12

Biocldmica et BiophysicaActa, 1136(1992) 12-16 © 1992ELsevierSciencePubl/sh~.~B.V.All rightsreserved0167-4889/92/$05.00

BBAMCR 13194

Identification of transglutaminase substrates in HT29 colon cancer cells: use of 5-(biotinamido) pentylamine as a transglutaminase-specific probe K y u n g N.

Lee, M e r l e

D . Maxwell, M a n f o r d K. P a t t e r s o n , Jr., P a u l J. B i r c k b i c h l e r and Eugene Conway

The SarmwlRoberts N,dde Foundario.L Bioraedicd Dit~sion, Ardmore, OK fUSAJ

(Recei'¢ed t0 December1991) (Revised manuscriptrecel,.-~d |2 ~,iarch l~t2_) Key winds: Ttamghttaminase:5-{Bk,tiasmidoMen~lamine:Fructnse-i.f-bisphosphatealdol~: {HT29celon cancer cell} A biotin~m.;ne probe, 54biotinarnidoMentylamine, was used for biotin-labeling of protcias in HT29 colon cancer cell extracts by endogenous transglumminase activity. The biotin-labeled protein substrates were isolated and recm,ered by avidin-affinity chromatography. The proteins were separated using SDS-polyacrylamide gel electrophoresis, eleclroblottcd onto a polwin),lidene difluoride membgmm,visual;Ted using Coomassic blue, cut out. and sequenced. Amino acid sequence data identified human fructost:-l,6-bisphosphat¢aldolasc A, an intracellu[ar prote[~ as a substrate for cellular transghttaminase.

lntmductien Transglutaminases are calcium-dependent acyltransferases which catalyze the post-translational modification of protein-bound glutamhie residues by the incorporation of primary amines, including the ~-amino group of protein-bound lvsine residues in approi~riate polypeptides [1-3]. Plasma (Factor Xllla) and epidermal (type 1) trans#utaminases are known to be involved in blood coagulation [4] and formation of the cornified envelope of terminally differentiated epidermal keratinocytes [5], respectively. The exact physioIGgical function of cellular transgiutaminase (type !!), however, is still an enigma although its proposed roles include growth regulation [6], differentiation [7], apoptosis [8], and GTP hydrolysis [9]. One of the crucial steps to clarify the role of the enzyme is to identify its cellular substrates. In this regard, fluorescent or radioactive amines have been used as probes for transglutaminase-~=atalyzed labeling of protein substrates [10-13L

Cortespottdence to: K.N. Lee, The Samuel RobertsNoble Foundation. llx, BiomedicalDivision.P.O. Box 2180. Ardmore.OK 7992. USA. Abbreviations:PVDF. pob'vinylidenedifluoride: EDTA. elhylenediamine tetraacetic acid: DTT. dithiothreitol: PMSF. phenylmelhylsulfonylfluoride: PNPG. p*nitrophenyl-O-o-galactoi~ranoside.

Recently, we developed a biotin-labeled amine probe, 5-(biotinamido)pentylamine (biotincadaverine) for transglu!aminase-directed labeling of substrates and used it for colorimeh~= transglutaminase assays [14,15], The biotin moiety of the compou.-x.d provides not only a mechanism for examining the incorporation of the amine into protein substrates, but also a mechanism for isolation and recovery of the labeled proteins by use of an avidin-affinity column. We report here that incubation of HT29 colon cancer cell extracts in the presence of Ca 2+ and biotincadaverine produces biotinylated proteins by endogenous transgiutaminasccatalyzed reaction. The labeled proteins were isolated and recovered by an avidin-affinity column. We also report the identification of one of the labeled substrate proteins by amino acid sequencing. Materials and Methods Cell culture and cellular protein labeling

HT29 human colon cancer cells (a gift from Dr. Bernard Weinstein, Columbia University) were grown in McCoy',. 5a medium, supplemented with 10% fetal bovine serum (Reheis), penicillin (50 ttg/ml) and streptomycin (100/~g/ml), in a humidified atmosphere at 7% CO, and 3T'C. CCells in T-75 flasks were rinsed four times with calcium-free Earle's solution, scraped into 50 mM Tris-HCi (pH 7.5L containing 250 mM sucrose, 0.2 mM MgSO4, 1 mM EDTA, 10 mM DTT,

