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Creating of GTP Cyclohydrolase‐1 Knock in Mouse
Author(s) -
Chen Wei,
Li Li,
Blinder Yelena,
Zhang Helen Heju,
Goldstein Anna,
Harrison David
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.lb642
Subject(s) - gtp cyclohydrolase i , tetrahydrobiopterin , phosphorylation , alanine , tyrosine hydroxylase , serine , chemistry , aspartic acid , nitric oxide synthase , biochemistry , biology , microbiology and biotechnology , enzyme , amino acid
Tetrahydrobiopterin (BH 4 ) is an essential cofactor for the nitric oxide synthase enzymes. We have shown that laminar shear stress dramatically increases BH 4 levels in human aortic endothelial cells. Oscillatory shear only modestly affected BH4 levels in the cells. We also demonstrated that laminar, but not oscillatory shear stress, stimulates phosphorylation of GTP cyclohydrolase‐1 (GTPCH‐1) the rate limiting enzyme for the BH 4 biosynthesis, at serine 81. To study this further, we have attempted to produce two mice in which serine 72 (the equivalent of S81 in humans) was replaced by aspartic acid or alanine, to respectively mimic or prevent phosphorylation, using knock‐in technology. The alanine substitution (GCH S72A ) proved to be lethal in that no viable agouti pups were obtained from chimeric founders and analysis of spermatic plugs showed no germ‐line transmission of the knock‐in transgene. In contrast, we have successfully obtained the aspartic acid substitution (GCH S72D ) heterozygotic mice, and found that they have increased aortic BH 4 levels than WT mice when aortas are maintained in no‐flow conditions for 24 hours (224±10 vs. 134 ± 2 pmols/mg protein). These experiments show that GTPCH‐1 phosphorylation is essential for survival and plays a critical role in modulating GTPCH‐1 function in vivo. Supported by NIH P01 HL058000.