Double-Lectin Site Ricin B Chain Mutants Expressed in Insect Cells

Nov 1, 1996 - ... Tagge,‡ John Chandler,‡ Mark Willingham,§ and Arthur Frankel*,† ... Pathology, Medical University of South Carolina, Charlest...
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Bioconjugate Chem. 1996, 7, 651−658

651

Double-Lectin Site Ricin B Chain Mutants Expressed in Insect Cells Have Residual Galactose Binding: Evidence for More Than Two Lectin Sites on the Ricin Toxin B Chain Tao Fu,† Chris Burbage,† Edward Tagge,‡ John Chandler,‡ Mark Willingham,§ and Arthur Frankel*,† Departments of Medicine, Surgery, and Pathology, Medical University of South Carolina, Charleston, South Carolina 29425. Received May 31, 1996X

Ricin toxin, the heterodimeric 65 kDa glycoprotein synthesized in castor bean seeds, contains a cell binding lectin subunit (RTB) disulfide linked to an RNA N-glycosidase protein synthesis-inactivating subunit (RTA). Investigations of the molecular nature of the lectin sites in RTB by X-ray crystallography, equilibrium dialysis, chemical modification, and mutational analysis have yielded conflicting results as to the number, location, and affinity of sugar-combining sites. An accurate assessment of the amino acid residues of RTB involved in galactose binding is needed both for correlating structure-function of a number of plant lectins and for the design and synthesis of targeted toxins for cancer and autoimmune disease therapy. We have performed oligonucleotide-directed mutagenesis on cDNA encoding RTB and expressed the mutant RTBs in insect cells. Partially purified recombinant proteins obtained from infected cell supernatants and cell extracts were characterized as to yields, immunoreactivities, asialofetuin binding, cell binding, ability to reassociate with RTA, and recombinant heterodimer cell cytotoxicity. Two single-site mutants (subdomain 1R or 2γ) and two double-site mutants (subdomains 1R and 2γ) were produced and studied. Yields varied by two logs with lower recoveries of double-site mutants. All the mutants showed immunoreactivity with a panel of anti-RTB monoclonal and polyclonal antibodies. Single-lectin site mutants displayed up to a 1 log decrease in asialofetuin binding avidity, while the double-site mutants showed close to a 2 log decrease in sugar binding. However, for each of the double-site mutants, residual sugar binding was demonstrated to both immobilized asialofetuin and cells, and this binding was specifically inhibitable with R-lactose. All mutants reassociated with RTA, and the mutant heterodimers were cytotoxic to mammalian cells with potencies 1000-fold or more times that of unreassociated wild-type RTA or RTB. These data support a model for three or more lectin binding subdomains in RTB.

INTRODUCTION

Lectins such as ricin toxin from the Ricinus communis plant mediate a wide range of biological effects due to binding cell surface carbohydrates. The B chain subunit of ricin (RTB)1 binds to mammalian cell membranes by recognizing galactose-containing receptors, and this reaction is the first necessary step for intoxication of cells (1). RTB binds β-galactosides (2) with association constants from 103 to 3 × 104 M-1 for simple sugars (3, 4) and 107-108 M-1 for free and cell surface-bound glycoproteins (5, 6). These results lead to the hypothesis that multiple low-affinity sugar binding on ricin interact with complex oligosaccharides and cells to yield high-affinity binding. Clustering of target sugars in the proper * Address correspondence to this author at the following address: Hollings Cancer Center, Rm 306, 86 Jonathan Lucas St., Charleston, SC 29425. Telephone: 803-792-1450. Fax: 803-792-3200. † Department of Medicine. ‡ Department of Surgery. § Department of Pathology. X Abstract published in Advance ACS Abstracts, November 1, 1996. 1 Abbreviations: RTB, ricin toxin B chain; RTA, ricin toxin A chain; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ELISA, enzyme-linked immunoassay; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; cDNA, complementary DNA; IC50, concentration of compound reducing cellular protein synthesis by 50%; moi, multiplicity of infection; EDTA, ethylenediaminetetraacetic acid; NTEAL, 50 mM NaCl/25 mM Tris (pH) 8/1 mM EDTA/0.01% sodium azide/ 25 mM lactose; NTEALT, 500 mM NaCl/25 mM Tris (pH) 9/1 mM EDTA/0.01% sodium azide/25 mM lactose/0.1% Tween 20.

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geometry to enhance lectin binding via interactions at multiple sites has been observed for the hepatic galactose N-acetylgalactosamine-binding receptor (7) and the macrophage mannose receptor (8). The X-ray crystallographic structure of ricin provides a structural basis for this hypothesis. RTB has two domains each with three subdomains (9). The six subdomains have similar folding and primary amino acid sequences and resemble the primitive galactose binding fold in discoidin I from the slime mold Dictyostelium discoideum. Tripeptide kinks in the loops from subdomains 1R, 1β, 2R, and 2γ may interact with galactosides. Each of these subdomains has aromatic residues which can interact with the nonpolar face of galactose, and three of the four subdomain folds (1R, 1β, and 2γ) have polar residues for hydrogen bond formation to the sugar hydroxyls. Cocrystallization of R-lactose at low concentrations (5 mM) with ricin permitted identification of bonds between lactose and amino acid residues of subdomains 1R and 2γ. Biochemical modification studies have identified one to three sugar binding sites per RTB molecule. NBromosuccinimide modification of Trp-37 reduced sugar binding, demonstrating a sugar-binding site in the subdomain 1R fold (10). N-Acetylimidazole O-acetylation of two tyrosines reduced sugar binding, implicating sites in subdomains 1β pocket and 2γ pocket (11). Further, three distinct sites on ricin were cross-linked by radiolabeled fetuin glycopeptide containing a dichlorotriazineactivated 6-(N-methylamino)-6-deoxy-D-galactose moiety, supporting the concept of three sugar-binding sites (12). © 1996 American Chemical Society

652 Bioconjugate Chem., Vol. 7, No. 6, 1996

Fu et al.

