(Received for publication, September 5, 1995; and in revised form, October 4, 1995)
From the
Four naturally occurring mutants with single amino acid
alterations in human 6-pyruvoyltetrahydropterin synthase (PTPS) were
overexpressed and characterized in vitro. The corresponding
DNA mutations were found in patients with hyperphenylalaninemia and
monoamine neurotransmitter insufficiency due to lack of the
tetrahydrobiopterin biosynthetic enzyme PTPS. To predict the structure
of the mutant enzymes, computer modeling was performed based on the
solved three-dimensional structure of the homohexameric rat enzyme. One
mutant (V57) is incorrectly folded and thus unstable in vitro and in vivo, while a second mutant (P87L) has substantial
activity but enhanced sensitivity to local unfolding. Two other
mutants, R16C and R25Q, form stable homomultimers and exhibit
significant activity in vitro but no activity in COS-1 cells. In vivo
P labeling showed that wild-type PTPS,
P87L, and R25Q are phosphorylated, while R16C is not modified. This
strongly suggests that the serine 19 within the consensus sequence for
various kinases, RXXS, is the site of modification. Our
results demonstrate that PTPS undergoes protein phosphorylation and
requires additional, not yet identified post-translational
modification(s) for its in vivo function.
6-Pyruvoyltetrahydropterin synthase (PTPS, ()EC
4.6.1.10) is required for the second step of the de novo biosynthesis of 5,6,7,8-tetrahydrobiopterin (BH
)
starting from GTP. PTPS converts the substrate 7,8-dihydroneopterin
triphosphate, the product from GTP cyclohydrolase I (EC 3.5.4.16), into
6-pyruvoyltetrahydropterin, which in turn is metabolized by sepiapterin
reductase (EC 1.1.1.153) to the pathway end product
BH
(1) . The BH
cofactor is involved in
various cellular processes. For instance, it increases the
proliferation rate of erythroid cells (2) and is a limiting
cofactor for the synthesis of nitric oxide by a number of recently
described nitric oxide synthases(3, 4) . The role of
BH
is best understood as redox cofactor for hydroxylation
of the aromatic amino acid phenylalanine in liver, but also of tyrosine
and tryptophan, which is rate-limiting for the dopamine and serotonin
biosynthesis in the brain(5) . In newborns, inborn errors of
BH
metabolism lead to a variant form of
hyperphenylalaninemia accompanied by severe monoamine neurotransmitter
shortage that results in progressive mental retardation due to limiting
cofactor availability for the tyrosine and tryptophan
hydroxylases(6) . For GTP cyclohydrolase I various autosomal
recessive and dominant mutations have been described that lead to a
limited BH
availability concomitant with mental retardation
and dopa-responsive dystonia,
respectively(7, 8, 9) . In contrast, PTPS
mutations have only been found to be
recessive(10, 11) . PTPS deficiency appears in three
different phenotypes, a central, a peripheral, and a transient form,
which may disappear during infant thriving(12, 13) .
Patients with the central type of PTPS deficiency exhibit a general
lack of BH
in all organs and monoamine neurotransmitter
shortage in the central nervous system, whereas patients with the
peripheral form do not synthesize BH
in peripheral organs
but have normal BH
and neurotransmitter levels in the
central nervous system. PTPS deficiency is thus a very heterogeneous
disorder with a hitherto unknown molecular basis.
Recent biochemical
and structural investigations of the human and rat enzymes have led to
a better understanding of PTPS structure-function on a detailed
molecular level(14, 15, 16) . The mammalian
PTPS requires two metals for activity, Zn and
Mg
, the former being an integral part of each active
site of the enzyme. Crystal structure analysis of the rat enzyme
confirmed the proposed homohexameric structure and furthermore revealed
that the enzyme is composed of two trimers with an overall toroidal
shape that has a diameter and height of 60 Å. One subunit folds
into a compact
+
structure comprising a four-stranded,
anti-parallel
-sheet onto which a pair of anti-parallel helices is
layered. Between strands
-1 and
-2 there is a 23-residue
insertion containing a short
-helical segment (
;
see Fig. 1). Three subunits are related by a 3-fold molecular
symmetry axis. Thereby, an unusual 12-stranded anti-parallel
-barrel is formed, which encloses a highly basic pore of
6-12 Å in diameter. The hexamer is formed by face-to-face
association of the two trimers. The putative catalytic sites, six per
active hexamer, are located at the trimer interface. Three subunits
take part in one catalytic site that is formed by two subunits from one
trimer and one subunit from the other trimer. The active center
contains the binding site for one Zn
ion and a
structural motif that resembles the acceptor site for the pyrimidine
ring system of GTP in G-proteins (16) .
