family of leukocyte-endothelial adhesion proteins, mediates the initial attachment of lymphocytes to lymph node high endothelial venules during lymphocyte recirculation. One of the endothelial-associated ligands for L-selectin is GlyCAM-1, a mucin-like glycoprotein, which presents novel sulfated, sialylated and fucosylated O-glycans. In order to understand the generation of these glycans, we have examined the biosynthesis of GlyCAM-1 in lymph node organ culture. Using peptide-specific antibodies, lectins, and recombinant L-selectin, we detected the following species of GlyCAM-1: unglycosylated (<28 kDa); modified with GalNAc only (28-33 kDa); modified with sialic acid, fucose, and sulfate but lacking L-selectin reactivity (40-50 kDa); and mature (L-selectin-reactive) ligand (50-60 kDa). Pulse-chase labeling at 15 °C suggested that GalNAc is added in a pre-Golgi compartment. Treatment with brefeldin A almost completely blocked sulfation, indicating that this modification occurs in the trans-Golgi network. Two distinct sialylation events occurred in the presence of brefeldin A, while fucosylation was partially blocked. We conclude that sialylation precedes both fucosylation and sulfation during biosynthesis. This ordering will help to identify the critical acceptor structures recognized by lymph node glycosyltransferases and sulfotransferases.
L-selectin is a lectin-like receptor that is widely expressed on
the surface of circulating leukocytes(1, 2) . It plays
a central role in lymphocyte-endothelial cell interactions in the
normal recirculation of lymphocytes between the blood and secondary
lymphoid organs(1, 3) . In addition, L-selectin, as
well as the other members of the selectin family, E- and P-selectin,
participate in the recruitment of various leukocytes to sites of
inflammation (reviewed in 4-7). In lymphocyte recirculation,
blood-borne lymphocytes interact with the specialized endothelial cells
of post-capillary high endothelial venules (HEV) ()and
ultimately extravasate across the endothelium into secondary lymphoid
organs. L-selectin is essential for the initial adhesive interaction of
lymphocytes with HEV of lymph nodes and also appears to be involved in
the recruitment of lymphocytes to Peyer's
patches(1, 3, 8, 9) . Leukocyte
integrins and their endothelial counter-receptors participate in later
steps of
recruitment(10, 11, 12, 13, 14) .
A fundamental property of L-selectin is that it recognizes
carbohydrate-based ligands via its lectin-like domain (reviewed in 7,
15). An L-selectin/Ig chimera has been used to identify two
HEV-associated ligands in mouse as GlyCAM-1 and CD34 (formerly
designated Sgp50 and Sgp90)(16, 17, 18) .
More recently, GlyCAM-1 has been observed in HEV-like vessels that are
induced at sites of chronic inflammation(19) . ()MAdCAM-1, a member of the Ig superfamily, exists in mouse
mesenteric lymph node HEV in a glycoform that is recognized by
L-selectin(20) . GlyCAM-1, CD34, and MAdCAM-1 are found at
other sites as glycoforms (19, 21, 22) that
do not exhibit high affinity binding with L-selectin. A fourth distinct
ligand, Sgp200 is also present in mouse lymph nodes (23) but
has not been identified at the molecular level. All of these
HEV-associated glycoproteins possessing ligand activity for L-selectin
are recognized by the function-blocking monoclonal antibody known as
MECA 79(16, 23, 24) . Finally, a heparin-like
ligand for L-selectin has been identified intracellularly in cultured
endothelial cells(25) .
Both GlyCAM-1 and CD34 are sialylated, fucosylated, and sulfated glycoproteins, and their primary sequence indicates that they are serine/threonine-rich mucin-like glycoproteins with many potential sites for O-linked glycosylation(16, 17, 18) . MAdCAM-1 also possesses a short mucin domain (26) which is proposed to bear the carbohydrate recognition determinants for L-selectin. In the case of GlyCAM-1, all of the oligosaccharides are O-linked, adding approximately 35 kDa to a predicted core protein of 14 kDa(17, 27) . GlyCAM-1 is present at high levels in the conditioned medium of lymph node organ cultures and in serum(17, 28) . By EM immunocytochemistry, GlyCAM-1 is undetectable on the apical plasma membrane of the endothelial cells of HEV but is found in large cytoplasmic vesicles (29) . Taken together, these observations suggest that GlyCAM-1 is a secreted product. In contrast, CD34 is an integral membrane protein (30) .
