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

[CANCERRESEARCH55,767â€”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 (4â€”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 (12â€”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(15,19â€”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 4Â°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 4Â°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-PAGEina4â€”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 4Â°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 to SDS-PAGE, as described by Laemmli (27), using a Ltd (Tokyo, Japan). The purified gp44 was denatured by boiling in the 10â€”20%gradientgel in a reduced condition. The gel was silver-stained with presenceof0.1%SDSin 15mt@isodiumphosphate(pH7.0).NP-40wasadded 2D-SilverStainII â€œDaiichiâ€•(DaiichiPureChemicalsCo.,Tokyo,Japan), tothemixtureatthefinalconcentrationof0.7%.Thedenaturedgp44wasthen according to the supplier's instructions. mixed with 0.3 units of N-glycanaseandincubatedfor 24 h at 37Â°C.The Preparation of Mouse PRP and Washed Platelets. PRP was prepared N-glycanase-treatedgp44was mixed with 0.01 units of neuraminidaseand from fresh heparinizedblood drawn from BALB/c mice by cardiacpuncture, incubatedfor1 h at 37Â°Cinthesamebuffer.TheN-glycanase-andnears followed by centrifugation at 230 X g for 7 mm at room temperature. minidase-treated sample was incubated with 2 miliunits of O-glycanase for an Platelet-poor plasma was obtained by centrifugation at 1500 X g for 10 mm of additional 24 h at 37Â°C. the remainingblood at room temperature.The plateletsin PRP were counted by a CoulterCounterandadjustedto 1 X 10@/mm3byaddingplatelet-poor RESULTS plasma.Washedplateletswerepreparedby the methodof Ardlie et a!. (28) with somemodifications.PlateletsinPRPwereseparatedfromplasmaby Purification of gp44. gp44 was isolated from NL-17 cell lysates centrifugationat 400 X g for 5 mm andthenwashedwith Ca2@-/Mg@-free through two sequential affinity chromatographies, described in â€œMa PBS.PlateletswereresuspendedinCa2@-fMg@'-freePBS,countedwitha terials and Methods.â€•Inthe first WGA-affinity chromatography, the CoulterCounter,andadjustedto 6 x 10@/mm3. presenceof gp44 in pass-throughfractions was not detectedby Platelet Aggregation and Inhibition. Platelet aggregation was measured Western blot analysis using mAb 8F11 (data not shown). Triton turbidometrically by an NKK HEMA Tracer I (Niko Bioscientific Co., Tokyo, Japan).A200-s.daliquotofPRPorwashedplateletswasincubatedinacuvette at 37Â°Cunderconstantstirringintheaggregometer.After5 mm,5 @dofthe reconstitutedgp44 solution in PBS was added,and the changesin light transmittanceweremonitoredfor 10mm.Thequantitationofgp44wascarried 123 outbymeasuringUVabsorbanceusingaDU64spectrophotometer(Beckman, Irvine, CA). Protein concentration was estimated according to the following formula(29): (kDa) Proteinconcentration(mg/mi)= 1.55Ans 0.76A@ Inhibitionof gp44-inducedplateletaggregationinhibitionwascarriedout 69@ using the purified mAb 8F11 andunimmunizedrat IgG (Zymed,SanFran cisco,CA). ThoseantibodiesweredialyzedagainstPBSat 4Â°Candfiltrated through0.22-pmfilter units(Millipore, Bedford,MA) beforehand.Thegp44 wasmixedwith eachantibodyfor 30 mmat roomtemperatureandthenadded 46Wâ€” to heparinized PRP. Radlolabeling CeH Proteins. NL-l7 cells grown in 100-mm-diameter tissueculture dishesto 70% confluencywere labeledwith 0.2 mCi/mi of [35S]methionine (TRAN35S-LABEL; ICN, Irvine, CA) in methionine-free RPM! 1640mediumfor8 h. Then,gp44waspurifiedfromthelabeledcells, asdescribedabove. Deglycosylatlon of gp44 by Trifluoromethanesulfonic Acid. Deglycosy 30â€” lationofgp44wasperformedaccordingtothemethodbySojarandBahi(30). Approximately 150 ng of gp44 was treated with 100 @.dofTFMS at 0Â°Cunder nitrogenfor 4 h in a l-ml screw-cappedvialwith occasionalshaking.Subse quently, the reaction mixture was cooled to below â€”20Â°Cbyplacing in dry ice-ethanolandthenneutralizedby the gradualadditionof 60% pyridine in 21.5â€” water. The neutralizedreactionmixture was dialyzed against0.01% NH.@HCO3.Thematerialswerelyophilizedandseparatedbyelectrophoresisin a 10â€”20%gradientSDS.polyacrylamidegelin a reducedcondition.The gel wasdried,andtheradioactivitywasdetectedusingKOdakx-omatAR ifim 14.3â€” (Kodak,Rochester,NY). Western Blot Analysis of Mouse Normal Tissues. A female mouse was perfused with PBS by cardiac puncture. The normal organ tissues, such as brain, heart, lung, spleen, liver, kidney, colon, lymph nodes, and ovary, were Fig.1. Identificationofgp44purifiedby two sequentialaffinitychroinatographies. Proteinspurifiedby chromatographiesusingWGA andmAn 8F11-affinitycolumnswere separated,washedwithPBS,andminced.Themincedtissuesweresuspended subjectedtoSDS-PAGEin a 10â€”20%gradientgel, followed by silver staining.Lane I, in lysis buffer for 30 mm, followed by centrifugationat 15,000X g. The molecular weight standard protein (Ameisham); Lane 2, WGA-affinity column elmnt proteincontentsofthetissueextractsweredeterminedbythemethodofLowry Lane 3, niAb 8F11 column eluent. 768

