[CANCER RESEARCH 48, 7173-7178, December 15, 1988]

Characterization of a Melanoma Antigen with a Mouse-specific Epitope Recognizedby a Monoclonal Antibody with Antimetastatic Ability1

Hisako Sakiyama,2 Eiki Matsushita, Ichiro Kuwabara, Mutsumi Nozue, Toshiko Takahashi, and Masaru Taniguchi

Division of Physiology and Pathology, National Institute of Radiological Sciences, Anagawa, Chiba, Japan 260 ¡H.S., M. N., T. T.], and Division of MolecularImmunology, Center for Neurobiology and Molecular Immunology, School of Medicine, Chiba University, Chiba, Japan 280 [E. M., l. K., M. T.]

ABSTRACT

We have characterized properties of a melanoma antigen with a mouse-specific melanoma epitope expressed on B16 melanoma by using synge-neic monoclonal antibodies with antimetastatic ability. The moleculerecognized by the antibody is a membrane glycoprotein with a molecularweight of 80,000. Studies on tunicamycin treatment indicated that thecore size of the molecule appeared to have a molecular weight of 69,000and also suggested that the carbohydrate moiety was greatly responsiblefor the conformation of the mouse melanoma epitope. The antigen wasreleased or shed into the culture medium from the cell surface, and theturnover rate of the antigen was within 1.5 h.

INTRODUCTION

Metastasis of tumor cells is known to be a complex phenomenon affected by a multitude of factors. These include loss ofadhesiveness to extracellular matrix and changes of cell surfacephenol ypos that influence immune responses and allow tumorcells to escape from host defense mechanisms (1). Cell surfacecomponents are thought to play a major role also in the interaction of tumor cells with their environment. In fact, severalinvestigators have suggested that changes in glycoproteins onthe cell surface are important in cancer metastasis (2). Inaddition to the above findings, recent studies on inhibition oftumor metastasis by monoclonal antibodies against cell surfacemolecules also provide clear evidence for the importance of cellsurface properties. Nicolson (3) has shown that the expressionof the corresponding antigen on tumor cells which was definedby monoclonal antibodies correlates with enhanced colonization of tumor cells. Moreover, Vollmers, Birchmeier, and co-workers (4-7) have described monoclonal antibodies that interfere with adhesion of tumor cells to the extracellular matrix,such as fibronectin and laminin, and inhibit lung colonizationof B16 melanoma.

Gunji and Taniguchi (8) have also reported that a singleinjection of a syngeneic monoclonal antibody (M562) recognizing a mouse-specific melanoma antigenic epitope significantlyreduced the numbers (1 of 10 of control) of lung metastaticcolonies of B16 melanoma cells in vivo. Therefore, it is quitelikely that the antigen recognized by the antibody might associate with some metastatic potential of B16 melanoma cells.Furthermore, Nozue et al. (9) have demonstrated that B16melanoma variant clones losing expression of the M562 andM622 antigens largely showed a reduced metastatic ability,correlating with a loss of adhesiveness to extracellular matrix.

Here, we describe the biochemical properties of a mousemelanoma membrane glycoprotein recognized by a monoclonal

Received 5/16/88; revised 9/1/88; accepted 9/14/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by Special Coordination Funds for Promoting

Science and Technology from the Science and Technology Agency of Japan and,in part, by Grants-in-Aid for Cancer Research and for Scientific Research onPriority Areas from the Ministry of Education, Science, and Culture, Japan; thePrincess Takamatsu Cancer Foundation; and the Uehara Memorial Foundation,Japan.

2To whom requests for reprints should be addressed.

antibody, which might play an important role in lung metastasisof B16 melanoma cells.

MATERIALS AND METHODS

Monoclonal Antibodies. Three antimelanoma monoclonal antibodies(M562, M622, and M2590) were established by fusion of P3U1 cellswith C57BL/6 spleen cells hyperimmunized with syngeneic B16 melanoma cells, and their properties have been described elsewhere (10).The isotype of these antibodies was IgM. M2590 was shown to recognize hematoside ((ÃŒM.I)(11). R-l, a monoclonal antibody (IgM) raisedagainst a protease of hamster fibroblasts,3 and anti-Thy-1 (IgM) wereused as controls for anti-mouse melanoma antibodies (M562 andM622).

