From the
B16 mouse melanoma cells adhere to and spread on laminin. We
have previously shown that cell spreading is uncoupled from adhesion
when unglycosylated laminin is used as a substratum; spreading was
restored by a Pronase digest of laminin which became inactive when it
was specifically depleted of its mannoside peptides; spreading was also
specifically restored by mannosides such as mannan, Man9, and Man6, but
not Man3. The effector mannosides bind to a cell surface receptor,
previously shown by direct and indirect methods. We have now identified
the receptor as cell surface calreticulin by isolating it via mannan
affinity chromatography and showing its sequence identity with mouse
calreticulin. Anti-calreticulin antibodies confirm this identity,
decorate the B16 cell surface, and block cell spreading. Purified B16
cell calreticulin from whole cell lysates successfully competes with
cell surface calreticulin and prevents cell spreading. The composite
data implicate cell surface calreticulin as a putative lectin that must
be occupied to initiate spreading of laminin-adherent B16 cells.
It is well established that components of the extracellular
matrix (ECM)
We have previously demonstrated that B16 mouse melanoma cells
plated onto laminin demonstrate rapid adhesion followed by extensive
spreading. A number of studies indicate that
Identification and characterization of the
responding B16 cell surface lectin became our next objective. Cell
surface lectins which recognize mannosides are potential candidates but
have not previously been implicated in cytoskeletal responses (Taylor et al., 1990). In the present study we have isolated the B16
surface putative lectin responsible for initiation of spreading of
laminin-adherent cells. The putative lectin is mouse calreticulin, as
shown by sequence identity, antibody immunostaining of calreticulin in
B16 cell lysates, on cell surfaces and of purified B16 cell
calreticulin. Antibodies block cell spreading, as does exogenous B16
calreticulin, serving as a competitor. These composite results indicate
that cell surface calreticulin must be engaged to trigger spreading of
laminin-adherent B16 mouse melanoma cells.
In order to purify the cell surface lectin responsible for
triggering cell spreading, an oligomannoside affinity column was
prepared by coupling yeast mannan to agarose beads. Mannan affinity
chromatography has been previously used successfully to purify plasma
lectins (Holmskov et al., 1993), and the methodology was
adapted to our needs. When a clarified lysate of B16 cells was passed
over the affinity matrix, we noted the retardation and significant
purification of a 60-kDa protein; the protein could be eluted with
suitable concentrations of either mannose or EDTA. As demonstrated in Fig. 1, biotinylation of cell surface proteins prior to cell
lysis and purification indicates that a portion of the 60-kDa
oligomannoside-binding protein resides on the cell surface. Cell
surface labeling using either NHS-biotin or sulfo-NHS-biotin yielded
similar results. Lane 6 serves as a control which shows that
the 60-kDa protein does not have intrinsic streptavidin binding
properties. Other controls were done which showed: 1) intracellular
actin was not biotinylated by the surface labeling procedure; and 2) a
pseudo-affinity column, prepared without mannan, did not bind the 60
kDa protein.
The composite results implicate B16 cell surface calreticulin
as the effector lectin that must be engaged by a suitable mannoside to
initiate spreading of laminin-adherent cells. Although calreticulin was
initially identified as a calcium-binding protein residing in the
endoplasmic reticulum, data from a variety of laboratories have
subsequently demonstrated its presence in a number of additional
cellular compartments; in some cells the molecule has also been
reported to be perinuclear, intranuclear, cytosolic, and/or to reside
on the cell surface (reviewed by Burns et al.(1994), Dedhar
(1994), Nash et al.(1994)). It is of particular interest to
these studies that calreticulin has previously been identified on the
surface of B16 cells where it serves as a major immunogen (Gersten et al., 1990, 1992). Our data suggest that the cell surface
molecule is similar, if not identical, to calreticulin previously
identified in other mouse cells and tissues (reviewed by Michalak et al.(1992)). Calreticulin also occurs on the surfaces of
human leukocytes, platelets, and endothelial cells; the leukocyte form
has been termed the collectin receptor, because it specifically binds
plasma proteins of the collectin category (Malhotra, 1993). Some data
suggest that leukocyte cell surface calreticulin may differ in
molecular properties from cell interior calreticulin; differences in
charge and size are reported (Eggleton et al., 1994).
