Originally published In Press as doi:10.1074/jbc.M910307199 on April 18, 2000
J. Biol. Chem., Vol. 275, Issue 25, 18926-18932, June 23, 2000
Development of the Pteridine Pathway in the Zebrafish,
Danio rerio*
Irmgard
Ziegler
§,
Thomas
McDonaldo¶,
Christian
Hesslinger,
Isabelle
Pelletier, and
Peter
Boyle¶
From the GSF Research Center, Institut für
Klinische Molekularbiologie und Tumorgenetik, 81377 München,
Germany and the ¶ University Chemical Laboratory, Trinity College,
Dublin 2, Ireland
Received for publication, December 22, 1999, and in revised form, April 18, 2000
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ABSTRACT |
In the zebrafish, the peripheral neurons and the
pigment cells are derived from the neural crest and share the pteridine
pathway, which leads either to the cofactor tetrahydrobiopterin or to
xanthophore pigments. The components of the pteridine pattern were
identified as tetrahydrobiopterin, sepiapterin, 7-oxobiopterin,
isoxanthopterin, and 2,4,7-trioxopteridine. The expression of GTP
cyclohydrolase I activity during the first 24-h postfertilization,
followed by 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase and
sepiapterin reductase, suggest an early supply of tetrahydrobiopterin
for neurotransmitter synthesis in the neurons and for tyrosine supply
in the melanophores. At 48-h postfertilization, sepiapterin formation
branches off the de novo pathway of tetrahydrobiopterin
synthesis. Sepiapterin, via 7,8-dihydrobiopterin and biopterin, serves
as a precursor for the formation of 7-oxobiopterin, which may be
further catabolized to isoxanthopterin and 2,4,7-trioxopteridine.
Neither 7,8-dihydrobiopterin nor biopterin is a substrate for xanthine
oxidoreductase. In contrast, both of these compounds are oxidized at
C-7 by a xanthine oxidase variant form, which is inactivated by KCN,
but is insensitive to allopurinol. The oxidase and the dehydrogenase
form of xanthine oxidoreductase as well as the xanthine oxidase variant
have specific developmental patterns. It follows that GTP
cyclohydrolase I, the formation of sepiapterin, and the xanthine
oxidoreductase family control the pteridine pathway in the zebrafish.
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INTRODUCTION |
The zebrafish has a number of features that have led to its
widespread use as a model system for genetic analysis and for the study
of vertebrate development (1). Several studies have been reported on
the staging, cell fate determination, lineage determination, and
patterning for the development of the peripheral neurons and the
pigment cells in this animal. Both of these types of cells are derived
from the neural crest (2-4). Large scale screens for embryonic and
early larval zebrafish mutations have enabled the analysis of different
aspects of neuronal and of pigment development and the identification
of the genes specifically required for them (5-8). In the zebrafish,
some of the pigments are believed to be pteridine-related, and
differences in their biosynthesis were suggested to occur in several
mutants (6).
The de novo biosynthesis of pteridines begins with GTP and
results in the formation of
H4biopterin.1 The
first committed step of this pathway is catalyzed by GTP cyclohydrolase
I (EC 3.5.4.16) and yields H2neopterin triphosphate (9).
6-Pyruvoyl-H4pterin synthase (EC 4.6.1.10) eliminates the
phosphate groups from this and catalyzes an intramolecular reaction,
yielding 6-pyruvoyl-H4pterin (10). Both keto groups of this
unstable intermediate are reduced by sepiapterin reductase (EC
1.1.1.153) via formation of
6-(1'-hydroxy-2'-oxopropyl)-H4pterin, isomerization to
6-lactoyl-H4pterin, and final reduction of this 1'-oxo
compound to H4biopterin; the necessary hydride equivalents come from NADPH (11; pathway reviewed in Ref. 12).
H4biopterin serves as the electron donor for the
hydroxylation of the aromatic amino acids phenylalanine, tyrosine, and
tryptophan and thus is the essential cofactor for the synthesis of the
neurotransmitters, catecholamine and serotonin (reviewed in Ref. 13).
H4biopterin is therefore synthesized in the neurons, and
its subsaturating levels, in some cases, control neurotransmitter
synthesis (14, 15). H4biopterin is also an essential
component of nitric oxide synthase (reviewed in Refs. 16, 17). The
diversity of nitric oxide function as a diffusible intercellular
messenger in fishes is, however, not yet explained in such detail as in
mammals (18) and not yet analyzed in the zebrafish.
The pigment cells of cyprinid fishes, including the zebrafish, comprise
three types. First there are the iridophores, which are packed with
purine crystals, identified as guanine and hypoxanthine (19). Second,
are the melanophores. They synthesize melanin from dopa, which in turn
is produced from tyrosine. The supply with this rate-limiting substrate
depends on the hydroxylation of phenylalanine and thus on the
availability of the essential cofactor H4biopterin. Several
years ago it was suggested that, in differentiating melanophores of
fish, H4biopterin is involved as the essential cofactor for
tyrosine formation (20), and more recently it has been demonstrated
that, in the human epidermis, H4biopterin does indeed
regulate the supply of tyrosine required for melanin synthesis (21).
