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*
Department of Pathology and Laboratory Medicine, University of California, Los Angeles, School of Medicine, and
Department of Molecular and Medical Pharmacology and Crump Institute for Biological Imaging, University of California, Los Angeles, CA 90095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the thymus, galectin-1 is produced by thymic epithelial cells (5). Galectin-1 induces apoptosis of CD3- and CD3dim immature thymocytes, but not of mature CD3bright thymocytes (3). The phenotype of the susceptible thymocytes implies that galectin-1 may participate in both nonselection, i.e., death of cells that are not positively selected, as well as negative selection (3, 6, 7). In the periphery, galectin-1 appears to eliminate Ag-specific T cells; in animal models of myasthenia gravis (8) and experimental autoimmune encephalitis (9), administration of galectin-1 abrogated the autoimmune response, and Ag-specific T cells could not be isolated from the lymph nodes of treated animals (9).
Elucidation of the mechanism of galectin-1-induced apoptosis is critical for understanding the role of galectin-1 in immune development and in the immune response. We have previously determined that galectin-1-induced apoptosis occurs via a mechanism distinct from that used in Fas or CD3-mediated apoptosis (4). Moreover, we have shown that the dimeric form of galectin-1 is required for induction of apoptosis (4), implying that cross-linking of cell surface receptors is involved in transducing the death signal. In the present study, we demonstrate that the initial steps in galectin-1 signaling involve binding to and segregation of specific T cell surface glycoproteins into discrete membrane microdomains. The composition of these microdomains is very different from that observed during signaling through the TCR (10, 11, 12, 13, 14, 15, 16). In addition, while mammalian lectins are known to participate in cell adhesion (17) and in phagocytosis of microorganisms and apoptotic cells (18, 19), induction of T cell apoptosis represents one of the first signaling functions identified for a mammalian lectin.
The specificity of glycoprotein receptor binding and segregation during galectin-1 signaling raises the question of how galectin-1 can discriminate among potential glycoprotein receptors, because the disaccharide ligands preferentially recognized by galectin-1 (Galß1,3GlcNAc and Galß1,4GlcNAc,3 type I and II lactosamine, respectively) are found on all complex N-linked and many O-linked glycoproteins (20, 21). Our results demonstrate that galectin-1 can discriminate among T cell surface glycoproteins and specifically bind to three T cell surface receptors, CD45, CD43, and CD7. Galectin-1 binding to CD45, CD43, and CD7 results in novel interactions among these glycoproteins during apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human T lymphoblastoid cell lines MOLT-4, CEM, and ARR (gift
of Dr. Ken Weinberg, Los Angeles Children’s Hospital, Los Angeles, CA)
were maintained in RPMI 1640 medium (Life Technologies, Gaithersburg,
MD), 10% fetal bovine serum, and 10 mM HEPES. mAbs were obtained from
the following sources: DFT1 (CD43), 2B11 (CD45), LCA (CD45), UCHL1
(CD45R0), 4KB5 (CD45RA), PD7/26 (CD45RB), UCHT1 (CD3), MT310 (CD4),
DK25 (CD8), MHM24 (CD11a), DAK-G01 (mouse IgG1), and rabbit
anti-mouse IgG1 from Dako (Carpinteria, CA); BL5 (CD18) and J-173
(CD44) from AMAC (Westbrook, ME); LT27 (CD27), 2H10 (CD120a), and
4D1B10 (CD120b) from Caltag Laboratories (South San Francisco, CA);
FN50 (CD69), M-T701 (CD7), and DX2 (CD95) from PharMingen (San Diego,
CA); T1 (CD5) from Coulter (Miami, FL); Leu-28 (CD28) from Becton
Dickinson (San Jose, CA); LT7 (CD7) from Advanced Immunochemical (Long
Beach, CA); DFT1-biotin (biotin-labeled CD43) from Ancell (Bayport,
MN); FS-7 (CD95) from Upstate Biotechnology (Lake Placid, NY);
fluorescein-conjugated goat anti-mouse IgG1 and Texas
red-conjugated goat anti-mouse IgG2a from Southern Biotechnology
Associates (Birmingham, AL); 9.4 (CD45) from the American Type
Culture Collection (Manassas, VA); T305 (CD43), a gift from R. Fox
(Scripps Clinic and Research Foundation, La Jolla, CA); PA2.6 (MHC
I), a gift from A. Jewett and B. Bonavida (University of California,
Los Angeles, CA); and rabbit polyclonal galectin-1
anti-serum, a gift from Incyte Pharmaceuticals (Palo Alto, CA).
Saccharides were obtained from the following sources: ß-lactose,
-D(+) fucose, and D(+)-mannose from Sigma (St. Louis,
MO); and NeuNAc2–6Galß1–4GlcNAc and
NeuNAc2–3Galß1–4GlcNAc from Oxford Glycosystems
(Rosedale, NY).
Recombinant galectin-1
The competent Escherichia coli line BL21DE3 was transformed with the plasmid which encodes galectin-1, pT7IML-1 (gift from Incyte Pharmaceuticals), and recombinant galectin-1 was produced and isolated as previously described (3). Before use, galectin-1 was dialyzed into 10 mM phosphate buffer, 140 mM NaCl (pH 7.4) (PBS), and 4 mM DTT and stored at -20°C.