Figure 1. Model of subdomains 1R and 2γ of ricin B chain showing amino acid residues altered in this study. Coordinates derived from Rutenber and Robertus (9). Ball and stick diagram display of Brookhaven coordinates on SYBYL software on Silicon Graphics Iris Indigo workstation with R carbon backbone displayed in gray and modified amino acid residues in black. Lactose molecules displayed in black also.

Mutational analysis of recombinant RTBs produced in Cos cells, bacteriophage, and Xenopus laevis ooctyes has suggested either one active lectin site in subdomain 2γ or two lectin sites in subdomains 1R and 2γ (13-15). RTB mutant N255A produced in Cos cells and RTB mutants K40M/N46G/N255G and D22Q/V23A/R24N/D234A/V235A/ R236T synthesized in Xenopus oocytes lacked sugar binding (13, 14). However, very small amounts of proteins were made in each case, and purification and immunologic characterization of products were not done. In all three mutational studies, decreased sugar binding due to misfolding or aggregation of recombinant RTBs could lead to an overestimation of the effect of individual modifications. To obtain more accurate quantitative information on the RTB lectin sites, our laboratory has expressed, partially purified, and characterized wild-type, singlesite, and double-site RTB mutants in insect cells (1619). We obtained microgram to milligram yields of recombinant proteins and were able to purify the lectins to 10-50% purity by Coomassie-stained SDS-PAGE. Wild-type recombinant RTB bound asialofetuin and cell surface oligosaccharides in a manner similar to that of plant RTB with half-maximal binding concentrations of about 5 × 10-9 M (16, 17). Single-site mutants of both the 1R subdomain and the 2γ subdomain had reduced binding affinity for asialofetuin and KB mammalian cells (18). However, the reduction in binding affinity was 1 log or less in each case. After reassociation with plant RTA, each single-site mutant retained HUT102 human leukemia cell cytotoxicity with ID50’s within 1-1.5 logs of that of the wild-type heterodimer. The minor effect of genetic modification of lectin sites in subdomains 1R and 2γ suggested incomplete inactivation of lectin sites or additional sugar-combining sites on RTB. We then expressed, partially purified, and characterized three double-site mutant RTBs and one additional single-site mutant RTB (19). Two of the double-site mutants had unique properties suggesting either persistence of two main galactose binding sites operating at reduced levels or the theory of three ricin lectin sites. To resolve the issue of incomplete inactivation of the lectin sites in the single- and double-site mutants, we now report the biological properties of two additional single-site mutants and two additional double-site mutants. Modifications at each site were chosen to maximally alter each lectin

pocket. Our findings support the hypothesis of at least three independent sugar-combining sites on ricin. EXPERIMENTAL PROCEDURES

Mutagenesis. pUC119-RTB plasmid containing a BamHI-EcoRI DNA fragment coding for ADP-RTB was propagated in INVRF′ Escherichia coli cells (InVitrogen, San Diego, CA) as previously described (16). Singlestranded DNA was produced by infection of transformants with M13K07 phage (Stratagene, La Jolla, CA) as previously described (20). Oligonucleotides were synthesized on an Applied Biosystems 380B DNA synthesizer and desalted with butan-1-ol. 39-mers were prepared with the modified codon flanked by 18 bases on each side matching RTB sequence and lacking an NciI site. Site-specific mutagenesis was performed by the Eckstein method using the Sculptor in vitro mutagenesis system (Amersham, Arlington Heights, IL) and the manufacturer’s instructions (21). Modifications were made at both the 1R and 2γ subdomains on the basis of the X-ray crystallographic model of the lectin binding sites (Figure 1) to either alter key polar residues which provide hydrogen bonds to sugar hydroxyls (D234E, N46G/K40M/N255G, and D22Q/V23A/R24N/D234A/ V235A/R236T) or change aromatic ring residues which provide van der Waals interactions between the protein and sugar (Y248H). Sequences of mutant RTB DNAs were confirmed by double-stranded dideoxy sequencing by the Sanger method using the Sequenase kit (USB, Cleveland, OH) (22). Construction of Transfer Vectors Encoding Mutant RTBs. Mutant RTB-encoding pUC119 DNAs were then restricted with BamHI and EcoRI, and the RTBencoding fragments were subcloned into pAcGP67A plasmid (PharMingen, San Diego, CA) and used to transform INVRF′ E. coli cells. Transfer vectors with mutant RTBs were then purified by cesium chloride gradient centrifugation. Isolation of Recombinant Baculoviruses. The Sf9 Spodoptera frugiperda ovarian cell line was maintained on TMNFH medium supplemented with 10% fetal calf serum and gentamicin sulfate (10 µg/mL). pAcGP67Amutant RTB DNAs (4 µg) were cotransfected with 0.5 µg of BaculoGold AcNPV DNA (PharMingen) into 2 × 106 Sf9 insect cells following the recommendations of the supplier. On day 7 post-transfection, media were cen-

