The differential catalytic activity of ribosome-inactivating proteins saporin 5 and 6 is due to a single substitution at position 162

Ghosh, Paroma; Batra, Janendra K.

doi:10.1042/bj20060895

Cited by 16 publications

(15 citation statements)

References 31 publications

(42 reference statements)

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“…In fact, Asn 162 lies in close proximity to three hydrophobic residues, Phe 149 , Ala 151 and Val 153 . The negative charge of the N162D substitution may affect the stability of the active cleft by introducing a local structural change [21]. …”

Section: Saporin-s6mentioning

confidence: 99%

Saporin-S6: A Useful Tool in Cancer Therapy

Polito

Bortolotti

Mercatelli

et al. 2013

Toxins

114

115

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Thirty years ago, the type 1 ribosome-inactivating protein (RIP) saporin-S6 (also known as saporin) was isolated from Saponaria officinalis L. seeds. Since then, the properties and mechanisms of action of saporin-S6 have been well characterized, and it has been widely employed in the construction of conjugates and immunotoxins for different purposes. These immunotoxins have shown many interesting results when used in cancer therapy, particularly in hematological tumors. The high enzymatic activity, stability and resistance to conjugation procedures and blood proteases make saporin-S6 a very useful tool in cancer therapy. High efficacy has been reported in clinical trials with saporin-S6-containing immunotoxins, at dosages that induced only mild and transient side effects, which were mainly fever, myalgias, hepatotoxicity, thrombocytopenia and vascular leak syndrome. Moreover, saporin-S6 triggers multiple cell death pathways, rendering impossible the selection of RIP-resistant mutants. In this review, some aspects of saporin-S6, such as the chemico-physical characteristics, the structural properties, its endocytosis, its intracellular routing and the pathogenetic mechanisms of the cell damage, are reported. In addition, the recent progress and developments of saporin-S6-containing immunotoxins in cancer immunotherapy are summarized, including in vitro and in vivo pre-clinical studies and clinical trials.

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Section: Saporin-s6mentioning

confidence: 99%

Saporin-S6: A Useful Tool in Cancer Therapy

Polito

Bortolotti

Mercatelli

et al. 2013

Toxins

114

115

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“…However, recent works have *Address correspondence to this author at the Institute of Biostructures and Bioimaging, CNR, Via Mezzocannone, n. 16. I-80134 Napoli, Italy; Tel: ++390812534507; Fax: ++390812536642; E-mail: rita.berisio@unina.it shown that DNase activity is observed even for highly pure RIPs [6,20,21] and that reduction in N--glycosidase activity results in a reduction of DNase activity [20], reinforcing the concept that RIPs act as N-glycosidases and as DNases using the same catalytic site [22].…”

Section: Introductionmentioning

confidence: 73%

Crystallization and Preliminary X-Ray Diffraction Analysis of PD-L1, a Highly Glycosylated Ribosome Inactivating Protein with DNase Activity

Ruggiero¹,

Maro²,

Mastroianni³

et al. 2007

PPL

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PD-L1 is a highly glycosylated type 1 ribosome inactivating protein, from Phytolacca dioica leaves, with the peculiarity to act also as a DNase. PD-L1 has been successfully crystallized using vapour diffusion and seeding techniques. Crystals belong to the monoclinic C2 space group, with unit cell dimensions a=161.01, b=34.73, c=120.63 A, beta=127.99 degrees . Two molecules are present in the asymmetric unit. Phase determination has been achieved using molecular replacement.

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“…The catalytic efficiency ( k cat / K m ) is 3.8 × 10 7 M −1 s −1 , which is ∼3.7-fold lower than values reported for RTA catalysis. 24 However, quantitation of the release of adenine from rabbit rRNA reveals that saporin L3 can depurinate up to 290 adenines/mol from rabbit rRNA (not shown). Moreover, saporin L3 also releases adenine from tRNA and luciferase mRNA at physiological pH.…”

Section: Resultsmentioning

confidence: 99%

Soapwort Saporin L3 Expression in Yeast, Mutagenesis, and RNA Substrate Specificity

Yuan

Sturm

et al. 2015

Biochemistry

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Saporin L3 from Saponaria officinalis (soapwort) leaves is a type 1 ribosome-inactivating protein. It catalyzes the hydrolysis of oligonucleotide adenylate N-ribosidic bonds to release adenine from rRNA. Depurination sites include both adenines in the GAGA tetraloop of short sarcin-ricin stem-loops and multiple adenines within eukaryotic rRNA, tRNAs, and mRNAs. Multiple Escherichia coli vector designs for saporin L3 expression were attempted but demonstrated high toxicity even during plasmid maintenance and selection in E. coli nonexpression strains. Saporin L3 is >103 times more efficient at RNA deadenylation on short GAGA stem-loops than saporin S6, the saporin isoform currently used in immunotoxin clinical trials. We engineered a construct for the His-tagged saporin L3 to test for expression in Pichia pastoris when it is linked to the protein export system for the yeast α-mating factor. DNA encoding saporin L3 was cloned into a pPICZαB expression vector and expressed in P. pastoris under the alcohol dehydrogenase AOX1 promoter. A fusion protein of saporin L3 containing the pre-pro-sequence of the α-mating factor, the c-myc epitope, and the His tag was excreted from the P. pastoris cells and isolated from the culture medium. Autoprocessing of the α-mating factor yielded truncated saporin L3 (amino acids 22–280), the c-myc epitope, and the His tag expressed optimally as a 32 kDa construct following methanol induction. Saporin L3 was also expressed with specific alanines and/or serines mutated to cysteine. Native and Cys mutant saporins are kinetically similar. The recombinant expression of saporin L3 and its mutants permits the production and investigation of this high-activity ribosome-inactivating protein.

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The differential catalytic activity of ribosome-inactivating proteins saporin 5 and 6 is due to a single substitution at position 162

Cited by 16 publications

References 31 publications

Saporin-S6: A Useful Tool in Cancer Therapy

Saporin-S6: A Useful Tool in Cancer Therapy

Crystallization and Preliminary X-Ray Diffraction Analysis of PD-L1, a Highly Glycosylated Ribosome Inactivating Protein with DNase Activity

Soapwort Saporin L3 Expression in Yeast, Mutagenesis, and RNA Substrate Specificity

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