Saccharide inhibition assay
Soluble, recombinant, biotin-labeled galectin-1 was preincubated with a competitive saccharide diluted in PBS, 1% BSA, and 1.2 mM DTT in a 96-well U-bottomed plate for 30 min on ice. MOLT-4 cells were washed and added to the plate for 1 h on ice. The final concentrations of biotinylated galectin-1 was 0.7 µM and saccharide inhibitor was 1 mM. The figure of 1 mM was chosen because the IC50 value for lactose was determined to be 1 mM using this assay. After washing, cells were incubated with HRP-conjugated streptavidin (Zymed Laboratories, South San Francisco, CA) for 1 h on ice. Following washing, cell-associated peroxidase activity was detected using the colorimetric substrate o-phenylenediamine dihydrochloride (Sigma). The plates were read at 492 nm using a Bio-Rad (Richmond, CA) ELISA reader with data reduction software.
Ab inhibition assay
mAbs specific for T cell surface glycoproteins were diluted in ice cold PBS and 1% BSA (pH 7.4) (PBS-BSA) and incubated with 5 x 106 MOLT-4 cells in a 96-well U-bottomed plate for 30 min on ice. Following washing, biotin-labeled galectin-1 was added to the cells for a final concentration of 0.7 µM and incubated for 1 h on ice. Unbound galectin-1 was removed and the cells were incubated with HRP-conjugated streptavidin for 1 h on ice. Following washing, cell-associated peroxidase activity was detected as described above.
Galectin-1 affinity chromatography
Cell surface glycoproteins were biotin-labeled as previously described (22) and membrane proteins were isolated (23). Isolated membrane proteins were solubilized in 0.1% Triton X-100. Solubilized membrane proteins were applied to a galectin-1 affinity column that was prepared by coupling galectin-1 to cyanogen bromide activated Sepharose 4B (Sigma) with a final concentration of 10 mg galectin-1/ml Sepharose 4B (24). After washing with PBS, 0.1% Triton X-100, 0.02% sodium azide (wash buffer), bound proteins were eluted with 0.1 M ß-lactose in wash buffer. Samples were resolved by 10% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and Western blotted with HRP-conjugated streptavidin (Zymed Laboratories). Proteins were visualized using Enhanced Chemiluminescence (Amersham, Arlington Heights, IL).
Immunoprecipitation
Immunoprecipitations were performed as previously described, with minor modifications (25). Fractions constituting the peak of the eluate from the galectin-1 affinity column were pooled. Depending on the Ab used, the samples were precleared twice with either protein A-Sepharose or with protein A-Sepharose saturated with rabbit anti-mouse IgG1 (Sigma) for 2 h, with rotating at 4°C. The precleared supernatant was collected and incubated with 5 µg mAb, recognizing the T cell surface glycoprotein of interest, or 5 µg of an isotype matched control, overnight at 4°C. To capture the Ab-protein complexes, protein A-Sepharose or protein A-Sepharose saturated with rabbit anti-mouse IgG1 was added and incubated for 2 h at 4°C with rotating. The beads were collected by centrifugation, washed, and boiled in SDS-PAGE sample buffer for 5 min. The samples were separated by SDS-PAGE under reducing conditions, transferred to nitrocellulose, and Western blotted as described above.
Coimmunoprecipitation of galectin-1 counterreceptors
A total of 1 x 107 MOLT-4 cells were washed and treated with 20 µM galectin-1, 1.2 mM DTT, or 1.2 mM DTT alone for 10 min on ice and 20 min in a 37°C water bath and cooled on ice. The cells were solubilized with 0.1% digitonin in PBS with protease inhibitors (solubilization buffer) for 1 h on ice. Insoluble extract was removed by centrifugation at 1500 rpm for 10 min. The supernatants were precleared twice with protein A-Sepharose for 2 h at 4°C with gentle rocking and incubated with 25 µl of galectin-1 antiserum for 2 h at 4°C with gentle rocking. The beads were collected and washed three times with solubilization buffer. Proteins bound to galectin-1 were dissociated with 0.1 M ß-lactose/solubilization buffer and immunoprecipitated using Abs against the glycoproteins of interest as described above.
Confocal immunofluorescence microscopy
A total of 1 x 107 MOLT-4 cells or human thymocytes were incubated with 20 µM galectin-1 and 1.2 mM DTT, or 1.2 mM DTT alone as a control, for 10 min on ice followed by 20 min in a 37°C water bath to allow migration of counterreceptors on the cell surface. Cells were cooled to 4°C, and bound galectin-1 was dissociated with ice cold 0.1 M ß-lactose. Paraformaldehyde (2%) was added for 30 min on ice to fix the cells, and the reaction was quenched with 0.2 M glycine for 5 min on ice. The cells were stained by incubating for 1.5 h at 25°C with 15 µg/ml each of the indicated combination of two Abs: 9.4 (CD45, mouse IgG2a), DFT1 (CD43, mouse IgG1), DFT1-biotin (CD43, mouse IgG1-biotin conjugated), M-T701 (CD7, mouse IgG1), LT7 (CD7, mouse IgG2a), or UCHT1 (CD3, mouse IgG1). The cells were washed and incubated at 25°C for 1.5 h with the appropriate secondary reagents: fluorescein-conjugated goat anti-mouse IgG1, fluorescein-conjugated goat anti-mouse IgG2a, Texas red-conjugated goat anti-mouse IgG2a, fluorescein-conjugated streptavidin, Texas red conjugated-streptavidin, or goat anti-mouse IgG1. The cells were washed and, when required, a tertiary reagent, fluorescein-conjugated rabbit anti-goat IgG, was added for 1.5 h at 25°C. The cells were washed, dropped on glass microscope slides, and mounted using Prolong Anti-fade mounting media (Molecular Probes, Eugene, OR). The fluorescently labeled cells were analyzed using the x100 objective on a Leica (Deerfield, IL) CLSM confocal laser scanning microscope. The cells were scanned by dual excitation of fluorescein (green) and Texas red (red) fluorescence. Dual emission fluorescent images were collected in separate channels at 0.5 µm optical slices. The images were processed on a Sun Workstation using AVS (Advanced Visualization Systems, Waltham, MA) image processing software. Areas of red and green overlapping fluorescence were represented with a yellow signal. The confocal images were printed on a Fuji (Tokyo, Japan) Pictography 3000 printer.