Cell Lines and Their Culture. B16 melanoma cells were maintainedin RPMI-1640 medium supplemented with 4% fetal calf serum. MEA-1, a subclone of B16, expresses melanoma antigen recognized by M562,M622, and M2590. A mutant clone, MEB-4, which does not expressthe antigen detected by either M562 or M622, was established bymutagenizing B16 melanoma cells with jV'-nitro-W-nitrosoguanidine

as described elsewhere (9).Immunoprecipitation. Subconfluent cell monolayers were cultured in

the presence of [3H]glucosamine (10 fiCi/ml) or [14C]glucosamine (3¿iCi/ml)for 3 days or [35S]cysteine (50 nCi/ml) overnight. The radio-

labeled cells were washed, harvested with EDTA, and solubilized bysonication in 50 mM Tris-HCl (pH 7.5) buffer containing 0.5% NP-40," 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, and 150

mM NaCl. Insoluble materials were removed by centrifugation at105,000 x g for 30 min, and the lysate was used for immunoprecipita-tion. The conditioned medium of the labeled cells was precipitated with70% ammonium sulfate. The precipitates were dissolved with waterand dialyzed against 50 mM Tris-HCl (pH 7.4): 150 mM NaCl buffer.NP-40 and deoxycholate were then added to make a final concentrationof 0.5%. The cell lysate and the conditioned medium were incubatedwith purified antibodies (50 ^g/ml) overnight at 4°C.The antigen-

antibody complexes were precipitated with anti-mouse IgM coupled toSepharose (Zymed Lab., Inc.) and washed with 50 mM Tris-HCl (pH7.5) containing 150 mM NaCl and 0.5% NP-40. The precipitates wereanalyzed by SDS-PAGE under reducing conditions. The gel was processed for fluorography using Enlightning (New England Nuclear, Boston, MA).

Cell Surface lodination and Affinity Purification. Cells were harvestedwith EDTA at subconfluency and iodinated using lactoperoxidase (0.2mg/ml), '"I (200 MCi/107 cells; Na'"l; New England Nuclear), and

0.06% H2O2 as described (12). Iodinated cells were washed 3 timeswith 10 mM KI in PBS. The cell lysates were incubated with antibody-coupled Sepharose overnight at 4°C.The antigen was eluted with 3 M

NaSCN in PBS (pH 7.4) and analyzed on SDS-PAGE.Treatment of gpSOwith Various Glycosidases. The antigen was treated

with sialidase (0.1 unit/ml; Nakarai Chem., Ltd., Tokyo, Japan) in 50mM sodium acetate buffer (pH 5.2), fucosidase (65 milliunits/ml) in100 mM sodium citrate phosphate buffer (pH 6.5), or Endo H (200milliunits/ml) in 150 mM citrate phosphate buffer (pH 5.0) containing100 mM NaCl and 0.1% bovine serum albumin. To treat the antigenwith Endo D (200 milliunits/ml) in 50 mM citrate phosphate buffer

3 H. Sakiyama, unpublished result.4 The abbreviations used are: NP-40, Nonidet P-40; TM, tunicamycin; gp,

glycoprotein; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; Endo H, endoglycosidase H; Endo D,endoglycosidase D; Endo F, endoglycosidase F; WGA, wheat germ agglutinin;GlcNAc, W-acetylglucosamine.

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(pH 6.S), it was first incubated with endogalactosidase (200 milliunits/ml) in 5 mM sodium acetate buffer (pH 5.8). The glycosidases (Endo Hand Endo D) were obtained from Seikagaku Kogyo Co., Ltd., Tokyo,Japan, and Endo F was purchased from Boehringer Mannheim Ya-manouchi Co., Tokyo, Japan.

Immunofluorescent Staining. Cells were inoculated on glass cover-slips. At various time intervals after inoculation, cells were fixed with10% formalin and stained with either M562 or M622 as describedpreviously (13).Antibody-binding Inhibition Assay. Melanoma cells (2 x 10") were

incubated with various doses of noniabeled monoclonal antibodies at4°Cfor 1 h, washed 3 times with PBS, and then incubated with 50 n\of '"I-labeled monoclonal antibodies (100 ng/m\; specific activities of

M562, M622, and M2590 were 1146, 606, and 957 cpm/ng, respectively) at 4°Cfor 1 h. The radioactivity was counted by an autogamma

counter (ARC500; Aloca Co., Tokyo, Japan).