Ricin Toxin B Chain Mutants

trifuged and the supernatants tested in limiting dilution assays with Sf9 cells. Sf9 cells (2 × 104) were incubated with 10-fold dilutions of supernatants in 96-well plates. Seven days postinfection, supernatants were saved and cells in each assay well were lysed with NaOH and the lysates transferred to nitrocellulose. The nitrocellulose was then blocked with Blotto and reacted with random primer [32P]-dCTP-labelled RTB DNA. After hybridization for 16 h at 67 °C, the dot blot membranes were washed with 0.1 × (150 mM NaCl/15 mM sodium citrate)/ 1% SDS, dried, and exposed to X-ray film. Positive wells were identified and supernatants reassayed by limiting dilution until all wells up to 10-8 dilution were positive. Two rounds of selection were required for each mutant. Recombinant viruses in the supernatants were then amplified by infecting Sf9 cells at a multiplicity of infection (moi) of 0.1, followed by collection of day 7 supernatants. Expression of Mutant B Chains in Sf9 Cells. Recombinant baculoviruses were used to infect 2 × 108 Sf9 cells at an moi of 5 in EX-CELL 400 media (JRH Scientific, Lexena, KS) with 50 mM R-lactose in spinner flasks. Media supernatants containing mutant RTBs were collected on day 6 postinfection. Purification of Mutant RTBS. Media supernatants were adjusted to 0.01% sodium azide and maintained through all purification steps at 4 °C. The supernatants were concentrated 15-fold by vacuum dialysis, centrifuged at 3000g for 10 min to remove precipitate, dialyzed against 50 mM NaCl, 25 mM Tris (pH 8), 1 mM EDTA, 0.01% sodium azide, and 25 mM R-lactose (NTEAL), ultracentrifuged at 100000g for 1 h, and loaded onto a P2 monoclonal antibody-acrylamide column previously described (16). The affinity column was then washed sequentially with NTEAL and 500 mM NaCl, 25 mM Tris (pH 9), 1 mM EDTA, 0.01% sodium azide, 25 mM R-lactose, and 0.1% Tween 20 (NTEALT), and mutant RTBs were eluted with 0.1 M triethylamine (pH 11). The alkaline eluants were immediately neutralized with 1 M sodium phosphate (pH 4.8) and stored at -20 °C until they were assayed. Optical densities at 280 nm were determined and aliquots mixed with reducing 2 × SDS sample buffer, boiled for 4 min, submitted to a 15% SDSPAGE, stained with Coomassie Blue R-250, and destained with acetic acid/methanol. Gels were scanned on an IBAS automatic image analysis system (Kontron, Germany) to estimate the fraction of the protein with a molecular mass of 32 kDa. Several preparations of mutant RTBs were made from cell pellets by dissolving pellets in 10 volumes of 20 mM Tris (pH 8), 50 mM NaCl, 1% NP40, 1 mM PMSF, 2 µg/ mL aprotinin, 1.5 µg/mL pepstatin, and 1.5 µg/mL leupeptin. The extracts were frozen at -70 °C, thawed, and centrifuged at 22000g for 15 min at 4 °C. Extracts were then dialyzed against NTEAL and treated in the same manner as dialyzed concentrated cell supernatants. Immunological Properties of Mutant RTBs. Aliquots of mutant RTBs, plant RTB (Inland Laboratories, Austin, TX), wild-type recombinant RTB, and prestained low-molecular mass standards (BioRad, Hercules, CA) were mixed with reducing 2 × SDS sample buffer, boiled for 4 min, submitted to a 15% SDS-PAGE, and electrophoresed for 90 min. Gels, Whatman 3M #1 paper, and nitrocellulose were equilibrated for 15 min in Towbin buffer (20 mM Tris/0.1 M glycine/20% methanol) and placed in a Semi-dry Trans-blot cell (BioRad). After electrophoresis at 15 V for 20 min, the nitrocellulose was blocked with 10% Carnation’s nonfat dry milk/0.1% BSA/ 0.1% Tween 20/0.02% sodium azide. The blots were then washed with PBS plus 0.05% Tween 20 and PBS,

