cross-linking of the two constituent polypeptide chains of ricin d - oda - agric biol chem 43 (1979)

8/13/2019 Cross-Linking of the Two Constituent Polypeptide Chains of Ricin D - Oda - Agric Biol Chem 43 (1979)

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Agric. Biol. Chem., 43 (3), 547 - 554, 1979 547

Cross-linking of the Two Constituent Polypeptide Chains of Ricin D with N,NL-o-Phenylenedimaleimide

Tatsuya ODA and Gunki FUNATSULaboratory of Biochemistry, Faculty of Agriculture,

Kyushu University, Fukuoka 812, JapanReceived August 21, 1978

Reaction of the reduced ricin D with N,NL-o-phenylenedimaleimide produced a cross-linked ricin D which could not be split into its constituent polypeptide chains with reducingreagents. This cross-linked ricin D was as stable as native ricin D and retained the fullcytoagglutinating activity, while the toxicity to mice and cultured cells was remarkably decreased.

The cross-linked ricin D was incorporated into cells in the same manner as native ricin D,but was not degraded by incubation with the lysate of rabbit reticulocyt es. From theseresults, it was inferred that ricin D is split into its constituent polypeptide chains by reductionof the inter-chain disulfide bond in cytoplasm and thus its toxic action is elicited.

Ricin D, a highly purified toxic lectin present in castor bean seeds (Ricinus communis), con

sists of an IIe-chain, which inhibits eukaryoticcell-free protein synthesis, and an Ala-chain,which agglutinates mammalian cells.1,2) In aprevious paper, we have shown that these twoconstituent polypeptide chains are linked witha single disulfide bond cleavable with reducingreagents such as 2-mercaptoethanol withoutdenaturation of ricin D and suggested that thisinter-chain disulfide bond plays an importantrole in the elicitation of the toxic action onthe basis of the fact that the toxicity of ricin Dis destroyed by splitting this inter-chain disulfide bond.

In order to investigate how the inter-chaindisulfide bond participates in the toxic actionof ricin D, we prepared a cross-linked ricin D,uncleavable by reduction, using N,NL-o-phe-nylenedimaleimide, a bifunctional reagent forneighboring sulfhydryl groups.

The present paper describes the preparationprocedure and some properties of the cross-linked ricin D.

MATERIALS AND METHODSMaterials. Ricin D and its Ala-chain were prepared

as described earlier.,? N,NL-o-Phenylenedimaleimide,abbreviated as o-Ph(Mal)2, was obtained from AldrichChemical Co. (U.S.A.). Trypsin, chymotrypsin andnagarse were purchased from Sigma Co., WorthingtonBiochemical Co. and Nagase Co., respectively.

All other reagents used were of analytical gradeunless otherwise specified.

Preparation of cross-linked ricin D. 2-Mercaptoethanol (0.1ml) was added to 10ml of ricin solution(25mg/ml of 5mM Tris-HCl buffer, pH 8.0). After2hr at room temperature, the reaction mixture wasapplied to a Sephadex G-25 column (3 x 40cm) whichwas previously washed with 5 mm phosphate buffer,pH 5.5, and eluted with the same buffer. By thisprocedure, 2-mercaptoethanol used for reduction wasremoved and 5mM Tris-HCl buffer of pH8.0, in whichricin D was dissolved, was replaced with 5 mm phosphate buffer, pH5.5. Two hundred milligrams ofcrystal o-Ph(Mal)2 (approximately 200 times mole ofricin D) was added to the reduced ricin D solution andthe reaction mixture was allowed to stand for 2hr withstirring in an ice-bath. After removal of unreactedcrystal o-Ph(Mal)2 on a glass filter, the protein wasprecipitated with ammonium sulfate.

Preparation of cross-linked cysteine. Cross-linkedcysteine was prepared as above. Thirty-six mg ofo-Ph(Mal)2 was added to the cysteine solution (0.6mg/ml of 5mM phosphate buffer, pH 5.5), and stood withstirring for 2 hr in an ice-bath.