Figure 1:
Fold of the human and rat PTPS
monomers. The amino acid sequences of human and rat PTPS are aligned, and identical amino acid residues or conservative
valine-leucine-isoleucine substitutions are boxed. Extensions
of the secondary structural elements deduced from the solved crystal
structure of rat PTPS are indicated, and numbering of -helices and
-strands is given below the amino acid sequence. Residues
directly participating in the active center and/or
Zn
-ion binding are dotted (His
,
Cys
, His
, His
, Asp
,
His
, and Glu
for the rat monomer). Single
amino acid exchanges found in human mutant PTPS that are described in
this work are indicated by arrows (R16C, R25Q,
V57, and
P87L). Positions of introduced factor Xa protease recognition sites for
cleavage of PTPS from the N-terminal MBP partner following purification
of recombinant MBP-PTPS fusion proteins are marked by triangles.
We previously
reported the mutation analysis from two patients with PTPS
deficiency(10) . In this study, we expanded the mutation
analysis to other patients and present the biochemical and structural
characterization of four particular mutant PTPS with single amino acid
alterations that were found to be responsible for BH deficiency in newborns. The model of the human enzyme structure
is based on computational modeling starting from the solved x-ray
structure of the rat PTPS. Based on the experimental observations with
recombinant mutant and wild-type proteins that suggested the need of
certain modification(s), we demonstrate that the native human PTPS is a
phosphoprotein potentially modified at serine 19, and we further
conclude that PTPS undergoes protein phosphorylation and other
post-translational modification(s) for normal in vivo function.
To express the mutant proteins R16C (C to T transition), R25Q (G
to A transition),
V57
(triplet deletion of G
to G
), and P87L
(C
to T transition), the corresponding fragments were
cloned into the pMal-c2 vector in a similar fashion as described above,
using the 5`-primer PTSP101 for R25Q and
V57, and the 5`-primer
PTPS201 for R16C and P87L. The templates for expression of the R16C and
R25Q mutant proteins were pHSY27 and pHSY29, respectively, and have
been described before (10) . Templates for PCR amplifications
to construct the corresponding expression plasmids for the mutant
proteins
V57 and P87L were generated by site-directed mutagenesis.
To perform oligonucleotide-directed in vitro mutagenesis, the
single-stranded bacteriophage M13 derivative pHSY22, an M13 mp19 clone
harboring the human liver PTPS cDNA was used as wild-type DNA
template(10) . Oligonucleotide PTPS200 was used for introducing
the C/G
to T/A
transition (P87L mutant),
and oligonucleotide PTPS16 was used for introducing the triplet
deletion (
V57 mutant) into wild-type cDNA. All constructs were
confirmed for correct sequences by DNA sequence analysis using the
AutoRead sequencing kit in combination with an A.L.F. DNA sequencer
(Pharmacia). The following pMal-c2 derivatives were generated:
pHSY2003, pHSY2004, pHSY2005, and pHSY2007, expressing the mutant PTPS
proteins R25Q,
V57, P87L, and R16C, respectively.
To express
various PTPS proteins in COS-1 cells, cDNAs were cloned into the
eukaryotic expression vector pSCT1 (a derivative of
pSCT-GAL556X(21) , having the GAL556X fragment replaced by a
synthetic polylinker fragment). ()PCR fragments were
generated with the 5`-primer PTPS21 and the 3`-primer PTPS102, each
containing a BamHI restriction site, and subsequently cloned
into the single BamHI site of plasmid pSCT1. For the standard
PCR reactions, the wild-type cDNA or the corresponding mutant DNAs
described above were used as templates. The following pSCT1 derivatives
were generated: pHSY2009, pHSY2010, pHSY2011, and pHSY2012, expressing
the mutant PTPS proteins R16C, R25Q,
V57, and P87L, respectively.
In addition, the wild-type sequence inserted in the correct orientation
(pHSY2013) and in the reverse orientation (pHSY2008) were used as
positive and negative controls, respectively.
The aim of this study was to characterize four mutant PTPS
proteins deduced from DNA mutation analysis from four patients with the
recessive inherited disorder of BH deficiency. A
description of the patients and primary fibroblast cell lines is
summarized in Table 1. More detailed information regarding case
reports and clinical symptoms of these patients has been the subject of
previous publications (see references in Table 1).
