804 cDNA CLONES FOR HUMAN SPHINGOMYELINASE ISOLATED USING THE λGT 11 SYSTEM

Abstract

Human acid sphingomyelinase activity is deficient in two variants of Niemann-Pick disease, called Types A and B. Type C variants have near normal enzyme activity. The genetic expression of each of these diseases is poorly defined. Our approach to date has been to characterize the structural and kinetic properties of human sphingomyelinase and we have recently characterized a monoclonal antibody against the protein. Using the monoclonal antibody and a λgt 11 expression library, prepared by Drs.J.S.O'Brien and J.de Wet, with cDNA against human hepatoma mRNA, we have succeeded in isolating 6 immunopositive clones. The initial screening used 17×106 recombinants per dish and immunodetection used hybridoma culture fluid (10-30 μg of monoclonal antibody/ml) and peroxidase labeled goat antimouse antibody. One clone produced a fusion protein of about 122,000 daltons while a second produced one of 124,000 daltons corresponding to about 6000 and 8000 daltons of sphingomyelinase protein respectively. These were detected using a anti-E.coli β-galactosidase serum. Immunological identity with sphingomyelinase was shown by direct immunoprecipitation of the fusion protein by anti-sphingomyelinase antibody and by inhibition of immunoreaction with native enzyme. The corresponding cDNA inserts, released by EcoR1 digestion, are 150 bp and 250 bp respectively. These data demonstrate the application of monoclonal antibodies to cDNA cloning and the successful isolation of cDNA clones for human sphingomyelinase.