Mutational analysis of the galactose binding ability of recombinant ricin B chain.
Ricin B chain (RTB) is an N-glycosylated galactose-specific lectin which folds into two globular domains. Each domain binds one galactoside. The x-ray crystallographic structure has shown that the two binding sites are structurally similar and contain key binding residues which hydrogen bond to the sugar, and a conserved tripeptide, Asp-Val-Arg. We have used oligonucleotide site-directed mutagenesis to change either the binding residues or the homologous tripeptide in one or other or in both of the sites. The 5' signal sequence and RTB coding region were excised from preproricin cDNA and fused in frame to generate preRTB cDNA. Transcripts synthesized in vitro from wild-type or mutant preRTB cloned into the Xenopus transcription vector pSP64T using SP6 RNA polymerase, were microinjected into Xenopus oocytes. The recombinant products were segregated into the oocyte rough endoplasmic reticulum and core-glycosylated, and the N-terminal signal peptide was removed. Mutating sugar binding sites individually did not abrogate the lectin activity of RTB. When both sites were changed simultaneously, RTB was produced which was soluble and stable but no longer able to bind galactose. Changing the Asn residues of the two RTB N-glycosylation sites to Gln showed that oligosaccharide side chains were essential for both the stability and biological activity of recombinant RTB.
Wales
R
R
Department of Biological Sciences, University of Warwick, Coventry, United Kingdom.
Richardson
P T
PT
Roberts
L M
LM
Woodland
H R
HR
Lord
J M
JM
D000818:Animals; D001212:Asialoglycoproteins; D004591:Electrophoresis, Polyacrylamide Gel; D060748:Fetuins; D005690:Galactose / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D007785:Lactose / Q000378:metabolism; D037102:Lectins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics; D012276:Ricin / Q000235:genetics*; D014158:Transcription, Genetic; D014981:Xenopus; D000509:alpha-Fetoproteins / Q000378:metabolism
D001212:Asialoglycoproteins; D060748:Fetuins; D037102:Lectins; D011994:Recombinant Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin; D012276:Ricin; D007785:Lactose; D005690:Galactose