1The Bone Biology Group, Division of Integrative Biology, Roslin Institute, Roslin, Midlothian EH25 9PS, 3Institute of Cell, Animal and Population Biology, Ashworth Laboratories, The University of Edinburgh, Edinburgh EH9 3JT and 4School of Chemistry, Joseph Black Building, The University of Edinburgh, Edinburgh EH9 3JJ, UK
2 To whom correspondence should be addressed. e-mail: alan.stewart{at}bbsrc.ac.uk
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Abstract |
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Keywords: haloacid dehalogenase superfamily/homology modelling/matrix mineralization/phosphatase/PHOSPHO1
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Introduction |
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PHOSPHO1 is a phosphatase first identified in chickens and has been implicated in the generation of inorganic phosphate for matrix mineralization. The PHOSPHO1 gene is expressed at levels 100-fold higher in differentiating chondrocytes than in non-chondrogenic tissues (Houston et al., 1999
), with the protein localized to sites of mineralization (Houston et al., 2004
). Orthologues of PHOSPHO1 have recently been identified in humans and mice, both sharing 62% identity at the amino acid level with the chick protein (Houston et al., 2002
). It has been postulated that in addition to TNAP other phosphatases are involved in matrix mineralization. Bone development and mineralization were found to be normal in newborn TNAP knockout mice, although hypomineralization and other abnormalities of the skeleton and dentition were observed subsequently (Waymire et al., 1995
; Narisawa et al., 1997
; Hessle et al., 2002
). Additionally, it has been shown that TNAP can be removed from some preparations of matrix vesicles without impairing their ability to mineralize (Register et al., 1986
). Specific inhibitory studies on TNAP also provide evidence that additional phosphatase activity is present within differentiating chondrocytes (Hsu and Anderson, 1996
). These studies demonstrate that TNAP is not essential for the initial events leading to bone mineralization and imply that other phosphatases are involved in matrix mineralization.
The PHOSPHO1 amino acid sequence contains three peptide motifs that are conserved within the haloacid dehalogenase (HAD) superfamily of magnesium-dependent enzymes (Figure 1). Much research has been targeted toward the study of the HAD superfamily, which catalyse the hydrolysis of C-Cl, C-OP and C-P bonds from a wide range of substrates and demonstrate remarkable functional diversity through a single protein fold (Ridder and Dijkstra, 1999). Interestingly, PHOSPHO1 shares no sequence similarity with almost all members of the HAD superfamily in the PDB outwith the three motifs, the exception being the phosphoserine phosphatases (PSPs) with which it shares
20% identity. X-ray crystallographic data have led to a rapid increase in our understanding of how these enzymes work, with the structural elucidation of a range of HAD proteins with metals, substrates, inhibitors and reaction intermediates bound (Wang et al., 2001
, 2002
; Lahiri et al., 2002
, 2003
). These structures show that the aforementioned motifs fold together to form the catalytic core of the enzyme. Studies involving site-directed mutagenesis have reinforced this observation and have shown that mutations within these motifs abolish or greatly reduce enzymatic activity in solution (Kurihara et al., 1995
; Collet et al., 1999
; Selengut, 2001
).
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Materials and methods |
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Searches were performed with the Ensembl Genome Browser available from the EMBL and Sanger Institute (http://www.ensembl.org) and with the BLAST algorithm (NCBI) (Altschul et al., 1997) using the human PHOSPHO1 protein sequence (TrEMBL code: Q8TCT1).
Homology modelling
The protein sequence of human PHOSPHO1 (Q8TCT1) was submitted to the 3D-PSSM www-server (http://www.sbg.bio. ic.ac.uk/3dpssm/) for fold recognition, secondary structure prediction and an initial pairwise sequence alignment between target and possible templates. For additional sequence alignments homologous sequences to PHOSPHO1 and potential templates were retrieved using BLAST against the non-redundant database from the NPS@ www-server (http:// npsa-pbil.ibcp.fr; Combet et al., 2000
) with a cut-off E-value of 1010. Multiple sequence alignments were generated using T-Coffee (Notredame et al., 2000
) and subsequently subjected to minor manual editing.