13

0_5 mM PMSF, and 500 /zg/ml leupeptin, and ruptured by sonication [16]. Ce!! extracts were prepared by centrifugation of the sonicates at 65 000 × g for 1 h and stored at -80°C. For endogenous transglutaminasemediated labeling of proteias, the cell extracts were incubated for 20 min at 37°C with 8 mM biotincadaverine (from E.IL Fujimoto, Pierce) and 5 mM CaCI2, then the reaction terminated by adding EDTA solution (final concentration 10 mM). For control reaction 5 mM EDTA replaced CaCi2, and for another control 10 mM cystamine, an enzyme inhibitor [2], was added in the presence of 5 mM CaCI 2.

Affinity chromatography Isolation and recovery of biotincadaverine-labeled proteins were carried out using Pierce Monomeric Avidin Column [17]. Biotincadaverine-labeled cell extract proteins were buffer-changed with buffer A (100 mM sodium phosphate, 150 mM NaCi, pH 7.2) using a PD-10 Sephadex column (Pharmacia), and 2.5 ml of the sample (I-~ rag protein/ml) loaded onto the affinity column (1.1 × 2.2 cm; 2.0 ml bed volume) which had been equil~rated with buffer A. Runthrough fractions were collected and reloaded. After the second loading, the column was washed with 13 bed volumes of buffer A and the biotinylated proteins were eluted from the column with 2 mm biotin in buffer A. During elution, fractious of 2 ml were collected. The first f'rce fractions were pooled, concentrated to a protein concentration of 2-5-3.0 m g / m l , and stored at -80°C. Parallel experiments, using biotincadaverine-labeling and avidinaffinity chromatography, were performed on rabbit muscle aldolase A (Sigma). The commercial aldolase A was labeled with biotincadaverine by purified human erythrocyte transglutaminase [18] under the same reaction conditions as those used for cellular protein labeling SDS-PAGE, electroblotting and protein sequencing SDS-PAGE was performed in a discontinuous buffer system using a 10% polyacrylamide separating gel [19]. Biotincadaverine-labeled proteins were blotted onto ProBlott membranes (Appfied Biosystems), which were then stained, as described by Matzud~ira [20]. In brief, the gels were soaked for 5 min in 10 mm 3-(cyclohexylamino)-l-prop;me sulfonic acid/10% methanol (pH 1!) (blotting buffer). ProBlott membranes were wetted with 100% methanol for a few seconds and rinsed with blotting buffer. Electroblotting was carried out in a Bin-Rad Mini Traus-Blot Electrophoretie Transfer Ceil at 60 V for 45 min. After transfer, the blots were rinsed with deinnized water, saturated with methanol for 3 s, stained for 1 min with 0.1% Coomassie blue in 40% m e t h a n o l / l % acetic acid, and then destained wkh 50% m e t h a n o l . After washing with deionized water, the blots were air-dried and stored at -2ff'C. The

TABLE I Characterization of HT29 cell cytosol transglutaminaseactit'i~' and biotincadacerine-labeling Preparation of cell extracts were carried out as described under Materials and Methods. Whenpre~nt, the Ca z+ concentrationwas

5 raM, EDTA was 5 mM+ and cystaminewas 10 mM+The value representsthe mean+ S+D.of three experiments,nd. activitywas t:ot detected+ Reaction conditions +Ca + EDTA, - Ca + Cy~tamine. +Ca

Transglutaminase Biotincadaverineactivity, labelingof (nmol putrescine/ cellularproteinsb rain per mg) (A~os) 1.1+_0.2 0.78,1±0.017 nd nd nd

nd

a Transglutaminaseactivitywas measuredby a publishedprocedure [181. b Biotincadaverine labeling and detection of tPe labeled proteins were carried out by a publishedmethod[14].3-;/zg of cell extract proteins and 8 mM biotincadaverim;were incubated for 30 rain at room temperature in wells of a 96.well mierotiterplate. B/otinylated proteinswere immobilizedonto the wellsand complexedwith slreptavidin-/3-galactosidase.The absorbanceat 405 nm was measured 60 rain after adding PNPG.