While mutations in the two patients U. T. and J. R. S. had
been screened and published before ((10) ; see Table 1),
mutation analysis on cDNA of cells derived from L. L. and S. S. was
done during this work. In all cases reverse transcription-PCR analysis
using PTPS-specific primers was performed starting from total RNA
isolated from cultured primary cells. PCR products were subjected to
direct sequence analysis (for experimental details see (10) ).
Patient L. L. showed on one allele a deletion from G to
G
, thus creating an in-frame deletion of valine codon 57
(
V57). The other allele had a deletion of 23 nucleotides extending
from T
to G
, leading to a frameshift after
lysine codon 54 and subsequent premature stop codon. The putative
truncated protein resulting from this allele was named K54X. Patient S.
S. had a homozygous C
to T transition, thereby expressing
a putative mutant protein having the proline at position 87 replaced by
a leucine (P87L; see also Fig. 1).
Immunoreactive material in
crude extracts from these fibroblasts was investigated using an
affinity-purified anti-human PTPS antibody, SZ28 (Fig. 2).
Western blot analysis showed two bands, an upper one at 16 kDa and
another band at 14 kDa. The 16-kDa band is clearly visible in
extracts from B. H. (wild-type control) and patient J. R. S. and
faintly visible from U. T.; whereas no 16-kDa band is present in
extracts from S. S. and L. L. The relative intensity of the 14-kDa band
is slightly stronger in all patients as compared with the healthy
control. In order to offer some explanations for the nature of the two
bands, this pattern was further investigated by first comparing the
band sizes with that of the recombinant wild-type PTPS purified from E. coli (see below). As can be seen from Fig. 2, the
recombinant PTPS comigrated with the upper, 16-kDa band. In another
series of experiments, we prepared crude extracts in the absence of any protease inhibitors from healthy control cells and tested
for the persistence of the two bands by Western blot and for PTPS
activity. The two immunoreactive bands remained stable and were still
detectable after keeping the extracts for 16 h at room temperature. In
parallel, PTPS activity dropped marginally to 63% of the initial
activity after 16 h of incubation (data not shown). We also transferred
the wild-type PTPS-cDNA by using a retroviral vector into primary
fibroblasts from patient S. S. and subsequently regained BH
biosynthetic activity. Upon examination by Western blot analysis
of crude extracts from these cells, the 16-kDa and 14-kDa
bands were visible only in cells with restored activity, thus
suggesting the upper band was responsible for PTPS activity. (
)From these observations we conclude that the 16-kDa band
represents the active form of PTPS and that the 14-kDa band is most
probably not a simple protease degradation product of PTPS but rather
an unknown protein that interacts with this purified antiserum.
Nevertheless, these experiments do not exclude the possibility that the
16-kDa band may represent a modified form of PTPS (see below).
Figure 2: Immunodetection of PTPS in primary fibroblasts from various patients. Western blot analysis of crude extracts from cultured fibroblasts is shown (see ``Experimental Procedures''). Proteins were separated under reducing conditions by 12.5% SDS-PAGE. Each lane contains approximately 0.1 mg of total protein. Names of patients and predicted amino acid exchanges for PTPS are indicated for each lane (see also Table 1). Purified human recombinant PTPS (1 ng) was added to crude extracts from patient J. R. S. (JRS + rec. hPTPS) or loaded on a separate lane (rec. hPTPS). Cross-reacting material was detected by using the affinity-purified rabbit anti-human PTPS antibody SZ28. BH, wild-type control; SS, LL, JRS, and UT, patients.
We
overexpressed and purified wild-type and the four mutant PTPS alleles
R16C, R25Q, V57, and P87L. The pMal-c2 vector was manipulated in
order to express recombinant MBP-PTPS protein fused by a factor Xa
protease recognition site (tetrapeptide IEGR; for details see
``Experimental Procedures''). The MBP-PTPS proteins were
affinity-purified, followed by protease factor Xa treatment, and were
subsequently separated from the MBP fusion partners as
described(14) . All PTPS proteins purified by this procedure
had the N-terminal sequence NH
-STEGGGR (with the exception
of hPTPS
; see Fig. 1) and were checked
by SDS-PAGE analysis (Fig. 3) and electrospray mass
spectrometry.