Sialic acid and sulfate are critical components of
the oligosaccharide ligands for L-selectin(16, 31) ,
and an essential role for fucosylation is strongly
suspected(32) . L-, E, and P-selectin recognize the sialyl
Lewis x tetrasaccharide (sLe,
Neu5Ac
2
3Gal
1-4(Fuc
1-3)GlcNAc), and
related structures (reviewed in Refs. 15, 33), although each selectin
has preferred biological
ligands(34, 35, 36) . There has been recent
interest in the possibility that sulfation may define a unique
modification of L-selectin ligands, which greatly enhances their
interaction with L-selectin. Direct structural analysis of GlyCAM-1 (37) has identified Gal-6-sulfate and GlcNAc-6-sulfate as the
major sulfated monosaccharides in the context of N-acetyllactosamine, i.e. Gal
1
4GlcNAc.
Further structural studies revealed that 6`-sulfo sialyl Lewis x, i.e. Sia
2
3(SO
-6)Gal
1
4[Fuc
1
3]GlcNAc
and 6-sulfo sialyl Lewis x, i.e. Sia
2
3Gal
1
4[Fuc
1
3]GlcNAc-6SO
are
major capping groups of this ligand(27, 38) .
Structures of two of the simplest O-glycans of GlyCAM-1 are
predicted (38) as Fig. S1and Fig. S2:
Figure S1: Structure 1.
Figure S2: Structure 2.
These oligosaccharides contain the T-antigen, i.e. Gal1
3GalNAc, which is incorporated into the core-2
structure (39) , i.e. Gal
1
3[GlcNAc
1
6]GalNAc.
Although there is increasing information about the carbohydrate structure of the biological ligands of L-selectin, as well as those for the two endothelial selectins(40, 41) , there have been no reports on the biosynthesis of these structures. The present study investigates the biosynthesis of O-linked oligosaccharides of GlyCAM-1 as it relates to the elaboration of functional ligand activity. We employ lectin analysis, pulse-chase labeling, and the inhibition of membrane transport (via reduced temperature and brefeldin A) for this analysis. We report the identification of GlyCAM-1 biosynthetic intermediates that represent distinct stages of the O-linked biosynthetic pathway. These studies provide a view of how L-selectin binding activity may be regulated at the level of O-glycan biosynthesis.
Figure 1:
Precipitation of GlyCAM-1 and
characterization of O-linked oligosaccharides. Lymph nodes
were metabolically labeled with [H]Ser/Thr, and
GlyCAM-1 was immunoprecipitated from the detergent lysate (A)
or conditioned medium (B) with anti-GlyCAM-1 anti-peptide 2
Ab. The material bound was eluted with peptide 2 Ab, dialyzed against
sialidase buffer, and treated with and without A. ureafaciens and V. cholerae sialidase. Equal
aliquots of sialidase treated (+) or untreated(-) GlyCAM-1
were then reprecipitated with preimmune serum, anti-GlyCAM-1 peptide 3
Ab, VVA, PNA, MAA, Limax agglutinin, AAA, SNA, or LEC-IgG. The material
bound was specifically eluted as described under ``Experimental
Procedures,'' acetone-precipitated, and solubilized in
Laemmli sample buffer. Aliquots of each sample were counted by liquid
scintillation (see Table 2) and subjected to analysis by SDS-PAGE
on 10% gels under non-reducing conditions (band profiles did not differ
with reduction), and fluorography using EN
HANCE. Discrete
low molecular mass proteins migrating between 28-33 kDa are
denoted by *. Although the spacing is the same, 28-33 kDa
proteins reprecipitated by VVA are shifted slightly upward in the gel
by 1 kDa. The VVA and AAA precipitates from desialylated GlyCAM-1 (data
not shown) were identical to the those shown for untreated GlyCAM-1.
Each lane contains intracellular (A) or secreted (B)
radiolabeled GlyCAM-1 from the lymph nodes (axillary, brachial,
cervical, and mesenteric) of one mouse. In both A and B, approximately 50% of the Limax agglutinin precipitate was
lost prior to SDS-PAGE. In A, the exposure time of the
anti-peptide 3 precipitates (lanes 2 and 3) was
one-third less than the rest of the precipitates to adequately
visualize the 28-33 kDa proteins. Molecular mass markers were
phosphorylase b (94,000), bovine serum albumin (67,000),
ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin
inhibitor (20,000); df, dye front.
