From the Departments of Pediatrics and Dermatology,
Children's Memorial Institute for Education and Research, Northwestern
University Medical School, Chicago, Illinois 60614 and the
§ Department of Tumor Immunology, Tokyo Metropolitan
Institute of Medical Science, Honkomagome,
Bunkyo-ku, Tokyo 113-8613, Japan
Received for publication, July 11, 2000, and in revised form, December 15, 2000
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ABSTRACT |
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Gangliosides GT1b and GD3, components of
keratinocyte membranes, inhibit keratinocyte adhesion to fibronectin.
Although ganglioside sialylation is known to be important, the
mechanism of inhibition is unknown. Using purified insect recombinant
Human keratinocyte motility on a fibronectin
(FN)1 matrix is critical for
the re-epithelialization of healing wounds, for the spread of cutaneous
malignancy, and in cutaneous embryogenesis. Although the molecular
events that influence this migration are poorly understood, the
interaction between keratinocyte Gangliosides are glycosphingolipids characterized by the presence of
one or more sialic acid moieties in the oligosaccharide chain (9). The
role of gangliosides, which are localized to the outer leaflet of the
plasma membrane of eukaryotic cells, is largely unknown, but studies
with cultured keratinocytes and keratinocyte-derived cells suggest that
gangliosides are involved in regulating cellular proliferation,
differentiation, and adhesion (10-13). The discovery that gangliosides
inhibit cell attachment and spreading on a FN matrix (14) led
investigators to consider gangliosides to be the cell receptors for FN
before integrin The possibility of a direct interaction between GT1b and
Cell Culture--
SCC12F2 (SCC12) cells, a generous gift from
James Rheinwald (Harvard Medical School, Boston, MA), were grown
in Dulbecco's modified Eagle's medium F-12 (1:1) supplemented
with 10% heat-inactivated fetal bovine serum (Life Technologies,
Inc.). Insect Sf9 cells were grown in TNM-FH serum-free
medium (Pharmingen, San Diego, CA), and Escherichia coli
cells were grown in LB medium (Life Technologies, Inc.).
Determination of Ganglioside Content in SCC12
Cells--
Gangliosides were extracted from SCC12 cell membranes using
chloroform/methanol as previously described (17). The aqueous phase was
separated and desalted, and the bands of gangliosides were separated by
thin-layer chromatography in chloroform/methanol/water with 0.02%
CaCl2 (55:45:10, v/v/v). Gangliosides were detected by
resorcinol staining. Band density was quantified by a Storm 800 fluorescence PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
The identity of the four bands of ganglioside was confirmed by
immunostaining with anti-GM3 antibody (courtesy of T. T.), anti-9-O-acetyl-GD3 antibody CDw60 (Sigma), anti-GD3
antibody R24 (Calbiochem), and anti-GT1b antibody (courtesy of T. T.). Binding was detected with horseradish peroxidase (HRP)-conjugated goat
anti-mouse IgG and enhanced chemiluminescence (ECL).
Construction of
The identity of the purified recombinant proteins was confirmed by
Western blotting. The purified recombinant proteins were boiled in
Laemmli sample buffer (19), and 10 ng/lane was applied to an 8%
SDS-polyacrylamide minigel. Protein was transferred to polyvinylidene
difluoride filters, and nonspecific binding was blocked by incubation
for 1 h with 5% dry milk in 50 mM Tris-HCl, pH 7.6, with 137 mM NaCl and 0.1% Tween 20. Lanes were treated with 50 ng/ml anti- Generation of Affinity-purified ELISA Binding Assays--
The ability of ganglioside to block
the binding of the Nickel-Agarose Binding Assay--
A novel assay, the NAB
technique, was used to assess whether the ability of GT1b to interfere
with integrin binding to FN specifically involves one of the integrin
subunits and to verify that the N terminus of integrin is critical.
This assay takes advantage of the fact that the His tag can bind to
nickel-agarose; thus, a His tag at the C-terminal end exposes the N
terminus with the integrin extracellular domains.
5 µg of purified insect recombinant Co-immunoprecipitation of Gangliosides with
Slot Blot Assays--
To further evaluate the ability of
gangliosides GT1b and GD3 to bind directly to
Effect of Ganglioside on the Expression of
The NAB technique was also used to investigate whether GT1b interferes
with the complex formation between Deglycosylation of Assessment of the Role of Glycosylation in the Ganglioside
Interaction with Integrin--
ELISAs were performed as described
above to examine the effect of gangliosides on the ability of
deglycosylated integrins and poorly glycosylated integrin from E. coli to bind to FN. In addition, bead binding assays and slot blot
assays were used to determine whether deglycosylation prevents the
direct binding of ganglioside to the integrin. Slot blot assays were
performed as described above, except that deglycosylated
In the bead binding assays, 50 µM GT1b or GD3 in PBS was
cross-linked to 250 µl of 0.2-µm amine-modified red
FluoSphere beads (1 × 105 particles/ml in PBS;
F-8763, Molecular Probes, Inc.) by mixing with 5 mg/ml EDAC in PBS
overnight at 4 °C. 50 µM GD1a, GM2, or GM3 in PBS or
PBS alone was coated onto FluoSphere beads as negative controls. To
detect the attachment of the ganglioside to the FluoSphere beads, 20 µl of GT1b-coated, GM3-coated, or uncoated red beads was washed and
loaded into the slots of a slot blot apparatus as described above. The
colored beads are easily visible under ultraviolet light as red bands.
The binding of gangliosides GT1b, GM2, and GM3 to beads was confirmed
by incubation with anti-GT1b, anti-GM2 (courtesy of Dr. P. Livingston,
Sloan-Kettering, New York, NY), and anti-GM3 monoclonal
antibodies, respectively, all applied at a ratio of 1:2 in PBS, and
detected by chemiluminescence as described above. Affinity-purified
Role of Specific Carbohydrate Moieties in the Interaction with
Ganglioside--
ELISAs were also used to examine the competitive
binding of concanavalin A (ConA) lectin, which recognizes mannose
residues, and GT1b or other gangliosides to
To further assess a preferential binding of GT1b and GD3 for
mannose-containing sugars, ELISAs were performed to examine the binding
of gangliosides to high mannose and reduced mannose forms of ovalbumin
(Sigma). Purification of "high mannose" ovalbumin protein cleaves
mannose residues and reduces content by ~30% to generate "low
mannose" ovalbumin (27). 50 µg/ml high or low mannose ovalbumin was
coated by overnight incubation in the presence of 10 mg/ml EDAC in PBS
at 4 °C onto a 96-well plate. After washing, 200 µl of 2-14
nM GM1, GM2, GD1a, GD3 or GT1b or 1% BSA was added to each
well and incubated overnight at 4 °C. The plate was then washed
vigorously with PBS and reacted with anti-GM1, anti-GM2, anti-GD1a,
anti-GD3, or anti-GT1b monoclonal antibody, followed by HRP-conjugated
secondary antibody and DAB reaction substrate. Binding was detected at
A450 nm. The experiment was performed at least
four times in triplicate.
Statistical Analysis--
All data were analyzed statistically
by Student's t test, with p < 0.05 considered significant.
