Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts

Agnes Zay, Francis Y M Choy, Chelsea Patrick, Graham Sinclair

Research output: Contribution to journalArticlepeer-review

Abstract

The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to interact with the L-protein, which is also part of the l-ketoglutarate dehydrogenase complex present in fibroblasts.

Original languageEnglish
Pages (from-to)299-307
Number of pages9
JournalBiochemistry and cell biology = Biochimie et biologie cellulaire
Volume89
Issue number3
DOIs
Publication statusPublished - 30 Jun 2011

Keywords

  • Amino Acid Oxidoreductases/biosynthesis
  • Amino Acid Sequence
  • Apoproteins/biosynthesis
  • Bacterial Proteins/biosynthesis
  • Carrier Proteins/biosynthesis
  • Chromatography, Affinity
  • Cloning, Molecular
  • DNA, Complementary/analysis
  • Dihydrolipoamide Dehydrogenase/biosynthesis
  • Escherichia coli/genetics
  • Fibroblasts/cytology
  • Histidine/metabolism
  • Humans
  • Hyperglycinemia, Nonketotic/enzymology
  • Molecular Sequence Data
  • Multienzyme Complexes/biosynthesis
  • Oligopeptides/metabolism
  • Peptide Synthases/biosynthesis
  • Pichia/genetics
  • Primary Cell Culture
  • Recombinant Proteins/biosynthesis
  • Sequence Alignment
  • Sequence Analysis
  • Skin/cytology
  • Transferases/biosynthesis

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