The third type of pigment cells found in the zebrafish are the
xanthophores, which contain pteridines serving directly as pigments.
Recently, it has been suggested that the yellow color of these cells is
due to sepiapterin (6), a compound that was first isolated from the
sepia mutant of Drosophila and was shown to be a
7,8-dihydropterin carrying a lactoyl group at position C-6 (reviewed in
Ref. 22). Accumulation of pteridines in the skin of various cyprinid
species has also been described (19, 23, 24), and the main component of
these, originally named ichthyopterin, was shown to be 7-oxobiopterin
(25, 26).
During the development of vertebrates, multipotent neural crest cells
give rise to peripheral neuronal cells as well as to pigment cells (27;
reviewed in Ref. 28). Although these two types of cells vary widely in
their phenotype and cellular function, it nevertheless seems clear that
both of them must express the enzymes that catalyze the de
novo synthesis of pteridines from GTP. So far, the numerous
genetic and developmental studies that address neuronal defects,
changes in pigment cell pattern, or both have focussed on the
description of the phenotype. The aim of the present work, therefore,
was to analyze the pattern of how pteridines occur in the zebrafish, to
elucidate the biosynthetic pathway leading to them, and thus to
identify committing steps in the biosynthesis of neurotransmitters, of
melanin, and of xanthophore pigments during the development of the organism.
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EXPERIMENTAL PROCEDURES |
Animals--
Wild type zebrafish of the AB background were
raised in the zebrafish facility of the Institute of Mammalian
Genetics, Neuherberg, and embryos were obtained from natural spawning.
They were grouped according to number postfertilization hours at
28.5 °C. The samples were collected in batches of 10-20 animals,
immediately frozen, and kept at 
85 °C until analysis.
Reagents--
Chemicals used for the determination of enzymatic
activities and for protein and pteridine determination were obtained as described previously (29, 30). Pterins were from Schircks (Jona,
Switzerland). 2,4,7-Trioxopteridine was prepared by a modification of a
previously described method (31) (see below). A sample of the same
compound, as well as 7-oxo-6-carboxypterin and 7-oxobiopterin isolated
from carp skin were donated by Dr. M. Nakagoshi (Kitasato). Construction of the plasmids used for expression of murine recombinant GTP cyclohydrolase I and for murine sepiapterin reductase followed the
procedures described earlier (11, 32). Recombinant
6-pyruvoyl-H4pterin synthase was a gift from Dr. A. Bacher
(München). Milk xanthine oxidoreductase was obtained from Sigma,
alkaline phosphatase from Roche Molecular Biochemicals, MicroSpin G25
columns from Amersham Pharmacia Biotech, Sep-Pak cartridges from Waters
Corporation, and cellulose thin layer chromatography plates from Merck.
Synthesis of 2,4,7-Trioxopteridine--
A suspension of
6-amino-5-nitroso-2,4(1H,3H)-pyrimidinedione (1.0 g; 6.4 mmol) in deaerated water (100 ml) containing sodium hydroxide
(0.26 g; 6.4 mmol) was hydrogenated over Raney nickel catalyst (1 ml)
at room temperature and pressure for 15 h. The reaction mixture
was filtered under nitrogen directly onto methyl glyoxalate hemiacetal
(1.57 g; 128 mmol) prepared from dimethyl L-tartrate (33),
and the resulting mixture was stirred in the dark at room temperature
for 10 h. Evaporation followed by recrystallization from water
using activated charcoal, gave 2,4,7-trioxopteridine as a light yellow
solid (1.05 g; 91%).
Identification of the Pteridine Pattern--
The samples
(batches of 50 animals, 96- to 120-h postfertilization) were extracted
by homogenization with a Wheaton Teflon homogenizer in 100 ml of Tris
buffer (50 mM, pH 6.9) and centrifuged at 13,000 × g for 10 min. The extract was oxidized by acidic iodine, deproteinized by trichloroacetic acid, and purified by cation-exchange chromatography, as described previously. The lyophilized material was
prefractionated by reverse-phase high pressure liquid chromatography (HPLC), using 3% methanol in water as the mobile phase, and a fluorescence detector, as described before (29, 30). The fractions were
concentrated in vacuo.
Three main fractions were obtained and identified using a combination
of physical and chemical methods. HPLC analysis using a Shimadzu model
RF 535 fluorescence detector was carried out on reverse-phase columns
using five different solvent systems, the compositions of which are
detailed below (see the legend of Table I). Mass spectrometric analysis
was carried out using a Micromass LCT instrument with electrospray
ionization and time-of-flight analysis. The source temperature was
30 °C, and the cone voltage was 30 V. Fractions I and II were
dissolved in 30 µl of 0.1 M ammonium hydroxide, and 30 µl of 15% methanol in water, respectively, and injected onto a C-18
reverse-phase HPLC column leading into the mass spectrometer. For
further identification of biopterin and of 7-oxobiopterin in Fractions
II and III, respectively, the fractions were oxidized by alkaline
permanganate, and the resulting pterin-6-carboxylic acids were
separated by HPLC and fluorometrically detected as described previously
(34).