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1995 American Association for Cancer Research. PLATELET-AGGREGATINGGLYCOPROTEINOF COLON CARCINOMA @ A a theloadingbuffer andthenwith 0.2% glycine-HC1(pH 4.3). The gp44 80 was eluted from the column by lowering the pH of the buffer to 2.3. (â€˜@ The presenceof 0.5% Triton X-100 in the elution buffer was also c60 essentialto efficiently elute gp44 from the column. The eluted pro 0 teinsfrom both columnswere separatedbySDS-PAGEfollowed by U) (I) silver staining to identify the purity (Fig. 1). No protein bands other E 40 U) than gp44 were present in the immunoaffinity eluate. The purified U) gp44 was detected as a broad yellowish band, as most glycoproteins @20 // tend to stain. at -a I @: c,d gp44-induced Platelet Aggregation. Fig. 14 shows the dose-dc ,@ @rtI -t-u@@L@rtr@ta@Ã¸@.*@ I pendentaggregationofmouseplateletsinducedby gp44.Theaddition 0 2 4 6 8 10 of 66 ng of gp44to 200 gil of heparinizedPRPdid not induceplatelet Time(mm) aggregationin 10 min. However,plateletaggregationwascausedby 330 ng of gp44 with a lag time of 2 mm and reached80% of the maximum aggregation by 7 mm. When the gp44 was doubled (660 B 80 e ng), the lag time was shortenedto 30 s. Furthermore,plateletawe .â€”@f gationwasobservedwhenPRP containing0.38% citric acidwas used C60 as a anticoagulant, although the lag time increased 5 times that for 0 â€”-,-.--@- --@, 9 heparinized PRP (data not shown). U, U, /â€˜_ We then carefully preparedwashed mouse platelets to exclude E40 / U) the possibility that gp44 acted on plasma components, such as C ,/ . .. U) / factor X, and hence induced platelet aggregation. No aggregation @20 was observedwith the washed platelets in a cuvette at 37Â°Cunder .C â€œ7 at i/i,., h constant stirring, showing that the isolated platelets were not @@ 0â€•@'?Â°@Â°â€•4 â€˜ir I i'o â€˜12 â€˜1@S activated during preparation.The gp44 also induced aggregationof mouse washed platelets in a dose-dependentmanner (Fig. 2B and Time(mm) 3). The lag time shortened as we increased the amount of gp44. These observations suggestedthat gp44 directly interacted with Fig. 2. Mouseplateletaggregationinducedbygp44in thepresence(A)or absence(B) ofplasma.A 5-,Llaliquotofreconstitutedgp44in PBSwasaddedto200p1of heparinized mouse platelets and induced aggregation. PRP(A) or washedplateletssuspension(B)at time 0, andthechangesinlight transmis Platelet aggregationwas inhibited by mAb 8F11 in a dose-depen sionweremonitored.Theamountsofgp44addedwere(a)660,(b)330,(c)66,(d)O,(e) 660,(f) 392,(g) 196,and(h) 0 ng, respectively. dentmanner.As shownin Fig. 4A, mAb 8F11 at 10 @g/mlprolonged aggregationlag time and at 100 @.tg/mlcompletelyinhibited gp44- induced aggregation. The unimmunized rat IgG did not suppress this x-100 at a concentrationof0.5% wasessentialtoelutegp44froma aggregationat any concentration(Fig. 4B). WGA-Sepharose column. The proteins eluted from the WGA-afflnity SF11 Antigens Expressed in Mouse Tissues. Lysates were pre column with 0.2 M N-acetyl-D-glucosaminewerethen appliedto the paredfrom mouse tissues, and the antigens recognized by 8F11 second 8F11-affinity column. A flow rate of less than 5 ml/h was were determined by Western blot analysis. Tissues used were requiredfor successfulbindingof the proteinsto mAb 8F11 coupled brain, heart, lung, spleen, liver, kidney, colon, lymph nodes, and to theSepharosebeads.Thecolumnwasfirst washedextensivelywith ovary. A single band was detected in the lysates from the lung,

Fig.3. gp44-inducedaggregationofmousewashedplate lets.Mousewashedplateletswereincubatedinthepresenceor absenceofgi'@@andexaminedby light microscopy.A,control mouse washed platelets;B, platelet aggregatesinducedby gp44.Bar, 100 @m.