GT1b and GD3 Are Ganglioside Components of the Membranes of the
Keratinocyte-derived SCC12 Cell--
We have previously shown that
gangliosides compose 0.1% of the lipids of intact epidermis (17), with
GM3 as the predominant ganglioside and smaller amounts of gangliosides
GD3 and GT1b. Studies with cultured keratinocytes (data not shown)
demonstrate a ganglioside profile that is similar to that of cultured
SCC12 cells (Fig. 1), with ~62.9% GM3,
16.9% 9-O-acetyl-GD3, 13.7% GD3, and 6.5% GT1b. The
presence of all four of these gangliosides in cultured SCC12 cells was
confirmed by immunostaining of the thin-layer chromatography plates
with anti-GM3, anti-9-O-acetyl-GD3, anti-GD3, and anti-GT1b
antibodies (Fig. 1).
GT1b Inhibits the Adhesion of
In previous studies (11), we bound FN or its RGD-containing fragment
directly to plastic wells to show by ELISA that GT1b and GD3 inhibit
the binding of cultured keratinocytes and SCC12 cells to native FN and
to the RGD-containing fragment of FN. Because the frequent and vigorous
washing of the plate led to detachment of variable amounts of FN, even
more when the RGD-containing fragment of FN was attached, we studied
methods to improve adherence of FN to the plate. When EDAC was used to
fix the FN or RGD-containing fragment of FN to the plate, results
paralleled those of studies without EDAC fixation, but the colorimetric
readings were higher, and the results were beautifully reproducible.
Thus, the binding of integrin to FN in the presence of GT1b was tested
using an ELISA technique with the RGD-containing fragment of FN fixed
to the plate with EDAC and with both Inhibitory Effect of GT1b Requires the Mechanism of Inhibition of Adhesion by Ganglioside Involves the
Direct Binding of GT1b to
Slot blot assays also showed significant binding of both
affinity-purified GT1b Does Not Affect the Expression of Integrins or the Ability of
Integrin Subunits to Form the Glycosylation of
Fluorescent bead binding assays were used to detect binding of
ganglioside to integrin by immunofluorescence. In this technique, the
ganglioside is initially bound to an amine-modified fluorescent FluoSphere bead with the carbodiimide reagent EDAC. Because the fixation by this method requires loss of a carboxyl group of the ganglioside, we were concerned about alteration in ganglioside function
and ability to bind. In several trials, whether binding GM1 to the
Deglycosylation of insect recombinant Extracellular N-terminal Region of Gangliosides Preferentially Bind Mannose Groups--
To address
the possibility that gangliosides recognize specific carbohydrate
moieties of the integrin glycoprotein, competition experiments were
performed between gangliosides and ConA and between gangliosides and
UEA-1 regarding their ability to bind affinity-purified During the last decade, several studies have shown the importance
of carbohydrate-carbohydrate interactions as the basis for cell
adhesion and recognition. For example, asparagine-linked oligosaccharides of Although the initial observation that polysialylated gangliosides
inhibit the binding of cells to FN was described in 1979 (14), the
mechanism of this effect has been unknown. The demonstration that
binding of Zheng et al. (21) clearly demonstrated that glycosylation of
integrin ELISAs with the deglycosylated forms of
The interference by GT1b in the binding of
Carbohydrate-carbohydrate interactions variably require the presence of
divalent calcium. For example, autoaggregation of mouse F9 embryonic
carcinoma cells is based on LeX-LeX interaction
in the presence of divalent calcium (30), but the carbohydrate-carbohydrate interaction of globoside (Gb4) with nLc4
(precursor of LeX), GalGb4, and LeX does not
require the presence of divalent cation (39). Although divalent calcium
is critical to the binding of The potential role of membrane gangliosides as "cofactors" that are
able to complex with receptors and facilitate or block function is also
suggested by ganglioside-receptor interactions other than GT1b
(GD3)- In contrast to the strong evidence that keratinocyte GM3 participates
in modulating EGFR activity, the role of the more complex gangliosides
GD3 and GT1b, present in lesser amounts in intact epidermis (17),
cultured keratinocytes, and keratinocyte-derived SCC12 cells, has been
unclear. The specific carbohydrate-carbohydrate interactions between
gangliosides GD3 and especially GT1b and integrin
5 and
1 proteins and
5
1 integrin from lysed
keratinocyte-derived SCC12 cells, we have shown that GT1b and GD3
inhibit the binding of
5
1 to fibronectin.
Co-immunoprecipitation of GT1b and
5
1
from SCC12 cells and direct binding of GT1b and GD3 to
affinity-purified
5
1 from SCC12 cells and
insect recombinant
5
1, particularly the
5 subunit, further suggest interaction between
ganglioside and
5
1. The carbohydrate
moieties of integrin appear to be critical since gangliosides are
unable to bind deglycosylated forms of
5
1
from SCC12 and insect cells or poorly glycosylated recombinant
5
1 from Escherichia coli
cells. The GT1b-
5
1 interaction is inhibited by concanavalin A, suggesting that GT1b binds to mannose structures in
5
1. The preferential
binding of GT1b to high mannose rather than reduced mannose ovalbumin
further implicates the binding of GT1b to mannose structures. These
data provide evidence that highly sialylated gangliosides regulate
5
1-mediated adhesion of epithelial cells
to fibronectin through carbohydrate-carbohydrate interactions between
GT1b and the
5 subunit of
5
1 integrin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5
1
integrin and the arginine-glycine-aspartic acid (RGD) site of FN is
known to be key (1). Increased expression of
5
1 has been shown in keratinocytes at the
migrating edge of wounds in vivo, in lesional skin of
patients with psoriasis, and in cultured keratinocytes (2-4). Both
receptor clustering on keratinocytes and ligand occupancy of
5
1 are required to activate intracellular
signal transduction components (5), including focal adhesion kinase,
phosphatidylinositol 3-kinase, protein kinase C, and integrin-linked
kinase, leading to cell adhesion to FN (6-8).
5
1 was identified (15,
16). Although these early studies showed that the terminal sialic acid
residues of gangliosides were critical for the inhibitory effect, the
mechanism of inhibition was unclear. More than 10 years later, our
laboratory used cultured keratinocytes and keratinocyte-derived cell
lines to demonstrate that the inhibition of migration and adhesion by
ganglioside GT1b is specific to plating on a FN matrix and is
competitively inhibited by RGDS peptide (11), suggesting that
ganglioside may abrogate the interaction between
5
1 and FN. SCC13 and HaCaT cells,
keratinocyte-derived cell lines with diminished expression of
5
1 in comparison with normal
keratinocytes and SCC12 cells, are not inhibited by GT1b in their
binding and migration on FN. The demonstration that treatment of HaCaT
cells with transforming growth factor-
1 not only increases the
expression of
5
1, but also restores the
responsiveness to the inhibitory effects of ganglioside further
supports a putative interaction between GT1b and
5
1 (13).