For the lectin and LEC-IgG binding
studies, [H]Ser/Thr-labeled GlyCAM-1, isolated
from detergent lysates or conditioned medium with anti-peptide 2 Ab,
was treated with or without sialidase. Equal aliquots were subsequently
incubated with the lectin panel, anti-peptide 3 Ab, or LEC-IgG, all
immobilized on agarose or Sepharose. The material bound was analyzed by
SDS-PAGE (Fig. 1) and was quantified by scintillation counting (Table 2). Fig. 1A presents the analysis of
precipitates of GlyCAM-1 from the detergent lysates. We established
that precipitation was efficient, since a second round of precipitation
yielded only 10-20% of the initial values (Table 2). VVA
precipitated proteins up to 45 kDa, including the discrete low
molecular mass species migrating between 28-33 kDa (denoted by
*). Without sialidase treatment of GlyCAM-1, PNA binding was
negligible. With prior desialylation, PNA binding increased
6-10-fold (Table 2), and the precipitated proteins ranged
from 34 to 45 kDa (Fig. 1A). As shown in Table 2,
24% of the total available GlyCAM-1 was precipitated by PNA after
desialylation, confirming that the sialylated T-antigen
(Sia
2
3Gal
1
3GalNAc) is a significant structure on
GlyCAM-1(46) . PNA did not bind the 28-33 kDa proteins
that were precipitated by VVA, suggesting that this cluster of proteins
contained only GalNAc and may represent the earliest glycosylated
precursors of GlyCAM-1. There are 38 potential sites for the initiation
of O-linked glycosylation of GlyCAM-1(17) , and it is
likely that the 28-33 kDa species contained different numbers of
GalNAc
1-Ser/Thr modifications. The precipitation of higher
molecular mass species (>33 kDa) by VVA suggested that non-extended
GalNAc residues existed on these proteins.
From both lysate and
conditioned medium, Limax agglutinin, MAA, and AAA precipitated only
those proteins migrating at 40-60 kDa, thus identifying the
sialylated and fucosylated forms of GlyCAM-1 (designated in Fig. 1A). The 40-60 kDa proteins also contained
the (SO
-6)Gal
1
4GlcNAc determinant as indicated
by SNA binding. It has been previously shown that SNA recognition of
GlyCAM-1 is inhibited by
2
3 sialylation of Gal in the SNA
epitope(27) . Without prior desialylation of GlyCAM-1, SNA
weakly precipitated the 40-60 kDa band (Fig. 1A and B). With sialidase treatment, SNA binding increased
by approximately 4-fold (Table 2) and precipitated a broad band
at 38 kDa (Fig. 1, A and B), representing the
desialylated form of the 40-60 kDa proteins. Thus, the >40 kDa
proteins contained the sialic acid, fucose, and sulfate modifications
found in the 6` sulfated, sLe
capping structure.
LEC-IgG precipitated a diffuse 50-60 kDa band (designated #, Fig. 1, A and B), indicating that only the highest molecular mass subset of the 40-60 kDa proteins possessed the features necessary for L-selectin binding. These results may indicate that the lower molecular weight forms of GlyCAM-1 contained incompletely processed oligosaccharides or an insufficient density of the mature oligosaccharide to support L-selectin binding. Secreted GlyCAM-1 was enriched in the more fully processed oligosaccharide structures (Fig. 1B, Table 2). This conclusion is supported by the higher proportion of AAA, MAA, Limax agglutinin, SNA and LEC-IgG binding, and the lower proportion of VVA binding. The representation of the T-antigen remained constant, which is consistent with the formation of this core oligosaccharide structure at an early stage of the O-glycan biosynthesis.
Figure 2:
Time course of the synthesis and
secretion of GlyCAM-1. Lymph nodes were pulse-labeled for 5 min with
[H]Ser/Thr and chased for various times in a 200
molar excess of unlabeled serine and threonine to give final chase
times of 5, 10, 20, 45, 90, 180, and 300 min. Detergent lysates (lys) and conditioned medium (CM) were generated for
each time point and pooled from two independent experiments. The
samples were normalized for total protein, and GlyCAM-1 was isolated by
immunoprecipitation with anti-peptide 2 Ab. The material bound was
eluted with peptide 2, dialyzed against PBS, and reprecipitated with
preimmune serum and anti-peptide 3 Ab. Aliquots of each sample were
counted in duplicate by liquid scintillation (see Fig. 3) and
subjected to analysis by SDS-PAGE. The anti-peptide 3 Ab
immunoprecipitates are shown; the preimmune serum immunoprecipitates
were completely negative (data not shown). Each lane contains
intracellular (lysates) or secreted (conditioned medium) pulse-labeled
GlyCAM-1 from the lymph nodes of 1.5 mice.