Apoptosis assay by confocal immunofluorescence microscopy
A total of 1 x 107 MOLT-4 or CEM cells were treated with 20 µM galectin-1 and 1.2 mM DTT, or 1.2 mM DTT as a control or 0.5 µg/ml FS-7 (CD95, IgM) for 20 min at 37°C as described above. Before fixation, the cells were resuspended in 1 ml binding buffer A (10 mM HEPES (pH 7.4), 150 mM NaCl, 2.5 mM CaCl2, and 1 mM MgCl2) and 5 µg biotin-conjugated Annexin V (Calbiochem, Cambridge, MA) and incubated on ice for 10 min. Paraformaldehyde (2%) was added for 30 min on ice, followed by quenching with 0.2 M glycine and calcium-free PBS. All washes used calcium-free PBS to assure that any unbound Annexin V could not bind to the cells during subsequent steps because Annexin V binding requires calcium. The mAb 9.4 (CD45, mouse IgG2a) was added for a 1.5-h incubation, 25°C. The cells were washed and incubated with fluorescein-conjugated anti-biotin Ab (mouse IgG1) for 1.5 h at 25°C. Following washing, the cells were incubated for 1.5 h with Texas red-conjugated goat anti-mouse IgG2a. Following a wash with calcium-free PBS, the cells were dropped on a slide and mounted with Prolong Anti-fade mounting medium. The fluorescently labeled cells were processed and analyzed as described above.
Apoptosis assay by flow cytometry
A total of 1 x 107 MOLT-4 cells were treated with galectin-1 as described above and incubated at 37°C for indicated time points. Following incubation, the cells were washed with 0.1 M ß-lactose to dissociate cell aggregates. The samples were resuspended in 1 ml binding buffer B (10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2) containing 5 µg fluorescein-conjugated Annexin V and 2.5 x 10-4 µg propidium iodide (R&D Systems, Minneapolis, MN), and incubated on ice for 10 min. The samples were immediately analyzed by flow cytometry. Flow cytometry data was acquired using a Becton Dickinson FACScan and analyzed using CellQuest software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify candidate glycoprotein receptors that bind galectin-1
on the T cell surface, we screened a large panel of mAbs to identify
Abs that would block galectin-1 binding to MOLT-4 human T cells. A
limited screen had previously determined that Abs recognizing CD45 and
CD43 inhibited galectin-1 binding to T cells (4, 5). In
this experiment, we included Abs to adhesion and homing receptors,
developmentally regulated T cell surface molecules, costimulatory
molecules, and members of the TNF receptor family. Table I
demonstrates that Abs recognizing CD45,
CD43, CD8, CD7, CD4, and CD3 inhibited galectin-1 binding to MOLT-4
cells. Abs to the adhesion molecules CD44, CD11a, and CD18 did not
inhibit galectin-1 binding, nor did Abs to the TNF receptor family
members CD95, CD120a, CD120b, or CD154 (Table I).
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, A and C). Based
on the relative mobilities of the bands and the Ab inhibition data
described above (Table I
), the relevant Abs were used to
immunoprecipitate and identify the eluted glycoproteins.
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A). The 40-kDa band was
identified as CD7 by immunoprecipitation (Fig. 1B). The
45-kDa band was identified as actin by Western blotting (data not
shown); galectin-1 has previously been shown to bind actin
(26). Three minor bands of 28, 20, and 16 kDa were also
isolated. The size of these three bands corresponded to the size of
components of the CD3 complex (27), and an Ab to CD3
inhibited galectin-1 binding to MOLT-4 cells (Table I), suggesting that
these bands were the components of the CD3 complex. However, we could
not immunoprecipitate these bands from the galectin-1 column eluate
because the CD3 Ab did not recognize the dissociated complex. To
confirm that these bands represented the members of the CD3 complex,
galectin-1 binding proteins were isolated from another T cell line,
ARR, which does not express CD3 (data not shown). We compared the
pattern of bands isolated from the MOLT-4 and ARR cell lines. Fig. 1C demonstrates that the 28-, 20-, and 16-kDa bands were not
isolated from the CD3- ARR cells, indicating
that the low m.w. bands isolated from MOLT-4 cells were components of
the CD3 complex. Thus, all of the galectin-1 binding proteins isolated
from MOLT-4 cells were identified, using Abs that blocked galectin-1
binding to the cell surface.
Although an Ab to CD8 also inhibited galectin-1 binding (Table I), CD8
was not isolated by galectin-1 affinity chromatography. A possible
explanation for this discrepancy is that CD8 associates with CD45 on
the T cell surface (27), so that steric hindrance by the
CD8 Ab may have blocked galectin-1 binding to CD45 on the cell
surface.