5
1 as the mechanism for ganglioside
action and its relevance for keratinocyte function have not been
explored. Using recombinant and affinity-purified forms of
5
1 and novel binding techniques, we
provide evidence that GT1b binds directly to the extracellular domain
of the
5 subunit through carbohydrate-carbohydrate
interactions, thereby inhibiting the interaction of
5
1 with FN.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5- and
1-containing
Plasmids and Generation of Recombinant Integrin
5 and
1 Subunits in Insect and E. coli Cells--
Several
human recombinant
5 and
1 integrins were
generated using both baculoviral and E. coli systems and
vectors with an N-terminal histidine tag that allowed cleavage or a
C-terminal His tag that could not be cleaved. For generating
5 integrin in insect cells with an N-terminal cleavable
His tag, the pAcHLT-C vector was cleaved by concurrent
incubation with StuI and SacI. The
5 cDNA (GenBankTM/EBI accession number
06256; courtesy of Dr. Erkki Ruoslahti, The Burnham Institute,
La Jolla, CA) (18) was cleaved with SacI and
SalI, and the SalI site was blunt-ended. The
cleaved
5 cDNA was ligated into the open pAcHLT-C vector. To
prepare integrin
1 in insect cells with an N-terminal
cleavable His tag, the pAcHLT-A vector was cut with StuI and
NcoI. The
1 cDNA (GenBankTM/EBI accession
number 07979; courtesy of Dr. Erkki Ruoslahti) (18) was cut with
NcoI and ApaI, blunt-ended at the ApaI
site, and ligated into the vector. Sf9 cells (2 × 106) were seeded into 60-mm Petri dishes and incubated for
2 h at 27 °C. BaculoGold virus DNA (Pharmingen) was mixed with
the recombinant
5 or
1 plasmid DNA and
transfected into insect cells following the manufacturer's
instructions. For generating recombinant integrins in both the insect
system and E. coli with a C-terminal non-cleavable His tag,
5 cDNA was cleaved by SalI and HindIII,
and the SalI end was blunted-ended. The
1 cDNA was
cut with ApaI and HindIII, and the
ApaI site was blunt-ended. These cDNAs were each ligated into a pTriEX vector, which had been cut at the
HindIII site and cut and blunt-ended at the BamHI
site. The
5- or
1-containing plasmid
(Life Technologies, Inc.) was transferred into E. coli cells
following the manufacturer's instructions or cotransfected with
BaculoGold virus DNA into insect cells. The recombinant proteins from
both insect cells and E. coli supernatants were purified by
passage over a Ni2+-nitrilotriacetic acid spin column
(QIAGEN Inc., Valencia, CA) following the manufacturer's instructions.
After purification, the N-terminal His tag was removed from
5 or
1 from insect cells, unless desired
for use in the nickel-agarose binding (NAB) technique. Thrombin
(Pharmingen) was added to the protein at a ratio of 1 unit of thrombin
to 10 µg of integrin subunit protein, run on a Sephacryl S-200HR
column (Amersham Pharmacia Biotech), and dialyzed against
phosphate-buffered saline (PBS), pH 7.2, following the manufacturer's
instructions. The C-terminal His tags were retained on the E. coli and insect cell integrin proteins. To make recombinant
5
1 complexes,
5 and
1 subunits were combined in a 1:1 molar ratio in the
presence of 30 µM CaCl2.
5 antibody (Transduction
Laboratories, Lexington, KY), 100 ng/ml anti-
1 antibody
(Transduction Laboratories), or 250 ng/ml
anti-
5
1 antibody (Zymed
Laboratories Inc., South San Francisco, CA) for 1 h, and
bands were detected by chemiluminescence using an ECL kit
(Amersham Pharmacia Biotech) with goat anti-mouse secondary antibodies
following the manufacturer's instructions.
5
1
from SCC12 Cells--
Intact integrin
5
1
was purified from SCC12 cells by affinity chromatography on
CNBr-activated Sepharose 4B (Sigma) coupled to RGD polymer and
anti-
5
1 antibody as a modification of the previously described techniques (20, 21). Briefly, RGD polymer (5 mg/ml
of gel) was covalently coupled to CNBr-activated Sepharose 4B according
to the manufacturer's instructions. SCC12 cells were lysed (20), and
the lysate was poured over the RGD column. After elution, the integrin
bound to RGD polymer was further purified by affinity chromatography
with anti-
5
1 antibody coupled to a
CNBr-activated Sepharose 4B column as described above for RGD polymer.
Fractions were assayed for integrin by Western blot assays using
anti-
5 or anti-
1 antibody and
chemiluminescence for detection.
5
1 integrin complex to
the RGD-containing fragment of FN in a cell-free system was assayed by
ELISA. 5 µg/cm2 RGD-containing FN fragment (Sigma) was
cross-linked to the wells of a 96-well plate by treatment with 10 mg/ml
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) (Molecular Probes,
Inc., Eugene, OR) in PBS overnight at 4 °C. To evaluate the effect
of gangliosides on the binding of
5
1 from
SCC12 cells to FN, cells were treated overnight with or without GT1b,
GD3, GM3, GM2, or GD1a (Calbiochem) at concentrations ranging from 1 nM to 50 µM. The SCC12 cells were lysed as
described by Pomies et al. (22), and the cell lysates in
lysis buffer at protein concentrations of 10 pg/ml to 1 mg/ml were
incubated in each FN fragment-coated well for 2 h at room
temperature. After washing, the wells were treated with
anti-
5
1 antibody, followed by
HRP-conjugated secondary antibody and 3,3'-diaminobenzidine tetrahydrochloride (DAB) reaction substrate. To test the ability of
gangliosides to block the binding to FN of a 1:1 molar mixture of
insect recombinant
5 and
1, 0.1 pg/ml to
0.1 µg/ml integrin (without the His tags) was preincubated with or
without gangliosides overnight at 4 °C and then applied to the FN
fragment-coated wells. Recombinant integrin bound to FN was
immunostained as described for
5
1 from
SCC12 cells. Binding was detected in a Vmax
kinetic microplate reader (Molecular Devices, Menlo Park, CA) at
A450 nm. In each case, uncoated wells and
omission of anti-
5
1 antibody served as
negative controls. All studies were repeated at least four times, with
triplicate wells for each condition.
5 or
1 protein with a C-terminal His tag (i.e. the
N terminus available) or, as a control, with an N-terminal His tag was
mixed with 100 µl of nickel-agarose beads (QIAGEN Inc.) for 1 h
at room temperature. After washing, the coated nickel-agarose beads
were mixed for 16 h in wash buffer (50 mM sodium
phosphate, 300 mM NaCl, and 10% glycerol, pH 8.0) containing 20 µg/ml RGD-containing 110-kDa FN fragment with 1-50 µM GT1b. Controls included mixing the integrin with
gangliosides without FN and mixing the integrin and FN without any
ganglioside. The bound bead was then washed and boiled in denaturing
Laemmli buffer. Separated proteins were evaluated by Western blotting as described above, except that proteins on polyvinylidene difluoride membrane were detected with a 1:1 mixture of anti-fibronectin antibody
(Zymed Laboratories Inc.) and anti-
5 or
anti-
1 antibody. As a positive control and to verify
results of the ELISAs, a 1:1 mixture of
5 and
1 (the
5
1 complex) was
bound to nickel-agarose and mixed with FN in the presence or absence of
GT1b. Similar techniques were used, except that the
nickel-agarose-bound C-terminal His-integrin
1 subunit
was first able to form nickel-agarose-bound C-terminal
His-
5
1 by incubation with the partner
5 subunit without its His tag, prior to mixing with FN
and ganglioside. To prevent separation of
5 from
1 and to allow detection with anti-
5
1 antibody, the complex of
nickel-agarose-bound His-
5
1 and FN was
incubated in Laemmli buffer containing 17.4% sucrose for 30 min at
room temperature without boiling, and the gel was treated with 2.5%
Triton X-100 before transferring to the polyvinylidene difluoride
membrane. NAB assays were performed three times.
