From the Department of Pathology and the
Jonsson Comprehensive Cancer Center, UCLA School of
Medicine, Los Angeles, California 90095
Received for publication, September 18, 2002, and in revised form, December 9, 2002
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ABSTRACT |
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The addition of sialic acid to T cell surface
glycoproteins influences essential T cell functions such as selection
in the thymus and homing in the peripheral circulation. Sialylation of glycoproteins can be regulated by expression of specific
sialyltransferases that transfer sialic acid in a specific linkage to
defined saccharide acceptor substrates and by expression of particular
glycoproteins bearing saccharide acceptors preferentially recognized by
different sialyltransferases. Addition of Glycosylation of cell surface proteins controls critical T cell
processes, including lymphocyte homing, thymocyte selection, the
amplitude of an immune response, and T cell death (1-9). The role of
glycosylation in these functions is specific, i.e. the
different functions require specific sugars on specific glycoprotein acceptors. Regulated glycosylation of specific acceptor substrates can
affect immune function by creating or masking ligands for endogenous
lectins. For example, modification of cell surface oligosaccharides by
the C2GnT and Fuc TVII glycosyltransferases results in specific
selectin-mediated trafficking patterns for Th1 and Th2 subsets (3).
Similarly, modification of CD45 by the C2GnT glycosyltransferase
regulates thymocyte susceptibility to cell death induced by galectin-1
(10).
During T cell development, expression of several glycosyltransferases
is temporally and spatially controlled (9, 11, 12). In the human
thymus, different members of the sialyltransferase family are expressed
in distinct anatomic compartments, so cells in those compartments
bear unique complements of sialylated oligosaccharides. For example,
the SA Reagents and Cells--
Galectin-1 was prepared as described
previously (13). Murine BW5147.3 (BW5147), PhaR2.1,
T200 Lectin Flow Cytometry--
Expression of cell surface
oligosacccharides was detected by flow cytometry with biotinylated
Phaseolus vulgaris agglutinin (PHA)1 and Sambuccus
nigra agglutinin (SNA) (E-Y Labs, San Mateo, CA) (10 µg/ml), as
described (12). For glycosidase inhibition, the cell lines were
cultured for 72 h with 2 mM deoxymannojirimycin (DMNJ)
(Oxford GlycoSystems, Inc., Rosedale, NY) or medium alone prior to
lectin analysis and cell death assays.
Transfection--
Rat ST6Gal I cDNA in the plasmid
STTyr-Myc-pcDNA 3.1 (17, 18) (gift of Dr. Karen Colley,
University of Illinois, Chicago, IL) or vector alone were transfected
into PhaR2.1 and T200 Galectin-1 Binding Assay--
5 × 105 cells
were suspended in PBS containing the indicated amount of biotinylated
galectin-1 (16) at 4 °C for 1 h. After washing, the cells were
incubated with streptavidin-fluorescein isothiocyanate (5 µg/ml)
(Jackson Immunoresearch Laboratories, West Grove, PA) for 45 min at
4 °C. After washing, the cells were analyzed by flow cytometry.
Galectin-1 Cell Death Assays--
Galectin death assays were
performed as described (16) with the following modifications.
105 cells were incubated with 20 µM
galectin-1 in 1.6 mM dithiothreitol/Dulbecco's modified
Eagle's medium or in 1.6 mM dithiothreitol/Dulbecco's modified Eagle's medium alone as a control for 4-6 h at 37 °C. 0.1 M Precipitation and Western Blot Analysis--
The cell lysates
from 4-9 × 106 cells were prepared as described
(12). To precipitate SNA-binding glycoproteins, the lysates were
precleared for 1 h with biotinylated bovine serum albumin (0.25 µg/225 µl cell lysate) and ImmunoPure Immobilized Streptavidin (Pierce). After centrifugation to remove insoluble material, the supernatants were incubated with SNA-biotin (5 µg/300 µl cell lysate) and ImmunoPure Immobilized Streptavidin overnight. The precipitates were washed four times with lysis buffer prior to SDS-PAGE. All of the steps were performed at 4 °C. To precipitate CD45, the supernatants were precleared with purified rat
IgG2b,
CD45 or SNA precipitates were separated by SDS-PAGE, blotted to
nitrocellulose, and probed with polyclonal goat anti-mouse CD45 (0.2 µg/ml) (Research Diagnostics Inc., Flanders, NJ) or SNA-biotin (1 µg/ml). Bound reagent was detected with horseradish peroxidase-labeled rabbit anti-goat IgG (Bio-Rad) or
streptavidin-horseradish peroxidase, respectively, and visualized by
ECL (Amersham Biosciences). ST6Gal I immunoblotting of whole cell
lysates was performed as described in Ref. 12, with rabbit anti-rat
ST6Gal I antiserum (gift of Dr. K. Colley).
PNGase F Digestion of CD45--
Cell lysates
(106 cells) were separated by SDS-PAGE, blotted to
nitrocellulose and probed with polyclonal goat anti-mouse CD45 (M-20)
(Santa Cruz Biotechnology, Santa Cruz, CA). The band corresponding to
CD45 was excised from the nitrocellulose, and the bound antibody was stripped with Restore buffer (Pierce). After washing two times with
25 mM Tris, 150 mM NaCl, 0.05% Tween, pH 7.5 (TBS-T) followed by two washes with 50 mM sodium phosphate,
pH 7.5, the membrane was incubated with 1.5 ml of 50 mM
sodium phosphate containing 10,000 units of PNGase F (New England
BioLabs, Beverly, MA) overnight at 37 °C with rocking. The
enzyme-treated membrane was washed with TBS-T and probed with
SNA-biotin, as described above.