Figure 3:
Electrophoretic analysis of purified
recombinant wild-type and mutant PTPS. Silver staining of 1.5 µg of
wild type (wt) and of each mutant PTPS (R16C, R25Q, P87L, and
V57) was performed after separation of proteins by 12.5% SDS-PAGE
in the presence of 2-mercaptoethanol (5% v/v) in the loading buffer.
The arrowheads indicate the position of the 16.3-kDa monomer,
and of the 32.5-kDa dimeric form of PTPS.
The measured average compound masses of the wild-type
(16,255.08 ± 3.71) and mutant proteins R16C (16,199.9 ±
1.42) and R25Q (16,224.24 ± 8.63) were in good agreement with
the calculated masses of 16,256 (wild-type), 16,203 (R16C), and 16,228
(R25Q), respectively. Wild-type PTPS revealed the monomeric as well as
the dimeric forms when inspected by SDS-PAGE under reducing conditions,
as shown before(14) . The same was true for the mutants with
single amino acid exchanges, R16C, R25Q, and P87L. The additional bands
below the 16.3-kDa monomer of mutant P87L were repeatedly found and are
due to moderate proteolytic degradation in the course of purification.
Separation by SDS-PAGE under nonreducing conditions revealed for P87L
the presence of a single monomeric band, two dimeric bands, and some
bands with higher molecular weight (not shown). These observations were
partially confirmed by mass spectrometry analyses, where three major
peaks of averaged compound masses were detected, one of 32,541 ±
6.15 which is the dimer of two full-length monomers (calculated mass of
32,544) and a second peak of 30,966.11 ± 2.76, which is
interpreted as a heterodimer of a full-length protein and a putative
truncated form lacking the first 15 amino acids (calculated mass of the
heterodimer is 30,970; putative PTPS derivative starting with RISFSA; see Fig. 1). A third peak with the mass of
29,391.94 ± 4.68 might be due to dimer formation of the
N-terminally truncated form (calculated mass of 29,395). Taken
together, wild-type, R16C, and R25Q exhibit a very similar behavior
upon overexpression and purification, with a yield of
50 mg of
protein from 0.5 liter of E. coli culture. Although
overexpression of the P87L allele yielded the same amount of protein,
this mutant exhibited a higher sensitivity with regard to degradation
during the purification procedure.
A completely different behavior
was observed for mutant V57. In comparison with wild-type PTPS,
this mutant protein eluted earlier from the gel filtration column and
was poorly cleaved by factor Xa protease, and only a minor fraction was
recovered upon size exclusion followed by affinity chromatography.
Consistent with these observations, purified
V57 appears as a
smear on the SDS-gel, with a major fraction migrating as monomer plus
several different aggregation and/or degradation products (see Fig. 3). Thus, mutant
V57 appears not to be stable as a
hexamer and has a tendency to aggregate into higher molecular
structures because it is probably not correctly folded during the
biosynthesis of the recombinant fusion protein or after cleavage from
the MBP partner. Since purification of this mutant protein ended up
repeatedly with a poor yield, we did not perform mass spectrometry
analysis.
Bearing in mind the very low activities
detected in primary fibroblasts from patients U. T., J. R. S., and S.
S. ( 1%; see Table 1), it was unanticipated to find such
significant activities for the overexpressed alleles R25Q, R16C, and
P87L, respectively. These mutant alleles needed to be further
characterized in order to explain the different PTPS activities between
fibroblasts in vivo and recombinant enzymes in vitro.
Figure 4: SDS-PAGE of purified wild-type and mutant PTPS upon (A) cross-linking in solution and (B) chymotrypsin digestion. A, 1.6 µg of each recombinant protein, treated with the cross-linker bis(sulfosuccinimidyl)suberate in solution, was separated by 12.5% SDS-PAGE under reducing conditions, and silver-stained to visualize multimerization of PTPS monomers. B, 11.6 µg of the different PTPS proteins were untreated (0 min) or treated (60 min) with chymotrypsin, separated by 17.5% SDS-PAGE, and stained with Coomassie Brilliant Blue.
We then analyzed the degradation pattern of the three mutant proteins with single amino acid exchanges following incubation with chymotrypsin (Fig. 4B). Upon protease digestion for 60 min, only P87L showed a relatively enhanced degradation pattern by chymotrypsin, thus indicating a reduced protein stability due to differential sensitivity to unfolding.