DEAE-cellulose column chromatography. The protein precipitate was dissolved in deionized water, dialyzed first against deionized water and then against 5mM

t Biochemical Studies on Ricin . Part XXIV. ForPart XXIII, see Agric. Biol. Chem., 42, 1927 (1978).


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548 T. ODA and G. FUNATSUTris-HCl buffer, pH 8.5. The protein solution wasapplied to a DEAE-cellulose column (2 x 40 cm) previously equilibrated with 5mM Tris-HCl buffer, pH 8.5.

After washing the column with the same buffer containing 18mM NaCl, the adsorbed protein was eluted bya linear gradient of NaCl from 18mM to 100mM in

the same buffer.

Disc-electrophoresis. Disc-electrophoresis was performed at pH 8.3 using 7.5% gel by the method ofOrstein.6) The protein in the gel was detected bystaining with amide black 10 B.

SDS-polyacrylamide gel electrophoresis. The molecular size of the protein was determined by SDS-polyacrylamide gel electrophoresis according to Weberand Osborn.7) Ten microliters of 20mM phosphatebuffer, pH7.2, containing 50% glycerol and 2% SDSwas added to 10l of the protein solution. Afterincubation at room temperature overnight, the proteinsolution was subjected to electrophoresis.

Amino acid analysis. The protein was hydrolyzedwith 5.7 N HCl containing 0.05% 2-mercaptoethanolin an evacuated sealed tube at 108C for 24hr. Theamino acid was analyzed with a JEOL amino acid analyzer (Type JLC-6AH).

Cross-linked cysteine in the cross-linked ricin Dwas determined as 2-amino-2-carboxyethylmercapto-succinic acid after hydrolysis with 5.7 N HCl.

Determination of pH and heat stabilities. One mlof protein solution (0.03% in 5 mm phosphate buffer,pH7.0) was dialyzed against 100 ml of 0.1 M KCl-HClbuffer, pH 1.0 or 0.1M Na2CO3-Na2B4O7 buffer, pH11.0, at 4C for 24 hr. After dialysis against a largeamount of 5 mm phosphate buffer, pH7.0, containing0.9% NaCl, cytoagglutinating activity was determined.For determination of heat stability, on the other hand,the protein solution (0.03% in 5mM phosphate buffer,pH 7.0, containing 0.9% NaCl) was heated at 50Cfor 1 hr. After cooling in an ice-bath, cytoagglutinatingactivity was determined.

Proteolytic digestion. Digestion of the protein wasperformed using trypsin, chymotrypsin and nagarse.Ten microliters of the protease solution (1 % in 50 mmTris-HCl buffer, pH 8.0) was added to 250l of theprotein solution (2 % in 50 mm Tris-HCl buffer, pH 8.0).After incubation at room temperature for 1 hr, thedigest was lyophilized.

Cytoagglutinating activity. Cytoagglutinating activity was determined with sarcoma 180 ascites (SA)tumor cells as previously described.2> To 0.2 ml ofSA cell suspension (1 x 107 cells/ml) were added 0.2mlportions of the protein solution at different concentrations. After incubation for 30min at room tem

perature, the agglutinating activity was measuredmicroscopically.

Toxicity. Toxicity of the protein toward mice wasdetermined with pure-bred male mice (ddN) as previously described.) Increasing amounts of the proteinwere injected intraperitoneally into groups of five mice ,weighing 25 +2 g, and the LD50 dose was calculated fordeaths occurring within 48hr.

Inhibitory activity of the protein toward the growthof the cultured cells was determined with XC andNIH3T3 cells as described previously.0) Trypsin treatedcells (3.5 x 106) were plated on to 35mm Falcon plasticdishes in I ml of Eagle's MEM supplemented with 10%fetal calf serum and 0.1g or 1g of ricin D, cross-linked ricin D or the isolated Ala-chain was added .After incubation for 2 hr at 37C, the medium wasremoved and replaced with fresh medium. After incubation for 24 hr at 37C, the cells were removed fromthe dishes by treatment with 0.1% trypsin solution andcounted after staining with trypan blue dye with ahemacytometer.