Sialyltransferase Activity Assay--
The cells were lysed in 50 mM sodium cacodylate, pH 6.5, 100 mM NaCl, 1 mM MgCl2, 1% Nonidet P-40, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride. 100 µl of lysates (3 × 106 cells) were incubated with 1.5 µM
CMP- CD45 Segregation Analysis--
The cells were treated with or
without galectin-1 as in the death assays. After treatment, the cells
were washed with PBS and fixed with 2% paraformaldehyde for 30 min at
4 °C. The reaction was quenched with 0.2 M glycine for 5 min at 4 °C, and the cells were blocked with in 10% goat serum for
1.5 h at room temperature. The cells were washed with PBS and
incubated with polyclonal goat anti-mouse CD45 conjugated to
fluorescein isothiocyanate (Pharmingen) in 2% goat serum for 1.5 h at room temperature in the dark. After washing, the cells were
mounted on slides with Prolong Anti-fade medium (Molecular Probes,
Eugene OR). CD45 cell surface localization was analyzed on a Fluoview
laser scanning confocal microscope (Olympus America Inc, Melville, NY),
at 100×. The number of cells demonstrating CD45 segregation or
clustering and the total number of cells were counted for six randomly
selected fields for each experiment. Approximately 50 cells were
counted in six fields. Percent CD45 segregation was calculated as
[100 × (number of CD45 segregated cells/total number of
cells)].
Protein-tyrosine Phosphatase Activity Assay--
4 × 106 cells were incubated at 37 °C in 400 µl of medium
containing PBS as a control (0 min) or 30 µg of galectin-1 for 1, 5, 15, or 30 min. At the indicated times, the cells were cooled on ice,
washed in PBS at 4 °C, and lysed (12), and the protein concentrations of the cell lysates were determined (protein assay kit;
Bio-Rad). Protein-tyrosine phosphatase (PTP) activity was measured
using p-nitrophenyl phosphate (Calbiochem) as a substrate in
the presence of okadaic acid to inhibit protein Ser/Thr phosphatases. Cell lysate (20 µg/25 µl) was incubated at room temperature for 4 h in 475 µl of PTP assay buffer (100 mM Hepes, pH
7.2, 150 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 10 mM p-nitrophenyl phosphate, 50 nM okadaic acid) either in the absence or presence of 50 µM bpV (phen) (potassium bisperoxo
(1,10-phenanthraline)oxo-vanadate(v)) as a specific PTP inhibitor (10).
The reaction was stopped by adding 500 µl of 1 N NaOH,
and released p-nitrophenol was measured at
A415 against appropriate blanks.
N-Glycans Are Essential for Galectin-1 Death--
Galectin-1
preferentially recognizes Gal
To determine whether N-glycans are necessary for galectin-1
induced death, human and murine T cell lines were treated with the
mannosidase I inhibitor DMNJ, to block trimming of terminal mannose
residues and subsequent elongation of N-glycans with LacNAc sequences. The effectiveness of DMNJ treatment was determined by
analyzing treated cells with the PHA, because inhibition of mannosidase
I activity would prevent elongation of the N-glycan chain
recognized by PHA (19). Cells treated with DMNJ showed a marked
reduction in PHA binding compared with cells cultured in medium alone;
importantly, DMNJ treatment did not affect the level of cell surface
expression of galectin-1 receptors CD43 or CD45, as determined by flow
cytometric analysis using the relevant antibodies (data not shown).
DMNJ-treated cells were examined for susceptibility to
galectin-1-induced cell death (Fig. 1).
The PhaR2.1, CEM, and MOLT-4 cell lines are all susceptible
to galectin-1-induced cell death, whereas the BW5147 cells are
resistant to galectin-1 because of the lack of core 2 O-glycans on cell surface glycoproteins (9, 10, 16). DMNJ
treatment resulted in a dramatic reduction in galectin-1-induced cell
death of the galectin-1-susceptible murine (PhaR2.1) and
human (CEM, MOLT-4) T cell lines. Although previous studies demonstrated that the glycosylation inhibitors benzyl- Expression of the ST6Gal I Reduces Galectin-1 Binding to T
Cells--
The addition of terminal
To directly examine whether addition of
We then determined whether addition of cell surface sialic acid by the
ST6Gal I enzyme would reduce galectin-1 binding to T cells. As shown in
Fig. 2C, galectin-1 binding to the SNA.9 cells (closed
circles) was markedly reduced compared with the level of binding
observed for the C.2 control cells transfected with vector alone
(open circles). However, the reduced, but not absent,
binding of galectin-1 to SNA.9 cells indicated that some of the
potential binding sites on these cells were not modified by the ST6Gal
I enzyme. For both SNA.9 and C.2 cells, galectin-1 binding was
completely inhibited in the presence of 100 mM lactose (squares), demonstrating that binding was
saccharide-dependent.
Expression of the ST6Gal I Reduces Susceptibility to
Galectin-1--
The SNA.1, SNA.9, and C.2 cells were examined for
susceptibility to galectin-1-induced death. As shown in Fig.
2D, the C.2 cells transfected with vector alone were
susceptible to galectin-1; ~50% of the cells underwent cell death,
determined by annexin V binding and PI uptake. In contrast, the SNA.1
and SNA.9 cells demonstrated only 10 and 18% galectin-1-induced death,
respectively. The resistance of the SNA.1 and SNA.9 cells to galectin-1
did not appear to result from a complete block of galectin-1 binding, as demonstrated by the binding curve in Fig. 2C; in
addition, all of the clones demonstrated cell agglutination when
galectin-1 was added, and the cell agglutinates were dispersable by the
addition of lactose (data not shown). These data demonstrated that
expression of the ST6Gal I, resulting in creation of SA ST6Gal I Expression Results in Increased Sialylation of
CD45--
Our laboratory has demonstrated that the T cell surface
glycoproteins CD7, CD43, and CD45 are receptors for galectin-1 (24). To
determine whether these glycoproteins were specifically modified by the
ST6Gal I, we examined CD7, CD43, and CD45 for increased sialylation of
N-glycans by SNA and antibody precipitation.
