Supplementary Table 1. Cα-RMSD values between RIP structures.

Protein Species RMSD1 PDB IDResolution

(Å) Reference

(a) Type I RIPs pokeweed antiviral

protein Phytolacca americana

3.41 1GIK 1.80 PubMed: 12615543

α-momordin Momordica charantia

1.62 1MRG 1.80 PubMed: 7619070

β-momordin Momordica charantia

1.65 1CF5 2.55 PubMed: 10329776

α-trichosanthin Trichosanthes

kirilowii 1.62 1MRJ 1.60

PubMed: 7619070

Gelonin Gelonium

multiflorum 1.45 3KTZ

1.60

PubMed: 19913503

bryodin I Bryonia dioica 1.70 1BRY 2.10 PubMed: 9115985

saporin-L1 Saponaria officinalis

4.07 3HIS 1.49 PubMed: 19920175

PD-L1 Phytolacca

dioica 3.31 3H5K 1.45

PubMed: 19452522

Bouganin Bougainvillea

spectabilis 3.17 3CTK 1.80

PubMed: 19616098

PD-L4 Phytolacca

dioica 3.32 2QES 1.24

PubMed: 17963235

Lychnin Lychnis

chalcedonica 3.82 2G5X 1.70

Dianthin 30 Dianthus

caryophyllus 4.05 1RL0 1.40

PubMed: 15681236

beta-luffin Luffa

aegyptiaca 1.63 1NIO 2.00

PubMed: 12876337

charybdin Charybdis

maritime agg 3.23 2B7U 1.60

PubMed:16817896

Alpha-momorcharin Momordica charantic

1.61 1F8Q 2.20 PubMed:11451448

(b) Type II RIPs

ricin Ricinus

communis 0 2AAI 2.50

PubMed: 1881880

Shiga toxin Shigella

dysenteriae 7.49 1DM0 2.50

PubMed: 7656009

Abrin-a Abrus

precatorius 1.32 1ABR 2.14

PubMed: 7608980

mistletoe lectin I Viscum album 1.41 1CE7 2.70 PubMed: 10198229

1  

ebulin I Sambucus

ebulus 1.81 1HWM 2.80

PubMed: 11288182

Agglutinin Abrus

precatorius 1.35 2ZR1 2.60

PubMed: 20433687

mistletoe lectin IV Viscum album 1.43 1YF8 2.80 PubMed: 15774467

Natural cinnamomin isoform III

Cinnamomum camphora

1.11 2VLC 2.95 PubMed:18837036

1The Main-chain atom RMSD was calculated by comparing RIPs with the reference protein ricin A chain.

2  

Supplementary Table 2. Specificity and protective effects of anti-ricin Abs

Abs Isotype1 A chain1 protective effects of Abs1

1B10 IgG1/IgG3 0.854 -

2D6 IgG1 0.781 +

2D12 IgG1/IgG3 0.57 -

2G9 IgM 0.995 -

3D10 IgG2a 0.853 -

4A5 IgG1/IgG3 0.865 -

4A12 IgG1/IgG3 0.956 -

4H12 IgG2a 0.998 -

5H11 IgG1 0.622 -

6C2 IgG1 0.744 ++

6C3 IgG1 0.733 -

6G3 IgG1 0.809 +

8H11 IgA 0.648 -

9E10 IgG2a 0.732 -

9G11 IgG1/IgA 0.551 -

13C10 IgG1 0.869 +

13G6 IgG1 0.929 ++

1The data were acquired from three different experiments. Data shown are representative of those experiments.

