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Purification and Characterization of the Platelet-Aggregating Sialoglycoprotein Gp44 Expressed by Highly Metastatic Variant Cells of Mouse Colon Adenocarcinoma 26'

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Purification and Characterization of the Platelet-Aggregating Sialoglycoprotein Gp44 Expressed by Highly Metastatic Variant Cells of Mouse Colon Adenocarcinoma 26' [CANCERRESEARCH55,767—773,February15,19951 Purification and Characterization of the Platelet-Aggregating Sialoglycoprotein gp44 Expressed by Highly Metastatic Variant Cells of Mouse Colon Adenocarcinoma 26' Minako Toyoshima, Motowo Nakajima, Takao Yamori, and Takashi Tsuruo2 Laboratory of Biomedical Research, institute of Molecular and Cellular Bioscience,The University of Tokyo. Yayoi i-i-I, Bunkyo-ku, Tokyo 113, (M. T., M. N.. T. Ti, and Cancer ChemotherapyCenter. JapaneseFoundationfor Cancer Research,Tokyo /70 fT. Y., T. Ti. Japan ABSTRACT aggregation mediated by trypsin-sensitive cell surface proteins (18). Neither the generation of thrombin nor the cellular efflux of ADP was A platelet-aggregatingsialoglycoproteiawitha molecularweightof involvedin the plateletaggregationinducedby NL-17 cells. We have 44,000 (gp44) was Immunochemically purified from highly metastatic generatedmAb 8F11 by immunizingratswith NL-17 cells to identify mouse adenocarcinoma cells. The rat monoclonal antibody (mAb) 8F11 the molecule (or molecules) involved in the induction of platelet used in the purification procedure has been generated previously against NL-17 cells derived from the mouse colon 26 cell line. mAb SF11 Inhibits aggregation.mAb 8F11 recognizeda membranesialoglycoprotein NL-17 cells from inducing platelet aggregation and suppresses their ex with a molecularweight of 44,000 (gp44) and inhibited the platelet perhnental metastasis to the lung. The purified gp44 induced mouse aggregation causedby NL-17 cells (18). The mAb 8F11 also inhibited platelet aggregation In a dose-dependent manner without any plasma the lung colonizationby NL-17 cells in experimentalmetastasis(25). component.ThisaggregationwascompletelyInhibitedbymAbSF11.The In the presentstudy, using affinity column chromatography,we gp44 was partially characterized by sequential enzymatic hydrolysis of purified the antigen recognized by mAb 8F11. The purified sialogly carbohydrates and was found to be O-glycans enriched. When gp44 was coprotein gp44 induced mouse platelet aggregation in a dose-depen sequentially treated with N-glycaaase and neuraminidase, It lost platelet dent manner,and this inductionwas inhibitedby mAb 8F11. Futher aggregation activity. Further treatment with O-glycanase resulted In a loss more, gp44 induced aggregation of washed platelets with no of the reactivity to mAb SF11.Theseresults suggestthat slalylated car requirementfor plasma components.We partially characterizedthe bohydrate chains of gp44 are Involved in the Induction of platelet aggre gation and may play an important role in the colonization of NL-17 cells properties of gp44 by chemical and enzymatic hydrolysis of carbo in the lung. hydrateslinked to the protein. We demonstratethatsialylatedcarbo hydrates of gp44 could be involved in the induction of platelet aggregationtriggeredby NL-17 cell surfacemolecules. INTRODUCTION Numerousstudieshave shownthat somehumanand animal tumor MATERIALS AND METHODS cells have platelet-aggregatingabilitiesthat correlatewith their met Animals and Tumor Cells. Female BALB/c mice were obtained from astatic potential (reviewed in Refs. 1 and 2). In ultrastructural studies, CharlesRiverJapan,Inc.(Tokyo,Japan).Miceaged8 to 12weekswereused plateletactivationis observedat the focal areaswhere the tumorcells in platelet aggregation experiments. The highly metastatic clone NL-17 was attachedto thevesselwall (3). The interactionsbetweenplateletsand established from mouse colon adenocarcinoma 26, as described previously tumor cells significantly contribute to hematogenousmetastasisin (24). NL-17 cells were cultured in RPMI 1640 containing 10% fetal bovine such ways as enhancementof tumor cell adhesionto extracellular serum and 2 mM glutamine. matrix (4—8),stimulationof tumor cell proliferation (9), evasionof mAb SF11. The mAb 8F11 (IgG2a) specific for gp44 was preparedby host immunosurveillance(10), and degradationof the proteoglycan immunizing rats with the crude membrane preparation of NL- 17 cells, as described previously (18). The purified antibody was dialyzed against PBS and scaffold of extracellular matrix (11). thenusedfor plateletaggregationexperiments. The mechanismsofplateletactivationby tumorcellsthathavebeen Lectin-Affinity Chromatography. Lectin-affinity chromatography was proposedare:(a) generationof thrombinthrougha coagulationpath carried out according to the method described by Bhavanandan and Katlic (26). way (2); (b) activationby ADP (12—14);(c)releaseof cathepsin-B WGA3 (Wako, Ltd., Osaka, Japan) was reconstituted in 0.1 M sodium bicar