Calreticulin immunostaining has been visualized in a variety of
cells (Opas et al., 1991). In those experiments the cells had
been permeabilized, and staining appears similar to that seen in Fig. 3B, where both cell surface and interior
calreticulin are seen. In fact, when the authors depleted the cells of
calreticulin by exposure to the calcium ionophore, A23187, the diffuse
immunostaining disappeared and the endoplamic reticulum network became
more prominent, suggesting that surface calreticulin may be shed; B16
cells and human leukocytes are known to shed calreticulin (Eggleton et al., 1994; Gersten et al., 1990, 1992). In
addition, immunostaining of myotubes showed prominent
``lacunae'' of calreticulin which seemed to be associated
with the cell surface (Opas et al., 1991). The different
immunostained cells displayed diffuse patterns which are consistent
with variable amounts of calreticulin on their cell surfaces. The
staining of surface calreticulin in our studies (Fig. 3A) resembles the previously observed pattern of
man-nan bound to the surface of the same line of B16 cells
(Chandrasekaran et al., 1994a).
B16 cells adhere to laminin
via several
In light of these observations, it is tempting to speculate
that calreticulin may interact directly with the integrin ectodomains
to mediate the cell signaling events which initiate cell spreading. In
fact, calreticulin of the endoplasmic reticulum has recently been shown
to be a molecular chaperone for immature N-linked
glycoproteins (Nauseef et al., 1995) and, based upon its
extensive similarity to the well characterized molecular chaperone
calnexin (Ware et al., 1995), calreticulin probably has
discrete binding sites for: 1) protein recognition and 2) recognition
of immature carbohydrates. Since B16 cell integrin ectodomains are N-glycosylated with mature glycosyl groups (Kawano et
al., 1993), cell surface calreticulin would probably bind to
integrins via its protein recognition domain, leaving its lectin
recognition domain available for binding to the laminin mannosides.
Direct protein-protein interactions between the integrins and
calreticulin have been demonstrated; Leung-Hagesteijn et al. (1994) have shown that calreticulin binds specifically to
integrins via its N-terminal region. This binding takes place via the
highly conserved KXGFFKR consensus sequence found in the
cytoplasmic domain of the
In summary, the ability of
laminin-adherent B16 mouse melanoma cells to spread is dependent upon
engagement of the ligand binding site of cell surface calreticulin.
Specific oligomannosides, namely Man6-8, present in mouse tumor
laminin and mouse cell-secreted laminin, must be recognized by B16 cell
surface calreticulin. Such recognition must either be at the time that
the integrin is engaged or subsequently, in order for the cells to
spread. Future studies should determine how cell surface calreticulin
communicates with
We are grateful to Dr. Ari Helenius for providing
purified rat liver calreticulin. We thank Dr. H.-D. Söling for the
generous gift of anti-calreticulin antiserum used in these studies. We
would also like to thank Dr. Gloria Gronowicz for helpful suggestions
concerning fluorescent labeling studies and Joe Leykam of the Michigan
State University Macromolecular Structure Facility for helpful
discussions concerning microsequencing of the purified protein.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)are able to support adhesion and
spreading of a variety of cell types. In many cases, the interaction of
cell surface integrins with specific peptide sequences in the ECM
molecules is responsible for triggering the adhesion and spreading
response (Diamond and Springer, 1994). Integrin ectodomains undergo
well characterized interactions with components of the ECM, whereas
integrin cytoplasmic domains are known to interact with a variety of
cytoskeletal components, including
-actinin and talin. Although
details of the signaling mechanisms remain to be elucidated,
integrin-ECM interactions result in the clustering of integrins into
focal contacts, where they provide a site on the cell membrane for
tethering the cytoskeleton, and for the subsequent initiation of
signaling events which mediate cell spreading (Schaller and Parsons,
1993).
