Effect of N- and C-terminal deletions on the RNA N-glycosidase activity and the antigenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica.
Karasurin-A, from root tubers of Trichosanthes kirilowii var. japonica, is a type I ribosome-inactivating protein (RIP) that displays activity of RNA N-glycosidase to remove an adenine in the conserved sarcin/ricin loop of the largest RNA in the ribosome. We expressed recombinant proteins of karasurin-A and its various mutants with N- or C-terminal deletions in Escherichia coli as fusion proteins with maltose-binding protein (MBP), and compared their enzymatic activities and antigenicities. Muteins of karasurin-A generated by deleting either the first 100 N-terminal or the last 30 C-terminal amino acid residues lost activity of RNA N-glycosidase. The mutant proteins whose 80 N-terminal or 20 C-terminal amino acids were deleted could depurinate rRNA although the activities were decreased drastically. The antigenicities of the recombinant proteins were considerably reduced by deleting 20 amino acid residues from either N- or C-terminal regions.
Kondo
Toshiya
T
School of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo 108-8641, Japan. kondout@pharm.kitasato-u.ac.jp
Kurihara
Satoko
S
Yoshikawa
Takafumi
T
Mizukami
Hajime
H
D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D017353:Gene Deletion; D058619:Maltose-Binding Proteins; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D054788:Ribosome Inactivating Proteins; D013329:Structure-Activity Relationship; D029778:Trichosanthes / Q000201:enzymology / Q000235:genetics
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C069870:karasurin protein, Trichosanthes kirilowii; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins