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

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Author(s)

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

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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.

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Original languageEnglish
Pages (from-to)299-307
Number of pages9
JournalInternational Journal of Biochemistry and Cell Biology
Volume89
Issue number3
DOIs
Publication statusPublished - Jun 2011

    Research areas

  • Amino Acid Oxidoreductases, Amino Acid Sequence, Apoproteins, Bacterial Proteins, Carrier Proteins, Chromatography, Affinity, Cloning, Molecular, DNA, Complementary, Dihydrolipoamide Dehydrogenase, Escherichia coli, Fibroblasts, Histidine, Humans, Hyperglycinemia, Nonketotic, Molecular Sequence Data, Multienzyme Complexes, Oligopeptides, Peptide Synthases, Pichia, Primary Cell Culture, Recombinant Proteins, Sequence Alignment, Sequence Analysis, Skin, Transferases, Journal Article, Research Support, Non-U.S. Gov't

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