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American Journal of Human Genetics logoLink to American Journal of Human Genetics
. 1993 Jan;52(1):144–151.

Cloning of functional alpha propionyl CoA carboxylase and correction of enzyme deficiency in pccA fibroblasts.

J Stankovics 1, F D Ledley 1
PMCID: PMC1682099  PMID: 8434582

Abstract

Propionyl CoA carboxylase (PPC) is a heteromeric enzyme composed of alpha subunits (PCCA) and beta (PCCB) subunits. We describe cDNA clones expressing human PCCA and complementation of the genetic defect in pccA fibroblasts by DNA-mediated gene transfer. Two cDNA clones were constructed. The first corresponds to the previously reported, putatively full-length, open reading frame. The second encodes a chimera composed of the mitochondrial leader sequence of human methylmalonyl CoA mutase and the mature PCCA protein. Both clones reconstitute propionate flux to normal levels in fibroblasts from patients genetically deficient in PCCA (pccA). The maximal level of propionate flux approached, but never exceeded, the levels seen in control plates of normal cells. In contrast, the maximal level of PPC holoenzyme activity reached only 10%-20% that of normal controls, which corresponded roughly to the fraction of cells actually transformed with the recombinant gene. These data suggest that the level of PCCA expression in fibroblasts does not normally limit PCC holoenzyme activity or propionate flux. The fact that a small fraction of cells reconstitutes propionate flux to normal levels suggests that metabolic cooperation between cells is capable of increasing the metabolic capacity of recombinant enzyme in a subpopulation of cells. These factors may have important implications for the rational design of somatic gene therapy for PCCA deficiency.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bailey J. E. Toward a science of metabolic engineering. Science. 1991 Jun 21;252(5013):1668–1675. doi: 10.1126/science.2047876. [DOI] [PubMed] [Google Scholar]
  2. Browner M. F., Taroni F., Sztul E., Rosenberg L. E. Sequence analysis, biogenesis, and mitochondrial import of the alpha-subunit of rat liver propionyl-CoA carboxylase. J Biol Chem. 1989 Jul 25;264(21):12680–12685. [PubMed] [Google Scholar]
  3. Gravel R. A., Lam K. F., Scully K. J., Hsia Y. Genetic complementation of propionyl-CoA carboxylase deficiency in cultured human fibroblasts. Am J Hum Genet. 1977 Jul;29(4):378–388. [PMC free article] [PubMed] [Google Scholar]
  4. Hsia Y. E., Scully K. J., Rosenberg L. E. Inherited propionyl-Coa carboxylase deficiency in "ketotic hyperglycinemia". J Clin Invest. 1971 Jan;50(1):127–130. doi: 10.1172/JCI106466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Jansen R., Ledley F. D. Heterozygous mutations at the mut locus in fibroblasts with mut0 methylmalonic acidemia identified by polymerase-chain-reaction cDNA cloning. Am J Hum Genet. 1990 Nov;47(5):808–814. [PMC free article] [PubMed] [Google Scholar]
  6. Kraus J. P., Williamson C. L., Firgaira F. A., Yang-Feng T. L., Münke M., Francke U., Rosenberg L. E. Cloning and screening with nanogram amounts of immunopurified mRNAs: cDNA cloning and chromosomal mapping of cystathionine beta-synthase and the beta subunit of propionyl-CoA carboxylase. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2047–2051. doi: 10.1073/pnas.83.7.2047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kwok S. C., Ledley F. D., DiLella A. G., Robson K. J., Woo S. L. Nucleotide sequence of a full-length complementary DNA clone and amino acid sequence of human phenylalanine hydroxylase. Biochemistry. 1985 Jan 29;24(3):556–561. doi: 10.1021/bi00324a002. [DOI] [PubMed] [Google Scholar]
  8. Lam Hon Wah A. M., Lam K. F., Tsui F., Robinson B., Saunders M. E., Gravel R. A. Assignment of the alpha and beta chains of human propionyl-CoA carboxylase to genetic complementation groups. Am J Hum Genet. 1983 Sep;35(5):889–899. [PMC free article] [PubMed] [Google Scholar]
  9. Lamhonwah A. M., Barankiewicz T. J., Willard H. F., Mahuran D. J., Quan F., Gravel R. A. Isolation of cDNA clones coding for the alpha and beta chains of human propionyl-CoA carboxylase: chromosomal assignments and DNA polymorphisms associated with PCCA and PCCB genes. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4864–4868. doi: 10.1073/pnas.83.13.4864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lamhonwah A. M., Gravel R. A. Propionicacidemia: absence of alpha-chain mRNA in fibroblasts from patients of the pccA complementation group. Am J Hum Genet. 1987 Dec;41(6):1124–1131. [PMC free article] [PubMed] [Google Scholar]
  11. Ledley F. D., Crane A. M., Lumetta M. Heterogeneous alleles and expression of methylmalonyl CoA mutase in mut methylmalonic acidemia. Am J Hum Genet. 1990 Mar;46(3):539–547. [PMC free article] [PubMed] [Google Scholar]
  12. MacGregor G. R., Caskey C. T. Construction of plasmids that express E. coli beta-galactosidase in mammalian cells. Nucleic Acids Res. 1989 Mar 25;17(6):2365–2365. doi: 10.1093/nar/17.6.2365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ohura T., Kraus J. P., Rosenberg L. E. Unequal synthesis and differential degradation of propionyl CoA carboxylase subunits in cells from normal and propionic acidemia patients. Am J Hum Genet. 1989 Jul;45(1):33–40. [PMC free article] [PubMed] [Google Scholar]
  14. Stephanopoulos G., Vallino J. J. Network rigidity and metabolic engineering in metabolite overproduction. Science. 1991 Jun 21;252(5013):1675–1681. doi: 10.1126/science.1904627. [DOI] [PubMed] [Google Scholar]
  15. Wilkemeyer M. F., Crane A. M., Ledley F. D. Differential diagnosis of mut and cbl methylmalonic aciduria by DNA-mediated gene transfer in primary fibroblasts. J Clin Invest. 1991 Mar;87(3):915–918. doi: 10.1172/JCI115098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wolf B., Hsia Y. E., Rosenberg L. E. Biochemical differences between mutant propionyl-CoA carboxylases from two complementation groups. Am J Hum Genet. 1978 Sep;30(5):455–464. [PMC free article] [PubMed] [Google Scholar]
  17. Wolf B., Paulsen E. P., Hsia Y. E. Asymptomatic propionyl CoA carboxylase deficiency in a 13-year-old girl. J Pediatr. 1979 Oct;95(4):563–565. doi: 10.1016/s0022-3476(79)80768-4. [DOI] [PubMed] [Google Scholar]
  18. Wolf B., Willard H. F., Rosenberg L. E. Kinetic analysis of genetic complementation in heterokaryons of propionyl CoA carboxylase-deficient human fibroblasts. Am J Hum Genet. 1980 Jan;32(1):16–25. [PMC free article] [PubMed] [Google Scholar]

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