â€˜ 4

769

A a untreatedsampleswere equally recognizedby mAb 8F11 (Fig. 7). 80 Then,N-glycanase-treatedgp44wascleavedby neuraminidasefor 1 -@ h at 37Â°C.The apparent molecular weight of the neuraminidase treatedgp44 slightlyincreasedbecauseof a decreasein its ionic â€¢@60 chargeasa resultofremoving sialicacids.The high-molecular-weight bandsshownin Fig. 7 areoligomersof gp44 thatwere producedin the concentrationprocess.No changein the reactivity to 8F11 was de tected in the N-glycanase and neuraminidase-treated gp44. Further treatmentof the productswith O-glycanaseresultedin a remarkable It: /C decrease in the molecular weight, accompanied by a loss of the @â€”@- d reactivity to mAb 8F11 (Fig. 7). The products from a prolonged @@@ 0 2 â€¢4 â€˜ 8 â€˜ incubationof gp44 with O-glycanasewere undetectableby Western blot analysisusing mAb 8F11 (datanot shown).Theseobservations TIme(mmn) indicatethatthemAb 8F11epitopeincludesa portionof the 0-linked carbohydrates. B Roleofgp44CarbohydrateChainsinPlateletAggregation.The products from enzymatic deglycosylations of the purified gp44 were examined for their ability to induce platelet aggregation. As e, f, 9, h shown in Fig. 8, N-glycanase-treated gp44 induced platelet C 0 aggregation as fast as did the intact gp44. However, sequential U) U) treatmentsof gp44 with N-glycanase and neuraminidaseresultedin E a loss of the platelet aggregation activity. The results suggest that U) C sialylated 0-linked oligosaccharides of gp44 may function in U) I. triggering platelet aggregation. .C at DISCUSSION 0 2 4 6 We haveshownpreviouslythatthehighlymetastaticcloneNL-17 Tmme(mm) derivedfrom mousecolonadenocarcinoma26possessesanability to Fig.4. Effectsof mAb 8F11 (A) and normal rat IgG (B) on gp44-inducedplatelet induce platelet aggregation (18). The aggregation was mediated by a aggregation.Thereconstitutedgp44(330ng) wasincubatedwith0â€”20@gofmAb 8F11 trypsin-sensitivecellsurfaceprotein,but neithergenerationof throm or normalrat IgG for 30 mm at roomtemperature,andthemixturewasaddedto 200 p1 bin through a procoagulantmechanismnor releaseof ADP was of heparinizedPRP.The changesin light transmissionweremonitoredfor 10 mm. The finalconcentrationsofIgGwere:aande,0 pg/mI;bandf 1 @g/ml;candg,10g.@g/ml;involvedin theaggregationinduction.Themonoclonalantibody8F11 andd andIt, 100p@Jm1. was raised against NL-17 cell membrane molecules. This antibody retarded platelet aggregation induced by NL-17 cells and showed a higher level of binding to NL-17 cells than the less metastatic NL-14 colon, lymph nodes, and ovary (Fig. 5). Expressionof the antigen cells. An antigen of mAb 8F11 has been detected as a membrane was particularly high in the lung compared with other organs. The glycoprotein with a molecular weight of 44,000 (gp44; Ref. 18). molecular weights of the antigens in normal tissueswere approx imately 41,000, slightly lower than that of gp44 expressed on NL-17 cell surfaces. 1 2 3 456789 10 Characterization of Carbohydrate Chains in gp44 as an 8F11 Antigen. TFMS treatmentof the radiolabeledgp44 for 4 h to hydro lyze both N- and O-glycosidiclinkagesresultedin a decreasein its (kDa) molecular weight from 44,000 to 36,000 (Fig. 6). However, the chemically deglycosylated protein band was still broad, suggesting the 97â€” molecularweight heterogeneityof gp44 was still present.Prolonged TFMS treatment did not produce a sharp, single band. Thus, TFMS may not be ableto cleaveall of the carbohydratemoietiesof gp44. I. - Next, we carried out an enzymatic deglycosylationof gp44 by sequentialtreatmentswith N-glycanase,neuraminidase,and O-gly @ canaseandthenexaminedtheirreactivityto mAb 8F11 usingWestern a blot analysis (Fig. 7). The N-glycanase is supposedto release all common classesof Mn-linked oligosaccharides, including sialylated, phosphorylated, or sulfated sugar chains. The neuraminidase we used @@ .4: cleaveda2-3-, a2-6-, and a2-8-linked sialic acids.The O-glycanase catalyzedthe releaseof the Galj3lâ€”3GalNAccoredisaccharidesat Fig.5. Westernblotanalysisofgp44-rclatedantigensinmousenormaltissuesdetected @ tachedto senneor threonineresiduesof glycopeptides.GlcNAcf3lâ€” by mAbSF11 Aliquotsofthenormaltissueextractscontaining4()jt@ofproteinwere 3GalNAc andotherstructuresfoundlinked to serineandthreomneare separatedbyelectrophoresisina4â€”20%gradientSDS-polyacrylamidegel(8.4x 9.0cm). Theelectrophoresedproteinsin thegelwereblottedontoa nitrocellulosemembrane.The not substrates.Theglycosidasesweusedhadno detectableactivityof blottedmembranewasincubatedwithmAt, 8F11(20 @g/ml),followedby incubationwith proteaseagainstfetuin as a control protein (data not shown). When peroxidase-linkedgoatanti-ratlgG antibodies.Themembranewassoakedintheenhanced chemiluminescencemixtureand exposedto a Kodak X-Omat AR film. Lane 1, brain; gp44 was treated with N-glycanase for 24 h at 37Â°C,no significant Lane 2, heart; Lane 3, lung; Lane 4. spleen; Lane 5. liver; Lane 6, kidney; Lane 7, colon; change of the molecular weight was detected, and the treated and Lane 8, lymph nodes; Lane 9. ovary; Lane 10. NL-17 cells. 770