Sepiapterin was identified by its enzymatic reduction to
H2biopterin. A centrifuged homogenate (Tris buffer, 50 mM, pH 6.9) of 40 animals (120-h postfertilization) was
concentrated by loading onto Sep-Pak cartridges as described before
(35), desorbed with methanol, concentrated in a vacuum, spotted on
cellulose thin layer plates, and developed with butanol/acetic
acid/water (4:1:5, v/v). The yellow fluorescent spots
(RF = 0.17) were extracted with Tris buffer (50 mM, pH 7.9), centrifuged, and incubated for 40 min with
NADPH (200 µM) and recombinant sepiapterin reductase for
40 min. After acidic iodine oxidation of the reaction product H2biopterin, the sample was analyzed by HPLC in solvent
systems I and II (see legend of Table I).
Quantitative Determination of the Pteridine Pattern during
Development--
Batches of 10-20 animals were extracted, oxidized,
deproteinized, and prepurified by cation-exchange chromatography as
described above. Because isoxanthopterin is only partially retained and also 7-oxobiopterin and 2,4,7-trioxopteridine are only partially retained, the lyophilized water washes were separately monitored and
their amounts combined. For quantitative determination by HPLC
analysis, the solvent systems I and II (see legend of Table I) were
used. Due to the different fluorescence characteristics of biopterin
and, conversely, of the pteridines substituted at the 7-position, the
samples were either monitored at two different wavelength settings
(350-nm excitation/450-nm emission and 340-nm excitation/420-nm
emission) or the relative emission characteristics were determined for
each series, and the samples were then monitored at only one wavelength
setting (350-nm excitation/450-nm emission) and the relevant factors
were applied for calculations. For the determination of sepiapterin,
the samples were homogenized in 1 ml of Tris buffer (50 mM,
pH 6.8), centrifuged, and then adsorbed onto reverse-phase C-18 Sep-Pak
cartridges (35). After washing, desorption with 3 ml of methanol, and
concentration by vacuum, sepiapterin was determined by its absorption
at 440 nm. The standard curve was linear in the range of 0-15
nmol/ml.
Enzyme Assays--
For all enzyme assays, batches of 10-20
animals were homogenized in Tris buffer (50 mM, pH 8.0) as
described above. The centrifuged homogenates (13,000 × g, 15 min) were passed through MicroSpin G25 columns. For
determination of GTP cyclohydrolase I activity, the reaction product
H2neopterin triphosphate was oxidized to neopterin
triphosphate by acidic iodine solution. After reduction of excess
iodine by ascorbic acid and dephosphorylation with alkaline phosphatase
(0.8 unit/200 µl), neopterin was determined by HPLC using solvent
systems I and II (see legend of Table I). For determination of
6-pyruvoyl-H4pterin synthase, H2neopterin
triphosphate was first generated by incubation of GTP with recombinant
murine GTP cyclohydrolase I. To determine the activity of the synthase,
the extract containing it (plus Mg2+, to achieve a final
concentration of 10 mM) was added, and the unstable
intermediate 6-pyruvoyl-H4pterin was converted to
H4biopterin by the addition of recombinant murine
sepiapterin reductase and NADPH (200 µM). After acidic
iodine oxidation, biopterin was separated by HPLC (36). The activity of
sepiapterin reductase was determined by reduction of sepiapterin to
dihydrobiopterin with NADPH, acidic iodine oxidation, and HPLC
separation of the resultant biopterin. The detailed conditions for all
enzyme assays were as described previously (30). For preparation of
6-pyruvoyl-H4pterin as a potential substrate for the
xanthine oxidoreductase variant, H2neopterin triphosphate
was first generated by incubation of GTP with recombinant murine GTP
cyclohydrolase I, and EDTA was quenched by addition of Mg2+
as described above; the reaction mixture was incubated with recombinant 6-pyruvoyl-H4pterin synthase for 20 min and then
immediately used. Due to the endogenous sepiapterin reductase activity
of zebrafish homogenates and the additional function of this enzyme as
an isomerase (11, 37), the intermediates 6-lactoyl-H4pterin
and 6-(1'-hydroxy-2'-oxopropyl)-H4pterin are transiently
formed also, so that these could also be considered as possible
precursors for 7-oxobiopterin formation. The assay for xanthine
oxidoreductase activity used the conversion of pterin to
isoxanthopterin, which was then separated by HPLC (solvent system IV;
see legend of Table I) and fluorometrically detected (340-nm
excitation/410-nm emission) (38, 39). The instrument was standardized
by measurements with the product (isoxanthopterin) and the substrate.
