Abrin and agglutinin-I from the seeds of Abrus precatorius are type II ribosome-inactivating proteins that inhibit protein synthesis in eukaryotic cells. The two toxins share a high degree of sequence similarity; however, agglutinin-I is weaker in its activity. We compared the kinetics of protein synthesis inhibition by abrin and agglutinin-I in two different cell lines and found that ∼200-2000-fold higher concentration of agglutinin-I is needed for the same degree of inhibition. Like abrin, agglutinin-I also induced apoptosis in the cells by triggering the intrinsic mitochondrial pathway, although at higher concentrations as compared with abrin. The reason for the decreased toxicity of agglutinin-I became apparent on the analysis of the crystal structure of agglutinin-I obtained by us in comparison with that of the reported structure of abrin. The overall protein folding of agglutinin-I is similar to that of abrin-a with a single disulfide bond holding the toxic A subunit and the lectin-like B-subunit together, constituting a heterodimer. However, there are significant differences in the secondary structural elements, mostly in the A chain. The substitution of Asn-200 in abrin-a with Pro-199 in agglutinin-I seems to be a major cause for the decreased toxicity of agglutinin-I. This perhaps is not a consequence of any kink formation by a proline residue in the helical segment, as reported by others earlier, but due to fewer interactions that proline can possibly have with the bound substrate. Abrin and agglutinin-I from the seeds of Abrus precatorius are type II ribosome-inactivating proteins that inhibit protein synthesis in eukaryotic cells. The two toxins share a high degree of sequence similarity; however, agglutinin-I is weaker in its activity. We compared the kinetics of protein synthesis inhibition by abrin and agglutinin-I in two different cell lines and found that ∼200-2000-fold higher concentration of agglutinin-I is needed for the same degree of inhibition. Like abrin, agglutinin-I also induced apoptosis in the cells by triggering the intrinsic mitochondrial pathway, although at higher concentrations as compared with abrin. The reason for the decreased toxicity of agglutinin-I became apparent on the analysis of the crystal structure of agglutinin-I obtained by us in comparison with that of the reported structure of abrin. The overall protein folding of agglutinin-I is similar to that of abrin-a with a single disulfide bond holding the toxic A subunit and the lectin-like B-subunit together, constituting a heterodimer. However, there are significant differences in the secondary structural elements, mostly in the A chain. The substitution of Asn-200 in abrin-a with Pro-199 in agglutinin-I seems to be a major cause for the decreased toxicity of agglutinin-I. This perhaps is not a consequence of any kink formation by a proline residue in the helical segment, as reported by others earlier, but due to fewer interactions that proline can possibly have with the bound substrate. Proteins capable of inactivating ribosomes, termed as ribosome-inactivating proteins (RIPs), 5The abbreviations used are: RIPs, ribosome inactivating proteins; APA-I, A. precatorius Agglutinin I; ER, endoplasmic reticulum; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell scan. are RNA N-glycosidases that depurinate the universally conserved α-sarcin loop of the 28 S rRNA of eukaryotes at the most widespread position A4324 (1Endo Y. Tsurugi K. J. Biol. Chem. 1987; 262: 8128-8130Abstract Full Text PDF PubMed Google Scholar, 2Sperti S. Montanaro L. Mattioli A. Stirpe F. Biochem. J. 1973; 136: 813-815Crossref PubMed Scopus (105) Google Scholar). The depurination prevents the binding of ribosomes to elongation factors thereby arresting protein synthesis causing cytotoxicity (3Endo Y. Misui K. Motizuki K. Tsurugi K. J. Biol. Chem. 1987; 262: 5908-5912Abstract Full Text PDF PubMed Google Scholar). RIPs are of two types: type I (single subunit) and type II (two subunits) (4Hartley M.R. Lord J.M. Biochim. Biophys. Acta. 2004; 1701: 1-14Crossref PubMed Scopus (164) Google Scholar, 5Olsnes S. Toxicon. 