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1995 American Association for Cancer Research. PLATELET-AGGREGATING GLYCOPROTEIN OF COLON CARCINOMA (kDa) resemblethe proteinsdescribedpreviouslyby other investigatorsin I 2 terms of molecular size and capability to induce platelet aggregation through a thrombin-independent mechanism. Moreover, we have dem onstrated that gp44 induces platelet aggregation without any plasma component,suggestingthat this protein may directly bind to its ligand(s) 200 â€” presenton the mouse platelet membraneand trigger aggregation. We examinedthepresenceofthe mAb 8F11 antigenin mouse tissues using Western blot analysis. High levels of the antigens with a molecularweight of 41,000were detectedin the lung, colon, and lymph nodes.The size of the normal tissueantigenswe detectedwas 69â€” 1234 46â€” j ::;@4_gp44

30â€”

21.5 â€” â€˜@â€” gp44 14.3 â€” 30

Fig. 6. Deglycosylationof gp44by TFMS. The purified gp44waschemicallydegly cosylatedby incubationin 100p1of TFMS at 0Â°Cundernitrogenfor 4 h. The reaction 21.5 â€” mixture was neutralizedwith 60% pyridine in water and dialyzed against 0.01% NH4HCO3.TheproteinsweresubjectedtoSDS-PAGEin a 10â€”20%gradientpolyacryl amidegel.TheradioactivityinthegelwasdetectedusingKOdakX-OmatAR film.Lane 1, control gp44; Lane 2, TFMS-treated gp44. 143