Labeling with 1211. Labeling of the protein with 125Iwas carried out according to Hunter.10> Fifty micro-liters of 5mM phosphate buffer, pH7.0, containing50g of the protein, was added to 110l of 0.5Mphosphate buffer, pH7.0, containing 1mCi of carrier-free Na125I (NEN) and then 50 ug of chloramine-Tsolution (100g) was added. After 1 min at roomtemperature with stirring, 100l (500g) of sodiummetabisulfite was added to stop the reaction. For thepurification of 125I-labeled protein, the reaction mixturewas applied to a Sephadex G-200 column (1.5 x 22 cm)previously washed with 5 mm phosphate buffer-0.9%NaCl, pH 7.2 (PBS solut ion), containing 1 % bovineserum albumin and eluted with PBS solution.

Irreversible binding of 125I-labeled protein to cells.0.3ml of 125I-labeled native ricin D solution (7 g,specific activity: 2.4 x 106cpm/g) or the cross-linkedricin D solution (8.8g, specific activity: 1.4 x 105cpm/

g) was added to 1.5ml of SA cell suspension (1x107cells/ml of PBS). After incubation for various timesat 0C or 37C, 0.3ml of the reaction mixture waspoured into 3ml of ice-cold PBS solution containing0.1 M lactose and stood for 10min at 0C. The cellswere washed three times with the same solution bycentrifugation (3000 rpm, 1min). The radioactivity offinal cell pellet was counted in a Aloka auto-wellgamma system (JDC-751).

Preparation of crude rabbit reticulocyte lysate.Crude rabbit reticulocyte lysate was prepared accordingto the method of Woodward et al.11) The reticulocytesobtained from an anematized rabbit were lysed bysuspension in an equal volume of distilled water andallowed to stand at 0C for 5 min. After removal of


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Cross-linking of Ricin D 549unlysed cells by centrifugation, the lysate was storedin aliquots of 3ml in dry-ice acetone.

In order to remove the materials of low molecularweight from the crude rabbit reticulocyte lysate, thelysate (1ml) was gel-filtered on a column (1 x 15cm)of Sephadex G-25 in PBS, and the fraction havinghemoglobin color eluted in the void volume wascollected.

Polyacrylamide gel electrophoresis of 125I-labeledprotein. After incubation of 125I-labeled protein inthe crude rabbit reticulocyte lysate at 37C for 30minor 2hr, 10l of the reaction mixture was subjected toSDS-polyacrylamide gel electrophoresis. After electrophoresis, the gel was quickly removed from the tube,frozen with dry-ice and uniformly sliced into pieces of1mm thickness. The radioactivity of each gel-slicewas measured with a Aloka auto-well gamma system(JDC-751).

RESULTS

Purification of cross-linked ricin DThe elution profile of the reaction mixtureof cross-linked ricin D on DEAE-cellulosecolumn chromatography is shown in Fig. 1;three fractions-F-1, F-2 and F-3-were obtained. As shown in Fig. 2, fraction F-1 gavea single band identical to that of native ricin Don disc-electrophoresis and gave two bandsafter treatment with 2-mercaptoethanol onSDS-polyacrylamide gel electrophoresis. Onthe other hand, fraction F-2 gave a single bandof faster mobility than native ricin D on disc-

FIG. 1. Chromatography of Cross-linked Ricin Don a DEAE-Cellulose Column.Reaction mixture of cross-linked ricin D was appliedto a column (2 x 40cm) of DEAE-cellulose previouslyequilibrated with 5mM Tris-HCl buffer, pH8.5.After washing with the same buffer containing 18mMNaCl, the adsorbed protein was eluted with a lineargradient of NaCl from 18mM to 100mM in the samebuffer. Each volume was 400ml.