5
1 and Detection of
Gangliosides--
SCC12 cells were treated for 48 h with or
without GT1b or GD3 at concentrations of 1 nM to 50 µM or, as a control, with 10, 50, or 200 µM
GM2, GD1a, or GM3. The cells were lysed in calcium-free buffer
containing 20 mM Hepes, pH 7.2, 1% Nonidet P-40, 10%
(v/v) glycerol, 50 mM NaF, 1 mM PMSF, 1 mM Na3VO4, and 10 µg/ml leupeptin (23). 5 µg of anti-
5
1 antibody was
added to 2.5 mg of cell-free lysates at 4 °C for 2 h. In some
studies, 5 µg/ml FN was added to determine whether the presence of FN
significantly altered the complex formation of ganglioside with
5
1 integrin. After treatment with 5 µg
of rabbit anti-mouse IgG antibody for 30 min, 10 µl of 50% protein
A-agarose was added, and the mixture was incubated for another 2 h
at 4 °C. One-eighth of the mixture was boiled in Laemmli buffer, and
the separated integrin
5 and
1 subunits
were detected by Western blotting with anti-
5 and
anti-
1 antibodies. Lipids were extracted from the
remaining samples with chloroform/methanol (2:1, v/v) (17), separated
by thin-layer chromatography as described above, and identified with
anti-GM3, anti-GM2, anti-GM1, anti-GD3, and anti-GT1b antibodies,
followed by enhanced chemiluminescence detection as previously
described (24). Band density was quantified by the Storm 800 fluorescence PhosphorImager.
5
1 or to one of its subunits, slot blot assays were performed. 20 µl of 50 µM GT1b, GD3, or, as
a control, GM3, GM2, GD1a, or 1% bovine serum albumin (BSA) was loaded
into each slot of a positively charged nylon membrane (Roche Molecular Biochemicals) on a Bio-Dot SF apparatus (Bio-Rad). The membrane was
air-dried and blocked by the addition of 1% BSA in PBS, pH 7.6. After
air drying, the membrane was incubated with 50 ng/ml affinity-purified
5
1 from the SCC12 cells, with 5 µg/ml
insect recombinant protein
5 or
1, or
with a 1:1 molar mixture of
5 and
1 in
PBS, pH 7.6, at 4 °C for 4-6 h. The membrane was washed with PBS,
and the binding was detected with anti-
5,
anti-
1, or anti-
5
1
antibody using Western blotting with an ECL kit as described above.
5
1 and on the Ability of
5
to Complex with
1--
SCC12 cells were incubated
overnight with GT1b at concentrations of 1 nM to 50 µM GT1b or, as a control, with 50 µM GM3,
GM2, or GM1 or medium without ganglioside. Cells were lysed, boiled in
Laemmli buffer, and run on an 8% SDS-polyacrylamide gel. After transfer to polyvinylidene difluoride membrane, the expression of
5 and
1 was detected with
anti-
5 and anti-
1 antibodies and an ECL kit.
5 and
1 subunits. 100 µl of nickel-agarose beads was mixed
for 1 h at room temperature with 5 µg of insect recombinant
5 with a C-terminal His tag or with 5 µg of insect
recombinant
1 with a C-terminal His tag, leaving the N
terminus free to interact. As a negative control, nickel-agarose beads
were also mixed with
5 or
1 subunits with N-terminal His tags. After washing, the
His-
5-nickel-agarose or
His-
1-nickel-agarose beads were mixed with GT1b at
concentrations of 1 nM to 50 µM in PBS for
4 h. Subsequently, 5 µg of the appropriate partner integrin
subunit without its His tag (e.g.
5 for
nickel-agarose-bound His-
1) was added for 1 h at
room temperature. After washing, the sample was boiled in Laemmli
sample buffer for 5 min, and the subunit combination was identified by
Western blotting as described above using a 1:1 mixture of
anti-
5 and anti-
1 antibodies. In all NAB
studies, the presence of GT1b was confirmed by loading an aliquot onto
a membrane for slot blot assays as described above, except that
anti-GT1b antibody was used to detect GT1b.
5
1--
To
assess the role of sugar moieties of
5
1
in the interaction with gangliosides,
5
1
from SCC12 cells and insect recombinant
5 and
1 proteins were deglycosylated by a modification of
previously described techniques (21, 25). In addition, poorly
glycosylated recombinant
5
1 was generated
in E. coli cells (26) as described above. After reaching
80% confluence, SCC12 cells were switched to serum-free medium with 2 µg/ml tunicamycin. 2 units/ml PNGase F (Calbiochem) was added to the
medium 22 h later, and the cells were incubated with the
combination of PNGase F and tunicamycin for an additional 2 h.
After washing with 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 mM MnCl2, and 0.2 mM PMSF to clear cleaved carbohydrate residues and residual
enzyme, the deglycosylated proteins were collected by cell lysis with
10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 mM MnCl2, 3 mM PMSF, and 0.1 M octyl glucoside. After centrifugation, the pellet was
discarded. Recombinant
5 and
1 were
treated with 0.4 units/ml PNGase F for 1 h at 37 °C (21) and
then lysed and purified by passage over a
Ni2+-nitrilotriacetic acid spin column as described above.
The resultant deglycosylated SCC12 cell
5
1, insect recombinant
5
1, and E. coli
5
1 were analyzed in comparison with the
original glycosylated forms of
5
1 by
electrophoresis on a 7.5% SDS-polyacrylamide minigel. Gels were
stained with Coomassie Blue or periodate-Schiff reagent (Sigma) and
compared with known molecular mass standards.
5
1 from SCC12 cells (50 ng/ml),
deglycosylated
5
1 from insect recombinant cells (5 µg/ml), and the poorly glycosylated
5
1 from E. coli (5 µg/ml)
were substituted for the glycosylated forms of
5
1.
5
1 integrin protein from SCC12 cells,
deglycosylated affinity-purified
5
1 from
SCC12 cells, recombinant
5 and
1 subunits
generated in insect cells and their 1:1 mixture, deglycosylated insect
recombinant
5 and
1 subunits and their
1:1 mixture, recombinant
5 and
1 from E. coli, and 1% BSA were coated onto 96-well plates
overnight at 4 °C as described for FN ELISAs. To ensure good binding
of integrin or integrin subunits to the plate, wells were washed and
then treated with anti-
5, anti-
1, or
anti-
5
1 antibody, and the integrin was
detected by immunofluorescence. After washing, the beads coated with
gangliosides were added to the integrin-coated plates. After a 2-h
incubation, the binding of the colored ganglioside-coated spheres to
integrin was visualized by immunofluorescence microscopy (Nikon Eclipse
TE300 immunofluorescence microscope linked to a computer with
Neurolucida software from MicrobrightField, Inc., Colchester,
VT) at magnification × 200 using a 580-nm filter. The number of
beads counted in 13 non-overlapping fields was expressed as a
percentage of the number of GT1b-coated beads bound to fully glycosylated affinity-purified
5
1 from
SCC12 cells.
5
1. In these studies, 96-well clear
polystyrene plates (Calbiochem) precoated with ConA by the manufacturer
were washed, and 200 µl of 10 nM GT1b, GM3, or GM2 or 1%
BSA was added to the plate concurrent with affinity-purified
5
1 from SCC12 cells at concentrations of
10
14 to 10
6 g/ml.