Figure 3:
Quantitation of the time course of
GlyCAM-1 secretion. Aliquots of the anti-peptide 3 immunoprecipitates
of Fig. 2pulse-labeled with [H]Ser/Thr
and chased in an excess of unlabeled Ser/Thr for 0-300 min were
subjected to scintillation counting. The values shown are the mean of
duplicate counts for each sample (standard deviations were less than 1%
of the mean value). CM, conditioned
medium.
We undertook
temperature block studies in order to localize the GlyCAM-1
glycosylation intermediates to particular intracellular compartments.
Lymph nodes were pulsed with [H]Ser/Thr for 5
min, as described for Fig. 2; however in this case, the chase
was performed at 37, 20, or 15 °C for both a short (20 min) and
long interval (90 min). As shown in Fig. 4, at 37 °C a small
fraction of the 28-33 kDa cluster was chased to 40-60 kDa
by 20 min. By 90 min, the 40-60 kDa proteins increased in the
lysate and accumulated in the conditioned medium, recapitulating what
was demonstrated in Fig. 2. Reducing the temperature to 20
°C significantly slowed the processing of the 28-33 kDa
proteins. At 20 °C, the 40-60 kDa proteins were completely
absent at 20 min and substantially reduced at 90 min. Secretion of
GlyCAM-1 was completely blocked. Furthermore, the level of the
50-60 kDa protein within the lysate was increased at 20 °C
relative to 37 °C at 90 min, suggesting that mature GlyCAM-1 was
accumulating, presumably in the TGN. With further reduction of the
temperature to 15 °C, processing of the 28-33 kDa proteins to
40-60 kDa, as well as secretion, were completely blocked at both
time points. The 28-33 kDa proteins were generated within the
5-min pulse-labeling period at 37 °C (see Fig. 2). The
demonstration that these proteins were not further processed at 15
°C strongly suggested that they acquired their additional molecular
mass before being transported to the Golgi cisterna. The 28-33
kDa proteins that accumulated at 15 °C comigrated precisely with
the GlyCAM-1 proteins precipitated by VVA in Fig. 1A.
These results suggest that the initiation of O-linked
glycosylation on the GlyCAM-1 core protein occurs in a pre-Golgi
compartment.
Figure 4:
The
effect of temperature on the oligosaccharide processing and secretion
of GlyCAM-1. Lymph nodes were pulse labeled for 5 min with
[H]Ser/Thr and chased at 37, 20, and 15 °C to
give final chase times of 20 and 90 min. Detergent lysates and
conditioned medium were obtained for each condition, and the samples
were normalized for total protein and subjected to immunoprecipitation
with preimmune serum and anti-peptide 2 Ab. The bound material was
solubilized in Laemmli sample buffer and analyzed by SDS-PAGE and
fluorography. The anti-peptide 2 immunoprecipitates and a
representative preimmune immunoprecipitate are shown; all preimmune
samples were completely negative.
Figure 5:
The maturation of O-linked
oligosaccharides of GlyCAM-1. Lymph nodes were pulse-labeled for 10 min
with [H]Ser/Thr and chased for various times.
Detergent lysates and conditioned medium were generated for each time
point, normalized for total protein, and subjected to
immunoprecipitation with anti-peptide 2 Ab and peptide elution. The
GlyCAM-1 recovered from the detergent lysate and conditioned medium
were combined for each time point, dialyzed against sialidase buffer,
and treated with or without A. ureafaciens and V. cholerae sialidase. Equal aliquots of sialidase
treated and untreated GlyCAM-1 were then reprecipitated with
anti-peptide 3 Ab, PNA, Limax agglutinin, AAA, SNA, or LEC-IgG. The
material bound was specifically eluted as described under
``Experimental Procedures'' and counted by liquid
scintillation. The data are plotted as the percent of total GlyCAM-1
bound by the lectins and LEC-IgG over time. The percent of GlyCAM-1
bound was calculated by dividing the counts bound with the lectin or
LEC-IgG by the counts bound with anti-peptide 3 Ab. For Limax
agglutinin, AAA, and LEC-IgG, data are shown for precipitates of
non-sialidase-treated GlyCAM-1, whereas for PNA and SNA, data are shown
for precipitates of sialidase-treated GlyCAM-1. Data are derived from
the mean of duplicate reprecipitations and have a standard deviation of
less than 5% of the mean value. Inset shows the percent of
GlyCAM-1 bound by AAA and SNA in an independent experiment with chase
times up to 360 min. These data are the mean of reprecipitations
performed in triplicate with a standard deviation of less than 1% of
the mean value.
