Low tetrahydrobiopterin biosynthetic capacity of human monocytes is caused by exon skipping in 6-pyruvoyl tetrahydropterin synthase.
Biosynthesis of (6 R )-5,6,7,8-tetrahydro-L-biopterin (H(4)-biopterin), an essential cofactor for aromatic amino acid hydroxylases and NO synthases, is effectively induced by cytokines in most of the cell types. However, human monocytes/macrophages form only a little H(4)-biopterin, but release neopterin/7,8-dihydroneopterin instead. Whereas 6-pyruvoyl tetrahydropterin synthase (PTPS) activity, the second enzyme of H(4)-biopterin biosynthesis, is hardly detectable in these cells, PTPS mRNA levels were comparable with those of cell types containing intact PTPS activity. By screening a THP-1 cDNA library, we identified clones encoding the entire open reading frame (642 bp) as well as clones lacking the 23 bp exon 3, which results in a premature stop codon. Quantification of the two mRNA species in different cell types (blood-derived cells, fibroblasts and endothelial cells) and cell lines showed that the amount of exon-3-containing mRNA is correlated closely to PTPS activity. The ratio of exon-3-containing to exon-3-lacking PTPS mRNA is not affected by differential mRNA stability or nonsense-mediated mRNA decay. THP-1 cells transduced with wild-type PTPS cDNA produced H(4)-biopterin levels and expressed PTPS activities and protein amounts comparable with those of fibroblasts. We therefore conclude that exon 3 skipping in transcription rather than post-transcriptional mechanisms is a major cause of the low PTPS protein expression observed in human macrophages and related cell types.
Leitner
Karin L
KL
Institute of Medical Chemistry and Biochemistry, University of Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria.
Meyer
Martina
M
Leimbacher
Walter
W
Peterbauer
Anja
A
Hofer
Susanne
S
Heufler
Christine
C
Müller
Angelika
A
Heller
Regine
R
Werner
Ernst R
ER
Thöny
Beat
B
Werner-Felmayer
Gabriele
G
D001483:Base Sequence; D001708:Biopterins / Q000031:analogs & derivatives* / Q000096:biosynthesis*; D015342:DNA Probes; D005091:Exons; D006801:Humans; D009000:Monocytes / Q000378:metabolism*; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D012333:RNA, Messenger; D001708:Biopterins; D019761:Phosphorus-Oxygen Lyases; C043497:6-pyruvoyltetrahydropterin synthase; C003402:sapropterin