Site-directed mutagenesis of hamster complement C1S: Characterization with an active form-specific antibody and possible involvement of C1S in tumorigenicity
✍ Scribed by Hisako Sakiyama; Munehiro Nishida; Norie Sakai; Ken Nagino; Shoichiro Miyatake; Takashi Saito; Shinobu Imajoh-Ohmi
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- French
- Weight
- 528 KB
- Volume
- 66
- Category
- Article
- ISSN
- 0020-7136
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✦ Synopsis
We have previously shown that non-transformed mouse A3 I cells became tumorigenic when they were transfected with hamster C I s cDNA expression plasmid BCMGSNeoCS. In the present study, mutations were introduced into the cDNA at the activation cleavage site, Arg423(AGG) and the active center Ser6 I'I(AGC). These amino-acids were replaced by His423(CAC) and Thr6 I7(ACC), respectively. The mutated cDNAs were inserted into BCMGSNeo and transfected to A31 and its polyoma-virus-transformed SEA7 cells. C I s produced from these transfectants lost their enzyme activity. Transfectants of these mutated C I s cDNA did not form tumors in nude mice. To distinguish between active and inactive C I s in situ. we have developed novel antibodies, one directed to the NH2-tenninal neoepitope of the L chain and the other specific for uncleaved inactive C I s. These antibodies were used to characterize C I s produced by transfectants, so as to determine whether or not it was cleaved at the right position.