In the presentstudy, we purified gp44 from NL-17 cells by two Fig. 7. Sequentialenzymaticdeglycosylationof gp44 with N-glycanase,neuramini affinity chromatogaphic methods, WGA affinity chromatography and dare,andO-glycanase.Thepurifiedgp44wasfirst incubatedwith0.3unit of N-glycanase immunochemical affinity chromatography using mAb 8F11. The pu for24 h at37Â°C,followedbyincubationwith0.01unitofneuraminidasefor1 h at37Â°C. rifled antigen was identified by SDS-PAGE as a single band with a TheN-glycanase-andneuraminidase-treatedsamplewasfurtherincubatedwith2 milli units of O-glycanase for 24 h at 37Â°C.The products were subjected to SDS-PAGE, molecular weight of 44,000. Using PRP, we first observedthat the followedbyWesternblotanalysisusingmAb8F11.Anequalamountofthegp44was ethanol-precipitatedand reconstitutedgp44 induced mouseplatelet loadedoneachlane.Lane1, untreatedgp44;Lane2. N-glycanase-treatedgp44;Lane3, aggregationin a dose-dependentmanner.Inductionof plateletaggre N-glycanase-andneuramimdase-treatedgp44;Lane 4. gp44 sequentiallytreatedwith N-glycanase,neuraminidase,andO-glycanase. gation in Pm' was not affectedby citric acid, which decreasedthe Ca2@level in plasmaby chelation.The purified gp44 also induced aggregation of mouse washed platelets without the addition of plasma components.The pretreatmentof gp44 with mAb 8F11 at the con centrationof 10 @.tg/mlprolongedthe lag time of the aggregation,and completeinhibition was observedat 100 @ig/ml. ab To reproducetheplatelet-aggregatingactivityof330ngof thepure C 0 gp44, 2.5 X 106 cells would be required: (a) considering the results (I) from the antibody binding study (18), we assumedthat 1 fmol of U) E antibodiesbind to 1 X iO@NL-17cells. Calculatingfrom our results, U) C 2.5 X 106 cells have 11 ng of gp44 on their cell surfaces. Therefore, â€˜U the amount of purified gp44 required to induce platelet aggregation a- was 30 times greaterthan the calculatedamounton the cell surface. .C Since the 8F11 antibody recognizes a sugar moiety of gp44, we 01 -J c,d assumedthat there might be severalantibodyrecognitionsitesper 1 moleculeof gp44; and(b) the gp44we usedfor platelet-aggregating 0 2 4@ 6 8 10 activity wasin a solubiized form, thustheconformationof theprotein may not be the sameasthe onepresenton the cell surface.For these Time (mm) two reasons, we do not think that the amount of gp44 used in our Fig. 8. Effects of enzymaticdeglycosylationsofgp44 on platelet-aggregatingactivity. platelet aggregationexperimentswas unreasonablyhigh. A 5-pi aliquotofuntreatedordeglycosylatedgp44(300ng)in PBSwasaddedto200 p1 of heparinizedPRPat time 0, andthe changesin light transmissionweremonitored.a, Many investigatorsisolated platelet-aggregatingsubstancesfrom intact gp44; b, N-glycanase-treatedgp44;c, N-glycanase-and neuraminidase-treated tumor cells (15, 19, 21â€”23,35). The gp44 that we purified does not gp44; d. N-glycanase-, neuraminidase-, and O-glycanase-treated gp44. 771

