192

Btoch#nmca et Bwphyswa Act& 1037 (Iq~)O) 192-,-199

Elsevier BBAPRO 33554

Purification and partial characterization of a novel cellular retinol-binding protein, type three, from the piscine eyes Shinji Nishiwaki t, Michimasa Kato t, Masataka Okuno ~, Masamitu Kanai 2 and Yasutoshi Muto I First Department o/Interred Medicine. Gifu Una,er~it)," School o/Medicine. Gifi~and " Deparm~nr o/Chnwal ~ o ~ " Shmshu Universiw ,~chool of Medwme. M ~ o /Japanl

.Me~cume.

IReceived 23 October 1989)

Key words: Cellular retmol binding protein, t)pe three; Cellular retinoid binding protein. F-type: Reunol; Cellular retino,c acid binding protein; (Piscin¢ retina)

A novel cellular refinol-binding larotein, termed t~pe three (CRBP !il), was isolated from eyes el the bigeye d trot.

CRBP I!! showed a molecular weight of 15400, an isoelectric point of 4.80, #.-mtdWlity in ek,t t t o ~ m s ~ and • ),..,! ol 350 rim, AII-tmm.rtqtin~ the emiogetmus ligand, could be cempetitively displaced by rtSlinmc acid lint not by CRBP ill was differenthtt~l from purified pisciue and rat cellular i~tineglbiading lWoteins (CP.JBIP) xml aeikmlm"rt.tim~ acid-binding pcoteins (CRABP) by its amino-acid compositkm, e k . t ' ~ I p ~ ¢ t ' ~ mobility, ~ spKqra

Ig,~mKm-bimli~s~eelr~.ity. Introduction The discovery, of a cellular binding protein specific for retinol [1], termed cellular retinol-binding protein (CRBPL seems to provide an intracellular counterpart to the plasma retinol-binding protein (RBP) and to prompt the research leading to the identification of the ~ o n d intracelhilar retinoid-binding protein, cellular retinoic acid-binding protein (CRABP) [2], which binds retinoic acid with high affinity and specificity. Recently. the tissue-specific cellular retinol-binding protein, type two (CRBP(II)L has also joined the family of cellular retinoid-binding protein [3,4]. Ik~th CRBP and CRABP are now available as a homogeneous preparation from several tissues, e.g.. rat tes~ts [1,2,5]. bovine retina I61 and human placenta [7]. and tissue levels of the proteins have been determined immuno~hemically in several rat tissues [8-10]. The binding activities of retinoi and retinoic acid have been

Abbresiattuns: CRBPIIIIL ceUular retmol-bmdmg protein, type threeCRBI~FL cellul,~r retinoid-binding protein. F-type: CRRP, cellular retinol-hinding protein: CRABP, cdtular retino/c acid-bindir,g protein: RBP. serum rettrK~l-binding protein; CRBINII), cellular nednoibinding protein, t>pe two. Corresp~mdence: Y. Muto. First Dcparlment of intcr~al Medicine, G.fu L:niscrstty S,:hc~.~l of Medicine, 40 Tsuga_gamachi. GII, 500. Japan.

detected in a variety of tumor tissues and cultured cell lines [11] mostly by a sucrose gradient analysis [12,13k which demaads a high ligand specificity of the retinoid-binding protein. CRBP and CRABP are dis, tinct from each other by a strict ligand specificity, either for retinol or retinoic acid, respectively. On the other hand, Shidoji and Muto found a unique displacement of label from the peak after gel fdtration of fish-eye cytosoi incubated with [3H]retinoic acid and unlabeled retinol [14]. The present study deals with the isolation of this new molecular species, which we proposed to designate cellular retinol-bindmg protein, type three (CRBP(lli)) from piscine retina m the absence of exogenous ligand and determines its endogenous refinoid.