2004; 44: 361-370Crossref PubMed Scopus (292) Google Scholar). Type I RIPs are single polypeptide proteins consisting of only the toxic A subunit with a molecular mass of ∼30 kDa, e.g. momordin (6Husain J. Tickle I.J. Wood S.P. FEBS Lett. 1994; 342: 154-158Crossref PubMed Scopus (72) Google Scholar) and gelonin (7Hosur M.V. Nair B. Satyamurthy P. Misquith S. Surolia A. Kannan K.K. J. Mol. Biol. 1995; 250: 368-380Crossref PubMed Scopus (43) Google Scholar). Type II RIPs are two polypeptide proteins with the toxic A subunit and a lectin-like B subunit, e.g. abrin (8Tahirov T.H. Lu T.H. Liaw Y.C. Chen Y.L. Lin J.Y. J. Mol. Biol. 1995; 250: 354-367Crossref PubMed Scopus (151) Google Scholar) and ricin (9Rutenber E. Katzin B.J. Ernst S. Collins E.J. Mlsna D. Ready M.P. Robertus J.D. Proteins. 1991; 10: 240-250Crossref PubMed Scopus (254) Google Scholar). The latter bind to the cell-surface receptors containing terminal galactose through the B subunit, enter the cells by receptor-mediated endocytosis (10Sandvig K. van Deurs B. EMBO J. 2000; 19: 5943-5950Crossref PubMed Google Scholar), and are transported to the endoplasmic reticulum (ER) by the retrograde pathway (11Day P.J. Owens S.R. Wesche J. Olsnes S. Roberts L.M. Lord J.M. J. Biol. Chem. 2001; 276: 7202-7208Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The reduction of the A-B intersubunit disulfide bond essential for the cytotoxicity takes place in the ER followed by translocation of the A chain to the cytosol. In addition to inhibition of protein synthesis, RIPs are also capable of inducing apoptosis (12Bolognesi A. Tazzari P.L. Olivieri F. Polito L. Falini B. Stirpe F. Int. J. Cancer. 1996; 68: 349-355Crossref PubMed Scopus (91) Google Scholar). The seeds of the subtropical leguminous climber Abrus precatorius contain the potent toxic lectin abrin and the relatively less toxic Abrus agglutinins (13Hegde R. Maiti T.K. Podder S.K. Anal. Biochem. 1991; 194: 101-109Crossref PubMed Scopus (104) Google Scholar). Abrin and agglutinin-I (APA-I) consist of a toxic subunit, the A chain of 30 kDa and the galactose-binding B subunit of 31 kDa (14Lin J.Y. Lee T.C. Hu S.T. Tung T.C. Toxicon. 1981; 19: 41-51Crossref PubMed Scopus (75) Google Scholar). A single disulfide bond covalently links the two chains. In our earlier studies we have demonstrated that apart from inhibiting protein synthesis abrin also triggers apoptosis via the mitochondrial pathway of cell death (15Narayanan S. Surolia A. Karande A.A. Biochem. J. 2004; 377: 233-240Crossref PubMed Scopus (77) Google Scholar). In spite of ∼70% overall sequence identity seen between abrin and APA-I, the striking differences in the toxicity exhibited by the toxin abrin and APA-I on different cells urged us to look at the structure-function relationship of these proteins. Studies were therefore initiated to compare the activities of abrin and APA-I with respect to the kinetics of protein synthesis inhibition and apoptosis. Data obtained showed that APA-I is manyfold less toxic than abrin on the cell lines tested. Apoptosis induced by APA-I follows the mitochondrial pathway of cell death like abrin. The three-dimensional structures of various type II RIPs have been reported, e.g. abrin (8Tahirov T.H. Lu T.H. Liaw Y.C. Chen Y.L. Lin J.Y. J. Mol. Biol. 1995; 250: 354-367Crossref PubMed Scopus (151) Google Scholar), ricin (9Rutenber E. Katzin B.J. Ernst S. Collins E.J. Mlsna D. Ready M.P. Robertus J.D. Proteins. 1991; 10: 240-250Crossref PubMed Scopus (254) Google Scholar), mistletoe lectin (16Niwa H. Tonevitsky A.G. Agapov I. Saward S. Pfuller U. Palmer R.A. Eur. J. Biochem. 2003; 270: 2739-2749Crossref PubMed Scopus (58) Google Scholar), ebulin (17Pascal J.M. Day P.J. Monzingo A.F. Ernst S.R. Robertus J.D. Iglesias R. Perez Y. Ferreras J.M. Citores L. Girbes T. Proteins. 2001; 43: 319-326Crossref PubMed Scopus (92) Google Scholar), and Himalayan RIP (18Mishra V. Ethayathulla A.S. Sharma R.S. Yadav S. Krauspenhaar R. Betzel C. Babu C.R. Singh T.