After washing, the binding of
5
1 integrin
to ConA was detected by incubation with
anti-
5
1 antibody, HRP-conjugated goat
anti-mouse secondary antibody, and DAB. Binding was measured as
described above at A450 nm. As a control, the
same experiment was performed with Ulex europaeus
agglutinin-1 (UEA-1) lectin, which preferentially binds fucose groups
rather than mannose, in substitution for the ConA lectin. The
experiment was performed at least four times in triplicate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Ganglioside content of keratinocyte-derived
SCC12 cells. Gangliosides were extracted from SCC12 cell membranes
using chloroform/methanol (2:1). The aqueous phase was separated and
desalted, and the bands of gangliosides were separated by thin-layer
chromatography in chloroform/methanol/water with 0.02%
CaCl2 (55:45:10, v/v/v). Gangliosides were detected by
resorcinol staining (not shown). The identity of each ganglioside was
confirmed by immunostaining with a mixture of anti-GM3,
anti-9-O-acetyl-GD3, anti-GD3, and anti-GT1b antibodies,
followed by HRP-conjugated goat anti-mouse IgG and ECL (second
lane). The first lane shows ganglioside standards GM3,
GD3, and GT1b.
5
1 to
FN in a Cell-free System--
Previous investigations have studied the
ability of ganglioside to block the binding and migration of intact
cultured keratinocytes and keratinocyte-derived cells on FN. To
eliminate the possibility that ganglioside-induced inhibition occurs
indirectly through alterations in other cellular components, we studied
the ability of GT1b to block the binding of
5
1 from lysed SCC12 cells and of
recombinant integrin
5
1 to the
RGD-containing cell-binding region of FN. Recombinant
5
and
1 integrin proteins were generated using an insect
system and a vector with a cleavable His tag. When combined in a 1:1
molar ratio, these purified
5 and
1
integrin subunits formed complexes that bound well to FN (Figs.
2B and 3).
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Fig. 2.
Effect of ganglioside on adhesion of
5
1
to FN. 5 µg/cm2 RGD-containing fragment of FN was
cross-linked to the wells of a 96-well plate by treating with 10 mg/ml
EDAC in PBS overnight at 4 °C. A, to test the effect of
gangliosides on the binding of
5
1 from
SCC12 cells, cells were treated overnight with or without 10 nM GT1b or GD3 or 50 µM GM3, GM2, or GD1a
(concentrations based on previous studies showing no inhibition or
toxicity at concentrations of 1 nM to 50 µM
GM3, GM2, and GD1a) and then lysed. Lysates at protein concentrations
of 10 pg/ml to 1 mg/ml were added to each FN fragment-coated well,
followed by anti-
5
1 antibody,
HRP-conjugated secondary antibody, and DAB reaction substrate.
B, to test the ability of gangliosides to block the binding
to FN of a 1:1 molar mixture of insect recombinant
5 and
1, 0.1 pg/ml to 0.1 µg/ml recombinant
5
1 was preincubated with or without
gangliosides overnight at 4 °C and studied as described above for
5
1 from lysed SCC12 cells. Binding was
detected in a Vmax kinetic microplate reader at
A450 nm. A:
, SCC12
5
1 without ganglioside;
, SCC12
5
1 + GD1a;
, SCC12
5
1 + GM2;
, SCC12
5
1 + GM3; ×, SCC12
5
1 + GD3; *, SCC12
5
1 + GT1b. B:
, insect
recombinant
5
1 without ganglioside;
,
insect recombinant
5
1 + GD1a;
, insect
recombinant
5
1 + GM2;
, insect
recombinant
5
1 + GM3; ×, insect
recombinant
5
1 + GD3; *, insect
recombinant
5
1 + GT1b.
5
1
from lysed SCC12 cells (Fig. 2A) and the recombinant
5
1 complex (Fig. 2B). 10 nM GT1b significantly decreased the binding to the
RGD-containing fragment of FN (p < 0.05 at 100 pg/ml
5
1 from lysed SCC12 cells and
p < 0.05 at 1 pg/ml insect recombinant
5
1). GM3, GM2, and GD1a at concentrations
as high as 50 µM and 1% BSA did not show a significant inhibitory effect on binding to FN of either
5
1 from lysed SCC12 cells or insect
recombinant
5
1. The effect of 10 nM GD3 on binding was intermediate, but significant for
both
5
1 from lysed SCC12 cells
(p < 0.05 at 10 ng/ml
5
1) and insect recombinant
5
1 (p < 0.05 at 10 pg/ml
5
1).
5 Subunit of
5
1--
The specificity of the
inhibitory effect of ganglioside on cell binding to a FN matrix, and
not to collagen I or other matrices (11), targeted the
5
subunit of
5
1 as critical since other
1 integrin keratinocyte complexes, such as
2
1 and
3
1,
promote adhesion to collagen I. Taking advantage of the known ability of nickel-agarose beads to bind proteins with a His tag, we used a
novel technique that directly assesses the ability of the His-tagged protein to bind other proteins, the NAB technique. Using the NAB technique to bind the His tag attached to the intercellular C terminus
of
5 to the nickel-agarose bead (i.e. the
extracellular N terminus of integrin is free to interact) and Western
blotting, we verified the results of ELISAs, showing that as little as
1 nM GT1b inhibits the binding of both the insect
recombinant
5
1 complex (Fig.
3A) and the
5
subunit (Fig. 3B) to the RGD-containing region of FN,
supporting
5 as the critical subunit for interaction with GT1b. The binding of the
1 subunit to FN was
overall much weaker, but was not diminished by incubation with
concentrations of GT1b as high as 50 µM (Fig.
3C).
5 and
1 subunits with
N-terminal His tags (i.e. the intercellular C terminus of
integrin is available to interact) did not bind to FN; and thus,
Western blots showed only bands of integrin (data not shown).
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Fig. 3.
NAB technique used to demonstrate the effect
of ganglioside GT1b on the binding of FN to recombinant
5
1
subunits. 5 µg of purified insect recombinant
5
or
1 protein with a C-terminal histidine tag was mixed
with 100 µl of nickel-agarose beads for 1 h at room temperature.
Binding of the C-terminal His tag leaves the N-terminal extracellular
portion of the integrin available for binding. After washing, the
coated beads with the free extracellular N terminus of integrin were
mixed for 16 h in wash buffer with 20 µg/ml 110-kDa FN
cell-binding fragment in the presence of up to 50 µM
GT1b. For evaluating the
5
1 complex, the
beads with integrin were first mixed with the partner integrin subunit
without its histidine tag before incubation with FN. The bound bead was
then washed and incubated with Laemmli buffer containing 17.4% sucrose
for the
5
1 complex at room temperature or
boiled in denaturing Laemmli buffer for
5 and
1 subunits. Bands were separated on an 8%
SDS-polyacrylamide gel and transferred to polyvinylidene difluoride
membrane for Western blotting. The presence of integrin or integrin
subunit and FN was detected with a 1:1 combination of anti-fibronectin
antibody and anti-
5
1 (A),
anti-
5 (B), or anti-
1
(C) antibody.
5
1 Integrin,
Particularly to the
5 Subunit--
The complexing of
GT1b with affinity-purified
5
1 from SCC12
cells was shown by co-immunoprecipitation of GT1b and
5
1 (Fig. 4A). Even without the addition
of supplemental purified GT1b, the presence of GT1b (Fig.
4B) and GD3 (data not shown) was detected in the complex
with
5
1 by thin-layer chromatography
immunostaining using anti-GT1b and anti-GD3 antibodies, respectively.