FIG. 2. Disc-electrophoresis and SDS-Polyacrylamide Gel Electrophoresis f the Fractions Obtained,see Fig. 1.Disc-electrophoresis (a) (d)) performed at pH 8.3using 7.5% gel, and SDS-polyacrylamideel electrophoresis ((e)-(h)). (a) native ricin D, (b) fractionF-1, (c) fraction F-2, (d) fraction F-3, (e) nativericin D, (f) 2-ME-treated fraction F-1, (g) 2-ME-treated fraction F-2, and (h) 2-ME-treated ractionF-3.electrophoresis and was not split into twochains by treatment with 2-mercaptoethanol.Fraction F-3 was not split into two chains bytreatment with 2-mercaptoethanol, but gavetwo bands on disc-electrophoresis. Fromthese results, it was revealed that fraction F-1is native ricin D and fractions F-2 and F-3are cross-linked ricin D. Yields of fractionsF-1, F-2 and F-3 were approximately 6%,80% and 11%, respectively.Amino acid composition of fraction F-2The amino acid composition of fraction F-2is given in Table I compared with that ofnative ricin D. Their compositions are veryclose to each other and no decrease of lysinewas found in fraction F-2. As shown in Fig.3,2-amino-2-carboxyethylmercaptosuccinic acidwas found in the acid hydrolysate of fractionF-2 and its amount was estimated to be approximately two moles per one mole of ricin D.This result clearly indicates that the cross-linking reaction occurred only with the cysteine


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552 T. ODA and G. FUNATSU

FIG. 7. SDS-Polyacrylamide Gel Electrophoretic Patterns of 125I-Labeled Native and Cross-linked RicinD after Incubation with the Lysate of Rabbit Reticulocytes.After incubation with the rabbit reticulocyte lysate, 125I-labeled ricin D was submitted to SDS-polyacrylamide gel electrophoresis. Details are in the text. (A), 125I-labeled native ricin D incubated with crudelysate; (B), 125I-labeled cross-linked ricin D incubated with crude lysate; (C), 125I-labeled native ricin Dincubated with gel-filtered lysate. -0, before incubation; x -- x, after 30min incubation; 0---0,after 2hr incubation.

to the cells at 0C was almost completely removed by washing with lactose, whereas ricinD incubated with SA cells at 37C for 30minwas irreversibly bound to the cells and theamounts of native and cross-linked ricin Dbound to the cells after 2 hr incubation werecalculated to 1.05g and 1.25g, respectively.The irreversible binding kinetics of the cross-linked ricin D were not distinguishable fromthose of native ricin D, suggesting that thecross-linked ricin D is incorporated into thecells in the same manner as native ricin D

. Integrity of cross-linked ricin D in the rabbitreticulocyte lysateFifty microliters of 125I-labeled native ricin D

(0.204g, specific activity: 6.2 x 106cpm/g)and 125I-labeled cross-linked ricin D (0.21g,specific activity: 9.1 x 106cpm/g) were addedto 200l of the crude lysate. After incubationat 30C for 1hr, 10l of the reaction mixturewas submitted to SDS-polyacrylamide gel elec-trophoresis. As shown in Fig. 7A, nativericin D incubated with the crude lysate gavefour peaks-P-1, P-2, P-3 and P-4. Theposition of P-1 was the same as that of nativericin D, and the positions of P-2 and P-3 wereidentical to those of the Ala- and Ile-chains of

ricin D which can be obtained by treatment ofnative ricin D with 2-mercaptoethanol. PeakP-4 was slightly smaller than the Ile-chain.Radioactivities of peaks P-1, P-2, P-3 and P-4after 30min incubation were approximately65%, 11%, 17% and 4% of that of peak P-1obtained without incubation, and 33%,28%,22 % and 16% after 2hr incubation, respectively. Total activities of these four peaks obtained from 30min and 2hr incubations wereapproximately 97% and 99%, respectively.

On the other hand, the cross-linked ricin Dincubated with the crude lysate and the nativericin D incubated with gel-filtered lysate werenot degraded into smaller molecules and gavea single peak at the position of native ricin D(Figs. 7B and 7C).

From these results, it was suggested thatnative ricin D is first split into Ala- and Ile-chains by reduction of the inter-chain disulfidebond and then the chains are limitedly degraded into smaller molecules in the crude lysate.