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2295-2304Crossref PubMed Scopus (22) Google Scholar). The complete amino acid sequence of APA-I has been determined (19Liu C.-L. Tsai C.-C. Lin S.-C. Wang L.-I. Hsu C.-I. Hwang M.-J. Lin J.-Y. J. Biol. Chem. 2000; 275: 1897-1901Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Crystals of Abrus agglutinin were obtained almost 30 years ago, and attempts to solve the crystal structure of APA-I were made 5 years ago by other groups (20Panneerselvam K. Lin S.-C. Liu C.-L. Liaw Y.-C. Lin J.-Y. Lu T.-H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 898-899Crossref PubMed Scopus (10) Google Scholar, 21Wei C.H. Koh C. Pfuderer P. Einstein J.R. J. Biol. Chem. 1975; 250: 4796-4801Abstract Full Text PDF PubMed Google Scholar). Here we report the three-dimensional structure of Abrin as a for molecular structure in of the sequence between APA-I and abrin. The structural of APA-I a at at a single The three-dimensional obtained has a structural for the decreased toxicity of APA-I in to abrin. The proteins were from the seeds of A. precatorius as (13Hegde R. Maiti T.K. Podder S.K. Anal. Biochem. 1991; 194: 101-109Crossref PubMed Scopus (104) Google Scholar). The were in acid and The to and the to Proteins were in containing and the The at at for and the with The bound proteins were with The containing abrin and APA-I were and with The to I and II were and The to APA-I to to by abrin, proteins were on reported earlier, the proteins Abrin and APA-I with and the has been used as a for the of Abrin and APA-I on the and of were in and the cell in and were with fetal bovine and at in a were in the of different concentrations of abrin APA-I in of the of and the cells were at for 30 S. Podder S.K. Karande A.A. Mol. Biochem. Scholar). The protein with acid addition of bovine as a protein and the at for at The with in of and the in a were at in the the of different concentrations of the toxins in of the the cells were for of and for The cell protein with with and in of in The in a The and were as J. Biol. PubMed Scopus Google Scholar). were and with PBS, and cells were with different concentrations of the on for The cells were in PBS, and bound by a fluorescence-activated analysis cells were in the of concentrations of abrin different and the cells were and at for 5 at the the in of of and in A. Y. Biol. 1995; Scholar). The cells were a J. F. Anal. Biochem. 1994; PubMed Scopus Google Scholar), cells were with of abrin in with the of as the cells were at for 5 in of PBS, and with for 30 at The cells were and with and with containing and for at The cells were by the cell earlier J. F. Anal. Biochem. 1994; PubMed Scopus Google Scholar), cells were in with in the of abrin various cells were at at for 5 in of PBS, and with for the cells were in and at for with the cell of and A and at for of and the for at of to the and a and at 30 for The a were by the and from A protein concentration of 5 in a of and a of of protein and were used for of for in from the from Crystals were with a of the were at The were on at of at the The of of for a of The in to as the were at with cell to be to a and The by the structure and The of J. Mol. Biol. PubMed Scopus Google Scholar) found to be is with the of in the crystal The is for APA-I the of abrin-a from the Data at and the molecular J. Acta Sect. A. 1994; Scopus Google Scholar). The and were that the APA-I two as by the abrin-a A chain and the other by abrin-a B chain. The to a high of and of used to the two of from to used for the of The disulfide bond A and B be in the in the of and and structure determined were with P. Acta Crystallogr. Sect. D Biol. Crystallogr. Scopus Google Scholar). of and were with P. K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: PubMed Scopus Google Scholar). The of the Crystallogr. Sect. D Biol. Crystallogr. 