Biological Toxins and Bioterrorism: Biological Toxins and Bioterrorism

81 Pages·2013·1.657 MB·English

by  P. Gopalakrishnakone (eds.)

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Abrin and Immunoneutralization: A Review
ShradhaBagariaandAnjaliA.Karande*
DepartmentofBiochemistry,IndianInstituteofScience,Bangalore,Karnataka,India
Abstract
AbrinisatypeIIribosome-inactivatingproteinobtainedfromthematureseedsofasubtropicalplant
named Abrus precatorius. It is a glycoprotein which arrests protein synthesis in eukaryotes by
inactivating ribosomes irreversibly. The heterodimeric protein comprises of the toxic subunit, the
AchainwhichisdisulfidebondedtotheBchain,agalactose-specificlectin,whichhelpsinbinding
andtraffickingofthetoxinincells.AsinglemoleculeoftheabrinAchainwhichreachesthecytosol
issufficienttokillthecell.Owingtoitsextremetoxicityandeaseofpurificationanddissemination,
abrinisconsideredasadreadedbioterroragent.Inspiteofseveralreportsonabrinpoisoning,there
isnoeffectiveantidoteorvaccineavailableagainstthelethaltoxin.Anactivesitemutantoftheabrin
A chain has been proposed as a potential vaccine candidate against abrin intoxication, though it
mightnotbeusefulforpublicatlarge.However,passiveadministrationofantibodieshasservedas
the primary mode of therapy against a large number of toxins. To date, the monoclonal antibody
D6F10istheonlyknownneutralizingantibodyreportedagainstabrin.Theantibodycanrescuecells
as well as mice challenged with lethal doses of the toxin. A recent study has demonstrated that the
epitopecorrespondingtotheantibodyispresentincloseproximitytotheactivesiteofabrinAchain
and the antibody can neutralize abrin-mediated cytotoxicity intracellularly. Humanization of the
antibodyandadetailedunderstandingofthetraffickingoftheabrin-antibodycomplexarerequired
for its development as therapy, pre- and post-abrin exposure.
Keywords
Abrin; RIPs; Neutralizing monoclonal antibody; Vaccine
List of Abbreviations
ABA Abrin A chain
APA Abrus precatorius agglutinin
BAT3 HLA-B-associated transcript3
DEAE Diethylamino ethyl cellulose
EF-2 Elongation factor 2
ER Endoplasmic reticulum
ERAD ER-associated degradation
ICP Intracranial pressure
Ig Immunoglobulin
ITs Immunotoxins
*Email:[email protected]
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JNK cJun N-terminal kinase
kDa kilo Dalton
LD 50 % lethal dose
50
mAb Monoclonal antibody
MAPK Mitogen activated protein kinase
MKK4 MAPK kinase 4
PCR Polymerase chain reaction
PDB Protein data bank
PDI Protein disulfide isomerase
RCA Ricinus communis agglutinin
RIPs Ribosome-inactivating proteins
rRNA Ribosomal ribonucleic acid
RTA Ricin A chain
SAPK Stress-activated protein kinase
SEK1 Stress signaling kinase1
TNF Tumor necrosis factor
Introduction to Abrin
Abrin, obtained from the Abrus precatorius plant, is a glycoprotein toxin that arrests protein
synthesis by inactivating ribosomes irreversibly (Stirpe 2004). It is a heterodimer comprising of
thecatalyticallyactiveAchainandalectin-likeBchainandisthereforeclassifiedasamemberofthe
typeIIribosome-inactivatingproteins(RIPs).TypeIIRIPsaremostlyofplantoriginanddifferfrom
bacterial toxins which inhibit protein synthesis through mechanisms other than ribosome inactiva-
tion. The heterodimeric toxins like abrin, its well-known sister toxin ricin, shiga toxin of bacterial
originarealsoreferredtoasA/Btoxinsastheyaremadeupoftwochains,A(foractive)andB(for
binding),linkedtogetherbyasingledisulfidebond(Stirpe2004).Thetoxicsubunit,i.e.,theAchain
of abrin, is an RNA N-glycosidase which depurinates the universally conserved a-sarcin loop of
ribosomal RNAs (Stirpe 2004). The A chain is known to depurinate only eukaryotic ribosomes
amongwhichmammalianribosomesaremostsensitive.Ontheotherhand,theBchainisinvolved
in the binding and transport of the toxins in cells (Stirpe and Battelli 2006).