Since it was described earlier that PTPS is relatively heat-stable (25) , susceptibility to heat pretreatment of mutant proteins R16C, R25Q, and P87L was assayed and compared with wild-type activity (not shown). Whereas wild type, R16C, and R25Q had still more than 80% of their initial activities following incubation at 50 °C, a different behavior was observed for P87L. For this mutant, activity dropped very sharply already when incubated at 50 °C, specifying enhanced thermolability at least under in vitro conditions.
Figure 5:
Relative activities of recombinant
proteins and of transfected alleles in COS-1 cells (A) and
visualization of PTPS cross-reacting material in crude extracts from
the transfected COS-1 cells (B). A, PTPS activity was
measured of affinity-purified PTPS proteins isolated from E. coli (open bars), and crude extracts from transiently
transfected COS-1 cells expressing various PTPS alleles (filled
bars). B, Western blot analysis of crude extracts from
COS-1 cells expressing the indicated PTPS proteins used in panel
A. The amount of total protein loaded in each lane was
normalized to equal amounts of -galactosidase activity (see
``Experimental Procedures''). control, COS-1 cell
extract containing the wild-type PTPS in the reverse orientation
(pHSY1008).
Figure 6:
Immunoprecipitation of phosphorylated
human PTPS expressed in COS-1 cells. A, transiently
transfected COS-1 cells expressing the indicated forms of PTPS were
labeled with [P]orthophosphate,
immunoprecipitated with the anti PTPS affinity-purified antibody
(SZ28), separated by 12.5% SDS-PAGE, and stained with Coomassie Blue.
The following controls were loaded: 5 µg of recombinant wild-type
PTPS purified from E. coli (rec. hPTPS), lysate
treated with the preimmune serum (pre), and precipitate from
COS-1 cells harboring pHSY2008, containing the wild-type PTPS sequence
in the reverse orientation (control). B,
autoradiography of the same gel as in panel A showing the in vivo
P-labeled wild-type (wt), R25Q,
and P87L PTPS. The arrowhead indicates the position of the
PTPS monomer.
In order to obtain a model of human PTPS, we started out with the structure of a rat liver PTPS subunit and replaced the relevant 15 amino acids using a molecular graphics system(24) . The modeling was restricted to the positioning of amino acid side chains in sterically reasonable conformations. We did not try to minimize the resulting structure, because the modeling did not produce strains in the structure with regard to the modified side chain positions. The following description of the mutation sites is based on the model of human PTPS (see Fig. 1and Fig. 7).
Figure 7:
Stereo picture of the human PTPS hexamer.
The subunits are shown in a ribbon-type presentation from the
N-terminal residue 8 to the C-terminal amino acid 145. The monomers of
each trimer are shown in different colors. The positions of
the amino acids affected in the mutants that were detected and analyzed
are indicated for one monomer in red. The positions of the
relevant active centers are indicated by the green spheres
that represent the Zn-ions. The 7,8-dihydroneopterin
substrate located in each active center is shown in a stick and
ball representation.
The side chain of
residue Arg lies at the entrance to the putative active
site pocket and could possibly bind the triphosphate moiety of the
substrate during catalysis. Since we do not know the exact nature of
this interaction, it was impossible to estimate from modeling the
effect of the R16C mutation. The distance of Cys
to the
active site residue Cys
is 12 Å, which makes a
disulfide bond formation unlikely.
Arg is located close
to the hexamer equator region on the protein surface and is only
sequentially close to the Zn
-binding residue
His
. Mutant R25Q should exhibit no gross structural
differences to wild-type PTPS and should behave accordingly in
catalysis.
Val is located on
-strand 2 and points
toward the hydrophobic interior of the subunit. In the deletion mutant
V57, the in-out
-strand pattern is perturbed, which
definitely will affect protein folding. Indeed, we did not see
expression of a correctly folded mutant protein.
Helix is an irregular four-turn helix in that it contains a kink
between the third and fourth turns. The latter is enlarged due to the
breakage of the H-bonding pattern through the presence of
Pro
. Exchange of this residue into leucine might lead to
structurally localized conformational changes, e.g. formation
of a regular four-turn helix. This could have an effect on the
positions of Asp
and His
, which are involved
in an intersubunit catalytic triad and thus implicated in the catalytic
process(16) .