Although six potential receptors were identified by Ab blocking
experiments, and five glycoproteins bound to a galectin-1 affinity
column, previous data demonstrated that all of these glycoproteins are
not required for galectin-1-induced apoptosis (3, 4). We
have demonstrated that the
CD3-CD4-CD8-
ARR cell line is susceptible to galectin-1 (4), as are
CD3- human thymocytes (3). Only
three galectin-1 binding proteins were isolated from ARR cells, CD45R0,
CD43, and CD7 (Fig. 1D). Because the ARR cell line is
susceptible to galectin-1, these data suggested that CD4, CD3, and CD8
are not involved in the death signal and, at most, CD45, CD43, and CD7
are directly involved in delivering the death signal following
galectin-1 binding.
Soluble glycoproteins may be more accessible to galectin-1 on an
affinity matrix than on the cell surface. Therefore, to identify the
glycoproteins that bound galectin-1 on the cell surface, we
coimmunoprecipitated galectin-1 and the candidate receptors that had
bound galectin-1 from MOLT-4 cells. Only CD45, CD43, and CD7
coimmunoprecipitated with galectin-1 (Fig. 1E), whereas CD3,
CD4 (Fig. 1E) and CD8 (data not shown) did not (Fig. 1E). Although Abs recognizing CD3 and CD4 inhibited
galectin-1 binding to the cell surface in the Ab inhibition assay,
these glycoproteins did not coimmunoprecipitate with galectin-1 from
the cell surface. As mentioned above for CD8, CD4 and CD3 also
associate with CD45 on the cell surface (28), so that
steric hindrance by Abs recognizing CD4 and CD3 may have blocked
galectin-1 binding to CD45. In addition, although CD4 and CD3 may have
oligosaccharide chains that are recognized by galectin-1 in solution,
these oligosaccharides may not be accessible to galectin-1 on cell
surface CD4 and CD3. Cell surface CD45 and CD43 are large, extended
molecules that may be more accessible to galectin-1 (29, 30). The coimmunoprecipitation experiment indicated that CD45,
CD43, and CD7 are the either the primary or the most accessible
glycoprotein receptors for galectin-1 on the cell surface, implying
that galectin-1 death involves one or more of these three
glycoproteins.
Receptor segregation on the cell surface following galectin-1 binding
The identification of three different glycoproteins that bound galectin-1 on the cell surface raised the question of how these glycoproteins interact to deliver the death signal. We previously determined that receptor cross-linking by dimeric galectin-1 was required to deliver the apoptotic signal (4). The different glycoproteins could be cross-linked via galectin-1 into a heterotypic complex, or galectin-1 could cross-link only identical glycoprotein receptors into a homotypic complex (31, 32). To examine the interaction of the glycoproteins following galectin-1 binding, cells were treated with galectin-1 for 20 min at 37°C. This length of time was chosen because we have previously demonstrated that the death signal is irreversible after this period (4), even if galectin-1 is removed from the cell surface (4). After 20 min, the cells were cooled to prevent further changes, galectin-1 was removed from the cell surface with lactose, and the cells were fixed. Because our results indicated that CD45, CD43, and CD7 were important for delivering the apoptotic signal, Abs to CD45, CD43, CD7 were added, and the cell surface localization of the glycoproteins was determined by confocal microscopy. In addition, although CD3 is not required for galectin-1-induced apoptosis, CD3 cross-linking augments galectin-1-induced death (3, 33), so the localization of CD3 was examined as well.
As shown in Fig. 2, galectin-1 binding
induced a dramatic redistribution of CD45 and CD43 on the surface of
MOLT-4 T cells. Before galectin-1 binding, there was a uniform
distribution of CD45 (red) and CD43 (green) on the cell surface, with
occasional colocalization (yellow) (Fig. 2A). On
galectin-1-treated cells, CD45 localized into one or two large islands
on the cell surface, virtually excluding CD43. CD43 aggregated into
multiple small clusters that segregated away from CD45 (Fig. 2). Thus,
the binding of galectin-1 to T cells induced redistribution and
segregation of CD45 and CD43, suggesting that this process was
important for inducing apoptosis.
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A demonstrates that, before
galectin-1 treatment, CD45 (red) and CD7 (green) were also uniformly
distributed over the cell surface, with some regions of colocalization
(yellow). Following galectin-1 treatment, CD45 again segregated into
one or two large islands on each cell, and CD7 aggregated into multiple
small clusters that segregated away from CD45. This pattern was
identical to that observed for CD45 and CD43. Indeed, before galectin-1
treatment, CD7 (green) colocalized with CD43 (red) (Fig. 3B). Following galectin-1 treatment, CD43 and CD7 remained
associated and moved into larger aggregates (Fig. 3B). These
data indicate that CD43 and CD7 may act as a complex during the
delivery of the galectin-1 death signal. It is important to note that
images of galectin-1-treated and control cells were collected by
confocal microscopy under exactly the same conditions, although the
staining of CD43 and CD7 appears brighter on the galectin-1-treated
cells. This is likely due to the movement of CD43 and CD7 into larger
and subsequently brighter clusters.