History of Abrin
AbrusprecatoriusisderivedfromtheGreekwordsabrus(meaningbeautiful)andprecare(meansto
pray)astheseedshaveaniceredcolorglossyappearancewithablackspot,asshowninFig.1,and
wereusedtomakerosaries(Olsnes2004).Owingtothedurabilityanduniformityofthehardshelled
seeds, they were often used for weighing gold and making cheap jewelry. Humans and birds, who
aidindispersalofabrinseeds,areusuallyunaffectedbythetoxicityastheseedspossessahardshell
whichremainsundigestedinthegutandisegestedintactfromtheintestinaltract(Olsnes2004).The
toxicity of the seeds of Abrus precatorius (jequirity bean) or Ricinus communis (castor bean) is
known since time immemorial (Olsnes 2004; Barbieri et al. 1993). It was later found that jequirity
beanandcastorbeanconsistofthetoxinsabrinandricinrespectively.Thesetoxinsareaccumulated
inthematuredseedsoftheir respective plantsourceandconstitutealmost5%ofthedryweightof
theseeds(LordandSpooner2011).TheearliestknownreportsonRIPscomefromstudiesonthese
twotoxins(Stirpe2004).ThetermRIPwascoinedafterthediscoverythatthesetoxinsareproteins
and act by inactivating ribosomes. In 1884, it was suggested that abrin is a protein. Later in 1891,
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Fig.1 Apictureoftheabrinseedpod.TheAbrusprecatoriusplantproducesmultiplepods,eachofwhichtypically
containsthreetofiveabrinseeds.Theovoid-shapedseedsarebrightredincolorwithablackspotatthebase(Photo
credit:NirmalyaBasu)
Hellin purified abrin and demonstrated that the purified abrin preparation resulted in the agglutina-
tion of red blood cells (Olsnes 2004). Further work on RIPs revealed that the observed toxic and
agglutinationactivitiesoftheseproteinsareduetothepresenceoftwodifferentpolypeptidechains.
OlsnesandPihldemonstratedthattheAchain(~30kDa)istheenzymaticsubunit,whiletheBchain
(~35 kDa) is a galactose-specific lectin (Olsnes 2004).
In the 1970s, two major events occurred in the history of RIPs that turned the attention of the
world towards these toxins. The first was the finding that RIPs are more toxic to tumor cells in
comparison to normal cells. Abrin and ricin were thus proposed as novel antitumor substances
(Olsnes 2004), and the toxic A chain was coupled to antibodies to specifically target cancer cells
(Kreitman1999).Theseantibodyconjugatedtoxins,referredtoasimmunotoxins(ITs),developed
intoanewclassofagentsforcancertherapeutics.TheITsinhibitedproteinsynthesisandinduced
apoptosis when targeted to malignant cells. In a recent study, the recombinant abrin A chain was
conjugatedtoanantibodyraisedagainsthumangonadotropin-releasinghormonereceptor,andthe
IT was shown to specifically induce cell death in cells expressing the receptor (Gadadhar and
Karande 2013). Many studies in the field of ITs have yielded promising results and clinical trials
havebeen carried out. It hasbeen proposedthat immunotherapyshould be used ascombinatorial
therapyinconjunctionwithradiotherapyandchemotherapy(Kreitman1999).However,itisalso
observed that ITs are less effective on solid tumors due to their poor penetration (Stirpe and
Battelli 2006).
Thesecondeventwaswhenricinbecameinfamousforitscriminaluse(refertochapteronRicin,
a review). Since then, several countermeasures have been developed against these deadly toxins
(Rainey and Young 2004).
Ricin is the most studied type II RIP, and the findings with ricin are often extrapolated to other
related toxins like abrin.
Synthesis and Role of Type II RIPs in Plants
The type II RIPs are synthesized in plants as single-chain polypeptide precursors referred to as the
prepro-form (Fig. 2). These precursors are inactive and consist of the endoplasmic reticulum
(ER) signal peptide followed by the A chain, a linker peptide, and finally the B chain (Barbieri
et al. 1993). The A and B subunits are connected by a disulfide bond formed between cysteine
residues at the C-terminus of the A chain and the N-terminus of the B chain (Barbieri et al. 1993).
