Characterization of a saporin isoform with lower ribosome-inhibiting activity

M. Serena Fabbrini, Emilia Rappocciolo, Daniela Carpani, Michela Solinas, Barbara Valsasina, Umberto Breme, Ugo Cavallaro, Anders Nykjaer, Ermanna Rovida, Giuseppe Legname, Marco R. Soria

Research output: Contribution to journalArticle

Abstract

We have expressed in Escherichia coli five isoforms of saporin, a single-chain ribosome-inactivating protein (RIP). Translation inhibition activities of the purified recombinant polypeptides in vitro were compared with those of recombinant dianthin 30, a less potent and closely related RIP,and of ricin A chain. Dianthin 30, and a saporin isoform encoded by a cDNA from leaf tissue (SAP-C), both had about one order of magnitude lower activity in translation inhibition assays than all other isoforms of saporin tested. We recently demonstrated that saporin extracted from seeds of Saponaria officinalis binds to α2-macroglobulin receptor (α2MR; also termed low density lipoprotein-receptor-related-protein), indicating a general mechanism of interaction of plant RIPs with the α2MR system . Here we report that SAP-C bound to α2MR equally well as native saporin. However, the same isoform had about ten times lower cytotoxicity than the other saporin isoforms towards different cell lines. This indicates that the lower cell-killing ability of the SAP-C isoform is presumably due to its altered interaction with the protein synthesis machinery of target cells. Since saporin binding to the α2MR is competed by heparin, we also tested in cell-killing experiments Chinese hamster ovary cell lines defective for expression of either heparan sulphates or proteoglycans. No differences were observed in cytotoxicity using native saporin or the recombinant isoforms. Therefore saporin binding to the cell surface should not be mediated by interaction with proteoglycans, as is the case for other α2MR ligands.

Original languageEnglish
Pages (from-to)719-727
Number of pages9
JournalBiochemical Journal
Volume322
Issue number3
Publication statusPublished - Mar 15 1997

Fingerprint

Ribosomes
Protein Isoforms
Ribosome Inactivating Proteins
Proteoglycans
Cytotoxicity
Saponaria
LDL-Receptor Related Proteins
Cells
saporin
Ricin
Cell Line
Macroglobulins
Heparan Sulfate Proteoglycans
Heparitin Sulfate
LDL Receptors
Cricetulus
Escherichia coli
Machinery
Seed
Heparin

ASJC Scopus subject areas

  • Biochemistry

Cite this

Fabbrini, M. S., Rappocciolo, E., Carpani, D., Solinas, M., Valsasina, B., Breme, U., ... Soria, M. R. (1997). Characterization of a saporin isoform with lower ribosome-inhibiting activity. Biochemical Journal, 322(3), 719-727.

Characterization of a saporin isoform with lower ribosome-inhibiting activity. / Fabbrini, M. Serena; Rappocciolo, Emilia; Carpani, Daniela; Solinas, Michela; Valsasina, Barbara; Breme, Umberto; Cavallaro, Ugo; Nykjaer, Anders; Rovida, Ermanna; Legname, Giuseppe; Soria, Marco R.

In: Biochemical Journal, Vol. 322, No. 3, 15.03.1997, p. 719-727.

Research output: Contribution to journalArticle

Fabbrini, MS, Rappocciolo, E, Carpani, D, Solinas, M, Valsasina, B, Breme, U, Cavallaro, U, Nykjaer, A, Rovida, E, Legname, G & Soria, MR 1997, 'Characterization of a saporin isoform with lower ribosome-inhibiting activity', Biochemical Journal, vol. 322, no. 3, pp. 719-727.
Fabbrini MS, Rappocciolo E, Carpani D, Solinas M, Valsasina B, Breme U et al. Characterization of a saporin isoform with lower ribosome-inhibiting activity. Biochemical Journal. 1997 Mar 15;322(3):719-727.
Fabbrini, M. Serena ; Rappocciolo, Emilia ; Carpani, Daniela ; Solinas, Michela ; Valsasina, Barbara ; Breme, Umberto ; Cavallaro, Ugo ; Nykjaer, Anders ; Rovida, Ermanna ; Legname, Giuseppe ; Soria, Marco R. / Characterization of a saporin isoform with lower ribosome-inhibiting activity. In: Biochemical Journal. 1997 ; Vol. 322, No. 3. pp. 719-727.
@article{c71676df567b426fb27ffff43eec622c,
title = "Characterization of a saporin isoform with lower ribosome-inhibiting activity",
abstract = "We have expressed in Escherichia coli five isoforms of saporin, a single-chain ribosome-inactivating protein (RIP). Translation inhibition activities of the purified recombinant polypeptides in vitro were compared with those of recombinant dianthin 30, a less potent and closely related RIP,and of ricin A chain. Dianthin 30, and a saporin isoform encoded by a cDNA from leaf tissue (SAP-C), both had about one order of magnitude lower activity in translation inhibition assays than all other isoforms of saporin tested. We recently demonstrated that saporin extracted from seeds of Saponaria officinalis binds to α2-macroglobulin receptor (α2MR; also termed low density lipoprotein-receptor-related-protein), indicating a general mechanism of interaction of plant RIPs with the α2MR system . Here we report that SAP-C bound to α2MR equally well as native saporin. However, the same isoform had about ten times lower cytotoxicity than the other saporin isoforms towards different cell lines. This indicates that the lower cell-killing ability of the SAP-C isoform is presumably due to its altered interaction with the protein synthesis machinery of target cells. Since saporin binding to the α2MR is competed by heparin, we also tested in cell-killing experiments Chinese hamster ovary cell lines defective for expression of either heparan sulphates or proteoglycans. No differences were observed in cytotoxicity using native saporin or the recombinant isoforms. Therefore saporin binding to the cell surface should not be mediated by interaction with proteoglycans, as is the case for other α2MR ligands.",
author = "Fabbrini, {M. Serena} and Emilia Rappocciolo and Daniela Carpani and Michela Solinas and Barbara Valsasina and Umberto Breme and Ugo Cavallaro and Anders Nykjaer and Ermanna Rovida and Giuseppe Legname and Soria, {Marco R.}",
year = "1997",
month = "3",
day = "15",
language = "English",
volume = "322",
pages = "719--727",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "3",