Bioconjugate Chem., Vol. 7, No. 6, 1996 653

incubated with rabbit anti-ricin antibody (Sigma, St. Louis, MO) at 1:400 in PBS plus 0.5% BSA plus 0.01% sodium azide for 1 h, washed again, incubated with alkaline phosphatase-conjugated goat anti-(rabbit IgG) (Sigma) at 1:1000 in PBS plus 0.5% BSA plus 0.01% sodium azide for 1 h, washed again, and developed with the Vectastain alkaline phosphatase kit, following the manufacturer’s recommendations (Vector Laboratories, Burlingame, CA). Blots were scanned as above to compare 32 kDa Mr band intensities. Monoclonal antibody P2, P8, or P10 (gifts of Dr. Walter Blattler, ImmunoGen, Cambridge, MA) (100 µL) at 5 µg/ mL in PBS was incubated in Costar EIA microtiter wells overnight at 4 °C. Samples of plant RTB, wild-type recombinant RTB, and mutant RTBs were treated for 20 min at room temperature with 5% β-mercaptoethanol and then dilutions made in EX-CELL 400. The antibodycoated microtiter wells were then washed with PBS plus 0.1% Tween 20, blocked with 3% BSA/PBS/0.01% sodium azide, rewashed and incubated with dilutions of the reduced RTB samples, rewashed and incubated with rabbit anti-ricin antibody at 1:400 in PBS plus 0.5% BSA plus 0.01% sodium azide, washed again, incubated with alkaline phosphatase-conjugated goat anti-(rabbit IgG) at 1:1000 in PBS plus 0.05% BSA plus 0.01% sodium azide, washed and developed with (p-nitrophenyl)phosphate at 1 mg/mL in 50 mM diethanolamine buffer (pH 9.8), and read on a BioRad 450 microplate reader at 405 nm. For each experiment, 12 different concentrations of plant RTB and recombinant RTBs were tested. A plot of absorbance versus dilution was made for plant RTB and recombinant proteins. Dilutions yielding halfmaximal binding were used to calculate concentrations. Lectin Activity of Mutant RTBs. Volumes (100 µL) of 1 µg/mL asialofetuin in PBS were added to wells of a Costar EIA plate and incubated overnight at 4 °C. Samples of plant RTB, wild-type recombinant RTB, or mutant RTBs in EX-CELL 400 were exposed to 5% β-mercaptoethanol for 20 min at room temperature to remove homodimers and dilutions made in EX-CELL 400 medium with or without 100 µg/mL asialofetuin or 100 mM R-lactose. The asialofetuin-coated microtiter wells were then washed with PBS/0.1% Tween 20, blocked with 3% BSA/PBS/0.01% sodium azide, and rewashed. The dilutions of various reduced RTBs were added to wells for 1 h and then removed and the wells washed again. Rabbit anti-ricin antibody was added (1:400 dilution in 0.5% BSA/PBS/0.01% sodium azide) for 1 h, and the wells were washed again; alkaline phosphatase-conjugated goat anti-(rabbit IgG) (1:5000 in 0.5% BSA/PBS/0.01% sodium azide) was incubated in the wells, and finally, the wells were washed and reacted with 1 mg/mL (p-nitrophenyl)phosphate in 50 mM diethanolamine buffer (pH 9.6) and measured in a microtiter plate reader at 405 nm. In each experiment, 12 different concentrations of plant RTB and recombinant protein were tested. As in the antibody ELISA, relative reactivity to plant RTB was calculated from concentrations giving half-maximal binding. The effects of 100 µg/mL asialofetuin or 100 mM R-lactose on half-maximal binding were calculated for plant RTB, wild-type recombinant RTB, and mutant RTBs. KB cells were washed with PBS and attached to polylysine-coated tissue culture dishes and centrifuged at 2000g for 10 min. The cells were then incubated live at 4 °C. The cells were washed with 2 mg/mL BSA in PBS and incubated in PBS plus BSA with or without 100 µg/mL asialofetuin and with 1 µg/mL freshly reduced plant RTB, recombinant wild-type RTB, or mutant RTB. The incubation was done at 4 °C. The cells were then

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washed with PBS and incubated with rabbit anti-ricin antibody at 1:400 in PBS plus BSA for 30 min at 4 °C. The cells were then washed with PBS and reacted with goat anti-(rabbit Ig) conjugated to rhodamine at 25 µg/ mL for 30 min at 4 °C. The cells were washed again in PBS, fixed in 3.7% formaldehyde in PBS, mounted under a #1 coverslip in glycerol-PBS (90:10), and examined under a Zeiss Axioplan epifluorescence microscope. Reassociation of Mutant RTBs with Plant RTA To Form Heterodimers. Portions (30 µg) of plant RTB and wild-type recombinant RTB and 1-5 µg of mutant RTBs were mixed with a 4-fold molar excess of plant RTA (Inland Laboratories) in a total volume of 0.5 mL of 0.1 M triethylamine/sodium phosphate (pH 7) shaking overnight at room temperature. The reaction mixture was then analyzed by a modified ricin ELISA. Wells of an EIA plate were coated with 10 µg/mL P2 monoclonal antibody to RTB diluted in PBS in a volume of 100 µL overnight at 4 °C. The wells were washed with PBS plus 0.1% Tween 20, blocked with 3% BSA in PBS plus 0.02% sodium azide, rewashed, and incubated with dilutions of ricin or reassociated heterodimers. The wells were again washed and incubated with biotin-conjugated RBR12 monoclonal antibody (RBR12 mouse monoclonal antibody reactive with RTA was a gift of Dr. Walter Blattler, ImmunoGen) at 5 µg/mL in PBS/0.5% BSA/0.01% sodium azide. Biotinylation was performed using (N-hydroxysuccinimido)biotin (Sigma) following the manufacturer’s instructions. Wells were washed and incubated with alkaline phosphatase-conjugated strepavidin (Sigma) at 1:1000 in PBS/0.5% BSA/0.01% sodium azide, washed again, and developed with (p-nitrophenyl)phosphate at 1 mg/mL in 50 mM diethanolamine (pH 9.8). Absorbance at 405 nm was read on a microtiter plate reader. Reassociated mixtures were also analyzed by nonreducing SDS-PAGE followed by immunoblots with RBR12 anti-RTA monoclonal antibody and P10 anti-RTB monoclonal antibody. Densitometric scanning using the IBAS 2000 automatic image analysis system was done to quantify shift of immunoreactive material from 30 to 60 kDa. Cytotoxicity of Recombinant Mutant Heterodimers. HUT102 human T leukemia cells (1.5 × 104) in 100 µL were placed in 96-well flat-bottomed plates in leucine-poor RPMI1640 containing 10% dialyzed fetal bovine serum. Fifty microliters of ricin (Inland Laboratories), recombinant wild-type RTB-plant RTA heterodimer, and mutant RTB-plant RTA heterodimers at varying concentrations were added in the same medium and the cells incubated at 37 °C in 5% CO2 for 24 h. [3H]Leucine, 0.5 µCi per well (120 mCi/mmol), in 50 µL of the same medium was added and incubated for 4 h. Cells were then harvested with a PhD cell harvester onto glassfiber filter mats. The filters were dried, mixed with 3 mL of liquid scintillation fluid, and counted in an LKBWallac liquid scintillation counter gated for 3H. Cells cultured with medium alone served as controls. All assays were performed in triplicate. The ID50 was the concentration of protein which inhibited protein synthesis by 50% compared with control. RESULTS

Yields and Immunoreactivity of Mutant RTBs. Two new single-site mutants were prepared (D22E and Y248H). Y248H was previously described as part of a single-domain RTB-gene 3 protein fusion on fd phage (23), but the properties of a full length RTB with the single mutation have not been reported. R. communis agglutinin B chain and ricin E B chain have histidines

Fu et al.

Figure 2. Insect-derived wild-type and mutant RTBs. (A) Coomassie-stained 15% reducing SDS-PAGE of mutant RTBs: lane 1, low-molecular mass prestained BioRad protein standards; lane 2, wild type; lane 3, D234E; lane 4, Y248H; lane 5, N46G/K40M/N255G; and lane 6, D22Q/V23A/R24N/D234A/ V235A/R236T. (B) Immunoblot using rabbit anti-ricin antibody of 15% reducing SDS-PAGE of wild-type and mutant RTBs. Lanes are the same as in panel A.

at position 248 which may contribute to their lower galactose avidities (24). The two double-site mutants, N46G/K40M/N255G and D22Q/V23A/R24N/D234A/V235A/ R236T, which were prepared had been previously expressed in Xenopus oocytes but have not been expressed and purified from insect cells (14). The yields were estimated from the optical density at 280 nm of neutralized alkaline eluants with postaffinity chromatography (plant RTB OD ) 1.44 for 1 mg/mL) and densitometry of Coomassie-stained reducing SDS-PAGE (10-30% of the protein migrated at 33 kDa, Figure 2A). These results were confirmed by densitometry of immunoblots with polyclonal rabbit anti-ricin antibody. As shown in Figure 2B, the single-site and double-site mutants were reactive with the polyclonal antibody. Finally, a monoclonal antibody anti-RTB ELISA was used to verify concentrations of each mutant. All three assays gave similar values. The yields of each of the four new

Bioconjugate Chem., Vol. 7, No. 6, 1996 655

Ricin Toxin B Chain Mutants Table 1. Yields of Mutant RTBs Produced in Insect Cellsa

protein

type of mutant (subdomain)

amount of partially purified mutant per liter of culture (µg)

reference

D234E Y248S Y248H wild-type W37S D234E/A237R W37S/Y248H N255G D22E Q35N N255A K40M/N46G K40M D22E/D234E W37S/Y248S N46G/K40M/N255G D22Q/V234A/R24N/D234A/V235A/R236T

2γ 2γ 2γ 1R 2γ 1R 2γ 2γ 1R 1R 2γ 1R 1R 1R, 2γ 1R, 2γ 1R, 2γ 1R, 2γ

1500 640 420 400 264 240 205 160 150 90 40 32 24 23 22 10 2

present study 18 present study 16 18 18 19 18 19 18, 19 18 18 18 19 19 present study present study

a Quantity of RTB based on P2 ELISA and product of densitometry of Coomassie-stained gels and absorbance at 280 nm of patially purified protein. At least four preparations of each mutant made. Values given are mean. Agreement between densitometry and ELISA within 30% in each case.

Table 2. Binding of Monoclonal Anti-RTB Antibodies to Mutantsa

protein D22E/D234E wild-type W37S/Y248H W37S/Y248S Q35N D22E D22Q/V23A/R24N/D234A/ V235A/R236T D234E D234E/A237R N46G/K40M N255G W37S N255A Y248H K40M N46G/K40M/N255G Y248S

type (subdomain)

% relative to monoclonal P2 P8 P10

1R, 2γ 1R, 2γ 1R, 2γ 1R 1R 1R, 2γ

60 88 90 94 96 100 100

100 20 710 160 250 100 250

2γ 2γ 1R 2γ 1R 2γ 2γ 1R 1R, 2γ 2γ

100 113 114 117 119 130 143 145 200 250

100 53 80 80 55 350 202 90 600 80

a Measured by P2, P8, and P10 ELISA calibrated with known amounts of plant RTB. Each assay employed 5% β-mercaptoethanol to reduce homodimers. Average of two to four assays on each mutant.

mutants and 12 previously prepared mutants are shown in Table 1. The lower yields of 3/11 single-site mutants (N255A, K40M/N46G, and K40M) and 4/5 double-site mutants (D22E/D234E, W37S/Y248S, N46G/K40M/ N255G, and D22Q/V23A/R24N/D234A/V235A/R236T) may be due to degradation of improperly folded proteins. Other investigators reported bioactivity of mutants without purification or characterization (13-15). Their assumption that modification of lectin site residues would not affect secondary structure may be inaccurate and overemphasize the role of individual residues and subdomains in sugar binding and cytotoxicity. Yields from cells extracts were similar to yields from supernatants in the four mutants tested (K40M, Q35N, N46G/K40M/ N255G, and D22Q/V23A/R24N/D234A/V235A/R236T). Reactivites of mutant RTBs with different monoclonal antibodies to RTB (P2, P8, and P10) were tested by substituting different monoclonal antibodies as capture reagents in the antibody ELISA. Equivalent results were observed for each antibody in most cases, suggesting similar folding of the mutants (Table 2).