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Fig. 2 Processing of prepro-RIPs into mature RIPs. The mature active form of type II RIPs is produced by the
excisionoftheleadersequence/ERsignalsequenceandthelinkerpeptidepresentintheprecursorpolypeptide.TheER
signal sequence is removed during translation, while the linker peptide is cleaved by an endopeptidase present in
vacuoles(FigureadaptedfromPh.D.thesisofDr.KalpanaSurendranath2007,IndianInstituteofScience,Bangalore)
In1991,thepreproabringenesequencewascharacterizedbasedonitssimilaritywithricin(Wood
et al. 1991). It was found that preproabrin consists of an N-terminal signal sequence of 34 amino
acids, followed by 251 amino acid long A chain, a 14 amino acid linker, and finally the B chain
consisting of 263 amino acids (Fig. 2).
TheroleofRIPsinplantshasbeenexploredwithrespecttoplantdefense.Thetoxinsareknownto
deter herbivory and possess antiviral, antifungal, and anti-insecticidal properties though the mech-
anismsoftheseeffectsstillremainunresolved(Stirpe2004).Intensiveeffortsweremadetoexplore
other activities of RIPs. It was found that in addition to the classical RNA N-glycosidase activity,
RIPs also cleave adenine from DNA or poly(A) tail containing RNAs. The polyadenosine glyco-
sidase activity was reported for many RIPs.
Intracellular Trafficking of RIPs
TypeIIRIPstakeadvantageofthecellularmachinerytogainaccesstotheirtargetincells(Sandvig
andvanDeurs2000;LordandSpooner2011).ThericinBchain,agalactose-specificlectin,bindsto
cell surface glycolipids and glycoproteins which end in terminal galactose. The toxin is then
endocytosed by clathrin-dependent and clathrin-independent mechanisms which contribute almost
equally in the uptake process. In addition to the endosomes and lysosomes, a relatively small
fraction (~5 %) of the internalized toxin is transported to the Golgi complex. Intracellular calcium
is believed to be involved in regulating the transport of ricin from the endosomes to the Golgi
apparatus.Henceforth,thetoxinexploitstheretrogradepathwayincellstoreachthecytosol.Inthe
absence of the KDEL sequence, required for transport to the ER, the toxin hitchhikes onto KDEL-
taggedproteinslikecalreticulinviathegalactose-bindingpropertyoftheBchain.Itisbelievedthat
theproteindisulfideisomerase(PDI)intheERlumenreducesthedisulfidebondbetweentheAand
B chains and facilitates partial unfolding of the A chain. The disassembly of the two chains is
essential for optimal enzymatic activity. Subsequently, the free A chain disguises as a misfolded
proteinandutilizestheER-associateddegradation(ERAD)pathwaytotranslocateintothecytosol.
The A chain partially unfolds and traverses through the Sec61p translocon to reach the cytosol.
AsignificantportionofthericinAchainescapesthenormaldegradativefateowingtoitslowlysine
content.Lysineresiduesarepotentialubiquitinationsites,andhencelowlysinecontentreducesthe
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risk of proteasomal degradation of the ricin A chain. Introduction of four additional lysines in the
ricin A chain dramatically reduces the cytotoxicity of ricin, and this effect is reversed on the
inhibition of proteasomes. It is hypothesized that ribosomes aid the refolding of ricin A chain in
thecytosol.Afterrefolding,thetoxicAchaininactivatesribosomesleadingtoinhibitionofprotein
synthesis and finally leads to cell death (Stirpe 2004). It is still not clear whether the B chain is
retained in the ER or traverses to the cytosol. Some reports indicate that the B chain might get
translocatedtothecytosolandexertsadditionaleffects.ItisobservedthattheBchainofshigatoxin
can induce DNA cleavage and apoptosis.
Numerousreviewsfocusonthetraffickingofricinandshigatoxininsidecells(Sandvigandvan
Deurs 2000; Lord and Spooner 2011). It is assumed that abrin travels inside the cell in a similar
manner (Fig. 3). Studies have reported that abrin enters cells via receptor-mediated endocytosis
and is processed through the cell’s vesicular system (Barbieri et al. 1993). Brefeldin A, a fungal
metabolite which blocks Golgi function, was shown to block intoxication by abrin and ricin,
confirming the involvement of Golgi in the intracellular processing of the toxins (Hudson and
Grillo 1991).