For the determination of the oxidase activity, the reaction mixture
(100 µl) contained the extract (30 µl), pterin (50 µM), and air-saturated Tris buffer (50 mM, pH
8.0). For the determination of combined dehydrogenase plus oxidase
activity, NAD (400 µM) was included. The samples were
incubated for 40 min at 37 °C, and the reaction was stopped by
addition of 2 N trichloroacetic acid (20 µl). Protein was
estimated by the Coomassie dye binding reagent (Bio-Rad protein assay
reagent) according to the manufacturer's instructions.
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RESULTS |
Identification of the Pteridine Pattern--
The extracts from 4- to 5-day-old zebrafish, which had been purified by cation-exchange
chromatography and then prefractionated by reverse-phase HPLC with 3%
methanol, yielded three major fractions, labeled I through III. Each of
these fractions was investigated separately, and the pteridines in them
were identified as described below.
Fraction I eluted at 9.8 min. On further reverse-phase HPLC analysis
with a fluorescence detector, it showed one peak that coeluted with
authentic isoxanthopterin in all of five different solvent systems
(Table I). Its maximum fluorescence
emission at 410 nm, and its UV spectra in both 0.1 M HCl
and 0.1 M NaOH were identical with those of authentic
isoxanthopterin. The two strongest peaks in its electrospray mass
spectrum occurred at m/z 180 (M + H+)
and 218 (M + K+) and were identical with the two strongest
peaks of authentic isoxanthopterin measured under the same
conditions.
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Table I
HPLC analysis of the zebrafish pteridine pattern
Extracts of zebrafish (96-120 h postfertilization) were prepurified as
described under "Experimental Procedures" and prefractionated by
reverse-phase HPLC with 3% methanol. The fractions (I-III) were then
analyzed in five solvent systems. The compositions of the solvent
systems were: I, 2-propanol 0.4%, triethylamine 1%, adjusted to pH
7.0 with H3PO4; II, 3% methanol, 1% acetonitrile,
0.02% phosphoric acid; III, 3% methanol, 1% acetonitrile; IV,
K2HPO4 3.5 mM, adjusted to pH 7.2 with
H3PO4; V, 5% methanol, NaH2PO4 100 mM, adjusted to pH 3 with H3PO4. The flow
rate was 1 ml/min; the numbers refer to the retention time (minutes).
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Fraction II eluted at 11.0 min. On further reverse-phase HPLC analysis
with a fluorescence detector, it showed two peaks in four out of five
solvent systems tested (Table I), and these were identified as
2,4,7-trioxopteridine and biopterin. First, the two peaks had the same
retention times as authentic samples of 2,4,7-trioxopteridine and
biopterin. Second, the excitation/emission wavelengths of the first
fluorescence peak were shifted from 350/450 nm to 340/420 nm, reaching
3-fold enhancement. This is typical of 7-oxopteridines (40-42). Third,
the mass spectrum of the first fluorescence peak showed a strong ion at
m/z 203 (M + Na+). Authentic
2,4,7-trioxopteridine, measured under the same conditions, also shows a
strong ion at m/z 203. Fourth, using liquid
chromatography-mass spectrometry, the compound in the second
fluorescence peak was found to have both UV and mass spectra identical
with those of authentic biopterin. The mass spectrum showed
m/z peaks at 238 (M + H+), 260 (M + Na+), and 276 (M + K+). Fifth, alkaline
permanganate oxidation of the second peak yielded pterin-6-carboxylic
acid (Fig. 1A). Alkaline
iodine oxidation of zebrafish extracts, which decomposes
H4biopterin (43), left no biopterin. This demonstrated that
the total biopterin fraction exists as H4biopterin.
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Fig. 1.
Reverse-phase HPLC of the pterin product
formed after alkaline permanganate oxidation of Fractions II and III
and after enzymatic reduction of the yellow pigment by recombinant
sepiapterin reductase. For preparation of Fractions II and III,
the extracts of 4- to 5-day-old zebrafish were subjected to acidic
iodine oxidation, prepurified by Dowex H+ exchange
chromatography, and prefractionated by reverse-phase HPLC with 3%
methanol as eluent. For preparation of the yellow pigment, the extract
was concentrated by Sep-Pak cartridges and then separated by cellulose
thin layer chromatography. A, permanganate oxidation product
of Fraction II. The compound eluting at 12.0 min with solvent V (see
legend of Table I) is identified as 6-carboxylic acid. A1,
elution profile of the sample; A2, elution profile of
synthetic 6-carboxylic acid. Both show a 140% relative fluorescence at
350-nm excitation/450-nm emission as compared with 340-nm
excitation/420-nm emission. B, permanganate oxidation
product of Fraction III. The compound eluting at 13.5 min with solvent
V (see legend of Table I) is identified with 7-oxo-6-carboxylic acid.
B1, elution profile of the sample; B2, elution
profile of synthetic 7-oxo-6-carboxylic acid. Both show a 150%
relative fluorescence at 340-nm excitation/420-nm emission as compared
with 350-nm excitation/450-nm emission. C, enzymatic
reduction of the yellow pigment. It was reduced to
H2biopterin by recombinant sepiapterin reductase and NADPH
and then oxidized by acidic iodine solution. The compound eluting at
7.4 min with solvent II (see legend of Table I) is identified as
biopterin by cochromatography with the synthetic compound.