The addition of FN to the mixture neither significantly increased nor
decreased the content of GT1b in the complex with
5
1 (Fig. 4B). As little as 1 nM exogenous GT1b, added to SCC12 cells to increase
membrane GT1b content, significantly increased the detection of GT1b
after extraction from the immunoprecipitated complex and separation by
thin-layer chromatography (Fig. 4B). The addition of 50 µM GD3 to cells before immunoprecipitation of
5
1 resulted in the detection of a strong
band of GD3 in the complex (Fig. 4A), but even the addition
of 200 µM GM3 did not lead to the detection of GM3 in the
complex with immunoprecipitated
5
1, as
shown by immunostaining with anti-GM3 antibodies (Fig. 4A).
Similarly, GM2 and GM1 did not immunoprecipitate with
5
1. Western blotting of the
immunoprecipitated complex confirmed the presence of the
5
1 proteins (Fig. 4C).
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Fig. 4.
Ability of GT1b to co-immunoprecipitate
with
5
1
from SCC12 cells. SCC12 cells were treated for 48 h with or
without gangliosides at concentrations as high as 200 µM.
The cells were lysed in buffer, and 5 µg of
anti-
5
1 antibody was added to 2.5 mg of
cell-free lysates at 4 °C for 2 h. In some studies, 5 µg/ml
FN was added to determine whether the presence of FN significantly
altered the complex formation of ganglioside with the
5
1 integrin. After treatment with 5 µg
of rabbit anti-mouse IgG for 30 min, 10 µl of 50% protein A-agarose
was added, and the mixture were incubated for another 2 h at
4 °C. Lipids were extracted from seven-eighths of the samples for
thin-layer chromatography with chloroform/methanol (2:1, v/v). The
aqueous phase was separated and desalted, and the bands were separated
by thin-layer chromatography in chloroform/methanol/water with 0.02%
CaCl2 (55:45:10, v/v/v). Gangliosides were detected by
immunostaining with anti-GM3 (A, second lane),
anti-GD3 (A, third lane), or anti-GT1b
(A, fourth lane; and B) antibody and
detected by enhanced chemiluminescence. The remaining one-eighth of the
mixture was boiled in Laemmli buffer, and the separated integrin
5 and
1 subunits were detected by Western
blotting with anti-
5 and anti-
1
antibodies (C).
5
1 (Fig.
5A) and the recombinant
5 subunit (Fig. 5B) to GT1b (both
p < 0.01 when compared by densitometric measurements with BSA and ganglioside controls) and to GD3 (both p < 0.05 compared with controls). No significant binding of integrin to
ganglioside was detected with GM3, GM2, or GD1a.
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Fig. 5.
Direct binding of GT1b and GD3 to
5
1
and the
5 subunit. 20 µl of
50 µM GT1b, GM3, GD3, GM2, or GD1a or 1% BSA was loaded
into each slot of a positively charged nylon membrane on a Bio-Dot SF
slot blot apparatus. After blocking with 1% BSA, the membrane was
incubated with 50 ng/ml affinity-purified
5
1 (A) or 5 µg/ml
recombinant protein
5 (B) at 4 °C for 4-6
h. Binding was detected with anti-
5
1 or
anti-
5 antibody by Western blotting with enhanced
chemiluminescence detection.
5
1
Complex--
Western blot analysis showed no detectable alteration in
the expression of
5
1 from lysed SCC12
cells that had been incubated in the presence of GT1b at concentrations
of 1 nM to 50 µM (Fig. 6A). Incubation of the SCC12
cells with other gangliosides at 50 µM, including GM3,
GM2, and GM1, similarly did not alter SCC12 cell expression (data not
shown). To address the question of the ability of ganglioside to block
integrin subunits from forming an
5
1
complex, we again used the NAB technique. Regardless of whether the
nickel-agarose beads bound the His tag of the C-terminal domain of
insect recombinant
1 or the His tag of the C-terminal domain of
5 (in both situations, the extracellular
N-terminal domains are free to interact), treatment with concentrations
of GT1b from 1 nM to 50 µM had no effect on
the ability of the integrin subunit to recognize and bind to its
partner (i.e.
5 to
1 and
1 to
5), as detected by equally dense
bands of
5 and
1 on Western blots after
subunit separation (Fig. 6B).
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Fig. 6.
Lack of effect of GT1b on the expression of
integrin
5
1
or the ability of the
5 and
1 subunits to complex with each
other. A, to study the effect of ganglioside on the
expression of
5 and
1, SCC12 cells were
incubated overnight with or without GT1b at concentrations of 1 nM to 50 µM. Cells were lysed, run on an 8%
SDS-polyacrylamide minigel, and transferred to polyvinylidene
difluoride membrane, and the expression of
5 and
1 was detected with a mixture of anti-
5
and anti-
1 antibodies and an ECL kit. B, to
determine the ability of GT1b to interfere with the complex formation
between
5 and
1 subunits, a NAB sandwich
technique was performed. 100 µl of nickel-agarose beads was mixed
with 5 µg of one of four recombinant integrin subunits:
5 with a His tag at the extracellular N terminus,
5 with its His tag at the intracellular C terminus,
1 with its His tag at the N terminus, or
1 with its His tag at the C terminus. After washing, the
His-
5-nickel-agarose or the
His-
1-nickel-agarose beads were mixed with 1-100
µM GT1b in PBS for 4 h, and 5 µg of the
appropriate partner integrin subunit without its His tag (for example,
recombinant
1 integrin for
5) was added
for 1 h at room temperature. After washing, the sample was boiled
in Laemmli sample buffer for 5 min, and bands were identified by
Western blotting as described above using a 1:1 mixture of
anti-
5 and anti-
1 antibodies.
5
1 Integrin Is
Critical for Interaction with GT1b--
Native affinity-purified human
5
1 and recombinant proteins generated in
insect cell systems are relatively well glycosylated (28). In contrast,
recombinant proteins generated in an E. coli system are
poorly glycosylated (26). To assess the importance of the integrin
carbohydrate groups for function and for binding to ganglioside, the
carbohydrate groups were stripped both from the insect recombinant
subunits and from
5
1 from SCC12 cells by
treatment with PNGase F alone or with PNGase F and tunicamycin, respectively. In addition,
5 and
1 were
generated in an E. coli system. The deglycosylated forms of
5
1 were compared with the poorly
glycosylated E. coli integrin and with the original
glycosylated forms of integrin. Treatment with PNGase F and tunicamycin
decreased the molecular mass of
5 from SCC12 cells from
145 to 100 kDa and that of
1 from SCC12 cells from 135 to 90 kDa (Fig. 7A). Similarly, deglycosylation with PNGase F decreased the molecular mass
of insect recombinant
5 from 135 to 100 kDa and that of
1 from 130 to 90 kDa. The molecular mass of E. coli
5 was 100 kDa, and that of
1
was 92 kDa. Use of a periodate-Schiff reagent stain to detect
carbohydrate groups showed that deglycosylation drastically reduced or
eliminated detectable carbohydrate residues on
5
1 (Fig. 7B) and verified the
poor glycosylation of the E. coli integrins. Deglycosylation
decreased the ability of
5
1 from lysed
SCC12 cells and recombinant
5
1 from
insect cells to bind to the RGD-containing fragment of FN. As detected
by ELISAs, deglycosylation reduced the binding of
5
1 from SCC12 cells to FN by 70% and
that of a 1:1 mixture of insect recombinant
5 and
1 integrins to FN by 65%. Similarly, a 1:1 mixture of
5 and
1 generated in an E. coli system bound weakly to FN (71% less than glycosylated
5
1 from SCC12 cells), verifying the
importance of integrin glycosylation for interaction with the
RGD-binding region of FN (21).