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Galectin-1 binding to thymocytes resulted in the same pattern of
receptor segregation that we have observed with MOLT-4 T cells. We
first examined the localization of CD3 and CD45 on both mature
CD3bright and immature
CD3dim thymocytes. The differences in
fluorescence intensity of CD3 on these two cell populations were
readily distinguishable by confocal microscopy. In addition, the number
of CD3bright cells determined by confocal
microscopy was approximately equivalent to the number of
CD3bright or single positive cells determined by
flow cyometry (3). On mature
CD3bright thymocytes, CD3 (green) and CD45 (red)
were colocalized and uniformly distributed over the cell surface before
galectin-1 binding (Fig. 4A).
On immature CD3dim thymocytes, the weak CD3
staining that was detectable was also distributed around the cell
surface and largely colocalized with CD45. A striking difference was
observed between CD3dim and
CD3bright thymocytes after galectin-1 treatment.
On the immature CD3dim cells, galectin-1 binding
resulted in the movement of all CD45 and CD3 into one or two islands on
the cell surface, exactly as we observed with MOLT-4 cells. However, on
CD3bright cells, the mature thymocyte subset that
is not susceptible to galectin-1 (3), no significant
movement of CD45 or CD3 was seen.
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B). Before
galectin-1 binding, immature CD3dim thymocytes
had a patchy distribution of CD3 and CD7 on the cell surface (bottom
two cells in field), while mature CD3bright
thymocytes showed an intense, uniform distribution of CD3 and small
clusters of CD7 (top cell in field). Following galectin-1 binding, CD3
and CD7 segregated on the immature CD3dim cells,
with the CD3 moving into one large island and the CD7 aggregating into
small clusters. However, on the mature CD3bright
cells, no significant movement of CD3 and CD7 was seen. These data
demonstrate that galectin-1 induces the same type of receptor
segregation on thymocytes as we observed with a human T cell line.
Importantly, this receptor segregation occurred only on the immature
CD3dim subset of thymocytes that we have
previously shown to be susceptible to galectin-1 (3)
and not on the mature CD3bright subset.
The absence of receptor segregation on mature thymocytes following
galectin-1 binding suggested that alterations in the saccharide profile
of these cells may have prevented galectin-1 binding. We have
demonstrated that CD45 on mature human thymocytes differs from CD45 on
immature human thymocytes by being terminally substituted with a
SA2,6Galß1,4GlcNAc sequence (34, 35). Previous
studies of galectin-1 binding specificity for soluble oligosaccharides
have shown that galectin-1 can bind lactosamine terminally substituted
with sialic acid in an 2,3 linkage, but not in an 2,6 linkage
(36); structural studies of galectin-1 revealed that
2,6-linked sialic acid would distort the binding pocket and prevent
binding of the lactosamine sequence (36). We examined
whether 2,6-linked sialic acid would prevent the binding of
galectin-1 to the T cell surface. Table II shows that 2,3 sialyllactose
(SA2,3Galß1,4GlcNAc) effectively competed for the binding of
galectin-1 to MOLT-4 cells, with a potency equivalent to lactose.
However 2,6 sialyllactose (SA2,6Galß1,4GlcNAc) failed to
compete for galectin-1 binding, suggesting that the presence of
2,6-linked sialic acid on CD45 of mature thymocytes would not allow
galectin-1 binding and may account for the resistance of these cells to
galectin-1.
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Although the images in Fig. 4 demonstrated that receptor
segregation occurred only on thymocyte subsets that are susceptible to
galectin-1, these results did not demonstrate that cell death occurred
during this process. To verify that cells that underwent receptor
redistribution and segregation also underwent apoptosis, we treated
MOLT-4 cells with galectin-1 for 20 min at 37°C and examined the
movement of CD45 and the binding of Annexin V, which binds to
externalized phosphatidylserine on apoptotic cells. By confocal
microscopy, 
70–80% of cells treated with galectin-1 at 37°C for
only 20 min bound Annexin V. Importantly, Annexin V bound only to
galectin-1-treated cells that demonstrated movement and patching of
CD45 (Fig. 5A).
Galectin-1-treated cells that did not display movement and patching of
CD45 did not bind Annexin V (Fig. 5A). We also examined the
localization of CD45 on CEM cells that were triggered to undergo
apoptosis by incubation with anti-Fas Ab for an identical time
period. CEM cells are susceptible to both Fas and galectin-1-induced
apoptosis, whereas MOLT-4 cells are not (4). As we
observed with MOLT-4 cells, galectin-1 treatment of CEM cells resulted
in the redistribution of CD45 into large patches on the surface of
apoptotic cells (Fig. 5B). In contrast, on anti-Fas
treated cells, CD45 remained uniformly distributed on the surface of
both apoptotic and nonapoptotic cells (Fig. 5C).
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A and B). These data revealed that movement of
phosphatidylserine to the external plasma membrane leaflet occurred in
discrete microdomains on the cell surface, rather than occurring
globally during the process of galectin-1-induced apoptosis. A similar
morphologic appearance of phosphatidylserine exposure on apoptotic
blebs has been observed to occur on HeLa cells induced to undergo
apoptosis by nutrient deprivation for 16 h (37). The
exposure of phosphatidylserine in discrete microdomains on apoptotic
blebs suggests that the scramblase, which has been shown to be
responsible for exposure of phosphatidylserine exposure on apoptotic
lymphocytes (38, 39), may be locally activated. Phosphatidylserine exposure is an early event in galectin-1-induced apoptosis
Annexin V binding was readily visible by confocal microscopy on
70–80% of cells that had been treated with galectin-1 for only 20
min at 37°C (Fig. 5, A and B). These findings
indicated that the local externalization of phosphatidylserine may be a
relatively early event in the process of galectin-1-induced apoptosis.