C1, elution profile of the sample at
t0; C2, elution profile of the sample
after 30 min of incubation.
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Fraction III eluted at 13.9 min. It showed a 1.5-fold increase in
fluorescence intensity at 420 nm as compared with 450 nm, indicating a
7-oxopteridine, and HPLC analysis in solvent systems I-IV (Table I)
suggested its identity with 7-oxobiopterin. This was confirmed by
alkaline permanganate oxidation of Fraction III to
6-carboxy-7-oxopterin (Fig. 1B).
The yellow pterin, isolated from the Sep-Pak cartridges, had an
absorption maximum at 340 nm in 0.1 M NaOH and at 410 nm in 0.1 M HCl, as is characteristic for sepiapterin (44).
Reduction of it with sepiapterin reductase plus NADPH yielded
H2biopterin, which was oxidized by acidic iodine to
biopterin, identified by HPLC analysis with solvent systems I and II
(Fig. 1C and Table I)
Development of the Pteridine Pattern--
The individual
pteridines, which constitute the final pattern of pigments in the
xanthophores, were found to have different developmental profiles (Fig.
2). Both H4biopterin and
sepiapterin were the first ones to form, appearing within 24-h
postfertilization. H4biopterin rapidly increased to a
maximum after 72 h, and finally stayed constant around 7 pmol per
animal. In contrast, sepiapterin continued to increase during the
following period up to a 10-fold level. Formation of the three
7-oxopteridines (7-oxobiopterin, isoxanthopterin, and
2,4,7-trioxopteridine) began after an initial time lag of 24 h,
but their concentrations then increased rapidly. This indicated that
the de novo biosynthetic pathway must first provide the
substrate for the subsequent formation of the 7-oxo derivatives.
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Fig. 2.
Pteridine pattern during the development of
the zebrafish.  , biopterin (left side scale);  ,
sepiapterin;  , total of 7-oxobiopterin, 2,4,7-trioxopteridine, and
isoxanthopterin (right side scale). Extracts were subjected
to acidic iodine oxidation, prepurified, and analyzed by reverse-phase
HPLC. Sepiapterin was determined in Tris buffer extracts (pH 6.9) after
concentration with Sep-Pak cartridges by its absorption at 440 nm.
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Development of H4biopterin Synthesizing
Enzymes--
The three enzymes that catalyze the biosynthesis of
H4biopterin from GTP jointly start to develop during the
first 24-h postfertilization (Fig.
3A). This agrees with the
early increase in H4biopterin levels (Fig. 2). However,
their activities diverge after 72 h: GTP cyclohydrolase I activity
reaches a maximum after 72 h and then declines, whereas both
6-pyruvoyl-H4pterin synthase and sepiapterin reductase
activities continue to increase after 72 h.
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Fig. 3.
Activities of the enzymes involved in
de novo H4biopterin synthesis of xanthine
oxidase and the xanthine oxidase variant during the development of the
zebrafish. A, enzymes of the de novo
H4biopterin synthesis. , GTP cyclohydrolase I (product:
H2neopterin triphosphate); ,
6-pyruvoyl-H4pterin synthase (product:
H4biopterin); , sepiapterin reductase (product:
H2biopterin). Extracts were assayed as described under
"Experimental Procedures." The products were subjected to acidic
iodine oxidation and analyzed by reverse-phase HPLC. B, ,
xanthine oxidase, product: isoxanthopterin; , xanthine oxidase + dehydrogenase, product: isoxanthopterin; , xanthine oxidase variant,
product: 7-oxobiopterin. Extracts were assayed for xanthine
oxidoreductase with pterin as substrate and O2 (oxidase
activity) or NAD+ as electron acceptor (oxidase + dehydrogenase activity) and for the xanthine oxidase variant with
biopterin as substrate and O2 as electron acceptor.
Isoxanthopterin and 7-oxobiopterin were analyzed by reverse-phase
HPLC.
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Pterin Metabolism by Xanthine Oxidoreductase and a Xanthine
Oxidoreductase Variant--
Xanthine oxidoreductase efficiently
converts pterin into isoxanthopterin (38, 45). This enzyme was
therefore a likely candidate for the generation of the 7-oxopterins
from intermediates that are synthesized along the route of the de
novo pathway in the zebrafish. The enzyme exists as two
interconvertible forms, xanthine dehydrogenase and xanthine
oxidoreductase, which use either NAD+ (the dehydrogenase)
or molecular oxygen (the oxidase) as the electron acceptor. In the same
way as purified milk xanthine oxidoreductase, extracts of zebrafish
(72-h postfertilization) were able to catalyze the oxygenation of
pterin to isoxanthopterin. The yield was 83.9 ± 13.1 pmol/mg/min
(O2 as electron acceptor) and 124 ± 20.8 pmol/mg/min (NAD+ as electron acceptor).