Biological Activities of Abrin
There are two major effects of the toxin on cells: inhibition of protein synthesis and apoptosis.
Inhibition of Protein Synthesis by Abrin in Cells
The B chain facilitates the trafficking of the toxic A chain to the cytosol where it inactivates
eukaryotic ribosomes and thereby inhibits protein synthesis. The inhibition of protein synthesis by
ricinincellswasdemonstratedin1971.TheobservationwaslaterconfirmedinvitrobyOlsnesand
Pihl(Stirpe2004).Theinvolvementoftheelongationfactor2(EF-2)andthekineticsofribosomal
depurinationbyabrinandricinwerealsoelucidated(Olsnes2004).Themechanismofinhibitionof
proteinsynthesiswasdelineatedbyEndoetal.in1987bystudyingtheactionofthericinAchainon
rat liver ribosomes (Endo et al. 1987). The study indicated that the A chain recognizes a highly
conservedregion,a“GAGA”sequenceinthe14nucleotidelonga-sarcinloop(Fig.3).Thea-sarcin
loopissometimesreferredtoasthe“Achillesheel”ofribosomesandisthetargetforseveraltoxins.
0
It is found approximately 400 nucleotides upstream from the 3 end of 28S rRNA of the 60S
ribosomalsubunit.The28SrRNAiscrucialforribosomefunctionasitssequenceformsapartofthe
binding site for eukaryotic as well as prokaryotic EF-2. The A chain irreversibly cleaves the
N-glycosidic bond between adenine and the sugar at position 4324 in the a-sarcin loop (Endo
etal.1987).Sincethetargetedadenineispresentinthemiddleoftheloop,itscleavagedestabilizes
theloopresultingininhibitionofbindingofEF-2andthusimpairsproteinsynthesis.Severalstudies
havebeencarriedouttodeterminethekineticsandmechanismofcatalysisoftheAchainofricin.It
has been reported that one molecule of ricin can inactivate up to 2,000 ribosomes/min. Autoradio-
graphicstudiessuggestthatpenetrationofasinglemoleculeofabrinissufficienttokillacell(Eiklid
et al. 1980). The halt in protein synthesis by RIPs is suggested to culminate in apoptosis-like cell
death, though the link between the two biological effects still remains elusive.
Abrin-Induced Programmed Cell Death
Inadditiontoinhibitionofproteinsynthesis,abrincantriggerapoptosisincells.In1980,astudyon
HeLa cells demonstrated that abrin, ricin, and modeccin toxins reach the cytosol and kill the cells
(Eiklidetal.1980).Itwasinitiallythoughtthatthetoxinsricinandabrininducenecroticcelldeath.
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Fig. 3 Schematic representation of the binding and subsequent intracellular trafficking of type II RIPs inside
acell.TheBchainbindstoterminalgalactoseoncellsurfacereceptorsandthewholetoxinisendocytosed.Thetoxin
reaches the Golgi complex and exploits retrograde transport to translocate to the ER. The separation of the A and
BchainsintheERbyproteindisulfideisomeraseisfollowedbytheescapeoftheAchaintothecytosolthroughthe
Sec61translocon.Inthecytosol,theAchaindepurinatesadenine4324ofthea-sarcinloopinthe60Sribosomalsubunit
andtherebyinhibitsproteinsynthesisirreversibly
One of the earliest reports that RIPs are capable of inducing programmed cell death or apoptosis
came from the studies carried out by Griffiths on rats (Griffiths et al. 1987). Typical markers of
apoptosis like chromatin condensation, membrane blebbing, and DNA fragmentation have been
observed in cells treated with the toxins (Hughes et al. 1996). Treatment with abrin results in cell
lysisofseveralcellsinamannercharacteristicofapoptosis.Severalstudieshaveestablishedthatthe
typeIIRIPslikericin, modeccin,andabrininducecelldeathbyapoptosis(Narayananetal.2005).