1994; PubMed Scopus Google Scholar). This the of 2003; PubMed Scopus Google Scholar). The APA-I I and II consist of and of chain A and groups and consist of and of chain B. of were J. Crystallogr. Scopus Google Scholar, Acta Crystallogr. A. Scopus Google Scholar) were for the and the the In the the and to and The to R.A. J.M. J. Crystallogr. Google Scholar) residue in the The are in The the from the Crystallogr. Sect. D Biol. Crystallogr. 1994; PubMed Scopus Google Scholar) of the were molecular and the high of in A. of protein of in in in in high in a and APA-I were by a as The of the two proteins is seen in The of proteins is in APA-I in the of the that is a The of APA-I is on the as compared with that of abrin, the molecular mass is of by Abrus the cytotoxicity of APA-I in comparison to abrin, and cells were with concentrations of the toxins for of The of protein by the of in the cells and as the of in the cells as compared with the cells. the kinetics of inhibition of protein Abrin protein synthesis in cell lines in a and However, as is toxicity seen in the cells at to of that the of protein is to the kinetics of inhibition APA-I also protein synthesis in the cell lines but the concentrations of the protein higher than that of abrin of the toxins showed cells to be to abrin and APA-I as compared with APA-I with Abrin on in the cytotoxicity of toxins is binding to the cell the of APA-I is due to of binding to binding of to and cells compared with that of were with different concentrations of the toxin for at Data in that the binding of toxin is in cell lines of Apoptosis by Abrin and have been to apoptosis in different cell lines Lin J.-Y. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), and studies from our have that cells apoptosis with abrin (15Narayanan S. Surolia A. Karande A.A. Biochem. J. 2004; 377: 233-240Crossref PubMed Scopus (77) Google Scholar). APA-I is similar to abrin with respect to as and cells were with the and by and that like abrin, APA-I also apoptosis in cell although the concentration of APA-I to apoptosis is than higher as compared with that of abrin. The of in cells by abrin and APA-I earlier than seen in the of in the of APA-I, the cells were with the with the toxins and for the by The the cell lines from apoptosis by abrin and APA-I like toxin J. A. Scholar) and toxin D. 2000; 68: PubMed Scopus Google Scholar) have been reported to apoptosis by but our earlier studies (15Narayanan S. Surolia A. Karande A.A. Biochem. J. 2004; 377: 233-240Crossref PubMed Scopus (77) Google Scholar) have that apoptosis not the death of cell-surface receptors has been to be essential for the of by K. PubMed Scopus Google Scholar). APA-I is a and there a that death receptors be and thereby the pathway initiated by APA-I, studies were on cells that the of cells were with APA-I and abrin for different and by and that abrin apoptosis in cell and the of apoptosis induced is with that of the cells that lectin is not with the apoptosis induced by APA-I, and therefore cell death is of with and were in the of abrin and APA-I for the cells were with of the and a the Abrin apoptosis in cells via the intrinsic pathway of cell death (15Narayanan S. Surolia A. Karande A.A. Biochem. J. 2004; 377: 233-240Crossref PubMed Scopus (77) Google we APA-I also apoptosis by the mitochondrial is protein that the intrinsic pathway of apoptosis. the of cells with different concentrations of APA-I, apoptosis as by for and that apoptosis follows the intrinsic pathway of cell of structure has been by molecular the of Abrus agglutinin as a Data The for the are for a of in the The of and are in The complete amino acid sequence of APA-I determined by Liu (19Liu C.-L. Tsai C.-C. Lin S.-C. Wang L.-I. Hsu C.-I. Hwang M.-J. Lin J.