3  

Supplementary Table 3. The epitope mapping of anti-RTA antibody

Clone Number Peptide Sequence Frequency6C2-A2 RVTQLELESLTH 136C2-C1 TQDSQEWATHRP 126C2-F6 MLSVQEHLGRWQ 9

Consensus TQ QE LTHRicn A chain (96)DNQEDAEAITHL

Corresponding (96) Q E ITHsegment of RA

Clone Number Peptide Sequence Frequency13G6-B8 FHWSWYTPSRPS 1013G6-D2 WHWSLWRPPYTL 7

Consensus FHWS W P PSRicn A chain (150)ALYYYSTGGTQLPTL

Corresponding (150) YYYS T PTsegment of RAClone Number Peptide Sequence Frequency

2D6-C4 AFDIPWLPSRVS 12D6-G3 QWLP

0QLPVRILG 9

Consensus IP LP RIRicn A chain (41) EIPVLPNRVGL

Corresponding (41) IP LP RVsegment of RA

4  

Supplementary Table 4.  Biacore analysis of RTA binding to ribosome with or without RTA mAb 6C2 and 6G3.

KDRTA-ribosome (nM) KD

RTA-ribosome with 6C2 (nM) KDRTA-ribosome with 6G3 (nM)

1.3 ± 0.34 1.43± 0.78 1.38± 0.45 Experimental error is the SD from three independent experiments.

5  

Supplementary Figure 1.

6  

7  

Supplementary Figure 1. Multiple sequence alignment of RIPs. The sequence

numbering for RIPs is indicated. Sequence alignments were carried out with the

software Discovery Studio 2.5. Amino acid color-coding is as follows: purple, neutral

side chains; yellow, hydrophobic side chains; green, hydrophilic side chains.

8  

Supplementary Figure 2

9  

Supplementary Figure 2.  Efficacy of RTA-specific mAb therapy at different

time points. A single dose (5ug) of mAb 6G3 was administered either 1h, 2h, 4h or

8h after Ricin injection. Data are mean ± SD of at least 3 experiments.

10  

Supplementary Figure 3

11  

Supplementary Figure 3. The difference in the protective mechanism of RTA

mAbs. (A-D) Block of RTA-slRNA interaction by RTA mAbs. After the

biotin-labeled slRNA was coated on the strepavidin (SA) chip, RTA and excessive

amounts of RTA mAbs were incubated at room temperature for 30 min and were then

flowed through the SA chip. The binding interaction was monitored in real time. (E)

Inhibition of RNA hydrolysis by RTA mAb. RTA was incubated with excessive

amounts of RTA mAb for 60 min and then mixed with ribosomes for another 60 min.

The extent of ribosome depurination was quantified using HPLC. Rib: ribosomes; RA:

ricin A chain. Data are mean ± SD (n=3).

12  

Supplementary Figure 4.

13  

Supplementary Figure 4. Uncompetitive inhibition of RTA as a function of

antibody concentration with a fixed concentration of 0.66 μM ribosome as a

substrate. Values for the inhibition constant (Ki) were calculated by fitting the initial

rates to the equation for uncompetitive inhibition. The graphs are representative of at

least 3 experiments, each of which showed similar results.

14  

Supplementary Figure 5.

15 

 

Supplementary Figure 5. The dihedral angle analysis (chi1/chi2) of the essential

amino acid residues for catalytic activity of ricin. The side-chain orientation of

E177 (A, B), R180 (C, D), Y80 (E, F) and Y123 (G, H) were analyzed by molecular

dynamics simulation for a period of 40 ns.

16  

Supplementary methods

Antibody preparation. Recombinant RTA was prokaryotically expressed in our

laboratory. Balb/c mice were immunized with 0.4 ml of purified RTA (100 μg per

mouse) emulsified in complete Freund’s adjuvant, and then boosted with the same

dose of RTA in incomplete Freund’s adjuvant at 2 week intervals, for two times.

Splenocytes from the immunized mouse were fused with SP2/0 myeloma cells.

Hybridoma cells secreting RTA mAbs were identified by Enzyme-linked

immunosorbent assay (ELISA) and then subcloned by limiting dilution. The RTA

mAbs were purified from ascites fluid using protein A (GE Healthcare). The detail

procedure was as follows (1, 2).