This period of time is very brief relative to the time required to
detect Annexin V binding by flow cytometry in apoptosis triggered by
other agents (40). However, a similar rapid time course of
phosphatidylserine exposure has been reported in cells that express
high levels of a phospholipid scramblase (39). To
determine the duration of galectin-1 treatment that was required to
detect phosphatidylserine exposure by flow cytometry, we performed a
kinetic analysis (Fig. 6). Incubation of
MOLT-4 cells with galectin-1 for only 20 min at 37°C resulted in
Annexin V binding to the majority of cells (Fig. 6), correlating with
Annexin V binding on 70–80% of cells by confocal microscopy. The
incremental shift in Annexin V binding intensity with increased
galectin-1 incubation time may be due to the initial exposure of
phosphatidylserine on discrete membrane microdomains, followed by a
more global or uniform exposure of phosphatidylserine at later time
points.
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A). These images
revealed that the islands of CD45 were localized on large membrane
blebs protruding from the cell surface. Little CD45 was visible on
other areas of the cell surface. The formation of membrane blebs is a
morphologic hallmark of apoptosis. Fluorescent microscopy of
galectin-1-treated cells also demonstrated that Annexin V bound to
blebs protruding from the cell surface, indicating that
phosphatidylserine exposure happened first on membrane blebs during
galectin-1-induced apoptosis (Fig. 7B). Thus, galectin-1
binding to T cells results in the rapid redistribution and segregation
of specific membrane glycoproteins, with a complex of CD45 and CD3
localized to the apoptotic blebs, and a complex of CD43 and CD7
localized in regions excluded from the apoptotic blebs. Moreover, we
have observed rapid colocalization of CD45 with externalized
phosphatidylserine during galectin-1-induced apoptosis, although it is
not yet clear if CD45 relocalization and phosphatidylserine exposure
occur in a specific temporal sequence.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown that cross-linking of galectin-1 receptors is essential for triggering galectin-1-induced apoptosis, as monomeric galectin-1 does not have apoptotic activity (4). In the present study, galectin-1 binding to human thymocytes resulted in the redistribution and segregation of CD45 together with CD3, and of CD43 together with CD7. These results demonstrated that galectin-1 cross-linking could separate a mixed population of glycoproteins on the cell surface into unique complexes in specific membrane microdomains. Brewer and coworkers (31, 32, 45, 46) have demonstrated that bovine galectin-1 can form ordered homotypic complexes composed of identical glycoproteins out of a mixture of glycoproteins in solution. The formation of homotypic complexes is thermodynamically driven, suggesting that the formation of such homotypic matrices may also occur on the surface of cells (31). In solution studies, the ability of galectin-1 cross-linking to form homotypic or heterotypic complexes of soluble glycoproteins relied solely on the number of oligosaccharide ligands per glycoprotein that could bind galectin-1, termed the valency of the glycoprotein. However, on the cell surface, the glycoprotein valency is not the only factor that will control the interaction and movement of galectin-1 receptors. The structure of the glycoprotein, protein-protein interactions of glycoproteins above and below the plasma membrane, and association with other cytoplasmic proteins will also determine how glycoproteins interact following galectin-1 binding.
As mentioned earlier, protein structure and conformation will contribute to the presentation of clustered lactosamine residues, promoting galectin-1 binding. In addition, protein structure will influence the ability of glycoproteins to pack into an ordered cross-linked lattice. In solution studies, the formation of lattice structures required identical repeating units of glycoproteins (31, 45, 47). The extended rod-like conformation of both CD43 and CD7, created by the high density of O-glycans on the peptide backbone (47, 48), may facilitate the packing of these two glycoproteins into a lattice on the cell surface.
Protein-protein interactions may also regulate the movement and
segregation of glycoproteins by galectin-1. Before galectin-1 binding,
CD45 and CD3 (Fig. 4A) and CD43 and CD7 (Fig. 3B)
were colocalized on the cell surface. The complex of CD45 and CD3 may
act as a single glycoprotein receptor, associating via protein-protein
interactions before galectin-1 cross-linking. Galectin-1 binding would
cross-link and segregate repeating units of CD45 associated with CD3
into a "homotypic" complex (Fig. 8).
The same may be true for CD43 and CD7 (Fig. 8). In contrast, CD7 and
CD3 co-localize before galectin-1 binding (Fig. 4B; Ref.
49). However, CD7 and CD3 separate into apparently more
stable complexes with CD43 and CD45, respectively, after galectin-1
binding. The same was seen for CD45 and CD7 (Fig. 3A), which
are known to associate on the T cell surface in the absence of
galectin-1 (50). These findings demonstrate that
galectin-1 binding results in selective maintenance of some
pre-existing glycoprotein associations and disruption of others.