-Y. J. Biol. Chem. 2000; 275: 1897-1901Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). A and B of APA-I have sequence identity with abrin-a A chain and with the abrin-a B However, the sequence with other type II RIPs are R. Podder S.K. Karande A.A. Eur. J. Biochem. PubMed Scopus Google Scholar). APA-I sequence identity with A chain of ricin and with the B chain. the with mistletoe lectin are and for A chain and B the sequence between of abrin and APA-I are the in are of in of the differences in of the three-dimensional structure for the major between abrin and in the of The overall folding of APA-I is similar to that of abrin. However, there major that for the less toxic of The only for of abrin-a and APA-I is is higher than that for A and B and The of APA-I is similar to that of other type II RIPs that abrin, and mistletoe the of the APA-I to the of the abrin-a A APA-I A chain of folding is of consisting of and two The of APA-I is residue than that of and the two are of from to and is the most conserved of the A chain of type II has a of with a of of two and two and a at the The of APA-I is than that of The for the terminal not the of APA-I be The structure of B the lectin is conserved the type II RIP The major differences in the the loop The lectin chain of APA-I is two and of of and The and a a This type of folding has been as the A.G. C. J. Mol. Biol. PubMed Scopus Google Scholar). APA-I B chain and The sequence to the in is In of APA-I sequence is is any amino The crystal structure the of two and holding in and APA-I B chain two at and in and The of in APA-I is similar to that of A chain of APA-I is with (1Endo Y. Tsurugi K. J. Biol. Chem. 1987; 262: 8128-8130Abstract Full Text PDF PubMed Google Scholar, 2Sperti S. Montanaro L. Mattioli A. Stirpe F. Biochem. J. 1973; 136: 813-815Crossref PubMed Scopus (105) Google Scholar). The overall structure of A chain of The is in the by these The of and are conserved the type II on crystal structure of various type II RIPs, has been that the in RIPs to (8Tahirov T.H. Lu T.H. Liaw Y.C. Chen Y.L. Lin J.Y. J. Mol. Biol. 1995; 250: 354-367Crossref PubMed Scopus (151) Google Scholar). of of the of and the overall in the (8Tahirov T.H. Lu T.H. Liaw Y.C. Chen Y.L. Lin J.Y. J. Mol. Biol. 1995; 250: 354-367Crossref PubMed Scopus (151) Google Scholar). In APA-I the position of is by to the chain to The position of is by chain with to and the chain bond with to The of is seen with of via and of is to of the to be at via a of The of two of and of the these The almost to be the for the of the The of these two is to a for the by to the of In of ricin (9Rutenber E. Katzin B.J. Ernst S. Collins E.J. Mlsna D. Ready M.P. Robertus J.D. Proteins. 1991; 10: 240-250Crossref PubMed Scopus (254) Google Scholar), the to Like in these are almost in The studies on of in the of ricin have that and the by and M.P. Y. Robertus J.D. Proteins. 1991; 10: PubMed Scopus Google Scholar). This that the of to in the to the the and of in the of and are the in the of Studies on of abrin-a C.H. Lee Lee T.C. Lin J.Y. J. Mol. Biol. PubMed Scopus Google Scholar) and the in the by and and galactose-binding and are in APA-I B chain as in abrin-a (19Liu C.-L. Tsai C.-C. Lin S.-C. Wang L.-I. Hsu C.-I. Hwang M.-J. Lin J.-Y. J. Biol. Chem. 2000; 275: 1897-1901Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). in the and In these the is to in a on by a kink in the and a conserved residue the of the E. Ready Robertus J.D. 1987; PubMed Scopus Google Scholar). APA-I the sequence of the kink in is for is The in abrin are and The in the two of APA-I are and as a binding for but not interactions with the between galactose and and in are by to amino and The of of abrin and agglutinin differences not The of APA-I are and in APA-I B chain and and in APA-I A chain (19Liu C.-L. Tsai C.-C. Lin S.-C. Wang L.-I. Hsu C.-I. Hwang M.-J. Lin J.-Y. J. Biol. Chem. 2000; 275: 1897-1901Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). at the and of the lectin in of the toxin subunit of for APA-I at than major with the other type II RIPs, APA-I be for less A different is seen at the of The of the residue at the is found to a major of the that in the binding of the in abrin, and APA-I that Asn-200 of to of ricin is for the binding to the in the α-sarcin a sequence at the of 28 S rRNA A.F. Robertus J.D. J. Mol. Biol. PubMed Scopus Google Scholar, Y. Eur. J. Biochem. PubMed Scopus Google Scholar). In of as a for the possibly by a with the of RNA S. S. U. S. A. PubMed Scopus Google Scholar). In the Asn-200 has a and can bind to the via However, a proline is at the position in by others earlier (19Liu C.