ELISA. Microtiter wells were coated with 100 μl of RTA (1 μg/ml) in PBS (PH 7.4)

overnight at 4 °C, washed three times with PBST (PBS-Tween20; 0.1%,vol/vol), and

blocked with 10% skim milk in PBST for 2 h at 37 °C before being probed with

primary antibodies. The wells were washed six times in PBST and HRP conjugated

goat anti-mouse IgG or IgG isotype-specific Ab was added and incubated for 1h at

room temperature. The plates were again washed, and the colorimetric HRP substrate

was added. The absorbance at 450 nm and 630 nm were read 20 min later. In

performing antibody competition ELISA, the ability of unconjugated mAbs to

compete with an HRP conjugated mAb for binding to RTA was measured. The

unconjugated mAb was added to Ag-coated wells, followed 1 h later by the

conjugated mAb. After overnight incubation the plates were washed, and substrate

was added (2).

17  

Epitope mapping. Peptide display phage libraries were selected for binding to mAbs

as previously described(2-4). The PhD-12 library (New England Biolabs) displaying a

12-amino acid linear peptide with a complexity of 2 × 109 phage was used. Antibody

was immobilized on 96-well plates at a concentration of 1 μg/well. Phage were

incubated with the Ab-coated wells at room temperature with gently rocking for 25

minutes and the unbound phage were washed away with TBS. Phage were eluted with

0.2 M glycine-HCl, pH 2.5, and reamplified to a titer of 1012 and reapplied to fresh

Ab-coated wells. Three or four successive rounds of amplification, adherence, and

elution resulted in the identification of phage that bound uniquely to the selecting Abs

and not to irrelevant controls. Phage was sequenced by automated methods using M13

primers. Epitopes were mapped onto the three-dimensional structure of ricin using

Discovery Studio 2.5 (Accelrys, San Diego, CA).

rRNA depurination assay. The extent of RNA hydrolysis by RTA was measured by

HPLC determination of the amount of adenine released(5, 6). Adenine used as

standard was purchased from Sigma. After pre-treated with RTA mAbs, 50ng of RTA

was incubated with 30 pmol of ribosomes in the reaction buffer (20mM Tris-HCl

pH7.4, 25mM KCl, 5mM MgCl2) at 30 °C for 60 min. Then, the reaction mixture was

injected into a reversed-phase C18 analytic column pre-equilibrated with isocratic

elution in 50 mM ammonium acetate (pH5.0) containing 10% methanol. The

toxin-mediated adenine release was quantitated by using an adenine standard

calibration curve.

Inhibition of ricin enzymatic activity. The ability of mAbs to inhibit the enzymatic

18  

activity of ricin was measured in a cell-free in vitro translation assay using rabbit

reticulocyte lysates (Promega) as both the source of mRNA and ribosomes. After

pre-incubated with 1 μg RTA, varying doses of RTA mAbs (from 0.1 μg to 10 μg) and

0.6 μg template mRNA were added to the rabbit reticulocyte lysates and incubated for

90 min at 30 °C. The reaction could be tested for the synthesis of functional luciferase

using Luciferase Reporter Assay System (Promega).

Neutralization of ricin-mediated cytotoxicity. The ability of Ab to protect against the

cytotoxic effect of ricin was measured using an MTT dye reduction assay. Ab and

ricin holotoxin were mixed and then added to target cells (5 × 103) in 96-well plates.

After 48 h, 20 μl of 5 mg/ml MTT was added into each well and the cells were

incubated for another 4 h. The supernatant was removed and 150 μl DMSO was added

to dissolve the formazan crystals. The absorbance of 490nm was determined by a

microreader. To get precise data, each concentration was set for three wells.

SPR Analysis. A BIAcore T100 SPR-based biosensor system was used to monitor the

dynamic interaction between RTA variants and the immobilized RNA oligo (slRNA)

(7). Briefly, the biotin-labeled slRNA was immobilized on the surface of the

streptavidin (SA) sensor chips. In mAbs blocking analysis, 240 μg/ml mAb was

incubated with 60 μg/ml RTA at 37℃ for 1 h. Then, the block of RTA-slRNA

interaction by RTA mAbs was monitored in real time.

