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

Ellen S. Vitetta, Nancy Yen

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

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

Keywords

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

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Genetics

Fingerprint

Dive into the research topics of 'Expression and functional properties of genetically engineered ricin B chain lacking galactose-binding activity'. Together they form a unique fingerprint.

Cite this