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1995 American Association for Cancer Research. PLATELET.AGGREGATINGGLYCOPROTEIN OF COLON CARCINOMA similar to the one found previously in highly metastatic mouse mel REFERENCES anoma B16-F10 cells (36). The results indicate that glycoproteins 1. Honn, K. V., Tang, D. G., and Crissman, J. D. Platelets and cancer metastasis: a immunochemically related to gp44 are present in normal and malig causal relationship? Cancer Metastasis Rev., II: 325â€”351,1992. nant mouse tissues. It is of interest whether the difference in molecular 2. Bastida, E., and Ordinas, A. Platelet contribution to the formation of metastatic foci: the role of cancercell-inducedplateletactivation.Haemostasis,18. 29-36, 1988. weight is a consequence of posttranscriptional modifications or not. 3. Chopra, H., Fligiel, S. E. G., Hatfield, J. S., Nelson, K. K., Diglio, C. A., Taylor, J. D., The purification of the mAb 8F1 1 antigens expressed in normal and Honn, K. V. An in vivostudy of the role of the tumor cell cytoskeleton in tumor tissues is in progress in our laboratory, and we are attempting to cell-platelet-endothelial cell interactions. Cancer Res., 50: 7686â€”7696,1990. 4. Varani, 3., Dixit, V. M., Fligiel, S. E. 0., McKeever, P. E., and Carey, T. E. determine their activity to induce platelet aggregation. Thrombospondin-inducedattachmentandspreadingof humansquamouscarcinoma The findings from a sequential enzymatic hydrolysis of carbohy cells. Exp. Cell Res., 167: 376â€”390,1986. drates linked to gp44 indicate that it is enriched with O-glycans. 5. Hawrylowicz, C. M., Howells, G. L, and Feldmann, M. Platelet-derived interleukin 1 induces human endotheial adhesion molecule expression and cytokine production. Futhermore, loss of the reactivity to mAb 8F1 1 was observed when J. Exp.Med.,174:785-790,1991. gp44 was treated with O-glycanase. Because of the inhibitory effect of 6. Skolnik,0., Bagge, U., Dahistrom,A., andAhlman,H. The importanceof 5-HT for mAb 8F1 1 on gp44-induced platelet aggregation, the carbohydrate tumor cell lodgement in the liver. hit. J. Cancer, 33: 519â€”523,1984. 7. Nierodzik, M. L, Plotkin, A., Kajumo, F., and Karpatkin, S. Thrombin stimulates chains linked to Ser,'Thrresidues were assumed to be responsible for tumor-plateletadhesionin vitroandmetastasisin vivo.J. Gin. Invest.,87: 229â€”236, the aggregation. 1991. Molecules responsible for the initial tumor cell-platelet interaction 8. Aruffo, A., Dietsch, M. T., Wan, H., Helistrom, K. E, and Hellstrom, I. Granule membrane protein 140 (GMP14O) binds to carcinomas and carcinoma-derived cell are totally unknown. At the molecular level, adhesion molecules such lines. Proc. Natl. Acad. Sci. USA, 89: 2292-2296, 1992. as integrins expressed on both tumor cells and platelets (37â€”40), 9. Tsuruo, T., Watanabe, M., and Oh-hara, T. Stimulation of the growth of metastatic platelet membrane glycoprotein Ib/IX (41), and P-selectin on platelets clones of mousecolon adenocarcinoma26in vitroby platelet-derivedgrowthfactor. Jpn.J. CancerRca.,80: 136â€”140,1989. (42â€”44)are candidates for irreversible formation of platelet-tumor 10. Cardinali, M., Uchino, R., and Chung, S. I. Interaction of fibrinogen with murine cell aggregates. Several reports suggested that ligands modulate the melanoma cells: covalent association with cell membranes and protection against recognition by lymphokine-activated killer cells. Cancer Res., 50: 8010â€”8016, affinity of integrins (45, 46). Therefore, resting integrmns may be the 1990. molecules responsible for the first contact between tumor cells and 11. Haimovitz-Friedman,A.,Falcone,D. J., Ekior,A., Schirrmacher,V.,Vlodavsky,I., platelets. P-selectin, on the other hand, needs activation for its ex and Fuks, Z. Activation of platelet heparitinase by tumor cell-derived factors. Blood, 78: 789â€”796,1991. pression, which indicates that this molecule is not involved in the 12. Bastida, E., Ordinas, A., Giardina, S. L, and Jamieson, 0. A. Differentiation of initial contact. Some other proteins on tumor cells described above platelet-aggregating effects ofhuman tumor celllines based on inhibition studies with function as thrombin generators, which alone are not physically in apyrase, hirudin, and phospholipase. Cancer Res., 42: 4348â€”4352 1982. 13. Grignani, G., Pacchiarini, L, Almasio, P., Pagliarino, M., Gamba, G., Rizzo, S. C., volved in the interactions of tumor cells and platelets. and Ascari, E. Characterization of the platelet-aggregating activity of cancer cells Another assumed mechanism of the initial contact between tumor with different metastatic potential. mt. J. Cancer, 38: 237â€”244,1986. 14. Longenecker, G. L, Sayers, B. J., Bowen, R. J., and King, T. Human rhabdosarcoma cells and platelets could be mediated by carbohydrates (1). Several cell-induced aggregation of blood platelets. Cancer Res., 49: 16â€”19,1989. studies supporting this hypothesis have been reported. For example, 15. Falanga, A., and Gordon, S. G. Isolation and characterization of cancer procoagulant: Pearistein et a!. (17) demonstrated that the level of tumor cell surface a cysteine proteinase from malignant tissue. Biochemistry, 24: 5558â€”5567, 1985. 