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Fig. 7.
Deglycosylation of
5
1.
SCC12 cells were treated with 2 µg/ml tunicamycin for 24 h, and
2 units/ml PNGase F was added to the medium for the last 2 h of
incubation. After washing with 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 mM MnCl2, and 0.2 mM PMSF to clear cleaved carbohydrate residues and residual
enzyme, the deglycosylated proteins were collected by cell lysis with
10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 mM MnCl2, 3 mM PMSF, and 0.1 M octyl glucoside. After centrifugation, the pellet was
discarded. Sf9 insect and E. coli recombinant
5 and
1 were treated with 0.4 units/ml
PNGase F for 1 h at 37 °C and then lysed and prepared as
described for
5
1 from SCC12 cells. The
resultant deglycosylated forms of
5
1
(+ lanes) were analyzed in comparison with the original
glycosylated
5
1 forms (
lanes) by electrophoresis on a 7.5% SDS-polyacrylamide
minigel. Gels were stained with Coomassie Blue (A) or
periodate-Schiff reagent (B) and compared with known
molecular mass standards.
subunit of cholera toxin, GM3 to the epidermal growth factor receptor,
or GT1b to
5
1, binding was beautifully
reproducible, and controls with other gangliosides and receptor
proteins were consistently negative (data not shown). Fluorescent bead
binding assays showed strong binding of gangliosides GT1b and GD3 to
native affinity-purified
5
1 from SCC12
cells (Table I), with 293.67 ± 18.65 beads bound per 13 non-overlapping fields within the wells. Consistent with the results of other experiments, the binding of the
GT1b-coated beads to native recombinant
5
1 was 84% that of affinity-purified
5
1 from SCC12 cells, and the binding of GT1b to the
5 and
1 subunits was 64 and
35%, respectively. The binding of the GD3-coated beads was 66-88%
that of the GT1b-coated beads. In contrast, the binding of beads coated
with GM3, GD1a, and GM2 was <9% that of GT1b to affinity-purified
5
1 with all forms of glycosylated
integrin.
Binding of gangliosides to deglycosylated 5
1
5
1
eliminated the ability of ganglioside GT1b or GD3 to bind the integrin
to the basal level of binding of other gangliosides (Table I) and of
beads coated with albumin alone. Similarly, both deglycosylated
5
1 from SCC12 cells and the poorly
glycosylated
5
1 generated in E. coli cells were not able to bind gangliosides in fluorescent bead
binding assays (data not shown). Slot blot assays also showed no
detectable binding of E. coli
5
1 or its subunits to gangliosides. These
data suggested the importance of the carbohydrate moieties of
5 in the interaction with the carbohydrate components of ganglioside.
5 Is Recognized
by GT1b--
To determine the site on
5 bound by
ganglioside, NAB assays were performed in which insect recombinant
5 integrin with either an intracellular C-terminal His
tag or an extracellular N-terminal His tag was linked to the
nickel-agarose bead. After the addition of
1 without its
His tag (for
5
1) or without the addition
of
1 (for studying the
5 subunit), the
free extracellular N-terminal region or intracellular C-terminal region
was allowed to interact with GT1b and with FN in solution. Recovery of
protein was determined by Western blotting, showing the binding of the
integrin to FN. The interaction of GT1b and the truncated termini of
5
1 or the subunit was determined by
thin-layer chromatography immunostaining with anti-GT1b antibody. GT1b
was detectable only when the C-terminal His-
5
1 or C-terminal His-
5
was studied, demonstrating the direct interaction of GT1b with the
extracellular N-terminal region of
5.
5
1 from SCC12 cells. Although
5
1 bound well to both ConA and UEA-1,
presumably by recognition of the integrin's mannose and fucose
residues, respectively, GT1b blocked the binding of
5
1 only to ConA (p < 0.001) (Fig. 8A), and not to
UEA-1 (Fig. 8B). Gangliosides GM3 and GM2 did not inhibit
the binding of
5
1 to either lectin. To
further consider that GT1b and GD3 recognize and bind to mannose
residues, ELISAs were performed with ovalbumin molecules with varying
contents of mannose. GT1b and GD3 selectively bound the high mannose
form of ovalbumin (p < 0.01 for GT1b and p < 0.05 for GD3) (Fig.
9, A and B).
Gangliosides GD1a, GM2, and GM1 showed weak binding, and no difference
in binding in relation to the mannose content of the sugars was noted
with these control gangliosides.
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Fig. 8.
Competitive binding of gangliosides and
lectins to
5
1.
96-well clear polystyrene plates precoated with ConA (A) or
UEA-1 (B) were washed, and 10 nM GT1b, GM3, or
GM2 or 1% BSA was added to the plate concurrent with affinity-purified
5
1 from SCC12 cells at concentrations of
10
14 to 10
6 g/ml.
Binding was measured at A450 nm after treatment
with anti-
5
1 antibody, HRP-conjugated
secondary antibody, and DAB reaction substrate.
,
5
1 without ganglioside;
,
5
1 + GM3;
,
5
1 + GM2; *,
5
1 + GT1b.
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Fig. 9.
Ganglioside binding to ovalbumin with
different mannose contents. ELISAs were performed to assess the
ability of gangliosides to bind the reduced mannose (A) and
high mannose (B) forms of ovalbumin. 50 µg/ml ovalbumin
was coated onto 96-well plates (50 µl/well) in the presence of 10 mg/ml EDAC in PBS overnight at 4 °C and washed. 2-14 nM
GM1, GM2, GD1a, GD3, or GT1b or 1% BSA was added to each well and
incubated overnight at 4 °C. The plate was then washed vigorously
and reacted with anti-GM1, anti-GM2, anti-GD1a, anti-GD3, or anti-GT1b
monoclonal antibody (or no primary antibody for BSA), followed by
HRP-conjugated secondary antibody and DAB reaction substrate. , GM1;
, GD1a;
, GM2; ×, GD3; *, GT1b.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6
1 integrin are known
to interact with metaperiodate- and N-glycanase-sensitive
components of laminin (29). Although the greatest interest has focused
on glycoprotein-glycoprotein interactions, glycolipids may also
participate in these carbohydrate-carbohydrate relationships and have
gained increasing attention as possible key modulators.
LeX-LeX glycolipid interactions are thought to
mediate at least the primary recognition and adhesion process among
mouse pre-implantation embryo cells (30). In addition, ganglioside GM3
can interact with several glycosphingolipids with a terminal
N-acetylgalactosamine and with lactosylceramide. GM3-Gg3
binding is thought to participate in the interaction of B16 melanoma
cells, which express high levels of GM3, and mouse L5178 lymphoma
cells, which express high levels of Gg3 (asialo-GM2) (31). The
interaction of GM3 with Gg3 or lactosylceramide, expressed on
endothelial cells, may also initiate metastasis (32). Most recently,
Zheng and Hakomori (33) demonstrated that the interaction of a
"disialyl-I" carbohydrate epitope on soluble FN and cell membrane
glycosphingolipid Gg3 promotes binding of the cell to soluble
fibronectin, suggesting that glycosphingolipid aggregates may
interconnect FN and the cell surface through an additional binding mechanism.