Analysis of the interaction between the RTA and ribosomes. The biaocore T100 (GE

Healthcare) was used to analyze the interaction between the ribosomes and RTA. The

RTA was used as the ligand and the monomeric ribosomes were used as the analyte.

19  

20  

After incubation with excessive amount (4:1 molar ratio) of RTA mAb 6C2 or 6G3,

ribosomes were then passed through both the sensor and the reference surfaces at

different concentrations at a flow rate of 30 μl per minute for 3 minutes to monitor the

association. The dissociation was recorded at the same flow rate for another 3 minutes.

The signal from the reference surface was subtracted from the signal from sensor

surface to correct for nonspecific binding. The sensor and the reference surfaces were

regenerated by injecting the regeneration solution for 1 minute followed by 1 minute

of 2 M NaCl solution in running buffer and 1 minute of running buffer at a flow rate

of 100 μl per minute. All interactions were analyzed at 25°C. The data was analyzed

using the BIAcore T100 Evaluation software version 2.0.

RTA kinetic assay. Varying concentrations of ribosome were incubated at 37℃ in a

solution containing RTA reaction buffer for 10 minutes. Reactions were initiated by

the addition of RTA. The solutions were quenched at timed intervals by the addition

of 100 μL 1M potassium phosphate (pH 8.0). The samples were then injected into a

reverse-phase C18 Waters Delta-Pak analytical column with isocratic elution in

50mM ammonium acetate (pH 5.0) containing 3% of 50% aqueous methanol, at a

flow rate of 1 mL/min. The catalytic release of adenine was monitored at 260 nm and

compared to a standard adenine concentration curve to calculate the quantity released

during catalysis. Adenine release per reaction time was used to determine the kinetic

parameters for RTA on ribosome. Initial rate kinetics were fit to the Michaelis-Menten

equation for the calculation of substrate Km, Kcat and Kcat/Km values.

RTA inhibition assay. Inhibition constants were determined using ribosome as the

21  

competitive substrate. In competitive inhibition assays, varying concentrations of RTA

mAbs were pre-incubated with 0.66 μM ribosome for 10 minutes in reaction buffer at

37℃. No adenine release was observed during this pre-incubation time. In a 100 μL

total volume, reactions were initiated by the addition of ribosome. Reactions were

quenched and adenine was quantified by the same HPLC method used in the catalysis

assays. Values for the inhibition constant (Ki) were calculated by fitting the initial

rates to the equation for noncompetitive inhibition.

In vivo protective effects. The mAbs were evaluated for their ability to protect mice

from the toxic effects of ricin. The mice were randomized into several groups (n = 10

mice/group) and intraperitoneally injected with ricin holotoxin diluted in 0.2 ml of

PBS (50 μg/kg). Passive immunity produced by mAbs was measured by premixing

ricin and mAb, then injecting mice with the mixture. For passive-transfer experiments,

we administered ricin-challenged mice with a single dose of RTA mAbs at indicated

time points (1 h, 2 h, 4 h or 8 h). The survival of mice was monitored until the

experiment was terminated.

Molecular Dynamics Simulations. All MD simulations presented in this work were

performed by using the AMBER 9.0 simulation package (8). The starting structure of

ricin is taken from the Protein Data Bank (PDB). We ran all production-phase

molecular dynamics simulations with a 2.0 fs time step under the isothermal-isobaric

ensemble (300 K or 500 K) with explicit solvent, using the TIP3P model(9) for water,

periodic boundary conditions, the particle mesh Ewald (PME) method(10) for

electrostatics, a 10 Å cutoff for Lennard-Jones interactions, and the use of SHAKE(11)

22  

for restricting motion of all covalent bonds involving hydrogen atoms. Water

molecules were added around the proteins using a 9 Å buffer from the edge of the

periodic box. The structures are minimized first by using the PMEMD module in

AMBER 9.0. MD simulations are carried out thereafter. The temperature of the

system is raised gradually from 0 to 300 K or 500 K in 50 ps and equilibrated for

another 100 ps. An additional 50 ns of MD simulation is performed for data

collection.

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