E,,~imenta proceawes Materials

80 heads of bigeye of tuna (Thunnus obesus), a generous gift from Tokai-Regional Fisheries Research Laboratory, Tokyo, were stored at - 2 0 ° C. Rat testes, epididymides and seminal vesicles were kindly provided by Department of Drug Safety Research. Eisai Co., Gifu. Sephadex G-50, SP-Sephadex (B.50) and standard proteins for isoelectnc focusing electrophoresis were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). DEAE-celluiose (DE-52) was product of Whatman (Clifton. U.S.A.). All-tram-{15-3Hlretino~

01674838/90/$03.50 ~Y 19'¢0 Flsesier St:ienc¢ Publishers BN. (Biomedical Di,,i~,c~a)

193 (20.4 Ci/mmol) and all-trans-[ll.13-3H]retinoic acid (46.0 Ci/mmoi) and Omniflour were obtained from New England Nuclear (Boston, U.S.A.). All-trans-[15l , ~ acid (8.4 mCi/mmol) was purchased from Amersham Co. (U.K.). All-trans-retinol was from Nakarai Chemc/al Co. (Kyoto. Japan) and all-trans-refanoic acid, retinaldehyde, retinyl acetate, retiny.~ palmilate and imidazole (grade Ill) were from Sigma Chemical Co. (.~. Louis. U.S.A.)` Protein standards for molecular weight analysis were purchased from BHD Ltd. (London, U.K.)` Ampholine* pH 4 - 6 and pH 3.5-10 were obtained from LKB Sweden AB ( S t o d d ~ n , Sweden).

Prepara'ion of piscine retina extract Both retina "and retinal pi~nent epithelium were readily removed from the half-thawed eyeballs, with no blood contamination. The preparation (wet weight 816 g) was honmgenized with 1700 ml of 50 mM Tris-HCl buffer (pH 8.4). containing 25 mM NaCI. 4 mM EDTA. 1 mM dithiothreitol. ! mM phenylmethanesulfonyl flmmd¢ and 0.004% NaN~ by a Polytron ~. The homogenate was centrifuged at 15000 x g for 60 rain and the supernatant wa~ subsequently ultracentrifuged at 105000 x g foe 90 min. The resulting supernatant was not incubated with exogenous retinoids, with a view to determine endogenous ligands bound by the binding proteins. Detection of retmotd-btndmg proteins CRBP (!!!). CRBP and CRABP were traced by monitoring the fluorescen~ : bound retinoids (excitation. 350 nm; emission. 475 nm) and by measuring the absorbance at 350 nm in each chromatographic procedure. The three binding, proteins were distinguished from each other by competitive binding assay on Sephadex G-50 {superfine) equilibriated with 50 mM Tr~i-HCI buffer (pH 6.4) containing 25 mM NaCI. 4 mM EDTA and 0.004~ NaN; {buffer A), as described previously [14]. Colunm chromatography Each colunm chromatographic procedure was carried out at about 4 ° C in a dim light. The supernatant derived from ultracentrifugation ~as applied to a !0 x 120 cm o~lnmn of Sephadex (3-.50 (fme) equilibrated with buffer A. Fractions of 30 ml were collected at a flow rate of 150 ml/h. Fractions containing the binding proteins were combined and concentrated to 150 ml using an Amicon cell with a YM-5 membrane. After re-chromatography of the resulting solution on the same column as above, fractions containing the binding proteins were com:entrated and dialyzed against 20 mM Tris-HC1 (pH 8.4). The preparation was subsequently submitted to DEAE-ceUulosc (1.6 x 40 cm) equilibrated with 20 mM

Tri~-HC1 buffer (pH 8.4) and etuted with a linear gradient of NaCI from 0 to 150 mM (total volume. 400 mi). 5 ml of each fraction were collected at a flo~ rate of 20

~/h. The sample containing CRBI~IlI) was applied to a column of Sephadex G-50 lsuperfine. 2.5 x 90 cm) equilibrated with buffer A and eluted at a flow rate of 15 ml/h with 4 ml of each fraction. Fractions correspooding to CRBP(lll) were collected, concentrated and dialyzed against 20 mM im/dazole acetate buffer (pH 6.4). The solution was submitted to a column of DEAE-cdlulose (i x 30 cm) equilibrated with 20 mM imidazol¢ acetate (pH 6.4). The column was eluted with a finear gradient of imidazole acetate from 20 to 200 mM (total volume 200 ml) at a flow rate of 6 mi/h. 1.5 ml fractions k o m the column were pooled and dialyzed against !0 mM sodium acetate buffer (pH 5.0) and applied to the final cluomatography on SP-Sephadex (1 × 30 cm). CRBP~III) was ehited with a linear gradi,..: "tom 10 to 80 mM of sodium acetate (total volume 160 ml). The flow rate was 6 m l / h and collected 2 ml in each fraction. CRBP and CRABP were also purified concurrently from the appropriate fractions from the step of ion-exchange chromatography on DEAE-cellulose (pH 8.4) by additional t~,o procedures, including chromatographies on Sephadex G-50 and DEAE-ceilulose (pH 6.4).