DISCUSSIONN,NL-o-Phenylenedimaleimide is an efficient

cross-linking reagent for joining two constituent polypeptide chains of ricin D at the inter-


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Cross-linking of Ricin D 553

chain disulfide bridge. Since it is known thatthis reagent reacts with amino groups, thereaction was performed at pH 5.5 in the presentexperiment. Amino acid analysis of the cross-linked ricin D revealed that s-amino groups oflysine residues in ricin D were not modifiedunder the conditions used. This cross-linkingreaction was successful only when o-Ph(Mal)2was added to the reduced ricin D in which bothIle- and Ala-chains coexisted.Cross-linked ricin D behaved as a moreacidic protein than native ricin D on DEAE-cellulose column chromatography at pH 8.5and easily separated from native ricin D.Since amino groups in ricin D were not modified in this reaction as mentioned above, thedifferent behavior in the chromatography maybe due to a conformational alternation. Detailsof the difference in the conformation betweennative and the cross-linked ricin D are underinvestigation.Cytoagglutinating activity of the cross-linkedricin D thus obtained was almost identical tothat of native ricin D, indicating that the Ala-chain in the cross-linked ricin D is intact,whereas the toxicity toward mice was remarkably reduced. Since the cross-linking reactionis mild and restricted to the inter-chain disulfidebridge, and the Ile-chain is very stable duringphysical treatments, ' it seems unlikely thatthe decrease of toxicity could be caused by themodification and/or denaturation of the Ile-chain. The results on the stability and digestibility of the cross-linked ricin D also show thatthe low toxicity of the cross-linked ricin D isnot due to inactivation and/or breakdown ofthe injected cross-linked ricin D on the wayto the cells in which the toxic action is exerted.Inhibitory activity of the cross-linked ricin Don the growth of cultured cells was much lowerthan that of native ricin D, suggesting that thedecrease of toxicity occurs at cell level.From biological and electronmicroscopicstudies, 141 t has beenn shown that the actionof ricin is elicited by following sequence ofevents: (a) binding of ricin to receptors on cellsurface; (b) clustering of ricin receptors; (c)endocytosis of the ricin molecule; (d) release

of ricin from endocytotic vesicles into the cellcytoplasm and activation of ricin; (e) interaction of activated ricin with ribosomes.To see the differences in these events betweennative and cross-linked ricin D, we used 125I-labeled ricin D and studied the kinetics ofirreversible binding to the intact cells and thedegradation in the rabbit reticulocyte lysate.The cross-linked ricin D was bound irreversibly to the cells in a slightly smalleramount than native ricin D and its bindingkinetics were almost identical to those of nativericin D. This result suggests that the cross-linked ricin D interacts with the ricin receptorand penetrates into the cells in the same manneras native ricin D.With regard to the activation of ricin by thecell-free system during protein synthesis,Lugnier et al.15)have shown by polyacrylamidegel electrophoretic analysis that native ricin issplit into its A (Ile)- and B (Ala)-chains in theincubation with reticulocyte lysate. We obtained a similar result for native ricin D incubated with crude lysate, but the cross-linkedricin D incubated with crude lysate and thenative ricin D incubated with the gel-filteredlysate remained intact. These results suggestthat the splitting of native ricin D into twochains could be caused by reduction of theinter-chain disulfide bond, but not by proteolytic hydrolysis, and that the smaller component appearing as a shoulder (peak P-4 inFig. 7A) in the peak of the Ile-chain may beproduced by proteolytic hydrolysis after thesplitting into two chains. Low toxicity of thecross-linked ricin D is due to unsplitting of theinter-chain disulfide bond, and this is consistent with the result that the toxicity of thecross-linked ricin D toward cultured cells wassimilar to that of the isolated Ala-chain whenthe Ile-chain is not involved in the toxic action.With regard to the susceptibility of ricin D toprotease, recently we found that native ricin Dis not susceptible to lysosomal protease, whilethe isolated chains are attacked and the degraded Ile-chain has a higher inhibitory activity for cell-free protein synthesis than nativericin D.16)


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cross-linking of the two constituent polypeptide chains of ricin d - oda - agric biol chem 43 (1979)

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