Identification of a non-purple tartrate-resistant acid phosphatase: an evolutionary link to Ser/Thr protein phosphatases?
© Schenk et al; licensee BioMed Central Ltd. 2008
Received: 9 June 2008
Accepted: 4 September 2008
Published: 4 September 2008
Tartrate-resistant acid phosphatases (TRAcPs), also known as purple acid phosphatases (PAPs), are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. The human enzyme is a major histochemical marker for the diagnosis of bone-related diseases. TRAcPs can occur as a small form possessing only the ~35 kDa catalytic domain, or a larger ~55 kDa form possessing both a catalytic domain and an additional N-terminal domain of unknown function. Due to its role in bone resorption the 35 kDa TRAcP has become a promising target for the development of anti-osteoporotic chemotherapeutics.
A new human gene product encoding a metallohydrolase distantly related to the ~55 kDa plant TRAcP was identified and characterised. The gene product is found in a number of animal species, and is present in all tissues sampled by the RIKEN mouse transcriptome project. Construction of a homology model illustrated that six of the seven metal-coordinating ligands in the active site are identical to that observed in the TRAcP family. However, the tyrosine ligand associated with the charge transfer transition and purple color of TRAcPs is replaced by a histidine.
The gene product identified here may represent an evolutionary link between TRAcPs and Ser/Thr protein phosphatases. Its biological function is currently unknown but is unlikely to be associated with bone metabolism.
Purple acid phosphatases (PAPs) are a diverse group of metalloenzymes that catalyse the hydrolysis of phosphate esters and anhydrides . PAPs are resistant to inhibition by L(+)-tartrate, a potent inhibitor of other acid phosphatases, and as such are also known as tartrate-resistant acid phosphatases (TRAcPs; alternative names include ACP5, TRAP) . They contain a bimetallic active site comprising seven coordinating amino acids that are conserved in all PAP isoforms identified to date [1, 2]. One metal site is invariably an Fe(III) and the characteristic purple color of TRAcPs arises from a tyrosine to Fe(III) charge transfer transition . The other site contains a divalent metal ion where M(II) = Fe, Zn or Mn depending on the source of the protein [1–5]. The X-ray crystal structures of TRAcPs from several sources, including human, pig, red kidney bean and sweet potato have been determined [6–9]. Notably, although their sequence identity is only < 20%, these enzymes have a common core structure with five motifs that contain the invariant seven metal coordinating amino acids in the catalytic site .
TRAcPs have been isolated from a range of plants, mammals and fungi, and TRAcP-like sequences have also been identified in a number of bacteria . Structural and biochemical characterisation of the TRAcPs from the red kidney bean, Phaseolus vulgaris, and sweet potato, Ipomoea batatas, have demonstrated their existence as homodimers with subunits of ~55 kDa [1, 5]. The plant isoforms may also exist as heterodimers of 57 and 63 kDa subunits . The catalytic centres of the red kidney bean, soybean and one isoform from sweet potato enzyme contain an Fe(III)-Zn(II) complex, whereas Fe(III)-Mn(II) is present in the other sweet potato form . Plant TRAcPs have been shown to exhibit an amino acid sequence similarity of > 70% . Mammalian TRAcPs have been characterised from multiple species including human, pig, cow, mouse and rat, and all exist as monomers of ~35 kDa, that share > 80% sequence identity and contain redox-active Fe(III)-Fe(III)/Fe(II) centers [2, 10]. A number of distinct TRAcP isoforms were identified in plants and bacteria, clearly illustrating the existence of multiple TRAcP genes in different kingdoms [1, 2]. This is further supported by the existence of a plantlike TRAcP in animals .
The biological roles for TRAcPs are diverse and species-dependent. Evidence has accumulated that links the mammalian enzymes to bone metabolism and bacterial killing, while plant enzymes maybe have a function in phosphate metabolism . Specifically, it could be shown that in transgenic mice the level of TRAcP expression correlates with the extent of bone resorption; TRAcP-knockout mice display symptoms characteristic for osteoporosis, while mice overexpressing TRAcP display an osteoporotic phenotype [11, 12]. TRAcP is a major histochemical marker for the diagnosis of bone-related diseases, and elevated serum concentrations of are also observed in patients with Paget's disease, osteosarcoma, breast and prostate cancer. Due to its role in bone resoption TRAcP has become a target for the development of anti-osteoporotic chemotherapeutics .