The development of the pteridine pattern (Fig. 2) suggests that at
approximately 48-h postfertilization there is a switch from production
of H4biopterin to production of the 7-oxopteridines. In
search of the actual precursor from which 7-oxobiopterin is formed
from an intermediate of the de novo pathway, zebrafish extracts were incubated with H4biopterin and with
6-pyruvoyl-H4pterin. The yield of 7-oxobiopterin from these
substrates, using either oxygen or NAD+ as electron
acceptor, was <0.5%. In the 6-pyruvoyl-H4pterin
experiment, only H4biopterin (30 ± 4.5 pmol/mg/min)
but no 7-oxobiopterin was formed, when NADPH (200 µM) was
included. This was to be expected, because it is known that endogenous
sepiapterin reductase activity (Fig. 3A), coupled to the
action of the enzyme as an isomerase (37), can convert
6-pyruvoyl-H4pterin into H4biopterin, through the intermediates 6-lactoyl-H4pterin and
6-(1'-hydroxy-2'-oxopropyl)-H4pterin. This shows that the
latter two compounds as well as 6-pyruvoyl-H4pterin and
H4biopterin do not serve as substrates for 7-oxobiopterin formation.
Although H2biopterin is not believed to be an intermediate
in the de novo route to H4biopterin, it is
nevertheless formed from sepiapterin. The reaction is also catalyzed by
sepiapterin reductase and requires NADPH (46). The resultant
H2biopterin can be oxidized to biopterin by dihydropterin
dehydrogenase (47) so that both H2biopterin and biopterin
become candidates for the biosynthetic precursor of 7-oxobiopterin.
Although it was found, as expected, that sepiapterin (40 µM), in the absence of NADPH did not serve as a direct
substrate, inclusion of NADPH (200 µM) in the experiment
resulted in the formation of 7-oxobiopterin. Furthermore, it was found
that the known inhibitor of sepiapterin reductase,
N-acetylserotonin (48) not only blocked the formation of
H2biopterin by endogenous sepiapterin reductase, but also
inhibited the formation of 7-oxobiopterin. In both of these reactions,
half-maximal inhibition occurred at 0.3 µM (data not
shown). These results show that formation of 7-oxobiopterin depends
upon the reduction of sepiapterin and suggest that either
H2biopterin or biopterin serve as the direct precursors. In
fact, it was found that with H2biopterin as a substrate,
zebrafish extracts (72-h postfertilization) yielded 8.8 ± 1.5 pmol of 7-oxobiopterin/mg/min. With biopterin, the yield of
7-oxobiopterin was 52.6 ± 5.5 pmol/mg/min (Fig.
4); inclusion of NAD+ had no
effect.
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Fig. 4.
Reverse-phase HPLC of the pterin product
after incubation of zebrafish extracts with biopterin. The
compound eluting at 11.4 min in solvent I (see legend of Table I), is
identified as 7-oxobiopterin. A, elution profile of the
sample at to; B, elution profile of
the sample after 40 min of incubation. The substrate fraction
(biopterin, elution at 8.3 min) shows a 150% relative fluorescence at
350-nm excitation/450-nm emission as compared with 340-nm
excitation/420-nm emission, whereas the product eluting at 11.4 min
(7-oxobiopterin) shows a 150% relative fluorescence at 340-nm
excitation/420-nm emission as compared with 350-nm excitation/450-nm
emission.
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It was also found, however, that purified xanthine oxidoreductase was
unable to oxidize either H2biopterin or biopterin to 7-oxobiopterin, neither with O2 nor with NADPH+
as electron acceptor (data not shown). Allopurinol is a potent inhibitor of xanthine oxidoreductase (49). Although this enzyme is
almost completely inhibited at concentrations of 1.25 mM,
the formation of 7-oxobiopterin from H2biopterin or
biopterin by zebrafish extract was found to be unaffected by
allopurinol at concentrations up to 1 mM (Fig.
5). These data indicate that the enzyme
that catalyzes the formation of 7-oxobiopterin from
H2biopterin or biopterin in the zebrafish, is related to
but is not identical to xanthine oxidoreductase. In keeping with this,
it was found that oxidation of H2biopterin or biopterin to
7-oxobiopterin by zebrafish extract was completely suppressed by
cyanide (5 mM), using either O2 or
NAD+ as electron acceptor (data not shown).All members of
the xanthine oxidase family of enzymes are known to be inactivated by
cyanide (50).
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|
Fig. 5.
Inhibition of xanthine oxidoreductase
activity and the activity of the xanthine oxidase variant by
allopurinol in extracts of zebrafish.  , xanthine oxidase; ,
xanthine oxidase variant. For activity determination of the xanthine
oxidase and the xanthine oxidase variant see the legend of Fig.
3B.
|
|
Development of Xanthine Oxidoreductase and the Xanthine
Oxidoreductase-related Enzyme--
The dehydrogenase form of xanthine
oxidoreductase can be converted reversibly to the oxidase by formation
of disulfide bridges from vicinal residues, or by irreversible
splitting through limited proteolysis (51, 52), but it is not yet
decided whether both entities may exist as such in the cell. We have
therefore analyzed separately the activities of xanthine oxidoreductase
and of xanthine dehydrogenase as well as the development of the
xanthine oxidoreductase-related enzyme in the zebrafish. Fig.