1 integrins are
involved in the adhesion process. Kramer et al.(1991) have
clearly described the involvement of
1 integrins in the adhesion
of mouse melanoma cells to the ECM. In addition, our laboratory has
demonstrated the clustering of
1 integrins at focal contacts when
B16 cells are seeded onto a laminin substratum (Chandrasekaran et
al., 1994a). Although integrin-ECM interactions are important for
cell adhesion in this system, cell adhesion and spreading are uncoupled
when these cells adhere to laminin which is devoid of its repertoire of N-linked carbohydrates (Dean et al., 1990). Those
carbohydrates span a range of structures from oligomannosides to
complex tri- and tetra-antennary substituents; relative abundance of
the different structures is quite variable (Arumugham et al.,
1986; Fujiwara et al., 1988; Knibbs et al., 1989). A
Pronase digest of laminin restores cell spreading, but it is
ineffective when specifically depleted of its mannoside peptides;
spreading is also specifically restored by suitable mannosides, e.g. mannan, Man9, and Man6, but not Man3 (Chandrasekaran et al., 1994a, 1994b). This response appears to be a surface
receptor mediated event, as demonstrated by visualization of mannoside
probes, and by binding studies with radiolabeled Man9 (Chandrasekaran et al., 1994a). Although interactions of
1 integrins with
the protein backbone of laminin are an important component in B16 cell
adhesion, our data clearly indicate that the carbohydrate moieties of
laminin play a critical role in triggering cell spreading. Furthermore,
binding of effector mannosides in the absence of integrin binding is
ineffective for initiating B16 cell adhesion or spreading
(Chandrasekaran et al., 1994b), implying that a specific order
of ligand occupancy must occur for such cellular responsiveness. Thus,
both integrin and lectin must either be occupied simultaneously, or
integrin occupancy must be followed by lectin occupancy, if cell
spreading is to occur.
Purification of Calreticulin by Mannan Affinity
Chromatography
Mannan affinity matrix was prepared by coupling
yeast mannan (Sigma) to CNBr-activated Sepharose 4B (Pharmacia Biotech
Inc.) using the protocol provided by the vendor. Routinely, B16 cells
from eight 245-mm plates were released at early confluence
by incubation with 2 mM EDTA in ice-cold Earle's basic
salt solution. Cells were washed three times with PBS (20 mM phosphate, 150 mM NaCl, pH 7.4) and subsequently lysed in
40 ml of a buffer composed of 0.02 M Tris, pH 7.4, 0.1 M NaCl, 0.5% Triton X-100, 5 mM CaCl
, 5
mM MgCl
, and 5 mM MnCl
In
addition, the lysis buffer contained the following protease inhibitors:
2 µg/ml leupeptin, 0.4 µg/ml antipain, 2 µg/ml benzamidine,
2 µg/ml aprotinin, 1 µg/ml chymostatin, 1 µg/ml pepstatin,
and 1 mM PMSF. Following lysis, the cell lysate was
centrifuged at 10,000
g for 30 min. The clarified
lysate was mixed with 3 ml of mannan affinity matrix and allowed to
rotate end-over-end at 4 °C overnight. Gel was subsequently
transferred to a column and washed with three column volumes of lysis
buffer, followed by elution with a buffer composed of 20 mM Tris, pH 7.4, 20 mM EDTA, 0.1 M NaCl, and 1
mM PMSF. Alternatively, 50 mM mannose was substituted
for EDTA in the elution buffer. Where indicated, cells were surface
labeled prior to lysis with NHS-biotin or sulfo-NHS-biotin (Pierce)
essentially as described by Hurley and Finkelstein(1990). Assessment of
cells before and after the labeling procedure indicated that both total
cell number and cell viability did not change significantly.
