Site-directed mutagenesis of hamster complement C1S: characterization with an active form-specific antibody and possible involvement of C1S in tumorigenicity.
We have previously shown that non-transformed mouse A31 cells became tumorigenic when they were transfected with hamster C1s 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 Ser617(AGC). These amino-acids were replaced by His423(CAC) and Thr617(ACC), respectively. The mutated cDNAs were inserted into BCMGSNeo and transfected to A31 and its polyoma-virus-transformed SEA7 cells. 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 neoepitope of the L chain and the other specific for uncleaved inactive C1s. These antibodies were used to characterize C1s produced by transfectants, so as to determine whether or not it was cleaved at the right position.
Sakiyama
H
H
Division of Biology and Oncology, National Institute of Radiological Sciences, Anagawa, Inage-ku, Chiba 264, Japan.
Nishida
M
M
Sakai
N
N
Nagino
K
K
Miyatake
S
S
Saito
T
T
Imajoh-Ohmi
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002461:Cell Line, Transformed / Q000637:transplantation; D002472:Cell Transformation, Viral; D003173:Complement C1s / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D008647:Mesocricetus / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009368:Neoplasm Transplantation; D011120:Polyomavirus / Q000502:physiology; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; D011994:Recombinant Proteins; D010450:Endopeptidases; D003173:Complement C1s