RESULTS

Immunoprecipitation

A B16 melanoma subclone (MEA-1) was labeled with glu-cosamine and immunoprecipitated by monoclonal antibodies.As shown in Fig. 1/4, both M562 and M622 specifically precipitated a glycoprotein with a molecular weight of 80,000 (gp80)from both cell lysates (Fig. I A, Lanes 2 and 3, respectively) andconditioned medium (Fig. \A, Lanes 5 and 6). The controlantibody did not precipitate gp80 (Fig. IA, Lanes 4 and 7). Thesame antigen was also immunoprecipitated from the parentB16 melanoma cells labeled with either [3H]glucosamine or[35S]cysteine (data not shown). Moreover, both M562 and

M622 also precipitated gp80 from the surface of iodinatedMEA-1 cells (Fig. \B, Lanes 1 and 2, respectively). This suggests that the gp80 is indeed expressed on the outer surface ofthe membrane.

As shown in Fig. 2, gp80 was not precipitated from MEB-4,a mutant subclone, which was negative for reactivity towardboth M562 and M622 antibodies. When NIH 3T3 or L-cells,negative for M562 or M622 reactivity, were labeled with['Hjglucosamine under the same conditions as described above,

gpSO was not precipitated by these antibodies (data not shown).

Effect of Tunicamycin on Antigen Expression

Study with Immunoprecipitation. When the cells were labeledwith [14C]glucosamine in the presence of TM (2 Mg/ml), no

radioactive molecule was specifically precipitated by the antibodies (Fig. 3A, Lanes 2 and 3). The same result was obtainedwhen the immunoprecipitation was carried out using externallylabeled cells that had been treated with TM (Fig. 3Ä,Lane 2).Meanwhile, when cells were labeled with [35S]cysteine in the

presence of TM, a M, 69,000 molecule was precipitated byM562 and M622 (Fig. 3C, Lanes 4 and 5), but not by thecontrol antibody (Lane 6) instead of gpSO precipitated fromTM-nontreated control cells (Fig. 3C, Lanes 1 and 2).

Study with Immunostaining. The antigen recognized by M622and M562 antibodies was found to be resistant to trypsintreatment (data not shown). We therefore treated cells with0.02% Pronase for 2 min at room temperature to remove theantigenic determinant from the cell surface. Cells were thencultivated in the presence or absence of TM (2 ¿ig/ml).Cellscultured in the absence of TM became stainable with bothM562 and M622 within 1.5 h after the treatment. The reactivityof the cells with the antibodies increased thereafter and reacheda normal level by 4 h after Pronase digestion (Fig. 4, A to D).On the other hand, when cells cultured in the presence of TMdid not spread out their cytoplasm even at 24 h after inoculationand under these conditions, no cells became significantly reactive with M562 and M622 by this time, If TM-treated cellswere made permeable by treatment with 0.2% Triton X-100, aslight increase in reactivity of the cells with M562 was observed(data not shown).

Biochemical Characterization of the Antigen

From the results that no specific molecule was immunoprecipitated from the cells labeled with radioactive glucosamine inthe presence of TM and also that TM-treated cells showed veryweak reactivity with either M562 or M622, we expect that thegpSO contains asparagine-linked oligosaccharides. To investigate what kinds of oligosaccharides were linked to the peptide,various glycosidases were tested for their abilities to remove thecarbohydrate chains. As shown in Fig. 5, treatment with EndoH and Endo D did not change the mobility of the molecule onSDS-PAGE. By the treatment with sialidase, the antigen lostapproximately 8,000 dallons. The experiments using [3H]fu-cose-labeled materials demonstrate that the antigen has fucosein the carbohydrate moiety (data not shown). However, fucosi-dase did not cause the detectable decrease of the molecularweight of the antigen on the SDS-PAGE.

BFig. 1. Immunoprecipitation of melanoma

antigen from radiolabeled Hid melanoma(MEA-1) cells. In A, extracts of [MC]GlcNAc-labeled MEA-1 cells (Lanes 1 to 4) or conditioned medium of MEA-1 cells (Lanes 5 to S)were immunoprecipitated with M622 (Lanes 2and 5), M562 (Lanes 3 and 6), or control IgMantibody (Lanes 4 and 7) as described in '•Materials and Methods." Immune complexes

were collected by centrifugation, solubilizedwith the gel sample buffer (IS), and subjectedto SDS-polyacrylamide gel (10%) electropho-resis. Lane I, cell Usate; Lane S, concentratedconditioned medium. The '4C-labeled proteins

were detected by fluorography. In B, solubilized '"I-labeled MEA-1 cells were incubatedwith M622 (Lane 1), M562 (Lane 2), or controlIgM antibody (Lane 3) without antibody (Lane4). Immune complexes (Lanes 1 to 3) and celllysate (Lane 4) were analyzed on SDS-polyacrylamide gel (10%) electrophoresis. '"I-labeled proteins were made visible by autoradi-ography.

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Since the antigen contains sialic acid and glucosamine in theoligosaccharide chains, we tested if the molecule binds to WGA.The antigen was first purified with a M622 antibody-conjugatedSepharose column and then applied to a WGA column (HonenOil Co., Tokyo, Japan). The gp80 was detected in the eluatefrom the lectin column with 0.2 M Ar-acetyl-glucosamine (Fig.

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5B). The mobility of the molecule on SDS-PAGE under reducing and nonreducing conditions is shown in Fig. 5C Themolecular weight of the antigen did not significantly changebefore and after reduction.

Inhibition of Antibody Binding with Monoclonal Antibodies

In order to investigate topographical localization of themouse melanoma epitopes recognized by two antibodies (M562and M622), melanoma cells were first incubated with one ofthe unlabeled monoclonal antibodies to block one epitope,followed by an incubation with radiolabeled antibodies whichare either identical to or different from the first antibody. Asshown in Fig. 6, the binding of the antibodies was mutuallyinhibited to the same extent as is caused by the same antibody.This binding inhibition assay was specific, because M2590recognizing the carbohydrate moiety of GMJ did not affect anybinding of M562 or M622 antibody.

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Fig. 2. Immunoprecipitation from [3H]GlcNAc-labeled M562 and M622 negative mutant B16 melanoma (MEB-4) cells. Cell lysate (Lanes 1 to 5) andconditioned medium (Lanes 6 to 10) of MEB-4 cells were immunoprecipitatedwith M622 (Lanes 1 and 6), M562 (Lanes 2 and 7), M2590 (Lanes 3 and «),control IgM antibody (Lanes 4 and 9), or none (Lanes 5 and JO), solubilized withgel sample buffer, and subjected to 5 to 15% gradient SDS-polyacrylamide gelelectrophoresis. 3H-labeled proteins were detected by fluorography.

DISCUSSION

We have identified and characterized a mouse melanoma-specific antigen recognized by syngeneic monoclonal antibodies, M562 and M622, obtained by fusion of P3U1 and C57BL/6 spleen cells primed with syngeneic B16 mouse melanoma(10). The melanoma specificity of these antibodies has beencharacterized and was found to be specific for mouse melanomacells (10). The antibodies react only with mouse melanomas,but not with other tumors, including neuroblastoma, fibrosar-comas, T-cell and B-cell lymphomas, etc., of mouse and humanorigin. Moreover, the antibody activities of M562 and M622were not absorbed with high doses of various normal tissues,including brain, skin, and eye, with normal melanocytes.

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Fig. 3. Immunoprecipitation of the antigen from TM-treated cells. B16 melanoma (MEA-1) cells were cultured in the presence or absence of TM. The cells werelabeled with [14C)GlcNAc (A), [Na'"I] (B), or ["Sjcysteine (C). Radioactive proteins were detected by autoradiography. A, MC-GlcNAc-labeled extracts of TM-nontreated cells precipitated with M622 (Lane 1). Extracts of TM-treated cells precipitated with M562 (Lane 2), M622 (Lane 3), and no antibody (Lane 4). B, '"I-labeled cell extract of TM nontreated (Lane 1) and TM treated (Lane 2) and precipitated with M622. In C, cells were labeled with ["Sjcysteine in the absence (Lanes

1 to 3) or presence (Lanes 4 to 6) of TM. Cell extracts were immunoprecipitated with M622 (Lanes I and 4), M562 (Lanes 2 and 5), and control antibody (Lanes 3ando).

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-TM +TM

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Fig. 4. EfTecIof TM on expression of the melanoma antigen. B16 melanoma (MEA-1) cells were treated with Pronaseand incubated on glass overslips as describedin "Results." At the times indicated after incubation in the presence (I to H) or absence (I to D) of TM, cells were fixed and immunostained with M562 as describedin "Materials and Methods."

With respect to the functional activity of the monoclonal to inhibit antimelanoma cytotoxic T-lymphocyte activity in theantibody, M562 possesses the ability to block experimental effector phase (14). These results indicate that the M562 mel-

lung metastasis of melanoma (8) and partially but significantly anoma epitope is important in tumor immune responses.7176

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Fig. 5. Characterization of the melanoma antigen.. I. glycosidase digestion ofthe antigen. The melanoma antigen labeled with [1H]GlcNAc was isolated byM622-conjugated Sepharose and was untreated (Lane 1) or treated with sialidase(Lane 2), fucosidase (Lane 3), Endo H (Lane 4), and Endo D (Lane 5). Electro-phoresis on a SDS-polyaerylamide gel (10%) was followed by fluorography. B,binding of the antigen to WGA. [3H]GlcNAc-labeled anitgen purifiai by M622antibody column was applied on a WGA-agarose column. It was eluted with 0.2M W-acetyl-glucosamine in PBS. The eluate was analyzed on SDS-PAGE (10%).C, effect of 2-mercaptoethanol on the mobility of the antigen. Immunoprecipitatesof ['HJGlcNAc-labeled antigen were electrophoresed under nonreducing (Lane 1)

and reducing conditions. (Lane 2).

The molecule recognized by these antibodies is a membraneglycoprotein (gp80), since the same molecule can be labeledwith radioactive cysteine, glucosamine, and fucose, and bysurface iodination with 125I(Fig. 1). As the antibody precipitateda [35S]cysteine-labeled M, 69,000 molecule from TM-treated

B16 melanoma cells (Fig. 3), this represents the size of the coreprotein. Therefore, about 14% of the molecular weight appearsto consist of carbohydrates. In our previous studies, we foundthat M 562 reacted with several proteins with different molecular weights including gpSO (9). In the present data we canclearly demonstrate that the epitope is on gpSO.

The results obtained from the TM treatment experiment(Figs. 3 and 4) also strongly suggest that the sugar moiety isimportant for transport of the gpSO to the cell surface and forthe conformation of the melanoma epitope, because the majority of antibody reactivity to the cells was greatly reduced afterTM treatment. Only a faint M, 69,000 band was detected byimmunoprecipitation from TM-treated cells (Fig. 3), while noimmunostaining was detected (Fig. 4). The discrepancy betweenimmunostaining and immunoprecipitation analyses seems tobe due to the sensitivity of these different methods used. Con-formational changes of the molecule by removal of the carbohydrate moiety are more likely to explain the decrease ofantibody reactivity after TM treatment. Change in molecularconformation of the antigen might also occur after its releasefrom cells, since antibody M562 effectively precipitated antigenfrom cell lysate but barely precipitated antigen released into themedium.

The metabolic rate of this molecule seems to be rather high,since the results of Pronase treatment demonstrated that thereexpression of the molecule occurred within 1.5 h and the levelof the expression became normal 4 h after Pronase treatment.

A similar melanoma antigen recognized by a syngeneic monoclonal antibody with antimetastatic activity has been reportedby Vollmers et al. (4-7). They reported that their antibody

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Fig. 6. Binding competition assay. Cells (2 x 10s) were incubated in a 96-well

flexible microtiter plate with nonlabeled M562 (A), M622 (B), or M2590 (C) atthe concentrations indicated and then incubated with 50 pg/ml of labeled M562(154,900 cpm; O), M622 (65.000 cpm; •¿�),or M2590 (140.200 cpm. x). Afterincubation, the cells were washed and dried, and the wells were cut off and countedwith an autogamma well.

detected the membrane glycoprotein with a molecular weightof 83,000. However, the molecular weight of their melanomaantigen was estimated to be 105,000 under nonreduced conditions, and the core protein had a molecular weight of 72,000which was estimated after endoglycosidase F treatment (7).Therefore, the glycoprotein does not seem to be identical to thegpSO described in this report.