Microsequence Analysis of Purified Protein
Mannan
affinity-purified calreticulin obtained from B16 cell lysates was
concentrated and subjected to SDS-polyacrylamide gel electrophoresis.
Protein was subsequently blotted onto Problot membrane (Applied
Biosystems) and visualized via staining with Coomassie Blue. Tryptic
digestion of the protein, HPLC purification of the subsequent peptides,
and N-terminal microsequencing of purified peptides was performed by
the Macromolecular Structure Facility, Michigan State University, East
Lansing, MI.
Antibodies
Rabbit anti-human calreticulin
antiserum was obtained from Affinity Bioreagents, Neshanic Station, NJ.
Sheep anti-human calreticulin antiserum was the generous gift of Dr.
H.-D. Söling, Georg-August-Universität, Göttingen,
Germany. Purified avian IgY was prepared from the egg yolks of a White
Leghorn hen immunized with mannan affinity-purified B16 calreticulin.
Prior to immunization, the protein was further purified by SDS-PAGE,
and immunization was achieved by intramuscular injection of 100 µg
of purified protein in a slurry of polyacrylamide and incomplete
Freund's adjuvant. An identical boost was administered 1 month
following initial immunization. Purification of IgY was achieved using
serial polyethylene glycol precipitations of yolk protein, as described
previously (Akita and Nakai, 1992). Control IgY was purified from the
yolks of eggs collected prior to immunization. Both preparations were
monitored for purity by SDS-PAGE.
SDS-PAGE and Immunoblotting
SDS-polyacrylamide gel
electrophoresis was carried out according to the method of Laemmli
(1970) under reducing conditions. For all polyacrylamide
electrophoretic experiments 5-15% gels were used. Where
indicated, electrophoretically separated proteins were blotted onto
supported nitrocellulose (Schleicher and Schuell) using the method of
Towbin et al.(1979). Immunodetection of calreticulin on
nitrocellulose filters was achieved essentially using the protocol
supplied by Affinity Bioreagents. Visualization of immunoreactive
proteins was achieved using the ECL detection kit (Amersham Corp.).
Immunocytology
B16 melanoma cells were seeded
under standard conditions onto glass coverslips that had been precoated
with purified EHS tumor laminin (Life Technologies, Inc.) at 50
µg/ml in PBS for 2 h at room temperature. Cells were allowed to
grow overnight under standard temperature and atmospheric conditions.
All labeling steps were carried out at room temperature and were
followed by three washes in PBS. Cells were rinsed with PBS, fixed with
3% fresh paraformaldehyde in PBS for 10 min, and, where indicated,
permeabilized with 0.1% Triton X-100 in PBS for 5 min. Fixed cells were
subsequently blocked by incubation with 20 mg/ml purified swine IgG
(Sigma) for 1 h, and calreticulin was labeled using sheep anti-human
calreticulin antibody at a dilution of 1:50 in PBS containing 1% BSA,
followed by incubation for 45 min. Samples were then blocked with a
solution of 4 mg/ml swine IgG in PBS containing 1% BSA for 1 h and
treated with fluorescein-conjugated rabbit anti-sheep IgG (Pierce) at a
concentration of 30 µg/ml for 45 min. Coverslips were mounted in a
solution of 2.5% n-propyl gallate in 1:1 PBS:glycerol, and the
samples were observed with a Nikon Optiphote microscope.
Inhibition of B16 Cell Spreading by Anti-calreticulin
Antibodies
Cell-spreading assays were essentially performed as
described previously (Chandrasekaran, 1994b). Purified preimmune and
postimmunization chicken antibodies were extensively dialyzed against
PBS prior to use in cell spreading assays. B16 cells at approximately
80,000 cells/ml of serum-free DMEM were incubated with indicated
concentrations of IgY for 30 min on ice. Cells were then seeded at 8000
cells/well, in the same solution, in non-tissue culture-treated plastic
96-well plates (Falcon) which contained a surface of 5 µg/well EHS
tumor laminin (Life Technologies, Inc.). Following 1 h of incubation at
standard temperature and atmospheric conditions, cells were fixed and
stained as described previously (Chandrasekaran et al.,
1994b).
