Restoration of lectin activity to an inactive abrin B chain by substitution and mutation of the 2 gamma subdomain.
Abrin is a heterodimeric plant protein that occurs in several isoforms (abrin-a, abrin-b, abrin-c and abrin-d), whose B chains are believed to either have (abrin-a and abrin-d) or lack (abrin-b and abrin-c) the ability to bind galactose. The 5' signal sequence and toxin B chain (ATB)-coding region were excised from a preproabrin cDNA [K. A. Wood, J. M. Lord, E. J. Wawrzynczak, and M. Piatak (1991) Eur. J. Biochem. 198, 723-732], tentatively identified as abrin-c, which was predicted to lack lectin activity, and fused in-frame to generate pre-ATB cDNA. Transcripts, synthesized in vitro from pre-ATB cloned into the transcription vector pSP64T, were expressed after microinjection into Xenopus oocytes. The recombinant ATB was shown, using a qualitative sugar-binding assay, to be devoid of lectin activity. Lectin activity could not be restored to this nonbinding ATB by replacing the 2 gamma subdomain with the corresponding galactose-binding 2 gamma subdomain from ricin B chain, but it was restored by replacement with the active galactose-binding 2 gamma subdomain from a different abrin isoform (abrin-a). The putative galactose-binding pocket of the nonbinding ATB 2 gamma subdomain contained a His residue at the position occupied by a residue with an aromatic side chain (Tyr or Trp) in functional 2 gamma subdomains. Mutationally converting this His to either Tyr or Trp restored lectin activity to the nonbinding ATB, emphasizing the contribution of an aromatic side chain in a functional 2 gamma subdomain galactose-binding site for members of this lectin family.
de Sousa
M
M
Department of Biological Sciences, University of Warwick, Coventry, UK.
Roberts
L M
LM
Lord
J M
JM
D000036:Abrin / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014981:Xenopus
D000036:Abrin; D014443:Tyrosine; D014364:Tryptophan