Moreover, galectin-1 binding results in the association of
glycoproteins, such as CD3 and CD45, that are segregated during TCR
interactions with an Ag-MHC complex (10, 12). This
demonstrates that galectin-1 binding creates a unique set of membrane
microdomains distinct from those created during other T cell signaling
events (10, 11, 12, 13, 14, 15, 16).
|
). Cytoskeletal changes during death may
influence the movement and segregation of CD45 and CD43 after
galectin-1 binding. Alternatively, disruption of cytoskeletal integrity
resulting from the movement of CD45 and CD43 may be involved in
triggering galectin-1 death, as has been described for endothelial
cells (52). Alterations in both fodrin and radixin have
been noted in apoptotic cells (53, 54), suggesting that
these proteins may play a role in the apoptotic pathway mediated by
galectin-1. It is not yet clear how CD45, CD43, and CD7 are involved in galectin-1-induced apoptosis. Both CD45 and CD43 have been implicated in triggering T cell apoptosis (55, 56, 57). Ab cross-linking of a specific glycoform of CD43 resulted in apoptosis of the Jurkat cell line (55). This apoptosis-inducing Ab specifically recognized a GalNAc-Ser containing epitope (55); because this monosaccharide is not a ligand for galectin-1, the relationship between this Ab and galectin-1 in triggering cell death is not yet clear. We are currently determining the role of CD43 in galectin-1-induced apoptosis. Although Ab cross-linking of CD45 has been shown to induce a pro-apoptotic signal in human T cells (56, 57), CD45 has also been shown to act as a negative regulator of T cell apoptosis (58). This may be relevant to galectin-1-induced apoptosis. Walzel et al. (59) have demonstrated that galectin-1 treatment of Jurkat T cells resulted in decreased phosphatase activity of immunoprecipitated CD45. In addition, we have recently found that protein tyrosine phosphatase inhibitors that block CD45 protein tyrosine phosphatase activity enhanced galectin-1-induced T cell death (J. T. Nguyen and L. G. Baum, unpublished data). These data support a model in which the galectin-1-induced CD45 cross-linking and sequestration that we have observed would functionally remove and/or inhibit the protein tyrosine phosphatase domain of CD45 from a pro-apoptotic signaling complex. Because galectin-1 binding to T cells results in tyrosine phosphorylation (33), segregation of the CD45 phosphatase may allow a kinase-dependent signal to be transduced.
CD7 is an intriguing candidate for a role in galectin-1-induced apoptosis. Galectin-1 is the first ligand to be identified for CD7, although previous studies have suggested that the CD7 ligand may be a lectin-like molecule (60). CD7 associates with the PI3 kinase, and ligation of CD7 results in tyrosine phosphorylation (49), as does galectin-1 binding (33). Moreover, CD7 is highly expressed on human thymocytes and activated T cells, populations that are susceptible to galectin-1 (3, 4). We have found that a CD7- human T cell line, HUT78, is resistant to galectin-1-induced apoptosis (data not shown), suggesting that CD7 is a necessary component of the galectin-1 death pathway. The galectin-1 death pathway may depend on concomitant signals from CD45, CD43 and CD7 to trigger death, signals that may result directly from the redistribution and segregation of these glycoproteins induced by galectin-1 binding.
The segregation of CD45, CD3, CD43, and CD7 into discrete complexes on the T cell surface may represent a paradigm for a variety of complex signaling pathways that involve matrix formation of cell surface receptors, as has been shown to occur during T cell activation (10, 11, 12, 13, 14, 15, 16). In addition to inducing apoptosis, galectin-1 is capable of exerting a mitogenic or cytostatic effect on cells, depending on the type and functional state of the target cells (61, 62, 63, 64). Subtle changes in receptor expression, glycosylation, stoichiometry, or cross-linking may determine which of these various effects are exerted by galectin-1.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Linda G. Baum, Department of Pathology, University of California, Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address:
3 Abbreviations used in this paper: Galß1,3GlcNAc, type I lactosamine; Galß1,4GlcNAc, type II lactosamine; NeuNAc 2,3 Galß1,4GlcNAc, 2,3 sialyllactose; NeuNAc 2,6Galß1,4GlcNAc, 2,6 sialyllactose.
Received for publication April 15, 1999. Accepted for publication July 19, 1999.