Total SNA-binding proteins were precipitated and probed with SNA. As
shown in Fig. 3A, there was
only one significant difference in the pattern of SNA-binding
glycoproteins precipitated from cells expressing the ST6Gal I (SNA.1)
compared with vector-transfected controls (C.2, C.4). In extracts of
SNA.1 cells, there was an obvious increase in SNA binding to a band of
approximate molecular mass of 200 kDa. Other SNA reactive bands
of various sizes were occasionally seen in different experiments (data
not shown), but these other bands were not consistently observed. In
contrast, the 200-kDa SNA+ band was consistently observed
in ST6Gal I-expressing clones. The relative mobility of the 200-kDa
band suggested that it could be CD45, a highly glycosylated protein
that is known to bear SA
To specifically determine whether the band exhibiting increased SNA
binding was CD45, both SNA and CD45 antibody were used to precipitate
material from vector transfected (C.4) and SNA.1 cells, and the
precipitates were probed with CD45 (Fig. 3B). The 200-kDa
band exhibiting increased SNA binding reacted with CD45 antibody,
demonstrating that CD45 was selectively hypersialylated in the SNA.1
cells. In addition, this band migrated with the same mass as
immunoprecipitated CD45. To determine whether the increased sialylation
of CD45 occurred on N-glycans, the preferred glycan acceptor
for the ST6Gal I, SNA.1 cells were pretreated with DMNJ prior to SNA or
CD45 precipitation. DMNJ treatment reduced SNA binding to protein
precipitated from SNA.1 cells to the level observed for control cells
(C.4) transfected with vector alone (Fig. 3B). PNGase F
treatment confirmed that, in cells overexpressing the ST6Gal I, sialic
acid addition to CD45 occurred on N-glycans. Whole cell
lysates of SNA.9 cells were probed with antibody to CD45. The CD45
bands were excised from the blot and incubated with or without PNGase
F, and the bands were reprobed with SNA. As shown in Fig.
3C, PNGase F dramatically reduced SNA binding to CD45 from
SNA.9 cells. Thus, the increased SNA binding to CD45 on cells
expressing the ST6Gal I resulted from the specific addition of
As mentioned above, the three primary receptors for galectin-1 on T
cells are CD7, CD43, and CD45 (24). We specifically precipitated CD7
and CD43 from SNA.9, SNA.1, and C.2 cells and saw no difference in SNA
binding to CD7 or CD43 (data not shown), indicating that the inhibitory
effect on galectin-1 cell death was not due to sialylation of CD7 or
CD43. We also did not detect CD7 or CD43 by immunoblotting SNA
precipitates with the respective antibodies (data not shown). To
further examine the acceptor substrate preference of the ST6Gal I, we
expressed the ST6Gal I in the murine T200 ST6Gal I Expression Inhibits CD45 Segregation on Galectin-1-treated
Cells--
We have demonstrated that galectin-1 binding to T cells
results in reorganization of the glycoprotein receptors CD45, CD43, and
CD7 into novel membrane microdomains (24). Specifically, CD45
segregates from CD43 and CD7 and localizes to membrane blebs on dying
cells. The segregation of CD45 caused by galectin-1 binding is
regulated in part by expression of the C2GnT glycosyltransferase that
creates branches on O-glycans bearing the LacNAc sequences recognized by galectin-1. Cells that do not express the C2GnT do not
demonstrate CD45 segregation after galectin-1 and are not susceptible
to galectin-1-induced cell death (10).
We examined the effects of ST6Gal I expression on CD45 segregation
after galectin-1 binding (Fig. 5). On C.4
cells transfected with vector alone, galectin-1 binding resulted in the
segregation of CD45 to membrane blebs on dying cells, exactly as
previously described (24). In contrast, galectin-1 binding to SNA.9
cells did not result in any detectable segregation of CD45. The diffuse distribution of CD45 on the cell surface was identical for SNA.9 cells
treated with either galectin-1 or buffer control. Thus, expression of
the ST6Gal I inhibited galectin-1-induced CD45 segregation on the
plasma membrane (Fig. 5A), as well as inhibiting
galectin-1-induced cell death (Fig. 2D). A comparison of the
effects of ST6Gal I expression on galectin-1-induced CD45 segregation
and on galectin-1-induced cell death is shown in Fig.
5B.
ST6Gal I Expression Abrogates Galectin-1-mediated Inhibition of PTP
Activity--
Previous work has demonstrated that binding of
galectin-1 to CD45 reduces the PTP activity of CD45 (25, 26). We asked whether ST6Gal I expression would modify the galectin-1-mediated effect
on PTP activity. In human cell lines, the galectin-1 effect on
immunoprecipitated CD45 has been examined (25, 26). However, because
all of the murine CD45 antibodies that we tested would not bind CD45 in
the presence of galectin-1, we measured the PTP activity of whole cell
lysates. The PhaR2.1 cell line, the parental line of the
SNA.1, SNA.9, and C.2 cells, demonstrated robust PTP activity (Fig.
6A). In contrast, the
T200
To assess the effect of ST6Gal I expression on PTP activity, we
examined the SNA.9 and C.2 cells at the indicated time points after
galectin-1 binding. As shown in Fig. 6B, galectin-1 binding to C.2 control cells resulted in a rapid and sustained decrease in PTP
activity (open circles). However, this effect was not seen when galectin-1 was added to SNA.9 cells (closed circles);
the PTP activity in lysates of SNA.9 cells treated with galectin-1 did
not differ appreciably from that observed for cells treated with buffer
alone (100%). All of the measurable p-nitrophenol release
was due to tyrosine phosphatase activity, because release was
completely inhibited by the addition of bpV (phen), a tyrosine phosphatase inhibitor (10).
Regulated expression of glycosyltransferases affects many cell
fate decisions. Altered glycosylation can directly modulate cellular
responses by creating or masking ligands for endogenous lectins. For
example, expression of specific glycosyltransferases creates potential
selectin ligands on peripheral T cells migrating to sites of
inflammation (3). Altered glycosylation can also indirectly modulate
cellular responses by affecting glycoprotein conformation or by
controlling intermolecular interactions. Expression of the GnT V enzyme
controls the amplitude of the T cell response to antigen (6), and
sialylation of cell surface glycoproteins regulates binding of MHC
class I molecules to thymocytes (4, 5).