The observations have been recorded in a variety of cell lines like U937, HeLa, and Jurkat
(Narayananetal.2004).InHeLaandMCF7cells,ricinwasshowntotriggermorphologicalchanges
characteristicofapoptosisbycaspase-3-mediatedproteolyticactivationofBAT3.Studieswithshiga
toxin also demonstrate enhanced Bax expression and activation of caspases. Similarly, abrin-
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induced apoptosis was observed to be caspase-3 dependent and followed the intrinsic pathway of
apoptosis (Narayanan et al. 2004). It is not yet clearly understood whether the inhibition of protein
synthesis by RIPs leads to apoptosis or whether these two are independent events. It was hypoth-
esizedthatdifferentregionsoftheAchaincanberesponsibleforinhibitionofproteinsynthesisand
apoptosis.Experimentalevidencedoesexisttosupportthespeculation.In2001,Shihetal.showed
that abrin inactivates a thiol-specific antioxidant protein, thereby resulting in the generation of
reactive oxygen species which are capable of participating in a wide variety of cellular functions
includingapoptosis(Shihetal.2001).Furthermore,reportshaveshownthatisolatedBchainofricin
hascytotoxiceffectsagainstepithelialcellsinratintestine.Iordanovetal.proposedthatthedamage
of28SrRNAbyricinresultedinanovelpathwayofkinaseactivationtermedasthe“ribotoxicstress
response,” resulting in the activation of SAPK/JNK and its activator, SEK1/MKK4 (Narayanan
etal.2005).ItwasalsoshowninthestudythatactivationofSAPK/JNK1isnotjustduetoinhibition
ofproteinsynthesisbutalsoduetosignalingfrom28SrRNAaffectedbyRIPs.Ricinisalsoknown
toinduceapoptoticcelldeathinthemacrophagecelllineRAW264.7withanincreasedsecretionof
TNF-a. Pretreatment with the p38 MAPK inhibitor, SB202190, resulted in appreciable drop in the
secretedTNF-alevelsandalsocelldeath(Narayananetal.2005).Anotherstudydealingwithabrin
treatment on a human B cell line, U266B1, revealed that abrin triggers programmed necrosis in
U266B1cellsinacaspase-independentmanner(Boraetal.2010).InU266B1cells,abrin-mediated
necrosisinvolvedproductionofreactiveoxygenspeciesleadingtolysosomalmembranepermeabi-
lization,resultinginreleaseofcathepsinsandsubsequentlycelldeath.Arecentreportalsorevealed
the involvement of the extrinsic pathway of apoptosis upon abrin treatment in Jurkat cells (Saxena
etal.2013).TheFas/Fasligandpathwaywasshowntogetactivatedleadingtocleavageofcaspase-
8 and the downstream effector caspase-3. Thus, it should be noted that though abrin inhibits
translation in all cell types, it can induce multiple signaling pathways resulting in cell death.
RIPs Obtained from Abrus precatorius Plant
A subgroup of type II RIPs, commonly referred to as four-subunit toxins, is tetrameric proteins
comprisingoftwoAchainsandtwoBchains(Fig.4).ThefoursubunittypeIIRIPshavealsobeen
foundtobelesstoxicandbetteragglutininswhencomparedtotheheterodimerictypeIIRIPs.Most
of the studies on RIPs have been conducted on ricin and abrin, whereas their corresponding
agglutinins, namely, Ricinus communis agglutinin (RCA) and Abrus precatorius agglutinin
(APA), have been ignored due to their lower toxicity. In terms of whole animal toxicity, ricin is
2,000 times more cytotoxic to mice than RCA. The 50 % lethal dose (LD ) for abrin is 20 mg/kg
50
bodyweight,whereasitis5mg/kgbodyweightforAPA(Bagariaetal.2006).Itwashypothesized
Fig.4 TypesoftypeIIRIPs.TypeIIRIPsareheterodimers(a)orheterotetramers(b)composedofthecatalytically
activeAchain(A)andthelectin-likeBchain(B)
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that the lower animal toxicity of the agglutinins might arise due to their aggregation with serum
galactoproteins and subsequent precipitation and destruction.
Various isoforms of abrin and the agglutinin (APA) have been purified from the seeds of Abrus
precatoriusplant(Hegdeetal.1991).Severalmethodslikeionexchange,gelchromatography,and
Sepharose affinity chromatography have been employed for the isolation of the isoforms. Lin
etal.in1981isolatedfourisoformsoftheheterodimericabrinandoneagglutinin(APA)andtested
for their toxicity. Out of the four isoforms of abrin named abrin-a, abrin-b, abrin-c, and abrin-d,
abrin-awasfoundtobethemosttoxic,whileabrin-bwasleastactive(Linetal.1981).TheBchains
oftheisoabrinsshowsubstantialconservation,whereasasmanyas46aminoacidsubstitutionswere
reported in the A chains (Hung et al. 1993). The complete amino acid sequences of the A chain of
abrin-a and B chains of abrin-a and abrin-b have been reported (Funatsu et al. 1988; Kimura
et al. 1993). Three isoforms of abrin A chains were cloned and expressed in E. coli in 1994
(Hung et al. 1994). However, APAwhich is a heterotetramer was not studied extensively owing to
its reduced activity (Lin et al. 1981).