-L. Tsai C.-C. Lin S.-C. Wang L.-I. Hsu C.-I. Hwang M.-J. Lin J.-Y. J. Biol. Chem. 2000; 275: 1897-1901Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) that the Pro-199 in APA-I cause of the and thereby be a for the decreased toxicity of The analysis of structure of a kink in the induced by the proline residue in The proline substitution takes place at the position of the from the In the has that the of of proline is at the of the PubMed Scopus Google Scholar). at the is perhaps as the than the PubMed Scopus Google Scholar). The of proline to chain and chain in binding of the in and is the major cause of the decreased toxicity of APA-I as compared with abrin-a and comparison of the in and APA-I of any kink in the by proline in APA-I not for APA-I as a the of APA-I the thereby its as a made of two A and two B chains. This is also from and in A and B. A-B are by a and other at the of A thereby a is a disulfide agglutinin K. Robertus J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar), APA-I has disulfide at the of the of in APA-I A chain. The formation and are at the The by the in the is is the for a a L. C. J. J. Mol. Biol. PubMed Scopus Google Scholar). However, are at the perhaps that the is of the type J.M. EMBO J. 2003; PubMed Scopus Google Scholar, H. I. T. PubMed Scopus Google Scholar). the of of the at the are in the of and The J. Mol. Biol. PubMed Scopus Google Scholar) is used to the of as obtained by The of the for the of APA-I is is the of to for and as by and J. Mol. Biol. PubMed Scopus Google Scholar). the factors as in high in the and the of and at the we are to a for be that the of APA-I from the crystal structure to that seen in the the of the of are not at and toxins are a single can a cell K. Olsnes S. A. PubMed Scopus Google Scholar). The protein synthesis of Abrus agglutinin reported to be weaker than abrin in in (19Liu C.-L. Tsai C.-C. Lin S.-C. Wang L.-I. Hsu C.-I. Hwang M.-J. Lin J.-Y. J. Biol. Chem. 2000; 275: 1897-1901Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). the have similar Google however, the for abrin is and agglutinin is 5 F. L. 10: PubMed Scopus Google Scholar). in our that the APA-I is less toxic to and the reduction in the of the be to the structural differences in the of abrin and The differences in protein synthesis inhibition between cell lines by APA-I is not due to binding of as by binding studies of apoptosis by RIPs has been for a H. K. S. Google Scholar). APA-I apart from protein synthesis inhibition also induced apoptosis. and cells showed addition of concentrations of APA-I, apoptosis only the addition of the of protein than to that of the that inhibition of protein synthesis is for apoptosis. the of APA-I, not any for apoptosis through the cell-surface death A cell not apoptosis on with APA-I, and can be that APA-I, like abrin, follows the mitochondrial pathway of cell the studies that abrin and APA-I are toxic in that inhibit protein synthesis and and the reason for the by APA-I is of the in the is in the crystal structure of The crystal structure of APA-I to the most factors for the less toxicity of APA-I than abrin. The structure The less toxic of APA-I is perhaps not of any kink in as (19Liu C.-L. Tsai C.-C. Lin S.-C. Wang L.-I. Hsu C.-I. Hwang M.-J. Lin J.-Y. J. Biol. Chem. 2000; 275: 1897-1901Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), but of the fewer interactions at the The of Pro-199 at the to Asn-200 in abrin-a and in the of the of interactions the chain and the chain with the The toxicity and the apoptosis studies the the of APA-I, three-dimensional structure is reported in the of studies are in in our for that the toxicity of The of in and and the are by the of of We and for the to the We S. K. Podder for
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