Previous work from our group demonstrated that O-glycans are
involved in galectin-1 induced cell death; specifically, addition of
core 2 O-glycans on CD45 was required for
galectin-1-mediated clustering of CD45, an initial step in the death
pathway (9, 10). The present work demonstrates that
N-glycans are also essential for galectin-1-induced cell
death, because treatment of murine and human T cells with the
mannosidase I inhibitor DMNJ, which blocks all complex
N-glycosylation, virtually abolished susceptibility to
galectin-1 (Fig. 1). The dramatic inhibition of cell death seen with
DMNJ treatment expands our previous work (16), demonstrating that
treatment of T cells with the mannosidase II inhibitor swainsonine, which prevents branching of N-glycans by the GnT V enzyme,
only partially inhibited galectin-1-induced cell death. Indeed, the murine PhaR2.1 cell line used in this study is highly
susceptible to galectin-1, although this cell line does not express the
GnT V (9). Thus, although the GnT V branch may augment galectin-1
susceptibility, other LacNAc sequences on N-glycans are
sufficient for galectin-1 binding to trigger the death signal.
In the T cell lines examined in this study, the preferred acceptor
substrate for the ST6Gal I was CD45. Preferred utilization of CD45 as
an acceptor substrate for the ST6Gal I is supported by our finding
that, despite expression of the ST6Gal I in the T200 Developmentally regulated changes in CD45 isoform expression may also
control recognition by or accessibility to the ST6Gal I during
glycoprotein synthesis. In human thymus, the SA Glycosylation of CD45 depends on a number of factors, including
lymphocyte subset and stage of maturation or activation. Differential glycosylation of CD45 is controlled in part by the repertoire of
glycosyltransferase enzymes expressed by the cell at each stage in T
cell development (11, 12, 32-35). Regulated expression of different
complements of glycosyltransferases during T cell maturation and
activation implies that different glycoforms of CD45 will interact with
different endogenous lectins, such as CD22, the cysteine-rich domain of
the mannose receptor, or galectin-1 (10, 27, 36).
Sialylation of CD45 has recently been shown to regulate
homodimerization of CD45 on the T cell surface (28). CD45
homodimerization is one mechanism to down-modulate the PTP activity of
the CD45 cytoplasmic domains, an effect that would reduce T cell
responsiveness to antigen. Although CD45 homodimerization has been
proposed to occur spontaneously (28), we and others have shown that
galectin-1 binding clusters CD45 and reduces PTP activity (Figs. 5 and
6 and Refs. 25 and 26), and the data presented here demonstrate that
this effect is negatively regulated by expression of the ST6Gal I and
sialylation of CD45. Galectin-1 clustering of cell surface receptors
has also been demonstrated to reduce T cell responsiveness to antigen
(37), suggesting that galectin-1 binding to CD45 and regulation of CD45
PTP may contribute to the observed anti-inflammatory properties of
galectin-1 in a number of animal models (reviewed in Ref. 31). The
addition of SA How does ST6Gal I expression inhibit galectin-1-induced cell death? One
possibility is that galectin-1 binds to LacNAc sequences on CD45
glycans; the addition of We are beginning to elucidate the critical roles played by specific
glycosyltransferases in lymphocyte development and function. C2GnT
transgenic mice demonstrated reduced T cell responses to antigen (2),
whereas GnT V null mice demonstrated increased T cell responses to
antigen (6). In ST3Gal I null mice, Marth and co-workers (7) found
increased apoptosis of peripheral CD8 cells. This group also found
profound defects in B cell function in ST6Gal I mice, although no
defects in T cell development or function were reported (38). However,
it is likely that complex interactions of glycosyltransferases may
govern thymocyte susceptibility to galectin-1. For example, in C2GnT
transgenic mice, we found increased susceptibility of immature
double-positive thymocytes to galectin-1 but no increase in galectin-1
susceptibility of mature, single positive thymocytes (9). Based on our
prior observation that medullary thymocytes expressed the SA It is increasingly apparent that post-translational modifications such
as glycosylation are essential in regulating cellular signaling in the
immune system, and that glycosylation is dynamically regulated during
immune system activation (39). Understanding the functions of specific
glycans, identifying the enzymes required to create or modify the
glycans and characterizing the glycoprotein substrates that bear the
glycans will provide new approaches to controlling lymphocyte
development and survival.
2,6-linked sialic acid to
the Gal
1,4GlcNAc sequence, the preferred ligand for galectin-1,
inhibits recognition of this saccharide ligand by galectin-1.
SA
2,6Gal sequences, created by the ST6Gal I enzyme, are present on
medullary thymocytes resistant to galectin-1-induced death but not on
galectin-1-susceptible cortical thymocytes. To determine whether
addition of
2,6-linked sialic acid to lactosamine sequences on T
cell glycoproteins inhibits galectin-1 death, we expressed the ST6Gal I
enzyme in a galectin-1-sensitive murine T cell line. ST6Gal I
expression reduced galectin-1 binding to the cells and reduced
susceptibility of the cells to galectin-1-induced cell death. Because
the ST6Gal I preferentially utilizes N-glycans as acceptor
substrates, we determined that N-glycans are essential for
galectin-1-induced T cell death. Expression of the ST6Gal I
specifically resulted in increased sialylation of N-glycans on CD45, a receptor tyrosine phosphatase that is a T cell receptor for
galectin-1. ST6Gal I expression abrogated the reduction in CD45
tyrosine phosphatase activity that results from galectin-1 binding.
Sialylation of CD45 by the ST6Gal I also prevented galectin-1-induced clustering of CD45 on the T cell surface, an initial step in galectin-1 cell death. Thus, regulation of glycoprotein sialylation may control susceptibility to cell death at specific points during T cell development and peripheral activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,6Gal sequence, the product of the ST6Gal I
sialyltransferase, is detected only on mature medullary thymocytes (12). Intriguingly, mature medullary thymocytes displaying SA
2,6Gal sequences are resistant to galectin-1-induced cell death (13, 14).
Because the addition of sialic acid in the
2,6 linkage to galactose
could mask terminal galactose residues required for galectin-1 binding
to T cell glycoproteins (15), we asked whether expression of the ST6Gal
I would control susceptibility of T cells to galectin-1-induced death.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and human CEM and MOLT-4 cell lines were propagated
as previously described (10, 16).
cells as described (9).