16. Grossi, I. M., Fitzgerald, L A., Umgarger, L A., Nelson, K. K., Diglio, C. A., Taylor, sialylation is positively correlated with the ability to aggregate plate J. D., and Honn, K. V. Bidirectional control of membrane expression and/or activation lets and to metastasize to the lung. Kijima-Suda et a!. (47) have shown of the cell IROlIb/IlIa receptor and tumor cell adhesion by lipoxygenase products of that a sialyltransferase inhibitor suppressed NL-17 cell-induced plate arachidonic acid and linoleic acid. Cancer Rca., 49: 1029â€”1037, 1989. 17. Pearlstein, E., Salk, P. L, Yogeeswaran, G., and Karpatkin, S. Correlation between let aggregation and experimental metastasis. Harvey et a!. (48) dem spontaneous metastatic potential, platelet-aggregating activity of cell surface extracts, onstrated a correlation of sialyltransferase activity of the tumor cells and cell surface sialylation in 10 metastatic-variant derivatives of a rat renal sarcoma cell line. Proc. Natl. Acad. Sci. USA, 77: 4336â€”4339, 1980. with their metastatic ability. These results suggest that sialoconjugates 18. Watanabe, M., Okochi, E., Sugimoto, Y., and Tsuruo, T. Identification of a platelet are involved in the tumor cell-platelet interactions. aggregating factor of murine colon adenocarcinoma 26: Mr 44,000 membrane protein In our study, N-glycanase-treated gp44 induced platelet aggre as determined by monoclonal antibodies. Cancer Rca., 48: 6411-6416, 1988. 19. Cavanaugh, P. G., Sloane, B. F., Bajkowski, A. S., Taylor, J. D., and Honn, K. V. gation as fast as did the intact gp44. However, gp44 completely Purification and characterization of platelet aggregating activity from tumor cells: lost platelet aggregation activity after sequential deglycosylations copurification with procoagulant activity. Thromb. Res., 37: 309â€”326,1985. with N-glycanase and neuraminidase. Therefore, sialylated 20. Chelladurai, M., Honn, K. V., and Walz, D. A. HLA-DR is a procoagulant. Biochem. Biophys. Res. Commun., 178: 467â€”473, 1991. 0-linked carbohydrate chains of gp44 may be mediators for the 21. Dvorak, H. F., Van-DeWater, L, Bitzer, A. M., Dvorak, A. M., Anderson, D., initial contact between NL-17 cells and platelets and may directly Harvey, V. S., Bach, R., Davis, G. L, Dewolf, W., and Carvalho, C. A. Procoagulant activity associated with plasma membrane vesicles shed by cultured tumor cells. trigger platelet aggregation. Cancer Res., 43: 4434â€”4441, 1983. In conclusion, metastasized tumor cells can be regarded as â€œthe 22. Bastida, E., Ordinas,A., Escolar, G., and Jamieson, 0. A. Tissue factor in ml survival of the fittest.â€•There are many steps to be overcome for crovesicles shed from U87MG human glioblastoma cells induces coagulation, platelet aggregation, and thrombogenesis. Blood, 64: 177â€”184,1984. tumor cells to accomplish metastasis (49, 50). Interactions between 23. Cavanaugh, P. G., Sloane, B. F., Bajkowski, A. S., Gasic, 0. 1., Gasic, T. B., and host platelets and tumor cells are thought to facilitate metastasis by Honn, K. V. Involvement of a cathepsin B-like cysteine proteinase in platelet enhancing tumor cell adhesion to the vessel wall. Thus, the ability aggregation induced by tumor cells and their shed membrane vesicles. Chin. Pip. Metastasis, 1: 297â€”307,1983. to aggregate platelets is thought to be one of the critical features of 24. Tsuruo, T., Yamori, T., Naganuma, K., Tsukagoshi, S., and Sakurai, Y. Character malignant tumor cells. We have isolated a sialoglycoprotein of M@ ization of metastatic clones derived from a metastatic variant of mouse colon adenocarcinoma 26. Cancer Rca., 43: 5437â€”5442,1983. 44,000, which is recognized by mAb 8F1 1, and this purified 25. Sugimoto,Y., Watanabe,M.,Oh-ham,T., Sato, S., lane,T., andTsuruo,T. Suppres protein alone induced mouse platelet aggregation without any sion of experimental lung colonization of a metastatic variant of murine colon plasma component. To our knowledge, this is the first report of the adenocarcinoma 26 by a monoclonal antibody 8F1 1 inhibiting tumor cell-induced platelet aggregation. Cancer Res., 51: 921â€”925,1991. purification of a tumor cell membrane protein responsible for 26. Bhavanandan, V. P., and Katlic, A. W. The interaction of wheat germ agglutinin with induction of platelet aggregation through a thrombin-independent sialoglycoproteins: the role of sialic acid. J. Biol. Chem., 254: 4000â€”4008, 1979. 27. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of mechanism. Further studies on the structures of gp44 core peptide bacteriophage T4. Nature (Land.), 227: 680â€”685,1970. and the 0-linked carbohydrates are necessary to understand the 28. Ardlie, N. 0., Packham, M. A., and Mustard, J. F. Adenosine diphosphate-induced molecular mechanisms of gp44-mediated platelet aggregation. In platelet aggregation in suspensions of washed rabbit platelets. Br. J. Haematol., 19: 7- 17, 1970. addition, studies of the presence of such glycoproteins in human 29. Warburg, 0., and Christian, W. Isolierung und kristallisation des ganmgsferments tumor cells are of great interest. enolase. Biochem. Zeitshrift., 310: 384â€”421, 1942. 772

