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

Abstract

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 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 T-->C or the IVS11-2A-->C 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+1G-->A) and c.1226 G-->A, 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 (IVS1-1G-->C)-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.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 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.

Figure 1

The relative abundance of mRNAs that carry premature termination codon. Top, Analysis of genomic DNA. Restriction-site analysis shows fragments of 122 bp, in the case of a T allele in position 699, and of 92 bp, if the C allele is present. The undigested 304-bp PCR products that contain a control RsaI are not shown. Lanes 1 and 9, Molecular-weight markers. Lane 2, Patient 4 [IVS7+1G→A; c.699C]/[IVS11−2A→C; and c.699T]. Lane 3, Patient 6 [c.1226G→A; c.699T ]/[IVS11−2A→C; c.699C]. Lane 4, Patient 9 [c.19_20insC; c.699C]/[IVS11−2A→C; c.699T]. Lane 5, Patient 10 [IVS1−1G→C; c.699C]/[c.28_29delG; c.699T]. Lane 6, Wild-type control c.699C/C. Lane 7, Wild-type control c.699T/T. Lane 8, Wild-type control c.699C/T. Bottom, RT-PCR/RFLP analysis of total fibroblast RNA. The presence of T and C in position 699 is signified by the 280-bp and 248-bp fragments, respectively. The uncut PCR products of 392 bp that contain a control RsaI are not shown. The lanes contain samples identical to those in top panel. The absence of a fragment signifying the C allele in lane 2 demonstrates that the mRNA that carries the c.828_931ins104 (IVS7+1G→A) is not present in the RNA preparation. Similarly, the absence of the T allele in lane 3 shows the virtual absence of the mRNA molecules carrying the c.1226G→A (W409X).

Figure 2

Western blot analysis of fibroblast extracts (native PAGE). The normally assembled tetramer is present only in control fibroblasts (10 μg of protein per lane) and is not detectable in any of the mutant cell lines (100 μg of protein per lane). Lane numbers correspond to patient numbers in table 2. Lanes 1, 2, 3, 4, 6 7, 9, 10, and 11 show results in patients who were not responsive to pyridoxine. Lane N shows results in a negative sample, mutant cell line 599 devoid of any CBS antigen (Skovby et al. 1984); lanes 13, 17, 19, and 20 show results in patients who were fully or partially responsive to pyridoxine. Lane C1 shows results in control fibroblasts 2047 with the sample frozen and thawed twice; lane C2 shows results in control fibroblasts 2047 analyzed in a separate experiment. The extract for lane C2 was prepared immediately before loading, with addition of β-mercaptoethanol and protease inhibitors.

Figure 3

Western blot analysis of mutants expressed in E. coli (SDS-PAGE). Lanes (50 μg of total protein each): A114V (c.341C→T); A155T (c.463G→A); E176K (c.526G→A); I278T (c.833T→C); del ex 12 (W409_G453del [IVS11−2A→C]) mutants; WT indicates wild-type pHCS3; NC indicates negative control (E. coli transformed with pKK 388.1 plasmid without the human CBS insert).

Figure 4

Oligomeric status and heme content of CBS mutants expressed in E. coli (native PAGE). Left, Immunodetection. The correctly assembled tetramers, marked as “4-mer,” are present in the wild-type lane and, in greatly diminished amounts, in A114V (c.341C→T) and E176K (c.526G→A) mutants. Aggregates of low mobility are present in all extracts as a smear. Lanes (each containing 50 μg of total protein) are as follows: A114V (c.341C→T); A155T (c.463G→A); E176K (c.526G→A); I278T (c.833T→C); W409_G453del (IVS11−2A→C) mutants cloned in pHCS3; WT50%, wild type pHCS3, 25 μg of total protein per lane; WT, wild type pHCS3, 50 μg; NC, negative control, E. coli transformed with pKK 388.1 plasmid without human CBS insert. Right, Heme staining. Non-CBS staining reaction of E. coli heme proteins was observed in all samples, including the negative control (top and bottom bands in all lanes). The protein loading is the same as in the panel at left. Heme bound to CBS is detectable only in the tetramers, both in the wild type and in the A114V (c.341C→T) mutant, but it is absent in the aggregates.