Following selection in G418, positive PhaR2.1 clones were
identified by SNA flow cytometry. Positive T200
clones
were identified by RT-PCR, performed essentially according to the
protocol provided in the Super ScriptTM One-Step RT-PCR
with Platinum Taq (Invitrogen, Carlsbad, CA), using
the primers 94 sense (TATGAGGCCCCTTACACTG) and 943A antisense (GCCGGAGGATGGGGGATTTGG) (18).
-lactose (final concentration) was added to dissociate galectin-1, and the cells were washed with PBS. Apoptotic cells were
identified using annexin V and propidium iodide as previously described
(10).
(0.25 µg/300 µl cell lysate) (Pharmingen, San
Diego, CA) and ImmunoPure Immobilized Protein G (Pierce), and CD45 was
precipitated with monoclonal antibody 30-F11 (3 µg/300 µl cell
lysate) (Pharmingen) and ImmunoPure Immobilized Protein G overnight.
-D-sialic acid (Calbiochem, San Diego, CA) and 500 µg of asialofetuin (Sigma) in 50 mM sodium cacodylate, pH
6.5, 1 mM MgCl2, for 2 h at 37 °C. To
stop the reaction, the mixtures were incubated for 10 min on ice.
Fetuin was precipitated with anti-fetuin antibody (Accurate Chemical Co., Westbury, NY) (5 µl/150 µl lysate), and immunoprecipitates were separated by SDS-PAGE, blotted to nitrocellulose, and probed with
SNA-biotin, as described above. Band intensity was determined using the
MultiImage Light Cabinet, model 2.1.1 (Alpha Innotech Corp., San
Leandro, CA) with ChemiImager 5500 software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,4GlcNAc (LacNAc) sequences that can
be presented on N- or O-linked glycans (15). Although prior work from our lab demonstrated that O-glycans
participate in galectin-1 T cell death (9), the role of
N-glycans in galectin-1 cell death is not clear. In
addition, the ST6Gal I enzyme preferentially sialylates terminal
galactose residues on N-glycans (12, 17); if the ST6Gal I
participated in regulating galectin-1 cell death in vivo, it
would likely occur through the modification of
N-glycans.
-GalNAc and
swainsonine reduced T cell susceptibility to galectin-1 (9, 16),
neither benzyl-
-GalNAc nor swainsonine had the dramatic inhibitory
effect on cell death that we observed with DNMJ treatment. These
results demonstrated that N-linked glycans are essential for
galectin-1-mediated T cell death.
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Fig. 1.
N-Glycans are essential for
galectin-1 induced death of murine (A) and human
(B) T cells. The mannosidase II inhibitor DMNJ
prevents mannose trimming and subsequent lactosamine elongation on
N-glycans. The cells were cultured in 2 mM DMNJ
(shaded bars) or in medium alone (open bars) for
72 h prior to galectin-1 death assays. Loss of
N-glycans was confirmed by PHA flow cytometry (not shown).
Cell death was detected as described under "Experimental
Procedures." In A, the PhaR2.1 murine cell
line is highly susceptible to galectin-1 prior to DMNJ treatment,
whereas the BW5147 cell line is minimally susceptible to galectin-1
death. In B, both MOLT-4 and CEM cell lines are susceptible
to galectin-1 prior to DMNJ treatment. In all cell lines that are
susceptible to galectin-1, death is abrogated by DMNJ treatment.
2,6-linked sialic acid can
block galectin-1 binding to the preferred saccharide ligand LacNAc.
This has been demonstrated for individual LacNAc units and for
poly-LacNAc chains (15, 20-22). The ability of a terminal SA
2,6Gal
sequence to block galectin-1 binding suggested that the addition of
2,6-linked sialic acid to T cell surface glycoproteins (12) could
regulate the susceptibility of thymocytes and T cells to
galectin-1.
2,6-linked sialic acid would
affect susceptibility to galectin-1, we expressed the ST6Gal I in the
galectin-1-susceptible murine T cell line PhaR2.1. The
plant lectin SNA recognizes the SA
2,6Gal sequence (12, 23). We used
SNA binding, detected by flow cytometry, to screen for clones
expressing the ST6Gal I. Two SNA+ clones, SNA.1 and SNA.9,
demonstrated increased SNA binding compared with a control clone
transfected with vector alone (C.2) (Fig. 2A). Both RT-PCR and
immunoblot analysis with anti-ST6Gal I serum demonstrated abundant
expression of ST6Gal I mRNA and protein in SNA.9 cells (Fig.
2B) and SNA.1 cells (data not shown), whereas no reactivity
was observed in control C.2 cells (Fig. 2B). ST6Gal I
expression did not affect the level of expression of galectin-1 receptors CD43 or CD45 on the SNA.1 and SNA.9 cells, determined by flow
cytometric analysis, nor the level of CD7 expression, detected by
immunoblotting (data not shown).
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Fig. 2.
Expression of the ST6Gal I in
PhaR2.1 cells confers resistance to galectin-1-induced
death. A, PhaR2.1 cells were transfected
with the cDNA encoding the ST6Gal I or with vector alone.
Expression of the ST6Gal I in isolated clones was confirmed by flow
cytometry with SNA, a lectin that recognizes the SA 2,6Gal sequence
created by the ST6Gal I. The SNA.1 and SNA.9 clones expressing the
ST6Gal I demonstrate increased SNA binding compared with the control
C.2 clone transfected with vector alone. B, RT-PCR
(top panel) and immunoblotting (bottom panel)
demonstrate expression of the ST6Gal I mRNA and protein in SNA.9
cells but not in control C.2 cells. C, expression of the
ST6Gal I reduces galectin-1 binding to the SNA.9 cells (filled
symbols) compared with the control C.2 cells (open
symbols). The cells were labeled with the indicated amounts of
biotinylated galectin-1 in the absence (circles) or presence
(squares) of 100 mM lactose, and bound
galectin-1 was detected with fluorescein isothiocyanate-avidin. The
cells were analyzed by flow cytometry, and the mean fluorescence
channel of each sample is shown. D, the SNA.1 and SNA.9
clones demonstrate reduced susceptibility to galectin-1-induced cell
death, compared with the control C.2 cells. Death assays were performed
as described under "Experimental Procedures," and the percentage of
cell death was determined by binding of annexin V.
2,6Gal
sequences on cell surface glycoproteins, reduced susceptibility to
galectin-1-induced T cell death and suggested that sialylation of
specific glycoproteins was responsible for resistance to
galectin-1-induced cell death.
2,6Gal sequences on both murine and
human T cells (12, 24).
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Fig. 3.
The ST6Gal I preferentially sialylates
N-glycans on CD45. A, total
SNA-binding glycoproteins were precipitated from control clones
transfected with vector alone (lanes C.2 and C.4)
or from the SNA.1 clone expressing the ST6Gal I. Precipitated
glycoproteins were probed with biotinylated SNA. The only significant
difference in the profile of SNA binding glycoproteins was an increase
in a band with a mass of ~200 kDa (arrow). B,
the SNA reactive band is CD45. The cells were cultured in 2 mM DMNJ, as above, or in medium alone. The cell lysates
were precipitated with CD45 antibody or SNA (indicated below) and
probed with CD45 antibody. The band with increased SNA staining reacts
with both SNA and antibody to CD45. In addition, the increased SNA
binding to CD45 is abolished by pretreatment with DMNJ, which blocks
synthesis of complex N-glycans. In both blots, the width of
the CD45 band is diminished in DMNJ-treated cells compared with cells
expressing the ST6Gal I, as a result of decreased complexity of
glycosylation. C, increased SNA binding to CD45 results from
sialylation of N-glycans. CD45 was detected in whole cell
lysates of SNA.9 cells by immunoblotting (top panel). The
CD45 bands were excised and incubated with or without PNGase F, as
indicated, and reprobed with SNA-biotin. Removal of
N-glycans from CD45 by PNGase F treatment reduced SNA
binding.
2,6-linked sialic acid to N-glycans on CD45. The
background level of binding of SNA to CD45 on control cells and on
DMNJ-treated cells may reflect SNA recognition of SA
2,6GalNAc
sequences on O-glycans on CD45 (23).
cell line, a
mutant of the BW5147 line that does not express CD45. Despite repeated
attempts, we could not isolate SNA+ clones from
T200
cells transfected with ST6Gal I cDNA (SNA.T1),
nor could we detect any increase in SNA binding to whole cell lysates
of SNA.T1 cells (Fig. 4, A and
B). Although RT-PCR analysis demonstrated that the ST6Gal I
mRNA was present in nine independent clones of ST6Gal I transfected
T200
cells (Fig. 4C), every clone was
SNA
by flow cytometry (Fig. 4A). In addition,
we detected ST6Gal I protein by immunoblotting in the ST6Gal I
transfected T200
cells (Fig. 4C), although the
cells were SNA
. Finally, to confirm that the ST6Gal I
expressed in T200
cells was enzymatically active, we used
asialofetuin as an acceptor substrate to assay sialyltransferase
activity. After incubation with cell lysate from either control cells
(C.T1) or SNA.T1 cells, fetuin was immunoprecipitated and subjected to
blotting with SNA-biotin to detect
2,6-linked sialic acid. As shown
in Fig. 4C, there was a significant increase in SNA binding
to fetuin incubated with SNA.T1 extract, compared with C.T1 extract. We
performed densitometric analysis of the SNA reactive bands; the ratio
of SNA binding to fetuin incubated with SNA.T1 cell extract compared with C.T1 cell extract was 6.3. This ratio was comparable with the
ratio we observed when asialofetuin was incubated with SNA.9 cell
extract compared with C.4 cell extract, 5.0 (data not shown). These
data demonstrated that equivalent ST6Gal I activity was present in the
SNA.9 and SNA.T1 cells, although only the SNA.9 cells that express CD45
became SNA+ by flow cytometry. Thus, CD45 is the primary
glycoprotein acceptor substrate for the ST6Gal I in these T cells, and
in the absence of CD45, there was no detectable sialylation of other
potential acceptors by the ST6Gal I in T200
cells.
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Fig. 4.
A, expression of the ST6Gal I in the
T200 cell line, which does not express CD45, did not
result in increased SNA binding. Nine clones expressing ST6Gal I
mRNA were examined, but none demonstrated increased SNA binding by
flow cytometry; clone SNA.T1 is shown for example. C.T1 is one of nine
control clones transfected with vector alone. B, SNA
blotting of whole cell extracts of C.T1 and SNA.T1 cells did not
demonstrate any differences in staining between the two clones.
C, T200
clones express ST6Gal I mRNA and
protein. RT-PCR and immunoblot analysis of nine clones demonstrated
ST6Gal I expression, as shown for the SNA.T1 clone, with no ST6Gal I
expression in any of the controls, as shown for the C.T1 clone. The
samples are representative of all 18 clones examined. The expressed
protein is enzymatically active, as demonstrated by the addition of
sialic acid to asialofetuin. Asialofetuin was incubated with lysates of
C.T1 or SNA.T1 cells and precipitated with anti-fetuin, and
2,6-linked sialic acid was detected by SNA blotting. Weak SNA
reactivity of fetuin incubated with extract of C.T1 cells may reflect
the addition of
2,6-linked sialic acid to O-glycans,
because no SNA reactivity was detected with the asialofetuin acceptor
substrate alone (not shown). Densitometric analysis of the SNA-binding
bands was performed; the ratio of SNA binding to fetuin incubated with
SNA.T1 extract compared with C.T1 extract was 6.3:1.
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Fig. 5.
ST6Gal I expression inhibits
galectin-1-induced segregation of CD45. C.4 or SNA.9 cells were
treated with galectin-1 or buffer control and fixed, and cell surface
CD45 was detected by immunofluorescence. A, cells were
analyzed by confocal microscopy to detect CD45 segregation.
B, the percentage of cells demonstrating segregation of CD45
was scored by counting ~50 cells in six fields. The cells were
treated with buffer control (open bars) or galectin-1
(shaded bars). The SNA.9 cells demonstrated a marked
reduction in CD45 segregation, compared with CD45 segregation in
control C.4 cells. The percentage of cell death for a parallel sample
is indicated by the numbers above each
bar.
cell line derived from the same precursor line as
the PhaR2.1 cells does not express CD45 and has
significantly reduced PTP activity (Fig. 6A). These results
indicate that CD45 accounts for the majority of PTP activity in the
PhaR2.1 cells.