like protease(15); (d) activationof the arachidonatemetabolism(16); bonate buffer (pH 8.3) containing 0.2 M N-acetyl-D-glucosamine as a hapten (e) interactions of tumor cells with platelets via a sialolipoprotein sugarfor the protectionof lectin-activesitesandwascoupledwith cyanogen and/or sialoglycoprotein (17, 18); or U) a combination of these. bromide-activatedSepharose4B(PharmaciaLKB Biotechnology,Inc., Up Among these mechanisms, several factors in tumor cells and mi sala, Sweden) suspended in the same buffer at the density of 2 mg/mI. crovesicles shedby tumor cells have been studied and found to induce Confluently grown NL-17 cells were harvestedby scrapingthem off the platelet aggregationthroughthrombin generationin the presenceof cultured dishes with a rubber policeman; the cells were washed with ice-cold PBS twice,suspendedinlysisbuffer[0.5% Triton X-100, 20 mM sodium plasmacomponents(15,19—23).However,plasmamembranefactors phosphate,0.15 M NaCl, 1 mM phenylmethylsulfonylfluoride, 1 kilounit that induceplatelet aggregationthroughtheir direct interactionswith aprotinin, and 5 mM N-ethylmaleimide(jH 73)], and homogenizedin a plateletshavenot beenidentified. Wheatonhomogenizer.Aftera 30-mmlysis at 4°C,unsolublematerialswere In previous studies,we establishedseveralclones with different pelleted at 15,000 x g. The cell extracts were pooled and applied to a 3 x metastaticabilitiesfrom murinecolonadenocarcinomacellline colon 20-cm WGA-Sepharose column equilibrated with loading buffer [0.2% Triton 26 and demonstratedagood correlationbetweentheir platelet-aggre x-100,20mMsodiumphosphate,0.15MNaCl,I mMphenylmethylsulfonyl gating activity and metastaticpotential (24). Among thoseclones,a fluoride,1 kilounitaprotinin,and5 mM N-ethylmaleimide(jH 7.3)]. The highly metastatic clone, NL-17, was found to induce platelet column was washed with the loading buffer to remove all unbound proteins. Bound proteins were then eluted with 0.2 M N-acetyl-o-glucosamine in loading buffer. All procedureswerecarriedout at 4°C. Received8/22/94;accepted12/9/94. Thecostsofpublicationofthisarticleweredefrayedinpartby thepaymentofpage Immunoaffinity Chromatography Using a mAb SF11 Column. Purified charges.Thisarticle mustthereforebeherebymarkedadvertisementinaccordancewith mAb 8F11 was dialyzed against 0.1 M sodium bicarbonate buffer (pH 8.3) and 18U.S.C.Section1734solelytoindicatethisfact. coupled to Affi Gel-lO (Bio-Rad, Richmond,CA) suspendedin the same I This work was supported in part by grants from the Ministry of Education, Science and Culture,Japan,andSpecialCoordinationFundof the ScienceandTechnology Agency,Japan. 3 The abbreviations used are: WGA, wheat germ agglutinin; PAGE, polyacrylamide 2 To whom requests for reprints should be addressed. gelelectrophoresis;PRP,platelet-richplasma;TFMS,trifluoromethanesulfonicacid. 767 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1995 American Association for Cancer Research. PLATELET-AGGREGATINGGLYtOPROThINOF @LONCARCINOMA bufferatthedensityof10mg/mI,accordingtothesupplier'sinstructions.The et al. (31). Tissue extracts that contained 40 @&gofproteins were subjected to unboundantibodieswerewashedfrom thegel with 0.2 M glycinebuffer (pH SDS-PAGEina4—20%gradientgel.Theelectrophoresedproteinsinthegel 2.3) containing 0.2% Triton X-100. The proteins isolated from the lectin wereelectrophoreticallytransferredtoa nitrocellulosemembrane(Scheicher column were applied on an immunoaffinity column equilibrated with the andSchuell,Dassel,Germany).Themembranewasincubatedinblocking loading buffer at a flow rate of less than 5 mI/h. The column was washed with buffer [10% skim milk in 150 mt.i NaCl-50 mi.sTris-Ha (pH 7.3)] for 2 h at the loading buffer and 0.2 M glycine-HCI (pH 4.3) containing 0.2% Triton room temperature and further incubated with 20 p.aJml of saAb 8F11 in the x-100toremoveunboundproteins.Boundproteinswereelutedwith0.2M samebuffer for 2 h. The membrane was washed four times with washing buffer glycine-HCI (pH 2.3) containing 0.2% Triton X-100. The eluted proteins were [0.05% Tween 20, 150 mM NaG, and 50 mt@iTTiS-Ha (pH 7.3)J, followed by concentratedbyusingCentricon-30concentrators(Amicon,Danvers,MA)and incubationwith peroxidase-conjugatedanti-ratIgO (Amersham,Buckingham thenprecipitatedbyaddingalOX volumeof100%ethanoltoremoveTriton shire,United Kingdom).After washingfour timeswith washingbuffer, the x-ioo.Theprecipitatedmaterialswerewashedwith80%ethanol(v/v)to proteins were detected by using the enhanced chemiluminescence detection remove salts and air-dried. The purified gp44 was reconstitutedin PBS for system(AinershamCorp.,ArlingtonHeights,IL) andKodakX-OmatAR film. plateletaggregationexperiments.Allprocedureswerecarriedoutat 4°C. Treatment of gp44 with Glycosldases N-glycanase (32), O-glycanase Electrophoresis. Proteins eluted from the 8Fll-affinity chromatography (33), andneuraniinidase(34)werepurchasedfromSeikagakUKougyouCo., column were subjected
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