Table 3. Binding of Mutant RTBs to Asialofetuina protein wild-type N255A Q35N D234E N255G Y248S D234E/A237R D22E W37S W37S/Y248S Y248H K40M K40M/N46G D22Q/V23A/R24N/ D234A/V235A/R236T K40M/N46G/N255G W37S/Y248H D22E/D234E

type of relative binding mutant to asialofetuin (%) reference 2γ 1R 2γ 2γ 2γ 2γ 1R 1R 1R, 2γ 2γ 1R 1R 1R, 2γ

83 70 50 33 33 32 30 25 24 20 17 15 12 10

16 18 18, 19 this report 18 18 18 19 18 19 this report 18 18 this report

1R, 2γ 1R, 2γ 1R, 2γ

4 2 2

this report 19 19

a Quantity of RTB based on P2 ELISA and asialofetuin binding measured by asialofetuin ELISA. Both assays employed 5% β-mercaptoethanol to reduce homodimers. Average of three to four assays run on each mutant. ∆32 nonsense mutant, DRA, and AcNPV antibody matrix eluants treated identically gave values of 0.01 µg/mL for both the P2 and asialofetuin ELISA. Experimentals had P2 values of 0.2-150 µg/mL and asialofetuin values of 0.02-58 µg/mL. Q35N assays within 1 week of preparation.

Sugar Binding of Mutant RTBs. Binding of partially purified mutants to immobilized asialofetuin was quantitated by ELISA, and the results are shown in Table 3. The two new single-site mutants showed a less than 1 log drop in binding relative to recombinant or plant RTB. The two new double-site mutants showed a close to 2 log drop in sugar binding. The limit of detection of the assay was a 2.5-3 log decrease in sugar binding avidity. An independent measure of mutant RTB binding to glycoproteins was made by detecting mutant RTB bound to cell surfaces. All mutants bound KB cells at 4 °C (Figure 3). Competition Experiments. Binding of recombinant proteins to immobilized asialofetuin was performed in the presence of 100 mM R-lactose or 100 µg/mL asialofetuin. In each case, the sugar binding of the RTB protein was inhibited by a competitor (Table 4). Lactose blocked wildtype and mutant RTB sugar binding between 2- and 81-

656 Bioconjugate Chem., Vol. 7, No. 6, 1996

Fu et al. Table 4. Competition of Mutant RTB Lectin Binding by Sugarsa protein Q35N Y248H K40M/N46G W37S D234E N255A D22E K40M wild-type W37S/Y248S N255G D22Q/V23A/R24N/D234A/ V235A/R236T K40M/N46G/N255G Y248S W37S/Y248H D234E/A237R D22E/D234E

type (subdomain)

fold inhibition lactose asialofetuin

1R 2γ 1R 1R 2γ 2γ 1R 1R 1R, 2γ 2γ 1R, 2γ

81 27 27 27 27 15 15 9 9 9 9 9

150 9 50 243 100 150 81 15 400 243 243 9

1R, 2γ 2γ 1R, 2γ 2γ 1R, 2γ

3 3 3 3 2

3 200 5 150 3

a Asialofetuin ELISA performed in the presence or absence of 100 mM R-lactose or 100 µg/mL asialofetuin. Twelve different concentrations of each protein tested and half-maximal binding concentrations compared to assess fold inhibition of binding.

Table 5. Reassociation of Mutant RTBs with RTAa protein

Figure 3. Binding of mutant RTBs to KB cells. Cells were attached to polylysine-coated tissue culture dishes, and all incubations were done at 4 °C. The cells were washed with 2 mg/mL BSA in PBS and the PBS plus BSA plus 1 µg/mL purified mutant RTB or wild-type RTB, rewashed, incubated with 1:200 rabbit anti-ricin antibody (Sigma) in PBS plus BSA, rewashed, incubated with affinity-urified goat anti-(rabbit Ig) coupled to rhodamine (Jackson ImmunoResearch) at 25 µg/mL, washed again, and fixed in 3.7% formaldehyde in PBS (magnification ) 250×; bar ) 20 µm): (A, C, E, G, and I) without 100 µg/mL asialofetuin and (B, D, F, H, and J) with asialofetuin. (A and B) wild-type RTB, (C and D) D234E, (E and F) Y248H, (G and H) N46G/K40M/N255G, and (I and J) D22Q/V23A/R24N/D234A/ V235A/R236T.