Although PTPS deficiency is well characterized in terms of
screening for metabolites based on the established BH biosynthesis pathway, the molecular consequences in vivo of mutant PTPS, and the different phenotypes of PTPS deficiencies
are not understood. Here we expressed recombinantly and characterized
four naturally occurring mutant PTPS with single amino acid alterations
that were detected in the PTPS-cDNA from four patients with
hyperphenylalaninemia due to BH
deficiency. Following, we
will first discuss some aspects of human wild-type PTPS in comparison
with the rat enzyme, followed by the consequences of the single amino
acid exchanges and implication of in vivo PTPS
phosphorylation.
Structural prediction of the human PTPS by homology
modeling is based on the high conservation of the primary sequence as
compared with the rat enzyme. Moreover, human and rat PTPS share not
only the homohexameric property as shown experimentally, but also all
the residues involved in active site formation and
Zn-ion binding (Fig. 1)(15, 16, 19) . The only
distinction is located at the very N-terminal part of the proteins,
which was not determined in the rat x-ray crystal
structure(15) . Nonetheless, attempts to crystallize the
recombinant 144-amino acid-containing human PTPS counterpart failed. We
also expressed and tried without success to crystallize a truncated
form of the active human enzyme, hPTPS
. The
rationale behind expressing this truncated version of PTPS was 2-fold.
On the one hand, we argued that a structurally nonlocalized domain such
as the N terminus may prevent hexamer assembly and growth of crystals.
On the other hand, we performed a computer search with the human PTPS
for potential protein patterns by using the PROSITE program (Wisconsin
GCG software, version 8). As a result of this pattern search, a
myristoylation site was predicted at glycine residue 6. However, an
internal glycine as a potential N-myristoylation site would
require a proteolytic processing to expose the Gly
as the
N-terminal residue(27, 28) . The rat PTPS isolated
from liver tissue was shown to be N-terminally truncated by having
cleaved off the first four amino acids and therefore exposing a valine
at the N terminus(29) , which is not a substrate for N-myristoylation. Whether the differences in the amino termini
between the human and rat enzyme have some functional meaning and
whether the human PTPS undergoes proteolytic processing too, followed
by potential modification(s), remains to be clarified. In this context
it is worth mentioning that the cysteine residue only present in the
human protein sequence at position 10 has been shown not to influence in vitro enzyme activity by assaying a PTPS mutant with a
cysteine to alanine substitution. (
)
Only those PTPS
mutants with a single amino acid difference as compared to wild-type
PTPS were chosen for recombinant expression and characterization, since
the C-terminally truncated allele K120X (patient J. R. S.) has
previously been shown to be completely inactive, and therefore K54X
(patient L. L.) must be inactive as well (10) . Overexpression
and purification of the second allele from patient L. L., V57,
revealed that this mutant form is incorrectly folded, and it was
expected to be unstable in vivo. Structure prediction and
biochemical and enzymatic tests, as well as the COS-1 cell expression,
are in agreement with the absence of any cross-reactive 16-kDa PTPS in
primary fibroblasts from patient L. L. due to rapid degradation of
V57 (Fig. 2).
Patient S. S. with the homozygous P87L
allele exhibits no 16-kDa cross-reactive PTPS in primary fibroblasts,
and consistently weaker bands were observed with Western blots from
extracts of overexpressing COS-1 cells (Fig. 5B). This
suggests that a Pro exchange to leucine induces modest
instability, and P87L is thus more sensitive to degradation in
vivo. A structurally localized conformational change was predicted
from computer modeling. Experimental evidence for local differences in
tertiary structure is the altered protease digestion pattern (Fig. 4B) and the enhanced sensitivity to thermal
denaturation of the otherwise normal hexameric mutant protein.
Interestingly, the P87L mutant has significant enzymatic activity as
recombinant protein in vitro as well as in COS-1 cells (Fig. 5A) and is phosphorylated in the eukaryotic cell
background. In contrast, the activity measured in fibroblast extracts
prepared from patients' primary cells was below the level of
detection. One explanation for this discrepancy is that a slightly
altered susceptibility to protease due to the single Pro
to leucine substitution leads to an enhanced degradation in
vivo, which is not compensated by the de novo biosynthesis of this mutant PTPS. Further investigations toward
characterizing the Pro
function in human PTPS will be of
interest at least due to the fact that from eight PTPS-deficient
patients analyzed for DNA mutations so far, four patients where found
to have one or both alleles with alterations at the Pro
site(11, 30) .