Structure and Mode of Action of Abrin
TheactivesiteresiduesofmostoftheRIPsareverywellconservedandsoistheirarchitecture.The
structureofricinwasthefirsttobesolvedinthefamilyofRIPs.Thecrystalstructureofabrin-awas
(cid:2)
solved at 2.14 A resolution by Tahirov in 1995 (Tahirov et al. 1995) (Fig. 5). The overall
architecture of abrin was found to be very similar to ricin. Abrin is composed of two polypeptide
subunits, the 30 kDa toxic A chain and the 33 kDa lectin-like B chain (Olsnes 2004). The two
subunitsofabrinaredisulfidebondedbytheCys-247residueoftheAchainwithCys-8ofBchain.
The B chain of abrin is glycosylated at two sites unlike the A chain which is not glycosylated
(Tahirovetal.1995).TheBchainbindstocellsurfaceoligosaccharidesendinginterminalgalactose
andaidstheinternalizationandintracellulartraffickingofthetoxininsidethecell.TheabrinAchain
Fig.5 CrystalstructureofabrinshowingtheheterodimericconfigurationcomprisingoftheAchain(green)and
theBchain(purple).TheactivesiteresiduesoftheAchain–Tyr-74,Tyr-113,Glu-164,Arg-167,andTrp-198–are
representedasballandsticksinpinkcolor,whiletheresiduesAsn-51andAsn-260nearthegalactose-bindingpocketsof
theBchainarerepresentedinyellow.Thefigurewasgeneratedusingtheopen-sourcePyMOLsoftware(PDBcodefor
abrin:1ABR)
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(ABA) is an RNA N-glycosidase and is proposed to have a well-defined active site cleft like ricin
(Tahirov et al. 1995). Once inside the cytosol, the A chain cleaves the C-N bond of an adenine at
position 4324 of the a-sarcin loop (Endo et al. 1987). Depurination of the adenine destabilizes the
a-sarcin loop in the 28S ribosomal RNA and results in loss of binding to the elongation factor
2 thereby stalling protein synthesis irreversibly (Endo et al. 1987).
Architecture of the Abrin A Chain
The three-dimensional structure of ABA is divided into three folding domains in accordance with
the structure of ricin (Tahirov et al. 1995). The domain 1, present towards the N-terminus, is
composed of the residues 1–109. It is rich in b- sheets and consists of a six stranded b- sheet and
two helices (Fig. 6). The domain 2, composed of residues 110–197, is the most conserved part
betweenabrinandricinAchainsandcomprisesoffivea-helices.Theaminoacids198–251towards
the C-terminus of the protein form the domain 3. A small b- sheet composed of two antiparallel
b -strands and two a- helices is present in the third folding domain of ABA (Tahirov et al. 1995).
The A chain of abrin consists of five invariant active-site residues, namely, Tyr-74, Tyr-113,
Glu-164, Arg-167, and Trp-198 (Fig. 5) (Chen et al. 1997). These residues are clustered together
aroundtheactivesitecleftandareknowntobeconservedthroughoutthewiderangeofRIPs.Hung
et al. in 1994 demonstrated that Glu-164 and Arg-167 are essential for catalysis. The protein
biosynthesis inhibitory activity of the mutants E164A, R167L, and the double mutant reduced to
25-fold,625-fold,and1,250-foldrespectivelyincomparisontowild-typeABA(Hungetal.1994).