Purification procedures of rat CRBP and CRA BP CRBP anti (. RABP were purified from rat .sex organs according to the ,nethod described by Kato et al. [5]. From 700 rat test,-'s. 700 epididyrmdes and 350 seminal vesicles. 4.7 mg of CRBP and 2.7 mg of CRABP were obtained and their purities were checked electrophoretically. Both proteins were stored at - 7 0 °C until u~. Gel ele(trcphoresis Polyaerylanude gel electrophoresis mith 0.1~ sodium dodecyl sulfate (SDS) was conducted using 14% polyacrylamide slab gel (pH 8.8). The following standard pro;eins were employed for the assessment of molecular weigh~ of the purified proteins: ovotransferrin (Mr 77000). bovine serum alburmn (M r 66290). ovalburmn {'~'tr 45000). bovine, ch2,motrypsinogen A ( M r 25700). horse myoglobin (M~ 17200) and horse ~'tochrome c (34, 12300). Non-denaturing polyacrylamide gel dectrophoresis was carried 3ut in 10% polyacrylamid¢ slab gel (pH 8.8). Anal)aical isoelectnc focusing in polyacr2,.lamide gel electrophoresis was carried out according to Righetti and Drysdale [15], using 6~ polyacrylamide disc gel containing Amphohne ~' at pH 4-6 (1.6%) and pH 3.5-10 (0.4q~). The isoelectric point of the proteins was estimated from the migration rates in comparison with standard proteins: human carbonic anhydrz,se B (pl 6.55). bovine carbonic anhydrase B (pl 5.85). /~-lacto-

194 globulin A (pl 5.20). soybean trypsin inhibitor (pl 4.55) and glucose oxidasc ( p / , ~ 15). Each gel was stained ac,;,,r,:,,~, ~'.~ the method of Reisner et al. 116].

High-performance size.exclusion chromatograph)' (HPSEC) system Molecular weight, binding affinity for various retiholds, and quantitation of three retinoid-binding proteins were analyzed by the HPSEC system, which consisted of a TSK G3000 SW column, a CCPM pump, a UV-8000 HPLC detector (Tc~yo Soda Kogyo Co.. Tokyo, Japan) set at 280 nm and a Chromatocorder 11 integrator (System Instniments Co., Tokyo, Japan). The column was equilibrated and eluted with 200 mM phosphate buffer (pH 7.2) at a flow rate of 1 m l / m i n and fractions of 0.5 ml were collected. The molecular weight estimation of each binding protein was based on a comparison of their retention time with that of the standard proteins: bovine serum albumin (M, 66 200), bovine chymotrypsinogen A (M, 25 700). horse myoglobin (M, 17 200), and horse cytochrome c (M, 12300). The binding affinity and quantitation of t,ae bimling proteins were determined by the method of competitive binding assay on this system, according to the method described by Shidoji et al. [14].

A mino-acid anal)'sis E~z.h of the purified proteins (20 /xg) were hydrolyzed with 6 M HCI for 21 h at 119°C, and analyzed on a Waters pico-TAG amino-acid analyzer.

standard. After mixing, the upper phase was evaporated with argon gas and dissoh'ed in 100 gl of ethanol. The extracted retinoids were analyzed by a Shimadzu gradient reversed-phase HPLC system consisting of a CLCODS column, two LC-6A pumps, a SPD-6AV spectrophotometric detector and a SCL-6A system controller. 85c~ of solvent A (methanol/ethanol = 1 : I) and i5% of solvent B (0.1~ phosphate) were followed by a gradient to 100~ of solvent A at 20 rain. Retinoids were detected by absorbance at 325 nm.

Other procedures Fluorescence and fluorescence spectra were analyzed with a Hitachi 204 fluorescence spectrophotometer. Absorbance measurement and absorption spectra were carried out with a Hitachi 220A spectrophotometer. Prior to me analysis of the spectrum, each of the bi~xling proteins were incubated overnight at 4 ° C with a 100 fold molar excess of ei,.her retinol or refinoic acid respectively, and were separated from respective free retiaoids by a gel filtration on a column of Sephadex G-50 (2.5 × 90 cm), equilibrated and eluted with 50 mM Tris-HCI buffer (pH 8.4). Protein concentrations were estimated by the method of Lov,~ et al. [181. with bovine serum albumin as a standard. Radioactivity was counted w~h a Beckmar, LS-7500 i;.... ~,4 r,cintillation counter. 7 hal of a scintillation cocktail (Triton X-100/toluene/Omniflour, 500 ml : 1000 m i : 6 g) were mixed with 0.2-0.5 ml of the samples. Results