30. Sojar,a T., and BahI, 0. P. Chemicaldeglycosylationof glycoproteins.Methods of platelet a granule proteins in platelet aggregation induced by human osteosarcoma EazymoL,138:341â€”350,1987. cells. Cancer Res., 53: 4695-4700, 1993. 31. Lowry,0. H., ROSCbTOUgh,N.J., Fan, A. L, andRandall,R. J. Proteinmeasurement 42. Weller, A., Isenmann, S., and Vestweber, D. Qoning of the mouse endotheial with the Folin phenol reagent. J. Biol. Chem., 193: 265â€”275,1951. selectins: expression of both E- and P-selectin is inducible by tumor necrosis factor 32. Plummer, T. H., Jr., Elder, J. H., Alexander, S., Phelan, A. W., and Tarentino, A. L a. J. Biol. Chem.,267: 15176â€”15183,1992. Demonstrationofpeptide:N-glycosidaseFactivityin endo-@-N-acetylglucosamini43. Larsen, E., Cell, A., Gilbert, 0. E., Furle, B. C., Erban, J. K., Bonfanti, R., Wagner, due F preparations. J. Biol. Chem., 259: 10700â€”10704,1984. D. D., and Furie, B. PADGEMprotein:a receptorthat mediatesthe interactionof 33. Koide,N.,andMuramatsu,T.Endo-@-N-acetylglucosamlmdaseactingoncarbohy activated platelets with neutrophils and monocytes. Cell, 59: 305â€”312,1989. drate moieties of glycoproteins: purification and properties of the enzyme from 44. Aruffo, A., Dietsch, M. T., Wan, H., HellstrÃ¶m,K. E., and HellstrÃ¶m,I. Granule D@coccus pneumoniae. 1. Biol. Chem., 249: 4897-4904, 1974. membraneprotein140(GMP-140)bindsto carcinomasandcarcinoma-derivedcell 34. Hayano, S., and Tanaka, A. Streptococcal sialidase. I. Isolation and properties of lines. Proc. NaIl. Acad. Sd. USA, 89: 2292-2296, 1992. sialidase produced by group K streptococcus. J. Bacteriol., 93: 1753â€”1757, 45. O'Toole, T. E., Loftus, J. C., Du, X., Glass, A. A., Ruggeri, Z. M., Shattll, S. J., Plow, @ 1967. E. F., and Ginsberg, M. H. Affinity modulation of the a Cli 3 integrin (platelet 35. Pearlstein,B.,Cooper,L B.,andKarpatkin,S.Extractionandcharacterizationofa GPIIb-IIIa) is an intrinsic property of the receptor. Cell Regal., 1: 883â€”893, plate1ct-aggre@g material from SV4O-transformedmouse 3T3 fibroblasts. J. Lab. 1990. am. Med.,93:332â€”344,1979. 46. Du, X., Plow, E. F., Frelinger, A. L, O'Toole, T. E., Loftus, J. C., and Ginsberg, 36. Watanabe, M., Sugimoto, Y., and Tsuruo, T. Expression of a Mr 41,000 glycoprotein M. H. Ugands â€œactivateâ€•integrina fib @33(plateletGPHb-ffla).Cell, 65: 409â€”416, assOciatedWiththrombin-independent platelet aggregation in high metastatic variants of murine B16 melanoma. Cancer Res., 50: 6657-6662, 1990. 1991. 37. Phillips,D. R., Charo,I. F., and Scarborough,R.M. GPIIb-ffla:the responsive 47. Kijima-Suda, I., Miyamoto, Y., Toyoshima, S., Itoh, M., and Osawa, T. Inhibition of integrin. Cell, 65: 359â€”362,1991. experimental pulmonary metastasis of mouse colon adenocarcinoma 26 subines by a 38. Boukerche, H., Berthier-Vergnes, 0., Tabone, E., Dare, J. F., Leung, L L K., and sialic acid: nucleoside conjugate having sialyltransferase inhibiting activity. Cancer McGregor,J.L Platelet-melanomacellinteractionismediatedbytheglycoprotein Res., 46: 858â€”862,1986. llb-ffla complex. Blood, 74: 658â€”663,1989. 48. Harvey, B. E., Toth, C. A., Wagner, H. E., Steele, G. D., Jr., and Thomas, P. 39. Ugen, K. E., Mahalingam,M., Klein, P. A., and Kao, K. J. Inhibitionof tumor Sialyltransferase activity and hepatic tumor growth in a nude mouse model of cell-induced platelet aggregation and experimental tumor metastasis by the synthetic colorectal cancer metastases. Cancer Res., 52: 1775-1779, 1992. Gly-Arg-Gly-Asp-Ser peptide. J. NatL Cancer Inst., 80:1461-1466,1988. 49. Nicolson, G. L Paracrine and autocrine growth mechanisms in tumor metastasis to 40. Karpatkin, S., Pearlstein, E., Ambrogio, C., and Caller, B. S. Role of adhesive specific sites with particular emphasis on brain and lung metastasis. Cancer proteins in platelet tumor interaction in vitro and metastasis formation in vivo.J. Clin. Metastasis Rev., 12: 325-343, 1993. Invest., 81: 1012â€”1019,1988. 50. Fidler, I. J., Fabra, A., Nakajima, M., and Radinsky, R. Genetic and epigenic 41. Qezardin, P., Drown, J., Morel-Kopp,M., Hanss,M., Kehrel,B., Serre,C., Kaplan, regulation of human colon carcinoma metastasis. Contrib. Oncol., 44: 13â€”26, C., and Delmas, P. D. Role ofplatelet membrane glycoproteins Ib/IX and Hbfllla, and 1992.

773

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1995 American Association for Cancer Research. 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, et al.

Cancer Res 1995;55:767-773.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/55/4/767

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/55/4/767. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.