Concerning the epitopes recognized by the antibodies, wecannot obtain a definite conclusion from the data shown hereof whether both or either one of them recognizes carbohydratemoieties or peptide determinants. Even if the antibodies recognize the peptide epitopes, the carbohydrate seems to belargely responsible for the conformation of the epitope and maybuild up the tertiary structure of the core protein architecture.

Experiments using these two monoclonal antibodies on inhibition of antibody competition binding suggest that these twodeterminants may be topographically closely located to eachother because one antibody (M562 or M622) competes andinterferes with the binding of the other. However, we havepreviously demonstrated that M562 but not M622 antibodydoes block experimental lung metastasis of B16 melanoma invivo (8). This suggests that M562 and M622 epitopes aredifferent from each other and also that only the M562 epitopeis a functional site and is responsible for lung metastasis. It isthus quite important to determine the primary sequence of the

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M562 epitope of the melanoma antigen in order to understandthe molecular basis of lung metastasis of melanoma cells. Thiswork is now in progress.

ACKNOWLEDGMENTS

We express our thanks to Chimi Saito and Kazuko Kuriiwa forpreparing this manuscript and Shigeru Sakiyama for reading over themanuscript.

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3. Nicolson. G. L. Organ colonization and the cell-surface properties of malignant cells. Biochim. Biophys. Acta, 695: 113-176, 1982.

4. Vollmers, P. H., and Birchmeier, W. Monoclonal antibodies inhibit theadhesion of mouse B16 melanoma cells in vitro and block lung metastasis invivo. Proc. Nati. Acad. Sci. USA, 80: 3729-3733, 1983.

5. Vollmers, H. P., and Birchmeier, W. Monoclonal antibodies that preventadhesion of B16 melanoma cells and reduce metastasis in mice: cross-reactionwith human tumor cells. Proc. Nati. Acad. Sci. USA, 80: 6863-6867, 1983.

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7. Wieland, I., Muller, G., Brann, S., and Birchmeier, W. Reversion of thetransformed phenotype of B16 mouse melanoma: involvement of an 83 Kdcell surface glycoprotein in specific growth inhibition. Cell, 47: 675-685,1986.

8. Gunji, Y.. and Taniguchi, M. Syngeneic monoclonal anti-melanoma antibodythat inhibits experimental lung metastasis of B16 melanoma. Jpn. J. CancerRes., 77:595-601. 1986.

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11. Hirabayashi, Y., Hamaoka, A., Matsumoto, M., Matsubara, T., Tagawa, M.,Wakabayashi, S., and Taniguchi, M. Syngeneic monoclonal antibody againstmelanoma antigen with interspecies cross-reactivity recognized GMj, a prominent ganglioside of BI6 melanoma. J. Biol. Chem., 260: 13328-13333,1985.

12. Phillips, D. R., and Marrison, M. Exposed protein on the intact humanerythrocyte. Biochemistry, W: 1766-1771, 1971.

13. Sakiyama, H., Takahashi, T., Hirabayashi, T., and Taniguchi, M. Changesof topographical distribution of GMJ during cell spreading and growth;examination by immunostainingwith monoclonal antibody against GM.i.CellStruct. Fund., 12: 93-105, 1987.

14. Ono, K., Takahashi, K., Hirabayashi, Y., Itoh, T., Hiraga, Y., and Taniguchi,M. Mouse melanoma antigen recognized by Lyt-2~, L3T4 cytotoxic T-

lymphocytes. Cancer Res., in press, 1988.15. Laemmli, U. K. Cleavage of structural proteins during the assembly of the

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1988;48:7173-7178. Cancer Res   Hisako Sakiyama, Eiki Matsushita, Ichiro Kuwabara, et al.   Antimetastatic AbilityEpitope Recognized by a Monoclonal Antibody with Characterization of a Melanoma Antigen with a Mouse-specific

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