Submitting the protein sequence of human PHOSPHO1 to the 3D-PSSM fold recognition server (Kelley et al., 2000) revealed the high-resolution (1.8 Å) X-ray structure of PSP from M.jannaschii (PDB code: 1F5S; Wang et al., 2001
) as the most suitable template for homology modelling. A pairwise alignment between the target and template sequences was manually adjusted taking into consideration multiple sequence alignments, structural alignments and the continuity of secondary structure elements. Twenty models were built using MODELLER v6.2 (Sali and Blundell, 1993
), keeping the active site Mg2+, and its bound phosphate and water molecules in the positions found in the template. Manual inspection of the five highest ranking models found Cys176 and Cys209, which are both conserved within the PHOSPHO1 multiple sequence alignment (Figure 2), to be in close proximity to each other, suggesting their involvement in a disulphide bond. Therefore, in a subsequent MODELLER run a disulphide bond between these two residues was enforced. Non-identical side chains between template and target were optimized using SCWRL (Bower et al., 1997
). As a final step, the model (residues 22255) was energy minimized in SYBYL v6.8 (Tripos Associates, St Louis, MO, USA) applying 75 steps of minimization. The protocol used the Powell algorithm and an improved version of the Tripos force field. The validity of the resulting model was assessed using PROCHECK v3.5 (Laskowsky et al., 1993
) and WHAT IF v4.99 (Vriend, 1990
), which are available on-line at the Biotech validation suite at http://biotech.embl-ebi.ac.uk. The binding site was analysed using the Binding Site module of InsightII v2000 (Accelrys Inc.).
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Results |
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The cDNA sequence corresponding to human PHOSPHO1 was first isolated from SaOS-2 osteoblast-like cells (Houston et al., 2002). Human PHOSPHO1 is a 267 amino acid single chain protein; analysis of the sequence using the web-based program, SignalP v1.1 (Nielsen et al., 1997
) predicts the absence of a signal peptide. This suggests PHOSPHO1 to be a soluble cytoplasmic phosphatase. The predicted molecular mass of PHOSPHO1 is 29.7 kDa; however, this does not take into account glycosylation or other possible post-translational modifications.
All members of the HAD superfamily contain three conserved peptide motifs, which fold to form the active site. Figure 1 shows an alignment of human PHOSPHO1 with a range of HAD superfamily enzymes at these three regions. Motif 1 [DXDX(T/V), often preceded by a Phe residue] contains two Asp residues involved in the binding of the catalytic magnesium ion, although only the first Asp is completely conserved in all HAD enzymes. It has been shown that the first Asp residue is the phosphorylation site in P-type ATPases (Asano et al., 1996), ß-phosphoglucomutases (Lahiri et al., 2002
), phosphotransferases (Collet et al., 2002
) and phosphatases (Collet et al., 1998
). Analysis of all known HAD superfamily crystal structures (with the catalytic metal bound) shows that the second Asp coordinates to the metal ion through the peptide backbone oxygen and not by the side chain carboxylate group. The first motif also contains a Thr or Val residue. The second motif contains a catalytically important Ser residue (Ser122 in PHOSPHO1) preceded in the chain by four bulky hydrophobic amino acids (Val118, Ile119, Leu120 and Ile121 in PHOSPHO1). The crystal structure of ß-phosphoglucomutase from Lactococcus lactis with the bound substrate analogue
-D-glucose-1,6-(bis)phosphate (PDB 1O08; Lahiri et al., 2003
) shows this corresponding Ser and the conserved Lys from motif 3 (Lys177 in PHOSPHO1) to interact directly with the oxyphosphorane group of a pentacovalent intermediate. Motif 3 also contains a third metal binding Asp residue (Asp203), which is a Ser in the HADs. This residue coordinates to the metal through its side chain in all of the catalytic metal-containing crystal structures within the HAD superfamily and is stabilized by an H-bond from the backbone amide of the conserved Thr/Val in motif 1.