ThecorrespondingaminoacidsGlu-177andArg-180inricinarehydrogenbondedtoeachotherand
involvedincoordinationofawatermolecule,importantfortheN-glycosidationreaction.Theamino
Fig.6 ThethreefoldingdomainsofABA.Domain1ofABAcomposedoftheaminoacids1–109iscoloredblue,
domain 2 composed of the amino acids 110–197 is colored pink, and the domain 3 composed of the amino acids
198–251iscoloredgreen.Thefigurewasgeneratedusingtheopen-sourcePyMOLsoftware
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acidsstabilizethetransitionstate,i.e.,theoxycarboniumringinthecatalyticreaction.Furthermore,
the residues Tyr-74, Tyr-113, and Trp-198 are involved in substrate binding. The aromatic rings of
both tyrosines lie almost parallel in a position appropriate for sandwiching the planar adenine
substrate (Tahirov et al. 1995). Mutagenesis of Tyr-74 or Tyr-113 increases the Km significantly,
while Kcat drops only slightly, confirming their role in substrate binding (Chen et al. 1997). The
authors also analyzed the mutants of the residues Tyr-74, Tyr-113, Glu-164, and Trp-198 for their
protein synthesis inhibitory activity and effect on conformation of the protein structure (Chen
et al. 1997). It was found that protein synthesis inhibitory activity of Y74F, Y113F, and W198F
decreased moderately but was almost abolished completely in case of E164Q. Moreover, the
mutants Y74F, Y113F, and E164Q attained almost similar conformation as the wild-type ABA
and were not involved in reassociation with the B chain of abrin. On the other hand, a change in
conformation was observed for W198F, and it could not reassociate with abrin B chain. The
observations clearly suggested that Trp-198 is involved in substrate binding and is required for
the structural integrity of the protein (Chen et al. 1997).
Three-Dimensional Structure of Abrin B Chain
ThecompleteaminoacidsequenceofabrinBchainwasobtainedin1993(Kimuraetal.1993).The
B chain is 267 amino acids long and consists of two homologous globular domains (Chen
et al. 1992). Each domain can bind one galactoside and comprises of four subdomains l, a, b,
andg(Tahirovetal.1995).Themainhydrophobiccylindricalcoreofthedomainsisformedbya,b,
and g subdomains, while the l subdomain connects the two domains. The a subdomain has all the
basicelementsforgalactosebinding,andanevolutionarytheoryforricinBchainstatesthattheabg
domain evolved by gene duplication events (Tahirov et al. 1995). The two galactose-binding
pockets for ricin have similar folding topologies. Each pocket consists of the conserved tripeptide,
Asp-Val-Arg,whichareisthekeyresiduesinvolvedinhydrogenbondingtothesugar.Theauthors
reportedthatsimultaneousmutationsoftheseresiduesinboththedomainswererequiredtoabolish
lectin activity of the B chain. It was observed that certain other residues, namely, Lys-40, Asn-46,
andAsn-255,aidinhydrogenbondingtothegalactosemoiety.Studieswiththesite-directedmutant,
N255AofricinBchainshowed~99%decreaseinbindingandtoxicitytocells.Chenetal.in1992
alsocommentedthatabrinBchainhastwopotentialgalactose-bindingsitescomprisingoftheamino
acids Asn-51 and Asn-260 (Chen et al. 1992).
Structural and Functional Details of Abrus precatorius Agglutinin
Abrus precatorius agglutinin or APA is a homologue of abrin obtained from the same plant source
(Lin et al. 1981). Unlike abrin, which is a heterodimer, APA is a heterotetramer comprising of two
abrin-like heterodimers. A study in 1991 documented the purification and characterization of APA
using lactamyl-Sepharose affinity chromatography followed by gel filtration and DEAE-Sephacel
column chromatography (Hegde et al. 1991). The overall folded architecture and all the catalytic
residues are completely conserved between abrin and APA A chains (Liu et al. 2000) (Fig. 7).
However,thereportedLD doseforabrinis20mg/kgbodyweightascomparedto5mg/kgincase
50
of APA (Bagaria et al. 2006). In spite of ~70 % sequence identity between the A chains, APA
exhibitsatleast200-foldlowertoxicitywhencomparedtoabrin(Bagariaetal.2006).Theprimary
structureandfunctionanalysisofAPAAchainsuggestedthatsubstitutionofAsn-200byPro-199in
APA can induce bending of the helical structure. This conformational change could presumably
lower the binding to the substrate, resulting in weak inhibitory activity (Liu et al. 2000). However,
thecrystalstructure ofthe~60kDaAPAheterodimer suggestedthatthedecreasedtoxicityofAPA
wasnotbecauseofakinkinducedinthehelix.Rather,thepresence ofPro-199insteadofAsn-200
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