Determination oi" endogenous ligand The endogenous ligands associated with each of the binding proteins were determined b ' , ;,:,,h-performance liquid chromatography (HPLC) of tat" extl a~., cd ligands, according to the method of Furr a,,d Oisor" !17]. With the exception of CRBP(IID from semi-final purification procedure, retinoids were extracted from the three isolated binding proteins with an c:'-~'d ~olt, me of n-hexane after addition of 20 gl of reti;', .~- "~ate as an internal

Purification procedures of ptsctne CRBP(Ili), CRBP and CRABP Table 1 presents datz~, from the purification of CRBP(II1), starting with 14 g of soluble extract from the homogenate of piscine retina. The extrac', was first submitted to gel filtration on Sephadex G-50 (fine), fi~r the purpose of excluding some of the retinoid-t)inding proteins with relati~dy

T,~BLF I Pur~ticatzon . ! C R B P t I I h from p~.~,me re. :a

Fraction

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CRBI~ i i i ) ! rag)

Pun ficati~-m {- fold)

Recover~ (q~)

Retina homogenate Sephadex G-50 (fine) (1) Sephadex G-50 (fine) ~11~ DEAE-cellulose (pH 8.4) Sephadex G-~0 (superfine) DEAE-¢ellulos¢ I p H 6.4) SP-Sephadex ~pH 5.0)

. ~-'~ ~ ~ 1 9o0 380 50 28 4.3 0.21

5.8 b 3.8 ~ 2.9 b 1.8 ~ 1.4 b 0.76 b 0.21 ~

7.4 37 250 _~0 3300 67 0C~)

06 50 31 24 13 3.6

J Estimated b} L o w ~ ' s meth.xt. h F..sumated b) competit|v¢ binding a~say of gel filtration on TSK G30(X) S ~ . • Assumed since no protein other than CRBP~III) , ~ demonstrable.

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F . g . I . Chromatography on DEAE-cellulos,e of the sample obtainod from the second gel rdtranon on Sephadex G-~O. The column ( 1.6 × 40 c-m) was equilibrated ~.~th ~ mM Tns-HC1 buffer (pFI 8 4) and etuted w~th a linear gradient of NaCI from 0 to 150 m M . Three pr~mntnent p¢:aks o4" fluorc~¢x~¢ w¢1"¢ o~'3s£'lt~c~L in fractions 39-48. fractions 53-59. :rod frm-ttons 68-79 Frcx,n the binding a x ~ . tbc f i r ~ peak pros-od to ¢omam CRBP~IIi). the ~ t ' t d peak to cot.tam CRBP. and tl~ third peak to ctmtam CRABP. F.a~ch fractlota v.a~ ptx~lod and subjected to further chromatograph~,.

large molecular sizes, i.e., inters'itial r e t i n o l - b m d t n g pt,.,tein ( M , 260000) [19] and cellular retinal-binding p r o t e i n ( M , 33000) [201. By an addition',d gel filtration o n the same column, the molecular weights of the p r o t e i n s in the s a m p l e ranged from Mr 10000 to 30000. T h e next c h r o m a t o g r a p h y on the DEAE--cellulos¢ ( p H 8.4) showed three p r o m i n e n t p e a k s of fluorescence (Fig. I). These peaks were a c c o m p a n i e d by the peaks of a b s o r b a n c e at 3.50 rim. The competitive binding a s ~ revealed that the first peak (fractions 3 9 - 4 8 ) was assignable to a binding protein h a - i n g an affinity for both retinol a n a retinoic acid. that is. C R B P ( I l l L On the other hand. the second peak (fractions 53-59) and the third peak (fraction:; 6 8 - lO) ~howed ,tri~.t ligand affinity either for retinol or for retinoic acid. respecttvcl~. indicating that the form,~r fract.~,[., contained C R B P a n d the latter CrG~BP. F u r t h e r purification of the fractions c o m p r i s i n g ti~e first fluorescent peak ~ a s achieved by g,I filtration on Scphadex G - 5 0 (superf'i-,,:~ and b)" ,on-exchange chrom a t o g r a p h y on DEAE--cellulose ( p H b.4) ( o a t a not shown). The final step of the purification of C R B P ( I I I ) was ion-exchange c h r o m a t o g r a p h y on SP-Sephadex ( p H 5.0) (Fig. 2). By elution with the sodium acetate buffer gradient, the fractions ( 5 2 - 5 5 ) c o m p r i s i n g the abs o r b a a c e p e a k a: 350 nm were cleariy separated from Mi r e m a i a m g impurities. CRP-P a n d C R A B P were isolated concurrently by the procedures described in Experimentzl p r ~ e d u : e s .