The design of such chemotherapeutics necessitates a high degree of specificity, in particular since enzymes closely related to TRAcPs may function in completely different roles in metabolism. We have thus extended our previous work on investigation of TRAcP and TRAcPlike protein content in animal genomes and identified a new gene product that is a remote homolog to both TRAcPs and Ser/Thr protein phosphatases.
Homolog identification and characterisation
The human TRAcP (ACP5) sequence (accession number NP_001602; unless stated otherwise accession codes are NCBI reference sequence numbers) was used to perform a five iteration PSI-BLAST search of the non-redundant database (the search conditions were the same as described previously ). This search identified a distantly related human sequence with the accession number NP_060810, that had 15% sequence identity and 29% similarity to the original acp5 query sequence. Related gene products from other eukaryotes were identified in the NCBI Homologene database http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene and ENSEMBL resources http://www.ensembl.org, and included Bos taurus (NP_001026941) Pan troglodytes (XP_001145620), Canis familiaris (XP_536969), Mus musculus (NP_666179), Rattus norvegicus (NP_001013985), Gallus gallus (XP_414732) and Plasmodium falciparum (XP_001348209) indicating that this new gene product is evolutionarily conserved. The new human sequence was used to query the nr database to search for the closest relative with known structure, and identified the catalytic domain of TRAcPs from red kidney bean (P. vulgaris), 4KBP , and sweet potato (I. batatas), 1XZW . Although sequence identities were low (18% across 246 residues as determined by PSI BLAST analysis) the E-values for the profile based search were 2 × 10-72 and 8 × 10-68 respectively, clearly indicating a significant relationship between these proteins and the novel sequence. Alternative transcripts for the mouse and human sequences were also included.
The intron-exon structure of the gene encoding this putative phosphatase (C530044N13Rik; ENSEMBL Gene ENSMUSG00000065979) comprises only 4 exons, spread over more than 100 kb of genomic DNA, a structure that is widely conserved in vertebrates. From analysis of RIKEN transcriptome data for the mouse homolog (GeneID 223978) using the CAGE analysis viewer http://fantom.gsc.riken.go.jp/, it is evident that the gene locus is actively transcribed in almost all tissues examined, including embryonic tissue as well as adult liver, lung, macrophages and neural tissue with little variation in CAGE Tag frequency (an index of gene expression). The promoter is conserved between mice and human, is relatively GC-rich, and initiates transcription at multiple sites in a 100 bp window around the site of the largest CAGE tag cluster, features consistent with a possible "housekeeping" gene function.
Structure prediction of Hsa _aTRAcP
To further assess the novel sequences as non-purple binuclear metallohydrolases, a structural model of Hsa _aTRAcP was constructed by comparative modelling using the sweet potato TRAcP coordinates . The only proteins with known structure identified from the PSI-BLAST search were the plant TRAcPs and the phosphodiesterase from Mycobacterium tuberculosis (Rv0805, 2HY1 ). An additional phosphodiesterase was identified from Enterobacter aerogenes (2dxn ), using the threading based approach mGenThreader . The prediction reliability scores for the bacterial diesterases were 114.5 and 105.8, respectively, with corresponding p-values (probabilities of false positives) of 1 × 10-10 and 1 × 10-9. These values are similar to those obtained for the closest TRAcP homologue, the enzyme from sweet potato (1XZW, with a reliability score of 87.3 and a p-value of 8 × 10-8. This strongly implies that Hsa _aTRAcP will adopt a fold similar to these proteins.
Previously we identified a high molecular weight human TRAcP . Here we have extended this study through the characterisation of a second transcript, Hsa _aTRACP, that is a remote relative of the PAPs, sharing 18% sequence identity with the plant enzymes as the closest relatives with known structure. Analysis of the active site of Hsa _aTRACP indicates that it is not likely to be a purple protein due to the absence of an essential tyrosine ligand (Figure 5). In this respect, Hsa _aTRACP resembles some cyclic nucleotide phosphodiesterases and novel Ser/Thr PPs. This may therefore represent an event of divergent evolution in the binuclear metallohydrolase family. Based upon the pattern of expression and putative cytoplasmic location, we speculate that Hsa _aTRACP is another member of the cytoplasmic protein phosphatase family that is likely to have a role in the regulation of signalling.
purple acid phosphatase
Ser/Thr protein phosphatase
tartrate-resistant acid phosphatase
root mean square deviation.
This work was funded by a grant from the Australian Research Council (DP0558652).
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