©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell Surface Calreticulin Is a Putative Mannoside Lectin Which Triggers Mouse Melanoma Cell Spreading (*)

Tracy K. White , Qiang Zhu , Marvin L. Tanzer (§)

From the (1)Department of BioStructure and Function, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030-3705

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

It is well established that components of the extracellular matrix (ECM)()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).

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 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.

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.


EXPERIMENTAL PROCEDURES

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.


RESULTS

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.


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.




DISCUSSION

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 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.

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 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.

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 1 integrins of B16 cells and how it reaches the cell surface from the interior.


FOOTNOTES

*
This research was supported by National Institutes of Health Research and Training Grants R01 AR17220 and T32 AR07340. A portion of this work was presented in poster form (White, T. K., Zhu, Q., and Tanzer, M. L. (1994) Mol. Biol. Cell5, 60a). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 203-679-2900; Fax: 203-679-2910.

The abbreviations used are: ECM, extracellular matrix; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; TBS, Tris-buffered saline; NHS, N-hydroxysuccinimide; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; IgY, immunoglobulin Y; BSA, bovine serum albumin; EHS, Engelbreth-Holm-Swarm; IgG, immunoglobulin G; DMEM, Dulbecco's modified Eagle's medium.

T. K. White and M. L. Tanzer, unpublished data.


ACKNOWLEDGEMENTS

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.


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