title: Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.
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.
authors.affiliation: Centre for Biomedical Research, Department of Biology, University of Victoria, Canada.
authors.forename: Agnes
authors.initials: A
authors.lastname: Zay
authors.forename: Francis Y M
authors.initials: FY
authors.lastname: Choy
authors.forename: Chelsea
authors.initials: C
authors.lastname: Patrick
authors.forename: Graham
authors.initials: G
authors.lastname: Sinclair
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001059:Apoproteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D008058:Dihydrolipoamide Dehydrogenase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D006639:Histidine / Q000378:metabolism; D006801:Humans; D020158:Hyperglycinemia, Nonketotic / Q000201:enzymology / Q000473:pathology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D009842:Oligopeptides / Q000378:metabolism; D010453:Peptide Synthases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D061251:Primary Cell Culture; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D016415:Sequence Alignment; D017421:Sequence Analysis; D012867:Skin / Q000166:cytology / Q000201:enzymology; D014166:Transferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; C471213:His-His-His-His-His-His; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011994:Recombinant Proteins; C064525:glycine cleavage system; D006639:Histidine; D000594:Amino Acid Oxidoreductases; D008058:Dihydrolipoamide Dehydrogenase; D014166:Transferases; D010453:Peptide Synthases; C072322:lipoate-protein ligase