}

TY - JOUR

T1 - Characterization of a saporin isoform with lower ribosome-inhibiting activity

AU - Fabbrini, M. Serena

AU - Rappocciolo, Emilia

AU - Carpani, Daniela

AU - Solinas, Michela

AU - Valsasina, Barbara

AU - Breme, Umberto

AU - Cavallaro, Ugo

AU - Nykjaer, Anders

AU - Rovida, Ermanna

AU - Legname, Giuseppe

AU - Soria, Marco R.

PY - 1997/3/15

Y1 - 1997/3/15

N2 - We have expressed in Escherichia coli five isoforms of saporin, a single-chain ribosome-inactivating protein (RIP). Translation inhibition activities of the purified recombinant polypeptides in vitro were compared with those of recombinant dianthin 30, a less potent and closely related RIP,and of ricin A chain. Dianthin 30, and a saporin isoform encoded by a cDNA from leaf tissue (SAP-C), both had about one order of magnitude lower activity in translation inhibition assays than all other isoforms of saporin tested. We recently demonstrated that saporin extracted from seeds of Saponaria officinalis binds to α2-macroglobulin receptor (α2MR; also termed low density lipoprotein-receptor-related-protein), indicating a general mechanism of interaction of plant RIPs with the α2MR system . Here we report that SAP-C bound to α2MR equally well as native saporin. However, the same isoform had about ten times lower cytotoxicity than the other saporin isoforms towards different cell lines. This indicates that the lower cell-killing ability of the SAP-C isoform is presumably due to its altered interaction with the protein synthesis machinery of target cells. Since saporin binding to the α2MR is competed by heparin, we also tested in cell-killing experiments Chinese hamster ovary cell lines defective for expression of either heparan sulphates or proteoglycans. No differences were observed in cytotoxicity using native saporin or the recombinant isoforms. Therefore saporin binding to the cell surface should not be mediated by interaction with proteoglycans, as is the case for other α2MR ligands.

AB - We have expressed in Escherichia coli five isoforms of saporin, a single-chain ribosome-inactivating protein (RIP). Translation inhibition activities of the purified recombinant polypeptides in vitro were compared with those of recombinant dianthin 30, a less potent and closely related RIP,and of ricin A chain. Dianthin 30, and a saporin isoform encoded by a cDNA from leaf tissue (SAP-C), both had about one order of magnitude lower activity in translation inhibition assays than all other isoforms of saporin tested. We recently demonstrated that saporin extracted from seeds of Saponaria officinalis binds to α2-macroglobulin receptor (α2MR; also termed low density lipoprotein-receptor-related-protein), indicating a general mechanism of interaction of plant RIPs with the α2MR system . Here we report that SAP-C bound to α2MR equally well as native saporin. However, the same isoform had about ten times lower cytotoxicity than the other saporin isoforms towards different cell lines. This indicates that the lower cell-killing ability of the SAP-C isoform is presumably due to its altered interaction with the protein synthesis machinery of target cells. Since saporin binding to the α2MR is competed by heparin, we also tested in cell-killing experiments Chinese hamster ovary cell lines defective for expression of either heparan sulphates or proteoglycans. No differences were observed in cytotoxicity using native saporin or the recombinant isoforms. Therefore saporin binding to the cell surface should not be mediated by interaction with proteoglycans, as is the case for other α2MR ligands.

UR - http://www.scopus.com/inward/record.url?scp=0030906147&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030906147&partnerID=8YFLogxK

M3 - Article

C2 - 9148741

AN - SCOPUS:0030906147

VL - 322

SP - 719

EP - 727

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 3

ER -