The arginine 16 and 25
substitutions, found in patients J. R. S and U. T., respectively, are
the most surprising PTPS alleles in terms of in vivo versus in
vitro enzymatic activities. In primary fibroblasts, R25Q seems to
be less stable than R16C and wild-type PTPS, based on Western blot
analyses, whereas all three proteins are equally stable when expressed
in COS-1 cells. Both recombinant arginine mutants exhibit similar
biochemical properties as compared with the purified wild-type PTPS,
including the significant activity in the in vitro assay with
the substrate 7,8-dihydroneopterin triphosphate (Fig. 5A). Only substrate binding seems to be somewhat
lowered in these two mutants as revealed by the increased K. However, in fibroblasts as well as in the COS-1
cells, the activity of both arginine mutants is absent or drastically
reduced. This suggested the occurrence of protein modification(s) of
wild-type PTPS that takes place in a eukaryotic cell background only
and is impaired in these two mutant PTPSs. Modification by protein
phosphorylation was an attractive hypothesis, because the other
BH
biosynthesis enzymes, GTP cyclohydrolase I and
sepiapterin reductase, have recently been shown to be
phosphorylated(31, 32) . Moreover, the residues
Arg
and Arg
are both located in potential
consensus recognition sequences for various kinases(26) ; i.e. residue Arg
overlaps with the motif
RXXS*, and Arg
overlaps with the motifs
RXXS* and S*XR, where the asterisk indicates the
phosphoserine (see also Fig. 1). Upon in vivo incubation with [
P]orthophosphate,
wild-type PTPS and the R25Q were found to be phosphorylated, whereas
the R16C mutant was not modified by
P. This has several
implications. Since the R16C mutant appears not to be a substrate for
phosphorylation in COS-1 cells and the
RRIS site is the
only motif that overlaps with a common consensus phosphorylation site
(RXXS*) described for many kinases(26) , we conclude
that serine 19, which is located within this motif and is on the
protein surface (not shown), must be the site of phosphorylation.
Direct proof for this has yet to be provided, especially since in
vitro attempts to phosphorylate with various commercially
available protein kinases, including cAMP-dependent protein kinase,
calmodulin-dependent protein kinase, and protein kinase C, revealed
that purified wild-type PTPS is not a substrate for any of
these enzymes. (
)All these protein kinases tested have the
common consensus phosphorylation site RXXS*, which overlaps
with the
RRIS site altered in mutant allele R16C. Another
consequence of modification by P
, as we speculate, is that
the phosphorylated form of PTPS might be more active than the
nonmodified protein. This is not only based on our measurements with in vitro purified enzymes compared with activity in COS-1
cells (Fig. 5A), but it is also suggested by Zhu et
al.(33) . In their report, they treated cultured rat
dopamine neurons with 8-bromo-cAMP, which stimulated BH
biosynthesis. This observation led them to suggest that BH
metabolism might be regulated through phosphorylation via
cAMP-dependent protein kinase. Since GTP cyclohydrolase I, the first
enzyme for BH
biosynthesis, has no consensus sequence for
this kinase, PTPS was speculated to be a substrate for the
cAMP-dependent protein kinase due to the presence of such consensus
sites(33) . Although based on our observations up-regulation of
PTPS through phosphorylation is also favored, the cAMP-dependent
protein kinase seems not to be the directly interacting
kinase, because PTPS was not labeled with
P by this kinase
in in vitro assays (see above). Thus, the corresponding
protein kinase that phosphorylates wild-type PTPS remains to be
identified.
The present results are complicated by the observation
that, although Arg is needed in PTPS for modification such
as phosphorylation, this modification may not be sufficient, as shown
by the inactive R25Q mutant that was found to be labeled with
P, presumably at Ser
. Arg
is
located in the motifs SHR
and
RLYS (Fig. 1) with the potential corresponding phosphorylation sites
serine 23 and/or serine 28, respectively. Although we cannot completely
rule out additional phosphorylation at sites other than the putative
phosphoserine at position 19, it seems unlikely that mutant R25Q is
partially phosphorylation-defective. Additional phosphorylation would
imply that the presence of Arg
is a prerequisite for the
phosphorylation of Ser
and/or Ser
, because
R16C was shown not to be labeled at all with
P. We
postulate that other not yet specified post-translational
modification(s) and/or interactions of wild-type PTPS may be required
for maximal in vivo activity and stability. Such additional
alterations or interactions are somehow prevented by the absence of
Arg
and not only lead to instability in vivo but
also render PTPS completely inactive. We are currently testing various
possibilities for additional post-translational modifications of PTPS.