PHOSPHO1 homologues
Homologous PHOSPHO1 genes were found in the vertebrates Rattus norvegicus (rat) and Takifugu rubripes (puffer fish). Analysis of rat and puffer fish genomic DNA sequences shows the PHOSPHO1 gene in both species to have a similar three exon gene structure to the mouse and human genes (Houston et al., 2002). Genomic DNA corresponding to part of the Danio rerio (zebrafish) PHOSPHO1 gene was also found. The partial zebrafish sequence most likely corresponds to the bulk of the protein encoded by the third exon. The position of rat PHOSPHO1 in the genome (RNO10q31) shows it to be located in the same syntenic region as the human, mouse and chicken genes (Houston et al., 2002
), strongly suggestive of orthology. The position of puffer fish and zebrafish PHOSPHO1 in their respective genomes could not be established as these genes were found on unassembled chromosomal fragments. Rat PHOSPHO1 only differs from the orthologous mouse protein by a single amino acid (Ala7 to Val) and shares 94 and 62% identity with human and chicken proteins, respectively. The partial zebrafish transcript shares 51% identity with the analogous human and mouse proteins and 45% identity with the chicken protein. Puffer fish PHOSPHO1 shares 43% identity with human and mouse proteins, 46% with the chicken protein and 55% identity with the zebrafish protein.
A BLAST search based upon the amino acid sequence of human PHOSPHO1 revealed several proteins to exist in other organisms with significant homology to PHOSPHO1. These were a putative phosphatase from Drosophila melanogaster (fruit fly) sharing 32% identity (TrEMBL code: Q9VWF0), two (A and B) from Arabidopsis thaliana (cress) sharing 29% (Q9FZ62) and 30% identity (Q9SU92), an Oryza sativa (rice) protein sharing 30% identity (Q94D09) and also three proteins from Lycopersicon ensculentum (tomato plant), which are annotated as LePS2 phosphatases and each share 30% identity (Q8GUC1, Q8GRL8 and Q9FPR1). The LePS2 phosphatases are highly expressed in the tomato plant during development and in response to low phosphate availability and infection (Baldwin et al., 2001
; Stenzel et al., 2003
). An alignment of PHOSPHO1 protein sequences from human, mouse, rat, chicken, zebrafish and puffer fish with the Drosophila and plant homologues is shown in Figure 2. All of the above proteins were found to contain the three sequence motifs conserved within the HAD superfamily. The degree of sequence homology suggests that these phosphatases and PHOSPHO1 homologues utilize the same or a highly similar substrate.
Tissue distribution of PHOSPHO1
At present, ESTs available in dbEST at the NCBI server indicate that human PHOSPHO1 is expressed in brain, B cells, cervix, colon, foetal liver and spleen, ovary, placenta, skin and in the sympathetic trunk. Expression of PHOSPHO1 has also been detected in cDNA from human SaOS-2 osteoblast-like cells (Houston et al., 2002). Murine (Mus musculus) ESTs show PHOSPHO1 to be expressed in brain, breast cancer cells, colon, heart, joints, mammary gland, organ of corti and salivary gland. ESTs corresponding to rat (R.norvegicus) PHOSPHO1 were identified in dorsal root ganglia and muscle. ESTs corresponding to chicken (Gallus gallus) PHOSPHO1 were identified in brain, bursa of fabricius, chondrocytes, limbs, liver, muscle and epiphyseal growth plate and small intestine. No ESTs corresponding to puffer fish (T.rubripes) or zebrafish (D.rerio) PHOSPHO1 were found.
Additionally, ESTs corresponding to the PHOSPHO1 homologue in D.melanogaster were found to be expressed in the head, ovary and salivary gland and also show expression at the embryonic and larval to early pupal stages of development. ESTs were also found in A.thaliana corresponding to the homologue, putative phosphatase B and also the homologues in O.sativa and L.ensculentum.