3B demonstrates that the activities of xanthine
oxidoreductase and of xanthine dehydrogenase diverge after 48-h
postferilization and that the dehydrogenase function dominates after
72 h. Additions to the assay of dithiothreitol (1 mM),
the protease inhibitor phenylmethylsulfonyl fluoride (200 µM), or the metalloprotease inhibitor EDTA (2.5 mM), separately or together, did not change the
developmental pattern (data not shown). This indicates that the
activities of the dehydrogenase and of the oxidase form are not caused
by interconversion of both enzyme forms during the assay. Both forms
rather appear to be present in the cell in a stage-specific fraction of
total oxidoreductase.
During the first 72-h postfertilization, the activity of the xanthine
oxidoreductase-related enzyme develops more slowly than the activities
of both xanthine oxidoreductase forms (Fig. 3B). Around the
hatching period, the increasing slope in its activity results in almost
the same activity levels at 120-h postfertilization.
| |
DISCUSSION |
In this study we have identified H4biopterin,
sepiapterin, 7-oxobiopterin, 2,4,7-trioxopteridine, and isoxanthopterin
as components of the pteridine pattern in zebrafish and analyzed their
biosynthesis during the first 120-h postfertilization. At this time,
the embryonic arrangement of the pigment cells is essentially
established (5). Furthermore, our results suggest that the pteridine
pattern involves two biosynthetic routes (Fig.
6). The first one leads from GTP to
H4biopterin (reviewed in Ref. 12), providing
H4biopterin as the essential cofactor for neurotransmitter
synthesis in the developing neurons (13, 15) and for phenylalanine
hydroxylation in the melanophores (21). This de novo
synthetic pathway is well understood in Drosophila (53) and
in mammalian cells (reviewed in Ref. 12), and it proceeds via
tetrahydropterin intermediates once the unstable
6-pyruvoyl-H4pterin is formed from H2neopterin triphosphate.
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|
Fig. 6.
Proposed pathway leading to the zebrafish
pteridines. The sequential action of GTP cyclohydrolase I,
6-pyruvoyl-H4pterin synthase, and sepiapterin reductase
catalyzes the de novo synthesis of the
H4biopterin cofactor from GTP. Sepiapterin takes a pool
position; it is accumulated as pigment and, after reduction of the C-6
side chain by sepiapterin reductase, additionally gives rise to
7-oxobiopterin, involving oxygenation at C-7 by a xanthine oxidase
variant. Isoxanthopterin and 2,4,7-trioxopteridine may arise from
either 7-oxobiopterin or from intermediates on its pathway by a series
of enzymatic and nonenzymatic reactions such as deamination, side chain
cleavage, and oxygenation of the pteridine core by xanthine
oxidoreductase. 1, GTP cyclohydrolase I; 2,
6-pyruvoyl-H4pterin synthase; 3, sepiapterin
reductase; 4, H2pterin oxidase; 5,
xanthine oxidase variant; 6, pterin deaminase; 7,
xanthine oxidoreductase; 8, nonenzymatic side chain
cleavage; asterisk, hypothetical enzymatic reaction.
|
|
The second route starts from sepiapterin, which accumulates during
the first 24-h postfertilization. The sepiapterin is reduced by
sepiapterin reductase to H2biopterin, which can be oxidized to biopterin by a dihydrobiopterin oxidase. This enzyme was isolated from Drosophila (47), but its occurrence in vertebrates has not been reported so far, nor are methods yet available that could enable studies of its synthesis in crude extracts. It is likely that
either H2biopterin or biopterin (which gives a 16-fold
higher yield) is oxidized at position C-7 by a xanthine oxidoreductase variant. It is not clear at what stage the 3-carbon side chain is lost
in the formation of the isoxanthopterin and the 2,4,7-trioxopteridine. It is also uncertain whether or not this side chain cleavage is enzymatically controlled (reviewed in Ref. 54). Possibly, the side
chain is lost prior to oxidation at position C-7, because both the
pterin and lumazine are well defined substrates for xanthine oxidoreductase (42, 45); this depends upon prior cleavage of the C-6
side chain and (in the case of lumazine) upon deamination at the C-2
position. The properties of pterin deaminase (EC 3.5.4.11) from
eukaryotes such as Dictyostelium discoideum, or rat liver, are poorly defined as yet (for review see Ref. 54)
The results suggest that the overall process of pteridine synthesis in
the developing zebrafish embryo is controlled by three reactions.