Figure 6:
The effect of brefeldin A on GlyCAM-1
synthesis, sulfation, and O-linked oligosaccharide structures.
Lymph nodes were pretreated with BFA at 0, 0.025, 0.25, or 2.5
µg/ml for 1 h and metabolically labeled with
[H]Ser/Thr or [
S]sulfate
in the continued presence of BFA. Detergent lysates and conditioned
medium were generated and and parallel samples were equalized for total
protein. A, GlyCAM-1 was immunoprecipitated from the
[
H]Ser/Thr-labeled lysate (⊡), conditioned
medium (
) or [
S]sulfate-labeled lysate
(&cjs3409;) with anti-peptide 2 Ab, peptide-eluted, and counted by
liquid scintillation. The data, reported as ``relative
biosynthesis'' are plotted as the percentage of GlyCAM-1 recovered
in the presence of BFA as compared to the absence of BFA. The data are
based on the mean of duplicate values from two independent experiments. B, aliquots of the GlyCAM-1 peptide 2 eluate from the
[
H]Ser/Thr-labeled lysate were treated with or
without Arthrobacter and V. cholerae sialidase and
reprecipitated with anti-peptide 3 Ab, PNA (
), MAA (
),
Limax agglutinin (
), AAA (⊡), SNA (
), or LEC-IgG
(&cjs3409;). The material bound was eluted as described under
''Experimental Procedures`` and counted by liquid
scintillation. The counts bound by the lectins and LEC-IgG were
normalized for the total available GlyCAM-1 at each BFA concentration
by dividing by the counts obtained with anti-peptide 3 Ab. The data,
reported as ``relative lectin reactivity,'' are plotted as
the percentage of GlyCAM-1 recovered in the presence of BFA normalized
to the percentage recovered in the absence of BFA. The percentages
recovered in the absence of BFA were as follows for each precipitating
reagent: PNA, 18.2; MAA, 3.8; Limax agglutinin, 25.9; AAA, 17.0; SNA,
13.1; LEC-IgG, 8.4. For MAA, Limax agglutinin, AAA, and LEC-IgG, data
are shown for precipitates of non-sialidase-treated GlyCAM-1, whereas
for PNA and SNA, data are shown for precipitates of sialidase-treated
GlyCAM-1. All values are derived from the mean of duplicate
reprecipitations (deviations were less than 5% of the mean
value).
The enhanced MAA reactivity with BFA treatment may represent the
combination of two factors. First, 2
3 sialylation of
Gal
1
4GlcNAc may be increased. Second, decreased sulfation of
Sia
2
3Gal
1
4GlcNAc at C-6 of Gal enhances
reactivity with MAA (27) . To determine the relative
contribution of these effects, a base line for maximal GlyCAM-1 binding
to MAA was established using sodium chlorate, which inhibits GlyCAM-1
sulfation by 90% without interfering with sialylation(31) . As
shown in Table 3, MAA precipitated 4.6% of the
[
H]Ser/Thr-labeled GlyCAM-1 from a detergent
lysate of untreated lymph nodes. With chlorate treatment, MAA binding
increased to 15.2%, while SNA binding decreased from 14.6 to 1.5%,
reflecting the decrease in sulfation. When BFA was added in combination
with chlorate, MAA still bound 10.4% of the available GlyCAM-1. Thus,
with the sulfation effect controlled for, MAA recognition of GlyCAM-1
was not substantially diminished (15.2 versus 10.4%). As
predicted from its binding specificity (Table 1), the enhanced
MAA binding seen with BFA, chlorate, or the two combined drugs was
prevented by sialidase treatment of GlyCAM-1 (Table 3).
LEC-IgG binding was completely inhibited by BFA treatment (Fig. 6B). The dramatic loss of LEC-IgG binding under this condition provides further evidence that sulfation is essential for recognition by L-selectin.