Inhibition of B16 Cell Spreading by Exogenous
Calreticulin
To assess cell spreading on laminin, 5 µg of
laminin/well in PBS was dried overnight in triplicate wells in plastic
non-tissue culture-treated 96-well plates. Subsequently, wells were
washed, and cells were seeded, as described previously (Chandrasekaran et al., 1994b). To assess the effects of purified calreticulin
on spreading induced by a laminin substratum, 5 µg/well laminin was
incubated with purified calreticulin at a molar ratio of 1:20
(laminin:calreticulin) in 20 mM Tris, pH 7.4, 0.1 M NaCl, 5 mM CaCl, 5 mM MgCl
, and 5 mM MnCl
at room
temperature for 2 h in triplicate wells in non-tissue culture-treated
96-well plates. Plates were subsequently treated as described above,
and assessment of cell spreading was performed as described previously
(Chandrasekaran et al., 1994b). For controls, hemoglobin
(Sigma) or BSA (Pierce) were substituted for calreticulin, at a molar
ratio to laminin of 20:1.
Figure 1:
Purification of a 60-kDa
oligomannoside-binding protein from cell surface-labeled B16 cells. B16
cells, surface-labeled with NHS-biotin, were subjected to mannan
affinity chromatography as described under ``Experimental
Procedures.'' Samples of whole cell lysate and column eluants were
subjected to SDS-PAGE and blotted onto nitrocellulose. Biotin labeled
proteins were visualized by treatment of blots with horseradish
peroxidase-conjugated streptavidin followed by ECL detection. Lane
1, initial cell lysate; lanes 2-4, successive
column washes showing retardation and significant purification of
60-kDa surface-labeled oligomannoside-binding protein; lane 5,
elution in the presence of 20 mM EDTA; lane 6, 60-kDa
protein purified from cells with no prior cell surface biotinylation
(this protein becomes visible following silver
staining).
Identification of the 60-kDa protein was achieved by
N-terminal microsequencing of tryptic peptides obtained from purified
protein. These analyses provided complete sequences of two different
tryptides and a partial sequence of a third tryptide (Fig. 2).
These fragments were identical to mouse calreticulin; although
calreticulin is a highly conserved protein, the lysine at position 63
is unique to mouse calreticulin. This result lends confidence that the
sequence is genuine and derives from the B16 mouse cells rather than
from the culture medium or some nonspecific contamination.
Figure 2:
HPLC profile and microsequencing of
purified 60-kDa oligomannoside-binding protein. The locations of three
sequenced fragments within the mouse calreticulin amino acid sequence
are shown. Incomplete sequence of the largest peptide was due to low
amounts of material.
Western
blot analyses revealed that B16 cell lysates showed a single
immunoreactive protein with anti-calreticulin antiserum. As expected
based on amino acid sequence identity, the mannan affinity-purified
molecule demonstrated strong immunoreactivity with the
anti-calreticulin antibodies that were tested (Fig. 3). We were
also able to demonstrate that the B16 protein purified via mannan
affinity chromatography migrated in register with calreticulin purified
from rat liver endoplasmic reticulum (Fig. 3). Immunostaining of
B16 cells demonstrated that surfaces become decorated with
anti-calreticulin antibody on both nonpermeabilized and permeabilized
cells. The surface decoration is more prominent in the former cells,
whereas the endoplasmic reticulum is more prominent in the latter
cells. In addition, bright punctate clusters of antibody are seen in
the circumnuclear region of permeabilized cells; double
immunofluorescence shows that 1 integrins co-localize with
calreticulin in these clusters (not shown).