Molecular weigh~ determination O n the H P S E C

system, that a b s o r b a n c e peak of

Fig. 2. Final chromatography oa SP-Sephadex Th,¢ column ~,as equdibrattd with 10 mM sodium acetate buffer (pH 5.0) and elmed with a linear gradient fcwm 16 to gO mM ~ l l u m acetate. By gel electrophoresis fraeticm~52-55 ~¢le revealed to he homogeno.xas~ C R B P ( I i l ) was equivalent ' a molecular wc,ght of 15400 and the peaks of pi:,~ine C R B P and C R A B P corresponded to those of 1 5 4 ~ and 15.~)O. respectively. The molecular weight was als, ,~tim~.d by SDSpolyac~'lamide gel elcctrophor~.'sls,I:%. 3:,j.fhe migration rate of C R B P ( I [ D was consistent with a molecular weight of 15300. w'. : pi.~ine C R B P and C R A B P shared the protein b a n d wi:h a migration rate corres p o n d i , ' 3 to a m o ' x u l a r ~',eight of 13700.

Gel eleclropho,esis All three r e t i n o i d - b i n d i n g proteins gave a single band when a n a l y z e d oy p o l y a c ~ l a m i d e gel electrophoresis in the a b ~ n c e of SDS (Fig. 3B). However. C R B P i I [ I )

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Fig. 3 Pol)at.'rylamMc gel ch.'ct,ophl,rc~ls ,,f purified pi~cmc CRBR Ill). CRBP anJ CIL,-~BPm the presence(A) and abstnc¢ (B) of SDS. A b ~ t 2 .ag of put]God protein v,a~ apphod to each ~.¢11. (A) From I¢[t t,, night: molc,:ular ~¢,ght ~t.mdard-~(MWL CRBI~ill)It i. CRISP (2) and ( R . ~ B P t3). From each >angle hand of the purified

prcWem, the m,ok~ular ~,elgh( ~,,L,,e~,limatod as 15300 (CI~BPIiI1)L 1~','OOq('RBP and (.'RABP) (B) Fr~,,m left to right: rat scram (RSI, CRBPII|i, (|). CRBP (2) and (. IL-XBP(3). Each i~-iatod prolcm ga,.e a ~ngl¢ band. CREl~lll) nugr,~tcd .o the avregion of rat so,rum pro[cm, and CRBP and (.'iL-.~BPmlgr.tLedLOthe p:calbumln region.

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Fig. 4. Analyses of pun~'ied piscme CRBI~III ). CRBP and CRABP b~ isoelectric focusing in polyacrylanude disc gels. About 2-5 pg of CRBPIIII) (1). CRBP (2) and CRABP (3) -~ere applied to the gels. CRABP dispIJycd two bands, the mare band was holo-CRABP and the rmnor band (arrow) ,,as apo-CRABP. The calculated is~a¢l¢ctric values ~rre 4.80 (for CRBP(II[) and CRABP) arid 4.73 (for CRBP).

alone migrated to the a:region of rat serum proteins, while the other binding proteins showed prealbumin mobility. On the other hard, similar pH values were obtained when the isoelectric points of the three proteins were analyzed by isoelectric focusing electrophoresis (Fig. 4). The apparent isoelectric pH values of the three binding proteins were 4.80 for CRBP(III), 4.73 for CRBP and 4.80 for CRABP, respectively. During dectrophoresis CRABP clearly resolved into two bands. By illuminating the gel in the dark with long-wave ultraviolet light, prior to the staining procedure, only the band on the anode side was found to have fluorescence associated with it. The above physiochemical properties of three retinoid-bmding proteins were summarized in Table 11 in comparison with rat CRBP and CR~ BP.

Determination of endogenous ligand The endogenous ligands associated with each of the binding proteins were analyzed by HPLC system of the extracted ligands. Only retinoi was identified in the hexane extract from the semi-final preparation of piscine

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