Interaction of human GTP cyclohydrolase I with its splice variants.
Tetrahydrobiopterin is an essential cofactor for aromatic amino acid hydroxylases, ether lipid oxidase and nitric oxide synthases. Its biosynthesis in mammals is regulated by the activity of the homodecameric enzyme GCH (GTP cyclohydrolase I; EC 3.5.4.16). In previous work, catalytically inactive human GCH splice variants differing from the wild-type enzyme within the last 20 C-terminal amino acids were identified. In the present study, we searched for a possible role of these splice variants. Gel filtration profiles of purified recombinant proteins showed that variant GCHs form high-molecular-mass oligomers similar to the wild-type enzyme. Co-expression of splice variants together with wild-type GCH in mammalian cells revealed that GCH levels were reduced in the presence of splice variants. Commensurate with these findings, the GCH activity obtained for wild-type enzyme was reduced 2.5-fold through co-expression with GCH splice variants. Western blots of native gels suggest that splice variants form decamers despite C-terminal truncation. Therefore one possible explanation for the effect of GCH splice variants could be that inactive variants are incorporated into GCH heterodecamers, decreasing the enzyme stability and activity.
Pandya
Maya J
MJ
Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria.
Golderer
Georg
G
Werner
Ernst R
ER
Werner-Felmayer
Gabriele
G
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D002850:Chromatography, Gel; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D006136:GTP Cyclohydrolase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D007527:Isoenzymes; D011994:Recombinant Proteins; D006136:GTP Cyclohydrolase