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M. Lanteri, V. Giordanengo, N. Hiraoka, J.-G. Fuzibet, P. Auberger, M. Fukuda, L. G. Baum, and J.-C. Lefebvre Altered T cell surface glycosylation in HIV-1 infection results in increased susceptibility to galectin-1-induced cell death Glycobiology, December 1, 2003; 13(12): 909 - 918. [Abstract] [Full Text] [PDF]
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M. Amano, M. Galvan, J. He, and L. G. Baum The ST6Gal I Sialyltransferase Selectively Modifies N-Glycans on CD45 to Negatively Regulate Galectin-1-induced CD45 Clustering, Phosphatase Modulation, and T Cell Death J. Biol. Chem., February 21, 2003; 278(9): 7469 - 7475. [Abstract] [Full Text] [PDF]
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S. G. Correa, C. E. Sotomayor, M. P. Aoki, C. A. Maldonado, and G. A. Rabinovich Opposite effects of galectin-1 on alternative metabolic pathways of L-arginine in resident, inflammatory, and activated macrophages Glycobiology, February 1, 2003; 13(2): 119 - 128. [Abstract] [Full Text] [PDF]
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T. A. Baldwin and H. L. Ostergaard The Protein-tyrosine Phosphatase CD45 Reaches the Cell Surface via Golgi-dependent and -independent Pathways J. Biol. Chem., December 20, 2002; 277(52): 50333 - 50340. [Abstract] [Full Text] [PDF]
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J. D. Hernandez and L. G. Baum Ah, sweet mystery of death! Galectins and control of cell fate Glycobiology, October 1, 2002; 12(10): 127R - 136R. [Abstract] [Full Text] [PDF]
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 L. Gauthier, B. Rossi, F. Roux, E. Termine, and C. Schiff Galectin-1 is a stromal cell ligand of the pre-B cell receptor (BCR) implicated in synapse formation between pre-B and stromal cells and in pre-BCR triggering PNAS, October 1, 2002; 99(20): 13014 - 13019. [Abstract] [Full Text] [PDF]
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M. Fortin, A.-M. Steff, J. Felberg, I. Ding, B. Schraven, P. Johnson, and P. Hugo Apoptosis Mediated Through CD45 Is Independent of Its Phosphatase Activity and Association with Leukocyte Phosphatase-Associated Phosphoprotein J. Immunol., June 15, 2002; 168(12): 6084 - 6089. [Abstract] [Full Text] [PDF]
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F. Yu, R. L. Finley Jr., A. Raz, and H.-R. C. Kim Galectin-3 Translocates to the Perinuclear Membranes and Inhibits Cytochrome c Release from the Mitochondria. A ROLE FOR SYNEXIN IN GALECTIN-3 TRANSLOCATION J. Biol. Chem., May 3, 2002; 277(18): 15819 - 15827. [Abstract] [Full Text] [PDF]
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G. A. Rabinovich, N. Rubinstein, and L. Fainboim Unlocking the secrets of galectins: a challenge at the frontier of glyco-immunology J. Leukoc. Biol., May 1, 2002; 71(5): 741 - 752. [Abstract] [Full Text] [PDF]
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K. E. Pace, T. Lebestky, T. Hummel, P. Arnoux, K. Kwan, and L. G. Baum Characterization of a Novel Drosophila melanogaster Galectin. EXPRESSION IN DEVELOPING IMMUNE, NEURAL, AND MUSCLE TISSUES J. Biol. Chem., April 5, 2002; 277(15): 13091 - 13098. [Abstract] [Full Text] [PDF]
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L. Cermak, S. Simova, A. Pintzas, V. Horejsi, and L. Andera Molecular Mechanisms Involved in CD43-mediated Apoptosis of TF-1 Cells. ROLES OF TRANSCRIPTION, Daxx EXPRESSION, AND ADHESION MOLECULES J. Biol. Chem., March 1, 2002; 277(10): 7955 - 7961. [Abstract] [Full Text] [PDF]
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I. Kuwabara, Y. Kuwabara, R.-Y. Yang, M. Schuler, D. R. Green, B. L. Zuraw, D. K. Hsu, and F.-T. Liu Galectin-7 (PIG1) Exhibits Pro-apoptotic Function through JNK Activation and Mitochondrial Cytochrome c Release J. Biol. Chem., January 25, 2002; 277(5): 3487 - 3497. [Abstract] [Full Text] [PDF]
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J. T. Nguyen, D. P. Evans, M. Galvan, K. E. Pace, D. Leitenberg, T. N. Bui, and L. G. Baum CD45 Modulates Galectin-1-Induced T Cell Death: Regulation by Expression of Core 2 O-Glycans J. Immunol., November 15, 2001; 167(10): 5697 - 5707. [Abstract] [Full Text] [PDF]
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C. D. Chung, V. P. Patel, M. Moran, L. A. Lewis, and M. C. Miceli Galectin-1 Induces Partial TCR {zeta}-Chain Phosphorylation and Antagonizes Processive TCR Signal Transduction J. Immunol., October 1, 2000; 165(7): 3722 - 3729. [Abstract] [Full Text] [PDF]
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K. E. Pace, H. P. Hahn, M. Pang, J. T. Nguyen, and L. G. Baum Cutting Edge: CD7 Delivers a Pro-Apoptotic Signal During Galectin-1-Induced T Cell Death J. Immunol., September 1, 2000; 165(5): 2331 - 2334. [Abstract] [Full Text] [PDF]
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H. Sano, D. K. Hsu, L. Yu, J. R. Apgar, I. Kuwabara, T. Yamanaka, M. Hirashima, and F.-T. Liu Human Galectin-3 Is a Novel Chemoattractant for Monocytes and Macrophages J. Immunol., August 15, 2000; 165(4): 2156 - 2164. [Abstract] [Full Text] [PDF]
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M. Galvan, S. Tsuboi, M. Fukuda, and L. G. Baum Expression of a Specific Glycosyltransferase Enzyme Regulates T Cell Death Mediated by Galectin-1 J. Biol. Chem., May 26, 2000; 275(22): 16730 - 16737. [Abstract] [Full Text] [PDF]
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T. A. Baldwin, M. Gogela-Spehar, and H. L. Ostergaard Specific Isoforms of the Resident Endoplasmic Reticulum Protein Glucosidase II Associate with the CD45 Protein-tyrosine Phosphatase via a Lectin-like Interaction J. Biol. Chem., October 6, 2000; 275(41): 32071 - 32076. [Abstract] [Full Text] [PDF]
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K. Hotta, T. Funahashi, Y. Matsukawa, M. Takahashi, H. Nishizawa, K. Kishida, M. Matsuda, H. Kuriyama, S. Kihara, T. Nakamura, et al. Galectin-12, an Adipose-expressed Galectin-like Molecule Possessing Apoptosis-inducing Activity J. Biol. Chem., August 31, 2001; 276(36): 34089 - 34097. [Abstract] [Full Text] [PDF]
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