Expression and functional properties of genetically engineered ricin B chain lacking galactose-binding activity

Ellen S. Vitetta, Nancy Yen

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Ricin is a potent plant toxin consisting of two disulfide-bonded subunits. The A chain of ricin is an N-glycosidase which inactivates 28 S RNA and inhibits protein synthesis. The B chain is a galactose-specific lectin with two galactose-binding sites. The genes encoding preproricin and its A and B chains have been cloned and expressed. In addition, X-ray crystallographic studies have identified the galactose-contract residues in both the high- and low-affinity galactose-binding sites of the B chain. In this study, the high-affinity galactose-contact residue of the B chain was changed from Asn-255 to Ala-255 by oligonucleotide-directed mutagenesis. The resulting mutant was sequenced to confirm the presence of a single mutation and was expressed in Cos-M6 cells. Both wild-type and mutant recombinant B chain could be immunoprecipitated with a heterologous anti-B chain antibody and both could form A-B heterodimers. However, as compared to the wild-type, the mutant B chain lacked more than 99% of its lectin activity and cytotoxicity as an A-B dimer. In conclusion, altering the contact residue of the high-affinity galactose-binding site of ricin B chain from Asn-255 to Ala-255 abrogates more than 99% of its lectin activity and the cytotoxicity of the A-B heterodimer to ricin-sensitive cells.

Original languageEnglish (US)
Pages (from-to)151-157
Number of pages7
JournalBBA - Gene Structure and Expression
Volume1049
Issue number2
DOIs
StatePublished - Jun 21 1990

Fingerprint

Ricin
Galactose
Lectins
Binding Sites
Cytotoxicity
Mutagenesis
Gene encoding
Glycoside Hydrolases
Contracts
Site-Directed Mutagenesis
Oligonucleotides
Disulfides
Dimers
X-Rays
RNA
X rays
Mutation
Antibodies
Genes
Proteins

Keywords

  • B chain
  • Galactose binding site
  • Gene expression
  • Plant toxin
  • Ricin
  • Site directed mutagenesis

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Genetics
  • Structural Biology

Cite this

Expression and functional properties of genetically engineered ricin B chain lacking galactose-binding activity. / Vitetta, Ellen S.; Yen, Nancy.

In: BBA - Gene Structure and Expression, Vol. 1049, No. 2, 21.06.1990, p. 151-157.

Research output: Contribution to journalArticle

@article{4c104b98e736472b9061e2b5e9155fb0,
title = "Expression and functional properties of genetically engineered ricin B chain lacking galactose-binding activity",
abstract = "Ricin is a potent plant toxin consisting of two disulfide-bonded subunits. The A chain of ricin is an N-glycosidase which inactivates 28 S RNA and inhibits protein synthesis. The B chain is a galactose-specific lectin with two galactose-binding sites. The genes encoding preproricin and its A and B chains have been cloned and expressed. In addition, X-ray crystallographic studies have identified the galactose-contract residues in both the high- and low-affinity galactose-binding sites of the B chain. In this study, the high-affinity galactose-contact residue of the B chain was changed from Asn-255 to Ala-255 by oligonucleotide-directed mutagenesis. The resulting mutant was sequenced to confirm the presence of a single mutation and was expressed in Cos-M6 cells. Both wild-type and mutant recombinant B chain could be immunoprecipitated with a heterologous anti-B chain antibody and both could form A-B heterodimers. However, as compared to the wild-type, the mutant B chain lacked more than 99{\%} of its lectin activity and cytotoxicity as an A-B dimer. In conclusion, altering the contact residue of the high-affinity galactose-binding site of ricin B chain from Asn-255 to Ala-255 abrogates more than 99{\%} of its lectin activity and the cytotoxicity of the A-B heterodimer to ricin-sensitive cells.",
keywords = "B chain, Galactose binding site, Gene expression, Plant toxin, Ricin, Site directed mutagenesis",
author = "Vitetta, {Ellen S.} and Nancy Yen",
year = "1990",
month = "6",
day = "21",
doi = "10.1016/0167-4781(90)90035-Z",
language = "English (US)",
volume = "1049",
pages = "151--157",
journal = "Biochimica et Biophysica Acta - Gene Structure and Expression",
issn = "0167-4781",
publisher = "Elsevier BV",
number = "2",

}

TY - JOUR

T1 - Expression and functional properties of genetically engineered ricin B chain lacking galactose-binding activity

AU - Vitetta, Ellen S.

AU - Yen, Nancy

PY - 1990/6/21

Y1 - 1990/6/21

N2 - Ricin is a potent plant toxin consisting of two disulfide-bonded subunits. The A chain of ricin is an N-glycosidase which inactivates 28 S RNA and inhibits protein synthesis. The B chain is a galactose-specific lectin with two galactose-binding sites. The genes encoding preproricin and its A and B chains have been cloned and expressed. In addition, X-ray crystallographic studies have identified the galactose-contract residues in both the high- and low-affinity galactose-binding sites of the B chain. In this study, the high-affinity galactose-contact residue of the B chain was changed from Asn-255 to Ala-255 by oligonucleotide-directed mutagenesis. The resulting mutant was sequenced to confirm the presence of a single mutation and was expressed in Cos-M6 cells. Both wild-type and mutant recombinant B chain could be immunoprecipitated with a heterologous anti-B chain antibody and both could form A-B heterodimers. However, as compared to the wild-type, the mutant B chain lacked more than 99% of its lectin activity and cytotoxicity as an A-B dimer. In conclusion, altering the contact residue of the high-affinity galactose-binding site of ricin B chain from Asn-255 to Ala-255 abrogates more than 99% of its lectin activity and the cytotoxicity of the A-B heterodimer to ricin-sensitive cells.

AB - Ricin is a potent plant toxin consisting of two disulfide-bonded subunits. The A chain of ricin is an N-glycosidase which inactivates 28 S RNA and inhibits protein synthesis. The B chain is a galactose-specific lectin with two galactose-binding sites. The genes encoding preproricin and its A and B chains have been cloned and expressed. In addition, X-ray crystallographic studies have identified the galactose-contract residues in both the high- and low-affinity galactose-binding sites of the B chain. In this study, the high-affinity galactose-contact residue of the B chain was changed from Asn-255 to Ala-255 by oligonucleotide-directed mutagenesis. The resulting mutant was sequenced to confirm the presence of a single mutation and was expressed in Cos-M6 cells. Both wild-type and mutant recombinant B chain could be immunoprecipitated with a heterologous anti-B chain antibody and both could form A-B heterodimers. However, as compared to the wild-type, the mutant B chain lacked more than 99% of its lectin activity and cytotoxicity as an A-B dimer. In conclusion, altering the contact residue of the high-affinity galactose-binding site of ricin B chain from Asn-255 to Ala-255 abrogates more than 99% of its lectin activity and the cytotoxicity of the A-B heterodimer to ricin-sensitive cells.

KW - B chain

KW - Galactose binding site

KW - Gene expression

KW - Plant toxin

KW - Ricin

KW - Site directed mutagenesis

UR - http://www.scopus.com/inward/record.url?scp=0025341724&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025341724&partnerID=8YFLogxK

U2 - 10.1016/0167-4781(90)90035-Z

DO - 10.1016/0167-4781(90)90035-Z

M3 - Article

VL - 1049

SP - 151

EP - 157

JO - Biochimica et Biophysica Acta - Gene Structure and Expression

JF - Biochimica et Biophysica Acta - Gene Structure and Expression

SN - 0167-4781

IS - 2

ER -