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Fig. 6.
ST6Gal I expression inhibits
galectin-1-induced modulation of CD45 protein-tyrosine phosphatase
activity. A, CD45 is the major PTP in
PhaR2.1 cells. Whole cell lysates of the CD45+
parental cell line, PhaR2.1, and the CD45
derivative T200
, were assayed for PTP activity in the
presence (solid bar) or absence (open bar) of the
PTP inhibitor bp V (phen). PTP activity was measured by the release of
p-nitrophenol, detected at 415 nm. B, ST6Gal I
expression abrogates the decrease in PTP activity triggered by binding
of galectin-1. C.2 cells (open symbols) and SNA.9 cells
(closed symbols) were incubated with 30 µg of galectin-1
for the indicated times at 37 °C. At the indicated times, the cells
were lysed, and PTP activity in whole cell lysates was measured as
described under "Experimental Procedures," in the presence
(squares) or absence (circles) of the PTP
inhibitor bpV (phen). C.2 cells demonstrate a 40% reduction in PTP
activity 1 min after galectin-1 binding. SNA.9 cells demonstrate no
change in PTP activity after galectin-1 binding.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cell
line that lacks CD45, we detected no increase in SNA binding to these
cells. Increased SNA binding to the other major galectin-1 receptors,
CD43 or CD7, was not detected in the PhaR2.1 or the
T200
cell lines. CD45 may be a preferred substrate
because of accessibility of CD45 glycans to the ST6Gal I during
synthesis or to recognition of peptide or conformational determinants
on the CD45 backbone by the ST6Gal I enzyme.
2,6Gal sequence was
only detected on the CD45RA isoform on mature thymocytes (12). In
murine thymus, CD45 on mature thymocytes also appears to be a preferred
acceptor for the ST6Gal I, because only mature thymocytes bound CD22, a
lectin that preferentially recognizes SA
2,6Gal (27). Recent work by
Xu and Weiss (28) has also demonstrated preferential sialylation of
high molecular weight isoforms of CD45, compared with the smallest
CD45RO isoform. Few examples of this degree of preferential acceptor
substrate recognition by sialyltransferases in vivo have
been reported. For example, polysialyltransferase enzymes are expressed
in a range of tissues, but polysialic acid is detected primarily on the
neural cell adhesion molecule NCAM (29, 30). Thus, tissue specificity
in both glycosyltransferase expression and in glycoprotein acceptor
substrate expression can control cell surface glycosylation. Because
galectin-1 is abundantly expressed throughout a variety of tissues, T
cells will encounter galectin-1 in many organs and at many points
during T cell development and peripheral activation. Thus, it is likely
that the T cell response to galectin-1 will be controlled at the level
of the T cell, i.e. by regulating glycosylation to control
susceptibility to cell death (31).
2,6Gal sequences to CD45 may be a mechanism to finely
tune immune regulation by galectin-1 and to prevent galectin-1-induced
apoptosis of specific populations, e.g. mature thymocytes.
2,6-linked sialic acid directly masks
LacNAc sequences, inhibiting galectin-1 binding to and clustering of
CD45 and initiation of cell death. Alternatively, the addition of
sialic acid to CD45 would also impart additional negative charge. Galectin-1 may bind to other LacNAc sequences on CD45 that are not
modified by sialic acid addition, but charge repulsion could prevent
close packing of CD45 required to initiate cell death. On the cell
surface, both direct masking of galectin-1 ligands on CD45 glycans and
increased charge repulsion among CD45 molecules may contribute to
inhibition of galectin-1-induced clustering, reduced PTP modulation,
and resistance to death.
2,6Gal sequence, we suggested that sialylation of single positive thymocytes could protect those cells from death.
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ACKNOWLEDGEMENTS |
---|
We thank Mabel Pang, Ivonne Diaz, and Karen Pace for advice and assistance; B. Kirk Allen and James C. Paulson for suggestions regarding sialyltransferase assays; Ingrid Schmid, Nathan Regimbal, and Steve Carbonne of the Jonsson Cancer Center Flow Cytometry Core Laboratory; Karen Colley, Michael Pierce, and Richard Hyman for reagents and cell lines; Karen Colley and Jamey Marth for helpful comments; and Leland Powell for valuable advice.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM63281 (to L. G. B.) and CA16042 (to the Jonsson Comprehensive Cancer Center).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported in part by a fellowship from the Lymphoma Research Foundation of America.
¶ Supported in part by a fellowship from the Japan Society for the Promotion of Science.
** To whom correspondence should be addressed. Tel.: 310-206-5985; Fax: 310-206-0657; E-mail: lbaum@mednet.ucla.edu.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M209595200
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ABBREVIATIONS |
---|
The abbreviations used are:
PHA, P.
vulgaris agglutinin;
DMNJ, deoxymannojirimycin;
LacNAc, lactosamine Gal1,4GlcNAc;
SNA, S. nigra agglutinin;
RT, reverse transcription;
PBS, phosphate-buffered saline;
PTP, protein-tyrosine phosphatase;
PNGase, peptide:
N-glycosidase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Lowe, J. B. (2001) Cell 104, 809-812[Medline] [Order article via Infotrieve] |
2. |
Tsuboi, S.,
and Fukuda, M.
(1997)
EMBO J.
16,
6364-6373 |
3. |
Blander, J. M.,
Visintin, I.,
Janeway, C. A., Jr.,
and Medzhitov, R.
(1999)
J. Immunol.
163,
3746-3752 |
4. | Moody, A. M., Chui, D., Reche, P. A., Priatel, J. J., Marth, J. D., and Reinherz, E. L. (2001) Cell 107, 501-512[Medline] [Order article via Infotrieve] |
5. | Daniels, M. A., Devine, L., Miller, J. D., Moser, J. M., Lukacher, A. E., Altman, J. D., Kavathas, P., Hogquist, K. A., and Jameson, S. C. (2001) Immunity 15, 1051-1061[Medline] [Order article via Infotrieve] |
6. | Demetriou, M., Granovsky, M., Quaggin, S., and Dennis, J. W. (2001) Nature 409, 733-739[CrossRef][Medline] [Order article via Infotrieve] |
7. | Priatel, J. J., Chui, D., Hiraoka, N., Simmons, C. J., Richardson, K. B., Page, D. M., Fukuda, M., Varki, N. M., and Marth, J. D. (2000) Immunity 12, 273-283[Medline] [Order article via Infotrieve] |
8. |
Kelm, S.,
Gerlach, J.,
Brossmer, R.,
Danzer, C. P.,
and Nitschke, L.
