Nagasawa Yoshimi, Fujii Kazuyuki, Yoshikawa Takafumi, Kobayashi Yoshinori, Kondo Toshiya
School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
Phytochemistry. 2008 May;69(8):1653-60. doi: 10.1016/j.phytochem.2008.02.012. Epub 2008 Apr 2.
Pokeweed antiviral protein (PAP) isolated from Phytolacca americana is a ribosome-inactivating protein (RIP) that has RNA N-glycosidase (RNG) activity towards both eukaryotic and prokaryotic ribosomes. In contrast, karasurin-A (KRN), a RIP from Trichosanthes kirilowii var. japonica, is active only on eukaryotic ribosomes. Stepwise selection of chimera proteins between PAP and KRN indicated that the C-terminal region of PAP (residues 209-225) was critical for RNG activity toward prokaryotic ribosomes. When the region of PAP (residues 209-225) was replaced with the corresponding region of KRN the PAP chimera protein, like KRN, was active only on eukaryotic ribosomes. Furthermore, insertion of the region of PAP (residues 209-225) into the KRN chimera protein resulted not only in the detectable RNG activity toward prokaryotic ribosome, but also activity toward the eukaryotic ribosomes as well that was seven-fold higher than for the original KRN. In this study, the possibility of genetic manipulation of the activity and substrate specificity of RIPs is demonstrated.
从美洲商陆中分离得到的商陆抗病毒蛋白（PAP）是一种核糖体失活蛋白（RIP），对真核和原核核糖体均具有RNA N-糖苷酶（RNG）活性。相比之下，栝楼素-A（KRN）是从日本栝楼变种中分离得到的一种RIP，仅对真核核糖体有活性。对PAP和KRN之间的嵌合蛋白进行逐步筛选表明，PAP的C末端区域（第209 - 225位氨基酸残基）对于其对原核核糖体的RNG活性至关重要。当将PAP的该区域（第209 - 225位氨基酸残基）替换为KRN的相应区域时，PAP嵌合蛋白与KRN一样，仅对真核核糖体有活性。此外，将PAP的该区域（第209 - 225位氨基酸残基）插入到KRN嵌合蛋白中，不仅使其对原核核糖体具有可检测到的RNG活性，而且对真核核糖体的活性也比原始KRN高7倍。在本研究中，证明了对RIPs的活性和底物特异性进行基因操作的可能性。