fold. Asialofetuin blocked sugar binding 3-400-fold. The inhibition of soluble carbohydrates of double-site mutant binding was a lower estimate as the assay sensitivity was only 5-10-fold less than the observed binding to immobilized asialofetuin in the absence of added sugars for four of the double-site mutants. Each experiment was repeated three times, and 12 different concentrations of recombinant protein and plant RTB were used to compare half-maximal binding concentrations. Binding of mutant RTBs to KB cells was blocked by 100 µg/mL asialofetuin (Figure 3). Reassociation of Mutant RTBs with Plant RTA. Incubation of mutant RTBs at 3 × 10-8 to 3 × 10-7 M with plant RTA at a 4-fold molar excess overnight at room temperature led to 11-100% reassociation (Table 5 and Figure 4). Similar levels of reassociation were seen using plant RTB or recombinant wild-type RTB with plant RTA at the same concentrations. The heterodimer

W37S N46G/K40M W37S/Y248S D22E D234E N255A Y248S Y248H wild-type D22E/D234E D234E/A237R Q35N W37S/Y248H K40M K40M/N46G/N255G D22Q/V23A/R24N/D234A/ V235A/R236T N255G

type (subdomain)

heterodimer formed (%)

1R 1R 1R, 2γ 1R 2γ 2γ 2γ 2γ 1R, 2γ 2γ 1R 1R, 2γ 1R 1R, 2γ 1R, 2γ

11 22 25 25 27 34 35 43 45 50 50 65 70 70 81 90

2γ

100

a

Assayed by modified ricin ELISA using P2 anti-RTB antibody capture and biotinylated RBR12 anti-RTA antibody detection reagent and confirmed by densitometry of immunoblots of nonreducing SDS-PAGE.

concentrations were quantitated by a modified ELISA which identified molecules with both RTA and RTB epitopes and by densitometry of 65 kDa bands of immunoblots with anti-RTB or anti-RTA antibodies. Both ELISA and immunoblots gave similar values and showed all mutants reassociated well with plant RTA and had minimal homodimer formation. Cytotoxicity of Mutant Heterodimers. The ID50 of ricin and recombinant mutant RTB-RTA heterodimers on HUT102 human leukemia cells is shown in Figure 5 and Table 6. The reduction in potency for single-site and double-site mutant RTB-RTA heterodimers compared to that for wild-type RTB-RTA and plant ricin was 1-2 log. Nevertheless, the potency for all the mutants was at least 3 log more than that for wild-type RTA or RTB alone. DISCUSSION

The molecular mechanism of interaction between ricin and its saccharide receptors on the cell surface is the first

Bioconjugate Chem., Vol. 7, No. 6, 1996 657

Ricin Toxin B Chain Mutants

Figure 5. HUT102 cell cytotoxicity. Assay as described in text: (b) ricin, IC50 ) 4 × 10-12 M; (O) D234E-RTA, IC50 ) 1 × 10-10 M; (9) Y248H-RTA, IC50 ) 8 × 10-11 M; (0) N46G/ K40M/N255G-RTA, IC50 ) 2 × 10-10 M; and (2) D22Q/V23A/ R24N/D234A/V235A/R236T-RTA, IC50 ) 5 × 10-11 M. Yield of reassociated heterodimer tested by modified ELISA. Each experiment performed in triplicate. Table 6. HUT102 Cell Sensitivity to Recombinant Heterodimersa protein wild-type W37S/Y248S-RTA W37S-RTA Q35N-RTA Y248S-RTA K40M/N46G-RTA D22Q/V23A/R24N/D234A/ V235A/R236T-RTA Y248H-RTA D234E/A237R-RTA N255G-RTA N255A-RTA D22E/D234E-RTA K40M-RTA D234E-RTA D22E-RTA K40M/N46G/N255G-RTA W37S/Y248H-RTA

Figure 4. Reassociation of mutant RTBs with plant RTA. (A) Immunoblots of 15% nonreducing SDS-PAGE of reassociated mutant RTB-plant RTA. Low-molecular mass BioRad protein standards are 106, 80, 49.5, 32.5, 27.5, and 18.5 kDa. The heterodimer appears at 60 kDa, and subunits appear at 30 kDa. Immunoblot uses monoclonal antibody P2 and P10 anti-RTB. (B) Same as panel A except immunoblot uses monoclonal antibody RBR12 anti-RTA: lane 1, low-molecular mass prestained BioRad protein standards; lane 2, D234E-RTA; lane 3, Y248HRTA; lane 4, N46G/K40M/N255G-RTA; lane 5, D22Q/V23A/ R24N/D234A/V235A/R236T-RTA; and lane 6, ricin.

necessary step in intoxication of cells. Determination of the number of lectin sites on RTB and their affinities and participating amino acid residues are important both to define the structure-function relationships of this and other plant lectins, to interpret intracellular routing signals, and to design and synthesize genetically engineered cell-selective toxins. While initial studies with ricin by equilibrium dialysis (25) and mutational analysis (13) suggested a single lectin site per molecule, subsequent equilibrium dialysis measurements (4, 26), X-ray diffraction analysis (9), chemical modification studies (10, 11), and mutational analyses (14, 15, 23) suggested two lectin sites in subdomains 1R and 2γ. However, for the mutational analy-

type (subdomain)

IC50 (M)

1R, 2γ 1R 1R 2γ 1R 1R, 2γ

5 × 10-12 1 × 10-11 2 × 10-11 2 × 10-11 5 × 10-11 5 × 10-11 5 × 10-11

2γ 2γ 2γ 2γ 1R, 2γ 1R 2γ 1R 1R, 2γ 1R, 2γ

8 × 10-11 8 × 10-11 8 × 10-11 8 × 10-11 9 × 10-11 1 × 10-10 1 × 10-10 2 × 10-10 2 × 10-10 2 × 10-10

a HUT102 cell cytotoxicity as described in text. Ricin IC 50 was 4 × 10-12 M.

ses, no quantitation of soluble immunoreactive proteins was reported, and hence, the true specific activities of the recombinant proteins were unknown. If misfolding with aggregation or proteolytic degradation occurred, no residual lectin activity would be seen even if three or more lectin sites were present. To more accurately measure sugar binding avidities of recombinant RTB proteins, we produced and purified wild-type and mutant RTBs from insect cells using the baculovirus system and monoclonal antibody affinity chromatography (16-19, present study). One log decreases in asialofetuin binding were observed with singlesite mutants, and in 4/5 cases, 2 log decreases in asialofetuin binding were seen with double-site mutants. The persistent lactose and asialofetuin-inhibitable sugar and cell binding of a number of double-site (subdomain 1R and 2γ) mutants suggests either incomplete inactivation of the two sites in the mutants or the existence of a third lectin site. Since seven different residues in one subdomain (1R) and seven different residues in the other domain (2γ) were tested (Figure 1), we believe the first hypothesis is unlikely and that ricin has three or more lectin binding sites. Possible third lectin sites include

658 Bioconjugate Chem., Vol. 7, No. 6, 1996

subdomain 1β with aromatic ring residue Y78 and a lectin pocket kink and subdomain 2R with aromatic residue W160, kink residues, and polar residues D153, E170, and Q171. Ongoing mutational analysis using the insect expression system should help address this question. Other observations support the hypothesis of three lectin sites on ricin. Three distinct sites on ricin were cross-linked by radiolabeled fetuin glycopeptides containing a dichlorotriazine-activated 6-(N-methylamino)-6deoxy-D-galactose moiety (12). When ricin molecules containing either two or three affinity cross-linkers were conjugated to monoclonal antibodies and tested for toxicity in vitro and in vivo, marked differences were observed (27). The residual nonspecific toxicity in vitro of the doubly blocked ricin conjugate could be blocked with excess lactose. The finding of three or more independent sugarcombining sites on a lectin is not unique to ricin and has been reported for hepatic galactose N-acetylgalactosamine receptor and macrophage mannose receptor (7, 8). The results also have implications in the role of the residual sugar-combining site of blocked ricin conjugates in intracellular trafficking (28) and in the genetic engineering of tumor cell selective ricin fusion toxins (29). ACKNOWLEDGMENT

We thank Joseph Vesely and Billie Harris for excellent technical assistance, Starr Hazard for molecular graphics analysis, James Nicholson for imaging analysis, Dr. Walter Blattler for the anti-ricin monoclonal antibodies, and Dr. Jerry Fulton for the plant ricin and ricin subunits. LITERATURE CITED (1) Olsnes, S., and Pihl, A. (1973) Different biological properties of the two constituent peptide chains of ricin, a toxic protein inhibiting protein synthesis. Biochemistry 12, 3121-3126. (2) Rivera-Sagredo, A., Solis, D., Diaz-Maurino, T., JimenezBarbero, J., and Martin-Lomas, M. (1991) Studies on the molecular recognition of synthetic methyl β-lactoside analogs by ricin, a cytotoxic plant lectin. Eur. J. Biochem. 197, 217228. (3) Olsnes, S., Sandvig, K., Refsnes, K., and Pihl, A. (1976) Rates of different steps involved in the inhibition of protein synthesis by the toxic lectins abrin and ricin. J. Biol. Chem. 251, 3985-3992. (4) Zentz, C., Frenoy, J., and Bourrillon, R. (1978) Binding of galactose and lactose to ricin: equilibrium studies. Biochim. Biophys. Acta 536, 18-26. (5) Baenziger, J., and Fiete, D. (1979) Structural determinants of Ricinus communis agglutinin and toxin specificity for oligosaccharides. J. Biol. Chem. 254, 9795-9799. (6) Sandvig, K., Olsnes, S., and Pihl, A. (1976) Kinetics of binding of the toxic lectins abrin and ricin to surface receptors on human cells. J. Biol. Chem. 251, 3977-3984. (7) Lee, Y. (1992) Biochemistry of carbohydrate-protein interaction. FASEB J. 6, 3193-3200. (8) Drickamer, K. (1995) Multiplicity of lectin-carbohydrate interactions. Nat. Struct. Biol. 2, 437-439. (9) Rutenber, E., and Robertus, J. (1991) Structure of ricin B chain at 2.5 Anstrom resolution. Proteins 10, 260-269. (10) Hatakeyama, T., Yamasaki, N., and Funatsu, G. (1986) Identification of the tryptophan residue located at the lowaffinity saccharide binding site of ricin D. J. Biochem. 100, 781-788. (11) Youle, R., Murray, J., and Neville, D. (1981) Studies on the galactose-binding site of ricin and the hybrid toxin Man6P-ricin. Cell 23, 551-559. (12) Lambert, J., McIntyre, G., Gauthier, M., Zullo, D., Rao, V., Steeves, R., Goldmacher, V., and Blattler, W. (1991) The galactose-binding sites of the cytotoxic lectin ricin can be chemically blocked in high yield with reactive ligands pre-

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