(2002)
J. Exp. Med.
195,
1207-1213 |
9. |
Galvan, M.,
Tsuboi, S.,
Fukuda, M.,
and Baum, L. G.
(2000)
J. Biol. Chem.
275,
16730-16737 |
10. |
Nguyen, J. T.,
Evans, D. P.,
Galvan, M.,
Pace, K. E.,
Leitenberg, D.,
Bui, T. N.,
and Baum, L. G.
(2001)
J. Immunol.
167,
5697-5707 |
11. | Baum, L. G. (2002) Immunity 16, 5-8[Medline] [Order article via Infotrieve] |
12. |
Baum, L. G,
Derbin, K.,
Perillo, N. L.,
Pang, M., Wu, T.,
and Uittenbogaart, C.
(1996)
J. Biol. Chem.
271,
10793-10799 |
13. |
Perillo, N. L.,
Uittenbogaart, C.,
Nguyen, J.,
and Baum, L. G.
(1997)
J. Exp. Med.
185,
1851-1858 |
14. |
Vespa, G. N. R.,
Lewis, L. A.,
Kozak, K. R.,
Moran, M.,
Nguyen, J. T.,
Baum, L. G.,
and Miceli, M. C.
(1999)
J. Immunol.
162,
799-806 |
15. |
Di Virgilio, S.,
Glushka, J.,
Moremen, K.,
and Pierce, M.
(1999)
Glycobiology
9,
353-364 |
16. | Perillo, N. L., Pace, K. E., Seilhamer, J. J., and Baum, L. G. (1995) Nature 378, 736-739[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Weinstein, J.,
de Souza-e-Silva, U.,
and Paulson, J. C.
(1982)
J. Biol. Chem.
257,
13845-13853 |
18. |
Ma, J.,
Qian, R.,
Rausa, F. M., III,
and Colley, K. J.
(1997)
J. Biol. Chem.
272,
672-679 |
19. |
Cummings, R. D.,
Trowbridge, I. S.,
and Kornfeld, S.
(1982)
J. Biol. Chem.
257,
13421-13427 |
20. |
Sparrow, C. P.,
Leffler, H.,
and Barondes, S. H.
(1987)
J. Biol. Chem.
262,
7383-7390 |
21. |
Barondes, S. H.,
Cooper, D. N.,
Gitt, M. A.,
and Leffler, H.
(1994)
J. Biol. Chem.
269,
20807-20810 |
22. |
Merkle, R. K.,
and Cummings, R. D.
(1988)
J. Biol. Chem.
263,
16143-16149 |
23. |
Shibuya, N.,
Goldstein, I. J.,
Broekaert, W. F.,
Nsimba-Lubaki, M.,
Peeters, B.,
and Peumans, W. J.
(1987)
J. Biol. Chem.
262,
1596-1601 |
24. |
Pace, K. E.,
Lee, C.,
Stewart, P. L.,
and Baum, L. G.
(1999)
J. Immunol.
163,
3801-3811 |
25. | Walzel, H., Schulz, U., Neels, P., and Brock, J. (1999) Immunol. Lett. 67, 193-202[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Fouillit, M.,
Joubert-Caron, R.,
Poirier, F.,
Bourin, P.,
Monostori, E.,
Levi-Strauss, M.,
Raphael, M.,
Bladier, D.,
and Caron, M.
(2000)
Glycobiology
10,
413-419 |
27. | Sgroi, D., and Stamenkovic, I. (1994) Scand. J. Immunol. 39, 433-438[Medline] [Order article via Infotrieve] |
28. | Xu, Z., and Weiss, A. (2002) Nat. Immunol. 3, 764-771[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Franceschini, I.,
Angata, K.,
Ong, E.,
Hong, A.,
Doherty, P.,
and Fukuda, M.
(2001)
Glycobiology
11,
231-239 |
30. |
Close, B. E.,
Tao, K.,
and Colley, K. J.
(2000)
J. Biol. Chem.
275,
4484-4491 |
31. | Rabinovich, G. A., Baum, L. G., Tinari, N., Paganelli, R., Natoli, C., Liu, F.-T., and Iacobelli, S. (2002) Trends Immunol. 23, 313-320[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Gillespie, W.,
Paulson, J. C.,
Pang, M.,
Kelm, S.,
and Baum, L. G.
(1993)
J. Biol. Chem.
268,
3801-3804 |
33. |
Kaufmann, M.,
Blaser, C.,
Takashima, S.,
Schwartz-Albiez, R.,
Tsuji, S.,
and Pircher, H.
(1999)
Int. Immunol.
11,
731-738 |
34. | Ohta, T., Kitamura, K., Maizel, A. L., and Takeda, A. (1994) Biochem. Biophys. Res. Commun. 200, 1283-1289[CrossRef][Medline] [Order article via Infotrieve] |
35. | Whiteheart, S. W., McLenithan, J. C., and Hart, G. W. (1990) Cell. Immunol. 125, 337-353[Medline] [Order article via Infotrieve] |
36. |
Martinez-Pomares, L.,
Crocker, P. R., Da,
Silva, R.,
Holmes, N.,
Colominas, C.,
Rudd, P.,
Dwek, R.,
and Gordon, S.
(1999)
J. Biol. Chem.
274,
35211-35218 |
37. |
Chung, C. D.,
Patel, V. P.,
Moran, M.,
Lewis, L. A.,
and Miceli, M. C.
(2000)
J. Immunol.
165,
3722-3729 |
38. |
Hennet, T.,
Chui, D.,
Paulson, J. C.,
and Marth, J. D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
4504-4509 |
39. | Comelli, E. M., Amado, M., Head, S. R., and Paulson, J. C. (2002) Glycobiology 12, 650 |