Impaired heme binding and aggregation of mutant cystathionine beta-synthase subunits in homocystinuria
Abstract
During the past 20 years, cystathionine β-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 about half of the 21 patients were not responsive to pyridoxine.
Twelve distinct mutations were detected in 30 independent homocystinuric alleles. One half of the alleles carried either the c.833 TRIGHTWARDS ARROWC or the IVS11-2ARIGHTWARDS ARROWC mutation; 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+1GRIGHTWARDS ARROWA) and c.1226 GRIGHTWARDS ARROWA, were severely reduced in the cytoplasm as a result of nonsense-mediated decay.
In contrast, the other three mRNAs-c.19_20insC, c.28_29delG, and c.210_235del26 (IVS1-1GRIGHTWARDS ARROWC)-were stable. Native western blot analysis of 14 mutant fibroblast lines showed a paucity of CBS antigen, which was detectable only in aggregates.
Five mutations-A114V (c.341CRIGHTWARDS ARROWT), A155T (c.463GRIGHTWARDS ARROWA), E176K (c.526GRIGHTWARDS ARROWA), I278T (c.833TRIGHTWARDS ARROWC), and W409_G453del (IVS11-2ARIGHTWARDS ARROWC)-were expressed in Escherichia coli. All five mutant proteins formed substantially more aggregates than did the wild-type CBS, and no aggregates contained heme.
These data suggest that abnormal folding, impaired heme binding, and aggregation of mutant CBS polypeptides may be common pathogenic mechanisms in CBS deficiency.