stained protein band was cut out with a razor blade, placed into the Blott cartridge, and sequenced using Applied Biosystems Model 477A pulsed liquid sequencer. Parallel blots of these samples were probed for biotincadaverine-labeled proteins using streptavidin-horseradish peroxidase conjugate system (BRL) according to the protocol supplied by the manufacturer. Assays Transglutaminase activity was measured by the incorporation of [l,4-t+C]putrescine into N,N-dimethylcasein [18]. Quantitation of biotincadaverine labeling assay in Table ! was carried out by a published procedure [14]. Protein concentratious were determined by the Bradford method [21], using bovine 3,-globulin as standard. Results and Discussion In preliminary, experiments, HT29 human colon cancer cell extracts showed high transgiutaminase activity. Table I also shows incorporation of biotiucadaverine into cellular proteins in the presence of Ca 2+, but not in the negative controls. Confirmation of the presence of transglutaminase in HT29 cell extracts was shown with monoclonal [22] and polyclonal [18] antibodies to cellular transglutaminase on Western blot (data not shown). These results suggested that the incorporation of biotincadaverlne into cellular protein in the HT29

:I °"1 o

+

+o

;=

|;,

;+

,,

B

+ii

1

2

--~+ ,0

¢ ii

~,

2

3

:Q~-4"

*" Ill

Fig. I. Isolation and recmery of biotincadaverine-labeled proteins by avidin-affinity chromatography.Conditions are desen'bedunder Materiala and Method';. "rh¢ inset shows a polyacrylarnid¢ gel stained with Cnoma_ssiebrilliant blue. Lane I. biotinylatet; nmlecular weight markers (Pierce):. lane ~ total proteios: lane 3. avidin-culumn-adsorbed proteins. Molecular weights are indicated as AI, × 10=5.

cell extracts was a transglutaminase-mediated reaction. A s a first step in ":dentifying the biotincadaverinelabeled proteins, we isolated the labeled proteins from reaction mixtures containing HT29 cell extracts, biotincadaverine, and Ca 2+, using avidin-agarose affinity chromatography [17]. Fig. 1 shows a representative elution profile of the proteins following the affinity chromatography. Most non-labeled proteins were removed from the affinity column during the washing procedures while the labeled proteins were retained on the column and later eluted with 2 mM biotin in washing buffer (buffer A). The eluted sample analysis was carried out by SDS-PAGE. The inset of Fig. I compares proteins in the reaction mixtures containing 1TI29 cell extracts, biotincadaverine, and Ca -'+ (Fig. 1 inset; lane 2) with proteins in the e l a t e d sample (lane 3). By Coomassie blue staining, the eluted proteins a p p e a r e d as several distinct bands, suggesting that these proteins are, indeed, biotincadaverine-labeled by transglutaminase. In o r d e r to clarify whether the proteins of the eluted sample from the affinity column were the result of biotincadaverine-labeling or nonspecific adsorption to the affinity matrix, we compared a Coomassie blueacfiqed SDS-polyacrylamide gel with a Western blot of a duplicate gel (Fig. 2). As shown in Fig. 2, all the Coomassie blue-stained protein bands (A; lane 3) matched with the protein bands detected by streptavidin-conjugated horseradish peroxidase system (B; lane 3). The SDS-PAGE and Western blot analyses, therefore, suggest that all the eluted proteins are labeled with biotincadaverine. A central issue of this report is the identification of the protein substrates labeled by endogenous transglutaminase-catafyzed reaction. The eluted proteins from

1

3

:~-'.~i

II 4 . Ill

"" ~

IV

~4-

IV

Fig. Z SD~PAGE and Western blot anab~is. (A) Coomassie bluestained pob'ac~-IamiOegel. Lanes I. biotinylated molecular weight markers (Pien:~k lanes Z biot~ncadaverine-labeledaldolase A prepared ffc~'n,rabbit muscle aldolase (Sigma) by' a~idin-affinitychromatography: lanes 3. biotin-~.~verine-labeled cellular proteins prepared by avldin-affini~.-chromarog~phy. L IL Ill. and IV indicate protein bands subjected to amino acid sequence anab~is. Molecular weights are indicated as ,*,f~x 10-3. (B) Western blot analysis of a duplicate polt,~ct3~la~'nidegel: electrobloned proteins are detected v,ilh su'eptat~din-hm3cradishperoxida~ system (BRL).

