SITE-DIRECTED MUTAGENESIS OF HAMSTER COMPLEMENT CLS - CHARACTERIZATION WITH AN ACTIVE FORM-SPECIFIC ANTIBODY AND POSSIBLE INVOLVEMENT OF CLS IN TUMORIGENICITY

We have previously shown that non-transformed mouse A31 cells became t umorigenic when they were transfected with hamster C1s cDNA expression plasmid BCMGSNeoCS. In the present study, mutations were introduced i nto the cDNA at the activation cleavage site, Arg423(AGG) and the acti ve center Ser617(AGC). These amino-acids were replaced by His423(CAC) and Thr617(ACC), respectively. The mutated cDNAs were inserted into BC MGSNeo and transfected to A31 and its polyoma-virus-transformed SEA7 c ells. C1s produced from these transfectants lost their enzyme activity . Transfectants of these mutated C1s cDNA did not form tumors in nude mice. To distinguish between active and inactive C1s in situ, we have developed novel antibodies, one directed to the NH2-terminal neoepitop e of the L chain and the other specific for uncleaved inactive C1s. Th ese antibodies were used to characterize C1s produced by transfectants , so as to determine whether or not it was cleaved at the right positi on. (C) 1996 Wiley-Liss, Inc.