First, an initial surge of GTP cyclohydrolase I activity promotes a
rapid increase in the synthesis of the H4biopterin cofactor. After 72-h postfertilization, however, GTP cyclohydrolase I
activity does not correlate with the onset of rapid production of
pteridine pigments. In mammalian cells, various cytokines and growth
factors regulate the activity of GTP cyclohydrolase I at the
transcriptional or post-transcriptional level (55-57), through post-translational modification by hyperphosphorylation (58) or by
association with a feedback regulatory protein (59). Research is under
way into the regulation of GTP cyclohydrolase I activity during the
development of the zebrafish and in particular to explain why this
activity starts to decrease at around 72-h postfertilization, which
coincides with the production of xanthophore pteridine pigments.
Second, the build up of sepiapterin provides the key precursor for the
7-oxopteridines in xanthophores. The precise origin of the sepiapterin
remains obscure. In Drosophila melanogaster it has been
proposed that sepiapterin may arise by air oxidation of the
6-lactoyl-H4pterin, which lies on the
H4biopterin pathway (53, 60). Another possibility is that
it could be formed from 6-pyruvoyl-H4pterin or
6-lactoyl-H4pterin under enzymatic control, e.g.
by a dismutase-type reaction, although this is still hypothetical. Sepiapterin assumed importance in earlier work because of its possible
role in a salvage pathway to H4biopterin in mammalian cells
(61). Here also, however, its origin remains obscure.
Third, a xanthine oxidase variant appears to control the formation of
7-oxobiopterin, which then may give rise to isoxanthopterin and
2,4,7-trioxopteridine. An enzyme related but not identical to xanthine
oxidase has already been observed in carp skin, and biopterin has been
suggested as its substrate (62). This variant form is grouped with the
xanthine oxidoreductase family because of its inactivation by cyanide,
which involves elimination of its sulfur in the vicinity of
Mo6+ (50). The xanthine oxidoreductase family of enzymes
has the same overall general structure but different substrate
specificities (reviewed in Ref. 63). The insensitivity of the zebrafish
xanthine oxidoreductase variant to allopurinol, whose "suicide"
product allo-xanthine binds to the active site (49), is consistent with its different substrate specificity. The three related enzymes, namely
the oxidase and dehydrogenase form of xanthine oxidoreductase, and the
xanthine oxidase variant, all have different developmental patterns.
This suggests that expression of different forms of xanthine
oxidoreductase in xanthophores is stage-specific. The xanthine
oxidoreductase gene is characterized by high rates of exon
multiplication during phylogeny, the functional meaning of which is
unknown. The expression is tissue- and cell-specific, and it is
regulated by different stimuli (reviewed in Ref. 63). Combined genetic
and biochemical approaches in the zebrafish may help to elucidate gene
structure and regulation of the xanthine oxidoreductase family.
In summary, our results suggest that GTP cyclohydrolase I, the xanthine
oxidoreductase family, and an as yet unexplained reaction leading to
sepiapterin, control the pteridine pathway in zebrafish embryos. This
may allow for a candidate gene approach to explain the mechanisms that
lead to pigment mutations by loss of pigment synthesis and to those
mutations that affect neuronal function.
| |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the substantial
support of Dr. Laure Bally-Cuif (München-Neuherberg) who supplied
us with the zebrafish embryos. We thank Dr. Motoko Nakagoshi (Kitasato)
for the generous gift of 7-oxobiopterin, 7-oxo-6-carboxypterin, and
2,4,7-trioxopteridine. We are indebted to Dr. Adelbert Bacher
(München) for supplying us with recombinant
6-pyruvoyl-H4pterin synthase and thank him, as well as Dr.
Wilf Armarego (Canberra), for most valuable suggestions. We thank Lutz
Weidner for technical assistance.
| |
FOOTNOTES |
*
The work was supported by the Training and Mobility of
Researcher's Network (Grant ERBFMRXCT-98-0204) of the European
Community.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Present address:
Anatomisches Institut der Technischen Universität München, Biedersteinerstr. 29, 80802 München, Germany. Tel.:
49-89-4140-3150; Fax: 49-89-397-035; E-mail: ziegler@gsf.de.
Published, JBC Papers in Press, April, 18, 2000, DOI 10.1074/jbc.M910307199
| |
ABBREVIATIONS |
The abbreviations used are:
H4biopterin, (6R)5,6,7,8-tetrahydrobiopterin;
pterin, 2-amino-4-hydroxypteridine;
neopterin, 6-(D-erythro-1',2',3'-trihydroxypropyl)pterin;
H2neopterin, 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin;
biopterin, 6-(L-erythro-1',2'-dihydroxypropyl)pterin;
H2biopterin, 7,8-dihydrobiopterin;
6-pyruvoyl-H4pterin, 6-(1',2'-dioxopropyl)-5,6,7,8-tetrahydropterin (also
6-lactoyl-5,6,7,8-tetrahydropterin and
6-(1'-oxo-2'-hydroxypropyl)-5,6,7,8-tetrahydropterin);
sepiapterin, 6-lactoyl-7,8-dihydropterin;
2, 4,7-trioxopteridine, 7-oxolumazine;
isoxanthopterin, 7-oxopterin;
xanthine oxidoreductase, xanthine:oxygen
oxidoreductase (EC 1.1.3.22).
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ABSTRACT
INTRODUCTION