the avidin affinity column were separated using SDSP A G E . The proteins were then transferred to a ProBIott membrane, visualized by Coomassie blue staining, cut out, and ~ q u e n c e d using an automated sequencer [20]. Initially, we a t t e m p t e d to sequence four TABLE It NH_,-temmml sequencing yield5 of band II protein Cycle I 2 3 4 5 6 7 8 9 10 iI 12 13 14 15

Amino acid Pro T~T Gin T.vr Pro ALl Leu Thr Pro Gin Gin L~ LYS Gin Leu

Yield a (pnml) 25.79 22.79 16~6 21.13 19.38 18.14 16.43 I 1.42 5.79 .-.46 4.82 4.54 9.48 4.29 4.90

a Phenylthiolrydantoin-derivatizcdamino acids were analy'~edon-line by reverse phase HPLC, utilizing 39% ef the product from each cycle.

15 TABLE !11 Comparison of the NH2-terminal sequences of band H protein and human aldolase isoz3"mes fA, B and C)

Hyphens indicale residues identical to those found in the correspondingpositionof band !1 protein. Protein Band II AldolasoA Aldolase 18 Aldolase C

NH2-terminal sequence PYOYPALTPEQg

............... AHR F .... -HS ....

S ......

sa

......

Y:EL

Reference Thiswork 25 26 27

major bands; band 1, II, !!1, and IV shown in Fig. 2. As shown in Tables II and 111, band I! protein, 40 kDa, was successively sequenced up to 15 NH2-terminal amino acid sequences and identified as fructose-l, 6bisphosphate aldolase A subunit, using an established database search program [23]. Additional evidence that this protein was aldolase A is shown in Fig. 2. The band 11 protein (lane 3) migrated on SDS-PAGE to the same position as biotincadaverine-labeled rabbit muscle aldulase A (lane 2). Rabbit aldolase A has the same number of amino acids and 98% homology [24] to auman aldolase A. Bands I, IiI, and IV did not show any amino acid signals in sequence analysis, suggesting that their amino termini might be blocked. Aldolase (fructose-l,6-bisphosphate aldolase, EC 4.1.2.13) is an enzyme that plays a pivotal role in glycolysis and fructose metabolism. Three isozymes have been found in muscle (aldulase A), liver (aldulase B), and brain (aldulase C) from healthy mammals [25]. Interestingly, the distn'butinn of the isozymes is changed during development or carcinogenesis [25]. In fetal liver aldolase A is predominantly expressed with a small amount of aldolase 13. During development aldolase B progressively increases and becomes a major form in a normal adult liver. In chemically-induced hepatoeareinoma, aldolase B expression is switched off, whereas aldolase A is increased, resulting in a pattern similar to the fetal one. In HT29 human colon cancer cells, we found only a!dolase A (Tables II and Ill) by mnino acid sequencing technique. It remains to be answered whether the colon cancer ceils express only A or whether aldolase A [26], not B [271 end C [28] i=: a transglutaminase substrate. It has been shown previously that aldolase is a substrate for cellular transglutaminase by measuring the incorporation of [t4C]methylamine into the protein in incubation of pure aldulase and etythrocyte transglutaminase [29]. However, the source and isozyme type of the aldulase were not reported in the paper [29]. Our study demonstrates that aldolase A is also labeled by a cellular system using endogenous transglutaminase. Thus, our results constitute the first demonstration that, among the various proteins in cell extracts, al-