Homology model of human PHOSPHO1
In the absence of structural data for PHOSPHO1, a three-dimensional model of human PHOSPHO1 (residues 22255) has been built using the crystal structure coordinates of MjPSP (PDB 1F5S; Wang et al., 2001). Despite the low sequence identity (19.4%), a more advanced bioinformatic analysis considering secondary structure prediction and fold recognition reveals an evolutionary relationship between PHOSPHO1 and PSPs, allowing us to build a meaningful structural model of PHOSPHO1. The structure of the human PHOSPHO1 model is shown in Figure 3A. The protein consists of two domains: the catalytic
/ß domain and a four-helix-bundle. The
/ß domain forms a typical Rossman fold structure consisting of a six-stranded parallel ß-sheet, surrounded by six
-helices. In the human PHOSPHO1 model, a hexadentate Mg2+ atom is bound in an octahedral geometry via three Asp residues [Asp32, Asp34 (backbone oxygen) and Asp203]; O-ligands are also provided by a phosphate anion and two water molecules (Figure 3B).
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Discussion |
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We have modelled the human PHOSPHO1 protein based upon the crystal structure of MjPSP. The model shows that the characteristic features of the catalytic site, with regard to the HAD superfamily, are all preserved. The three structural motifs fold to form the catalytic site. All residues involved in the recognition of the phosphate group are fully conserved, with the exception of Gly100, which is replaced by Asp123. In addition, these residues are also involved in the hydrolysis reaction, as is a fully conserved Ser (99 in MjPSP, 122 in PHOSPHO1). Consequently, the modelled catalytic site in PHOSPHO1 closely resembles the catalytic site in PSP, suggesting that PHOSPHO1-catalysed phosphotransfer occurs by the same well defined stepwise Mg2+-dependent mechanism. This is a dynamic process involving the reversible phosphorylation of the first Asp residue (11 in MjPSP, 32 in PHOSPHO1) in motif 1.
In contrast, none of the residues primarily responsible for substrate specificity in PSP is present in PHOSPHO1. The crystal structure of MjPSP (mutant Asp11Asn) with bound phospho-L-serine (PLS) (PDB 1L7P; Wang et al., 2002) reveals that Glu20 and Arg56 are particularly important residues with regard to the specificity of the enzymesubstrate interaction. Arg56 forms H-bonds between the side chain guanidinium group and the two carboxylate oxygens of PLS. Glu20 forms an H-bond with the substrate amino group. Additionally, Met43 and Phe49 are in van der Waals contact with the serine side chain. Neither of the aforementioned four residues are conserved in PHOSPHO1; we therefore conclude that PHOSPHO1 is not a member of the subfamily of PSPs but belongs to a novel, closely related enzyme group that utilize a different substrate.
One of the most testing problems in modern biochemistry is in the identification of the natural substrate of a novel enzyme. The range of potential substrate molecules for PHOSPHO1 is large. Phosphoethanolamine, pyridoxal 5'-phosphate and inorganic pyrophosphate are known substrates of TNAP (Whyte, 1994) and may also be substrates of PHOSPHO1, whilst the addition of ß-glycerolphosphate to bone marrow cells in culture increases their ability to mineralize (Coelho and Fernandes, 2000
). The utilization of a bulky substrate molecule such as a sugar phosphate or pyridoxal 5'-phosphate is supported by the observation that chicken PHOSPHO1 can hydrolyse p-nitrophenol phosphate (Farquharson et al., 2002
). Analysis of the binding pocket of our model supports the idea that PHOSPHO1 can accommodate a larger substrate than PSPs (Figure 4). Glu20 in MjPSP is replaced by the non-conserved Asn41 in human PHOSPHO1, and Arg56 is replaced by Tyr78, which is conserved within the PHOSPHO1 homologues, but not in the putative phosphatases from Drosophila and plants. Both replacements imply a slightly more spacious binding pocket.
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Acknowledgements |
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Received July 9, 2003; revised September 26, 2003; accepted October 21, 2003