Figure 7:
The
effect of BFA on the maturation of O-linked oligosaccharide
structures. Lymph nodes were preincubated with (+) or without
BFA(-) at 2.5 µg/ml for 1 h and then metabolically labeled
with [H]Ser/Thr in the presence or absence of
BFA. Detergent lysates were generated and equalized for total protein.
GlyCAM-1 was isolated from the lysates with anti-peptide 2 Ab, eluted
with peptide, and dialyzed against sialidase buffer. The GlyCAM-1
preparations were treated with (+) or without(-) Arthrobacter and V. cholerae sialidase.
Equal aliquots of each sample were reprecipitated with anti-peptide 3
Ab, PNA, MAA, Limax agglutinin, SNA, or LEC-IgG, and eluted as
described under ``Experimental Procedures.'' The eluates were
acetone-precipitated and subjected to analysis by SDS-PAGE and
fluorography.
GlyCAM-1 is an HEV-derived, secreted ligand for L-selectin. Its functional role in lymphocyte-HEV binding has not been determined as yet. Nonetheless, detailed biochemical analysis of GlyCAM-1 is warranted, since it shares a carbohydrate-based recognition determinant with the other known HEV ligands for L-selectin (23) and is associated with sites of chronic inflammation(19) .
We have defined the following discrete stages in biosynthesis of GlyCAM-1: 1) unglycosylated species of <28 kDa; 2) discrete 28-33 kDa proteins containing GalNAc-terminating chains; 3) a broadly migrating 40-50 kDa species containing the T-antigen, sialic acid, fucose, and sulfate, but not reactive with LEC-IgG; and 4) a 50-60 kDa sialylated, fucosylated, and sulfated protein, reactive with LEC-IgG.
By pulse-chase analysis, we established that the first three groups
of proteins were biosynthetic intermediates of mature GlyCAM-1. The low
molecular mass proteins (28-33 kDa) were synthesized within 5 min
and processed to 40-50 kDa with a half-time of approximately 30
min. The half-time for the acquisition of L-selectin binding was 65
min. An unexpected finding was that up to 75% of the 40-50 kDa
species was secreted into the medium without attaining the capacity to
bind L-selectin.
Using temperature blocks, we have gained information about the initiation of O-glycosylation in GlyCAM-1. A reduction of the temperature to 15 °C during the chase period completely blocked the processing of the rapidly synthesized 28-33 kDa cluster. In multiple cell types, membrane transport into the Golgi stacks is blocked at 15 °C, and glycoproteins accumulate in pre-Golgi transitional elements of the endoplasmic reticulum(48, 49, 52, 53, 54) . Since the 28-33 kDa proteins contain GalNAc-terminating chains, our findings indicate that the initiation of glycosylation on GlyCAM-1 occurs in a pre-Golgi compartment. In some systems, the addition of GalNAc to nascent proteins occurs in the endoplasmic reticulum or transitional elements of the endoplasmic reticulum(55, 56, 57) , whereas in others, initiation appears to take place in the Golgi apparatus (58, 59, 60) . Thus, the site for the initiation of O-linked glycosylation appears to differ for different cell types and perhaps for different core proteins.
The metabolic inhibitor BFA is a valuable experimental tool that permits discrimination of processing events in the ER/Golgi compartment from those in the TGN. We employed this drug to dissect the terminal processing events for GlyCAM-1, which could not be adequately resolved by pulse-chase analysis. As expected, BFA completely blocked the secretion of GlyCAM-1 into conditioned medium. BFA caused the accumulation of biosynthetic intermediates of GlyCAM-1 with oligosaccharides that were efficiently sialylated, partially fucosylated, and almost completely lacking in sulfate.
The epitope
for PNA (the T-antigen) increased approximately 5-fold with BFA
treatment, and it was fully sialylated since prior desialylation was
required for binding. The increased level of sialylated T-antigen with
BFA may have been due to the increased contact of GlyCAM-1 with the
appropriate glycosyltransferases in the BFA-induced compartment. BFA
treatment also allowed the formation of N-acetyllactosamine
(Gal1
4GlcNAc) within GlyCAM-1 and its efficient sialylation.
Thus with BFA, the total amount of [
H]Gal
released by Diplococcus exo-(
1
4)galactosidase
decreased only marginally relative to the control. Additionally, the
release of [
H]Gal completely depended upon the
prior desialylation of GlyCAM-1, consistent with a fully sialylated
state of the terminal Gal
1
4 residues. Finally, MAA reacted
with BFA-generated GlyCAM-1 comparably to chlorate-generated GlyCAM-1,
indicating the formation of Sia
2
3Gal
1
4GlcNAc.