dolase A is a transglutaminase substrate. Aclditionally, a series of techniques: (a) endogenous transglutaminase-mediated biotincadaverine labeling of cellular proteins; (b) recovery of the labeled proteins by avidin-affinity column; and (c) identification of the protein by sequence analysis was established as a powerful tool for the identification of potential physiological substrates for transglutaminase. The physiological function of cellular transglutaminase in posttranslational modification of aldolase A is currently unknown. Transglutaminase may catalyze incorporation of polyamines into aldolase A, resulting in modulation of its enzymatic activity and interaction with other biological molecules [30-31]. Future studies will be directed toward this issue. Acknowledgments We would like to thank Joe Clouse, Molecular Analysis and Synthesis Section of The Samuel Roberts Noble Foundation, Inc. for the protein sequencing, and Laura Smith for typing this manuscript. References I Folk, .I.E. and Finlayson, LS. (1977) Adv. Protein Chem. 31. !-133. 2 Lorand, L. and Conrad, S.M (1984) Mol. Cell. Biochem. 58, 9-35. 3 Chang, SA. (1975) in lsozwae (Markert, C.L, ed.), Vol. !. Pp. 259-273, Academic Press.Hew York. 4 Pisano.JJ., Finlayson,J.S, and Peytone.M.P.(1968) Science 160, 892-893. 5 Rice, R.H. and Green, H. (1979)Cell 18, 681-694. 6 Birckbichler, PJ. and Patterson, M.K., Jr. (1978) Ant.. N.Y. Acad. Sci. USA 312, 354-365. 7 Murtaugh,M.P., Mehta, K., Johnson,J., Myers,M., Juliano,R.L and Davies, PJ-A. (1983).L Biol. Chem. 258, 11074-11081. 8 Fesus, L., Thomazy, V. and Falus, A. (1987) FEBS Len- 224, 104-108. 9 Lee, ILN., Birckbichler, PJ. and Patterson, M.g, Jr. (1989) Biochem. Biophys.Res. Commun.162,1370-1375. 10 Lorand,L., Rule, lq.G.,Ong, H.H., Furlanetto, R., Jacobsen,A., Downey,J., Oner. N. and Braner-Lorand,J, (1968)Biochemistry 7, 1214-1223. 11 Lee, ILN., Fesus, L., Yancey, S.T., Girard, .I.E. and Chang, S.l. (1985) .I. Biol. Chem. 260,14689-14694. 12 Lee, K-N.,Chung,S.L, Girard. J.E. and Fesus,L. (1988) Biochim. Bioph~,~.Acta 972,120-130. 13 Prince, C.W., Dickie, D. and Krumdieck, C.L. (1991) Biochem. BiophysRes. Commun.177,1205-1210. 14 I¢¢, K.N., Birekbiehler, PJ. and Patterson, M.K.,Jr. (1988)Clin. Chem. 34, 906-910. 15 Jeon,W.M., Lee, K.N., Birckbichler,PJ., Conway,E. and Patterson, M.K., Jr. (1989)Anal. Biochem. 182, 1"/0-175. 16 Lee, K.N., Birekbichler, PJ., Patterson, M.K., Jr., Conway, E. and Maxwell,M. (1987)Biochim.Biophys.Acta 928, 22-28. 17 Henrikson, K.P., Allen, S.H.G. and Maloy, W.L (1979) Anal. Biochem. 94, 366-370. 18 Lee, ILN.,Birckbiehler, PJ. and Fesus,L (1986) Prep. Biochem. 16, 321-335. 19 Laemmli,U.IL (1970) Nature 227, 680-685.

20 Matsudaira, P. (1987) J. B/eL Chem. 262, 10035-10038. 21 Bradford, M.M. (1976) Anal. Biochem. 72. 248-~--54. 22 Birckbichler. PJ _ UpchutclL tl.F, Patterson, M.K., Jr. and Conway, E. (1985) Hforidoma 4.179-186. 23 Pearson, W.IL and Lipman, DJ. (1988) Proc. Natl. Acad. Sci. USA 85, 2444-2448. 24 Kukita, A.. MukaL T , Mijata, T. and Hori, IL (1988) Eur. J. Bioch~m. 171,471~,78. 25 Horecker. B.I_, Tsolas, O. and Lai, C.Y. (1972) in The Enzymes (Boyer, P-D_ ed.), Vol. 7. pp. 213-258. Academic Press, New York.

26 Sakaldba.,~a.M., MukaL T. and Hori. K. (1985) Biochem. Biophys. Res. C3mmun. 131.413-429. -r/ Paolclla. G~ Santamaria, R . lzzo, P., Costanzo, P. and Salvatorc. F. (1984} Nucl¢/c Ac/d Res. 12, 7401-7410. 28 Buono. P_ MancinL E P . Izzo, P. and Salvator¢. F. (19~0) Eur. J. Biochem. 192, 805-81 I. 29 Brenner, S.C. and Wold, F. 0978) Biochim. Biophys. Acta 522, 74-83. 30 FoIL J.F-. Park, M.H. Chung, S.L Schrode..I., Lcstcr, E.P. and Cooper. H.L (1980)J. Biol. Chem. 255. 3695-3700. 31 PiacentinL M. and BeninatL S. (1988) Biochem. J. 249, 813-817.