Impaired heme binding and aggregation of mutant cystathionine beta-synthase subunits in homocystinuria

During the past 20 years, cystathionine beta -synthase (CBS) deficiency has been detected in the former Czechoslovakia with a calculated frequency of 1:349,000. The clinical manifestation was typical of homocystinuria, and ab out half of the 21 patients were not responsive to pyridoxine. Twelve disti nct mutations were detected in 30 independent homocystinuric alleles. One h alf of the alleles carried either the c.833 T -->C or the IVS11-2A -->C mut ation; the remaining alleles contained private mutations. The abundance of five mutant mRNAs with premature stop codons was analyzed by PCR-RFLP. Two mRNAs, c.828_931ins104 (IVS7+1G -->A) and c.1226 G -->A, were severely redu ced in the cytoplasm as a result of nonsense-mediated decay. In contrast, t he other three mRNAs-c.19_20insC, c.28_29delG, and c.210_235del26 (IVS1 1G -->C)-were stable. Native western blot analysis of 14 mutant fibroblast lin es showed a paucity of CBS antigen, which was detectable only in aggregates . Five mutations-A114V (c.341C -->T), A155T (c.463G -->A), E176K (c.526G -- >A), I278T (c.833T -->C), and W409_G453del (IVS11-2A -->C)were expressed in Escherichia coli. All five mutant proteins formed substantially more aggre gates than did the wild-type CBS, and no aggregates contained heme. These d ata suggest that abnormal folding, impaired heme binding, and aggregation o f mutant CBS polypeptides may be common pathogenic mechanisms in CBS defici ency.