5
1 to FN is inhibited in a
cell-free system provides further evidence of a direct role of
ganglioside interaction with integrin, rather than triggering an
inhibitory effect through an additional cellular component. The strong
correlation between the results of slot blot, ELISA, and bead binding
assays with affinity-purified
5
1 from
SCC12 cells and the results with insect recombinant
5
1 suggests that the recombinant proteins
generated in insect cells represent a good model of
5
1 generated endogenously in human
keratinocytes. Furthermore, the preferential binding of ganglioside to
the
5 subunit and, similarly, the specific inhibition of
5 binding to FN by ganglioside are consistent with the
previously demonstrated specificity of the inhibitory effect of
ganglioside GT1b on binding to a FN matrix and not to several other
matrices that are recognized by keratinocyte integrins that share the
1 subunit (11). The ability of a particular ganglioside to bind to
5
1 correlates well with the
inhibitory effect of that ganglioside on
5
1 binding to the RGD-containing fragment of FN. Thus, GT1b binds most strongly and shows the greatest inhibitory effect, whereas GD3 shows both weaker binding to integrin and a lesser
effect on inhibition. Other tested gangliosides, including GM3, did not
show any inhibitory effect or binding. Given that the carbohydrate
moieties distinguish these gangliosides, these data not only show a
direct interaction between the gangliosides and integrin, but also
suggest that differences in both the degree of sialylation and the
pattern of glycosylation affect the ability to bind integrin.
5
1 is critical for binding to
FN; however, the importance of integrin glycosylation for binding of
ganglioside to
5
1 has not been addressed.
To consider the role of sugar moieties on the integrin in the
interaction with ganglioside, we deglycosylated native
5
1 from SCC12 cells prior to extraction
from the cells and also cleaved the sugar moieties from recombinant
5 and
1 that we generated in insect
cells. In addition, we generated poorly glycosylated recombinant
5 and
1 in E. coli. All three
of these sugar-poor forms of
5
1 showed
poor to no staining with periodate-Schiff reagent and an ~30%
reduction in molecular mass after deglycosylation.
5
1 and the poorly glycosylated
5
1 from E. coli reiterated the
findings of Zheng et al. (21), demonstrating that binding to
FN of deglycosylated integrin decreases by at least 80% in comparison
with glycosylated
5
1. We have now shown
that the ganglioside-integrin complex formation, which may participate
in the regulation of cell-matrix interactions, also requires the
recognition by ganglioside of integrin glycosylation sites in the
extracellular N-terminal region, the region of
5
1 involved in RGD binding. Although
deglycosylation of integrin also dissociates the
5 and
1 subunits from each other (19), GT1b does not affect
the ability of integrin subunits to associate.
5
1 to Con A, which is known to bind
mannose residues (34), and not to UEA-1, which is known to bind to
fucose residues, suggests that GT1b recognizes a mannose-containing
region of the extracellular domain of N-glycan. The
demonstration that GT1b and GD3 bind preferentially to high mannose
ovalbumin, but not to ovalbumin with a lower content of mannose,
further supports the recognition by these gangliosides of mannose
structures. Although the carbohydrate structures of
5
1 from human keratinocytes are unknown,
at least 35 different types of N-linked oligosaccharides
have been separated and characterized from the
5
1 receptor from human placenta (28). The
most common sugar group is the biantennary
di-
-(2,3)-sialylfucosyl residue, and >50% of the sugars are
fucosylated at the N-acetylglucosamine residue at its
reducing end; all contain an oligomannose core typical of
N-glycans. High mannose residues compose only 1.5% of the
sugars of human placenta
5
1. In contrast,
the N-glycosylation patterns of recombinant glycoproteins
expressed in insect cells, although variable, show mostly high
mannose-type (Man5-9-GlcNAc2) and short
truncated structures with fucose
1,6-linked to the asparagine-bound
GlcNAc residue (35). The binding of ConA to mannose does not
distinguish among different manno-oligosaccharides. In contrast, other
lectins show greater specificity. For example, Tulipa
gesneriana lectin prefers manno-oligosaccharides with
Man(
1-6)Man linkage rather than
1-2 or
1-3 linkage (36),
Galanthus nivalis agglutinin prefers terminal
Man(
1-3)Man (37), and Crocus sativus lectin prefers
Man(
1-3)Man(
1-4)GlcNAc (38) in the N-glycan core
structure. Further study, including competition assays with these
lectins that recognize more specific mannose structures, will be
required to explore the specificity of ganglioside binding and to
explain the recognition by specific gangliosides of the carbohydrate
moieties of specific glycoproteins.
5
1 and the
5 subunit to fibronectin and to the ability of the
insect recombinant
5 and
1 subunits to
complex,2 our
co-immunoprecipitation studies of ganglioside and
5
1 were not performed in the presence of
calcium, and ELISA studies in the presence of 1 mM EDTA
have shown no effect of depletion of divalent cation on the ability of
GT1b to bind to either the 1:1 mixture of insect recombinant
5 and
1 subunits or to the
5 subunit itself.2 Although these results
suggest that the carbohydrate-carbohydrate interaction of complex
gangliosides with
5
1 integrin does not require divalent cation, further studies are in progress.
5
1. For example, GD3 and GD2
co-immunoprecipitate with
v
3 (40); given
the structural similarities between the
5
1 and
v
3
integrins, it is likely that the
ganglioside-
v
3 interaction similarly
involves carbohydrate-carbohydrate recognition of the extracellular
N-terminal region of
v. Studies in 1988 first
demonstrated that ganglioside GM3 co-immunoprecipitates with the
epidermal growth factor receptor (EGFR) (41), and this ability to
complex was further suggested by ELISAs (42). In fact, we have recently
provided evidence that the relationship between GM3 and the EGFR that
is required for inhibition of EGFR activation involves
carbohydrate-carbohydrate
interaction.3 Furthermore, we
have determined that the basis for the inhibitory effect of GM3 on EGFR
activity in SCC12 cells involves, at least in part, the decreased
availability of receptors for ligand binding, resulting in decreased
phosphorylation of several components of the EGFR signal transduction
pathway (43). The lack of any direct interaction of GM3 with integrin
5
1 suggests that the regulation by GM3 of
5
1/FN-mediated adhesion as described by
Zheng et al. (44) relates to other effects of GM3, such as
those on signal transduction pathways, rather than a direct binding of
ganglioside to the integrin.
5
1 indicate a potentially critical role
for these gangliosides in the function of human keratinocytes, which
are highly dependent upon the interaction of
5
1 and FN for regulation of cell
adhesion, motility, and the ability to undergo apoptosis. Given the
potential importance of these sialylated gangliosides in modulating
biologic behavior in vivo, further studies on the role of
gangliosides in disorders with aberrant adhesion and motility on
fibronectin, such as psoriasis and cutaneous carcinomas, are warranted.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Eric Bremer for support.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant R01 AR44619.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.
¶ To whom correspondence and reprint requests should be addressed: Div. of Dermatology 107, Children's Memorial Hospital, 2300 Children's Plaza, Chicago, IL 60614. Tel.: 773-880-4698; Fax: 773-880-3025; E-mail: apaller@northwestern.edu.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M006097200
2 X. Wang, P. Sun, A. Al-Qamari, T. Tai, I. Kawashima, and A. S. Paller, unpublished data.
3 X. Q. Wang, P. Sun, M. O'Gorman, T. Tai, and A. S. Paller, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: FN, fibronectin; HRP, horseradish peroxidase; NAB, nickel-agarose binding; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; DAB, 3,3'-diaminobenzidine tetrahydrochloride; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; PNGase F, peptide N-glycosidase F; ConA, concanavalin A; UEA-1, U. europaeus agglutinin-1; EGFR, epidermal growth factor receptor.
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