Taken together, these results argue that the sialyltransferases that
form the Sia2
3Gal
1
3GalNAc and
Sia
2
3Gal
1
4GlcNAc structures are localized in a
pre-TGN compartment. This conclusion is consistent with previous
studies on several glycoproteins in which BFA treatment does not impede
sialylation of O-linked
oligosaccharides(61, 62, 63) . The
1
4 galactosyltransferase involved in the formation of N-acetyllactosamine is localized to the trans-Golgi
cisternae in a number of cells(64, 65) . Thus, the
Gal
1
4GlcNAc
2
3 sialyltransferase pertinent to
GlyCAM-1 is likely to reside in the trans-Golgi cisternae, in
distinction to the apparent TGN localization of sialyltransferases that
act on N-linked
oligosaccharides(63, 66, 67) . Our data
cannot distinguish the subcellular localization of the
Gal
1
3GalNAc
2
3 sialyltransferase relative to the
Gal
1
4GlcNAc
2
3 sialyltransferase. However, the
T-antigen-specific
2
3 sialyltransferase involved in the
synthesis of another sialomucin has been mapped to a compartment
proximal to the trans-Golgi cisternae(65) .
Fucose
is added in an 1
3 linkage to GlcNAc in the N-acetyllactosamine of GlyCAM-1. BFA inhibited fucosylation of
GlyCAM-1 by
50% as determined by direct precipitation with AAA and
by the defucosylation requirement for exo-(
1
4)galactosidase
action. Thus, the accessibility of nascent oligosaccharides to the
fucosyltransferase was clearly affected by BFA. In contrast to the
sialyltransferases, the fucosyltransferase appears to reside in a
compartment that was partially redistributed by BFA. Given the apparent
greater efficiency in the redistribution of the sialyltransferases, the
fucosyltransferase is likely to reside in a more distal region of the
biosynthetic pathway. This conclusion is consistent with the general
finding that
2
3 sialylation precedes
1
3
fucosylation during the synthesis of
sLe
(68, 69) .
Gal-6-sulfate and
GlcNAc-6-sulfate occur equally in GlyCAM-1(37) . In the
presence of BFA, the sulfation of GlyCAM-1 was almost completely
suppressed, as demonstrated by the 85% reduction in [S]sulfate labeling of GlyCAM-1 and the 99%
reduction in SNA binding. Since BFA allowed the synthesis of the
Gal
1
4GlcNAc structure, the inhibition of sulfation is likely
attributable to the inaccessibility of GlyCAM-1 biosynthetic
intermediates to the TGN where the relevant sulfotransferases reside.
In a number of other systems, BFA has been employed to reach the same
conclusion about the subcellular localization of sulfotransferases that
modify O-glycans(70, 71, 72, 73) .
Taken together, our biosynthetic analysis of GlyCAM-1 argues that
the sialylation events precede both fucosylation and sulfation. As
noted above, the ordering of sialylation versus fucosylation
is consistent with previous studies. The relationship of fucosylation
to sulfation is more problematic. BFA produced partial inhibition of
fucosylation (50%) and almost complete inhibition of sulfation,
which would argue for fucosylation occurring before the two sulfation
modifications. In support of this possibility, Jain et al.(74) have reported that several of the known
1
3/4 fucosyltransferases are unable to fucosylate
Sia2
3(SO
-6)Gal
1
4GlcNAc to form the
6`-sulfo sLe
capping structure, whereas these enzymes are
active on the non-sulfated structures. However, Scudder et al.(75) have reported that a lymph node N-acetylglucosamine-6-O-sulfotransferase is unable to
add sulfate to GlcNAc-containing oligosaccharides if the C-3 position
of GlcNAc is substituted with fucose, arguing that sulfation cannot
precede fucosylation on this sugar. Clearly, further studies are
necessary to define the temporal relationship of the two sulfation
events to fucosylation during the biosynthesis of GlyCAM-1.
The present study has identified glycosylation intermediates of GlyCAM-1 as it is synthesized in mouse peripheral lymph nodes. Our analysis helps to elucidate acceptor structures for the endothelial enzymes that form the ligand. An important future challenge is to determine the molecular identity of these enzymes and to understand how their activities are regulated in lymphoid organs and at sites of inflammation.