Restoration of lectin activity to a non-glycosylated ricin B chain mutant by the introduction of a novel N-glycosylation site.
Ricin B chain (RTB) is an N-glycosylated, galactose-specific lectin. Removal of the two native N-glycosylation sites at Asn95 and Asn135 by site-directed mutagenesis generated a recombinant protein devoid of lectin activity. Two novel N-glycosylation sites were introduced into RTB at Asn42 and Asn123, either singly or in combination. Microinjection of pre-RTB transcripts into Xenopus oocytes showed that these novel sites became glycosylated in vivo. The single oligosaccharide site chain at Asn42 restored lectin activity to RTB, whereas glycosylation at Asn123 or simultaneous glycosylation at Asn42 and Asn123 failed to do so.
Zhan
J
J
Department of Biological Sciences, University of Warwick, Coventry, UK.
de Sousa
M
M
Chaddock
J A
JA
Roberts
L M
LM
Lord
J M
JM
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D005260:Female; D006031:Glycosylation; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012276:Ricin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D011994:Recombinant Proteins; D012276:Ricin