Figure 3:
Immunostain analyses of B16 cells and
cellular calreticulin. A-C represent immunocytological
staining with sheep anti-human calreticulin antibody of B16 cells
adherent and spread on a laminin substratum. A,
nonpermeabilized cells; B, cells permeabilized with 0.1%
Triton X-100; C, control for A using secondary
antibody alone. Controls for B were identical to this panel
(not shown). Magnification, 600. D, Western blot
analysis of B16 cell lysate and mannan affinity-purified calreticulin
with the same antibody used in A-C. Lane 1, B16
cell lysate stained with Coomassie Blue; lanes 2 and 3, Western blot of B16 cell lysate and mannan
affinity-purified calreticulin, respectively. E, Western blot
analysis of mannan affinity-purified calreticulin (lane 1) and
calreticulin purified from rat liver endoplasmic reticulum (lane
2). This analysis was performed using rabbit anti-human
recombinant calreticulin antiserum as described under
``Experimental Procedures.''
Competition experiments
showed that purified B16 cell calreticulin successfully prevented cell
spreading (Fig. 4). Anti-calreticulin antibodies also prevented
cell spreading; a preparation of avian immune IgY had an effective
range of 0.8 through 1.4 µg/µl for complete inhibition of
spreading (Fig. 4). Partial spreading was not scored in this
assay; presumably the effective range will be broader if partial
spreading is scored.
Figure 4:
Inhibition of B16 cell spreading. B16
cells were seeded under conditions described in detail under
``Experimental Procedures.'' a, cells adherent on a
glycosylated laminin surface containing preadsorbed B16 cell
calreticulin, at a 20-fold molar excess over laminin. b, cells
adherent and spread on a glycosylated laminin surface. Identical
results were seen when a 20-fold molar excess of hemoglobin or bovine
serum albumin has been preadsorbed to the laminin surfaces. c,
cells adherent and spread on a glycosylated laminin surface in the
presence of preimmune IgY, in the same concentration range as in d. d, cells adherent on a glycosylated laminin
surface in the presence of immune IgY, effective range 0.8-1.4
µg/µl. Magnification, 300.
1 containing integrins (reviewed by Kramer et
al.(1991)). We have previously demonstrated that integrins must
either be engaged simultaneously with or prior to lectin engagement in
order for cells to spread (Chandrasekaran et al., 1994a,
1994b), suggesting there must be cross-talk between cell surface
calreticulin and integrins. The notion that integrins must work in
concert with other cell surface receptors in order to mediate specific
signaling events has been well established (Gingell, 1993;
Schweighoffer and Shaw, 1992; Damsky and Werb, 1992). For example,
Shattil et al.(1994) have recently demonstrated that tyrosine
phosphorylation of focal adhesion kinase, a cytosolic tyrosine kinase
that has been increasingly implicated in integrin-mediated cell
signaling (Schaller and Parsons, 1994), requires simultaneous
occupation of both integrin and agonist receptors on the surface of
platelets. In light of these and other data, it is of interest that we
observe significant increases in tyrosine phosphorylation of focal
adhesion kinase when adherent B16 cells commence spreading, i.e. upon occupation of both integrin and lectin receptors.
(
)It is of particular interest that calreticulin has
previously been demonstrated to co-localize with
1 integrins in
clusters (focal contacts) in PC-3 cells (Leung-Hagesteijn et
al., 1994); those clusters appear similar to the ones seen in Fig. 3B, which are probably focal contacts on the lower
surface of the B16 cells where they are adherent to the laminin
surface.
integrin subunits. Although these
observations are not compatible with the present findings, which would
implicate calreticulin interactions with integrin ectodomains, the
composite data are suggestive that cell surface calreticulin may
interact directly with the integrins on the cell surface and thereby
mediate signal transduction events.
1 integrins of B16 cells and how it reaches the
cell surface from the interior.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.