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Molecular evolution of the tissue-nonspecific alkaline phosphatase allows prediction and validation of missense mutations responsible for hypophosphatasia.

ABSTRACT: ALPL encodes the tissue nonspecific alkaline phosphatase (TNSALP), which removes phosphate groups from various substrates. Its function is essential for bone and tooth mineralization. In humans, ALPL mutations lead to hypophosphatasia, a genetic disorder characterized by defective bone and/or tooth mineralization. To date, 275 ALPL mutations have been reported to cause hypophosphatasia, of which 204 were simple missense mutations. Molecular evolutionary analysis has proved to be an efficient method to highlight residues important for the protein function and to predict or validate sensitive positions for genetic disease. Here we analyzed 58 mammalian TNSALP to identify amino acids unchanged, or only substituted by residues sharing similar properties, through 220 millions years of mammalian evolution. We found 469 sensitive positions of the 524 residues of human TNSALP, which indicates a highly constrained protein. Any substitution occurring at one of these positions is predicted to lead to hypophosphatasia. We tested the 204 missense mutations resulting in hypophosphatasia against our predictive chart, and validated 99% of them. Most sensitive positions were located in functionally important regions of TNSALP (active site, homodimeric interface, crown domain, calcium site, …). However, some important positions are located in regions, the structure and/or biological function of which are still unknown. Our chart of sensitive positions in human TNSALP (i) enables to validate or invalidate at low cost any ALPL mutation, which would be suspected to be responsible for hypophosphatasia, by contrast with time consuming and expensive functional tests, and (ii) displays higher predictive power than in silico models of prediction.

SUBMITTER: Silvent J

PROVIDER: S-EPMC4148848 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Molecular evolution of the tissue-nonspecific alkaline phosphatase allows prediction and validation of missense mutations responsible for hypophosphatasia.

Silvent Jérémie J Gasse Barbara B Mornet Etienne E Sire Jean-Yves JY

The Journal of biological chemistry 20140714 35

ALPL encodes the tissue nonspecific alkaline phosphatase (TNSALP), which removes phosphate groups from various substrates. Its function is essential for bone and tooth mineralization. In humans, ALPL mutations lead to hypophosphatasia, a genetic disorder characterized by defective bone and/or tooth mineralization. To date, 275 ALPL mutations have been reported to cause hypophosphatasia, of which 204 were simple missense mutations. Molecular evolutionary analysis has proved to be an efficient met ...[more]

PMID: 25023282

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Project description:Tissue-nonspecific alkaline phosphatase (TNAP) is an enzyme present on the surface of mineralizing cells and their derived matrix vesicles that promotes hydroxyapatite crystal growth. Hypophosphatasia (HPP) is an inborn-error-of-metabolism that, dependent upon age of onset, features rickets or osteomalacia due to loss-of function mutations in the gene (Alpl) encoding TNAP. Craniosynostosis is prevalent in infants with HPP and other forms of rachitic disease but how craniosynostosis develops in these disorders is unknown.Because craniosynostosis carries high morbidity, we are investigating craniofacial skeletal abnormalities in Alpl(-/-) mice to establish these mice as a model of HPP-associated craniosynostosis and determine mechanisms by which TNAP influences craniofacial skeletal development.Cranial bone, cranial suture and cranial base abnormalities were analyzed by micro-CT and histology. Craniofacial shape abnormalities were quantified using digital calipers. TNAP expression was suppressed in MC3T3E1(C4) calvarial cells by TNAP-specific shRNA. Cells were analyzed for changes in mineralization, gene expression, proliferation, apoptosis, matrix deposition and cell adhesion.Alpl(-/-) mice feature craniofacial shape abnormalities suggestive of limited anterior-posterior growth. Craniosynostosis in the form of bony coronal suture fusion is present by three weeks after birth. Alpl(-/-) mice also exhibit marked histologic abnormalities of calvarial bones and the cranial base involving growth plates, cortical and trabecular bone within two weeks of birth. Analysis of calvarial cells in which TNAP expression was suppressed by shRNA indicates that TNAP deficiency promotes aberrant osteoblastic gene expression, diminished matrix deposition, diminished proliferation, increased apoptosis and increased cell adhesion.These findings demonstrate that Alpl(-/-) mice exhibit a craniofacial skeletal phenotype similar to that seen in infants with HPP, including true bony craniosynostosis in the context of severely diminished bone mineralization. Future studies will be required to determine if TNAP deficiency and other forms of rickets promote craniosynostosis directly through abnormal calvarial cell behavior, or indirectly due to deficient growth of the cranial base.

Project description:Tissue-nonspecific alkaline phosphatase (TNAP) is necessary for skeletal mineralization by its ability to hydrolyze the mineralization inhibitor inorganic pyrophosphate (PPi), which is mainly generated from extracellular ATP by ectonucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Since children with TNAP deficiency develop bone metaphyseal auto-inflammations in addition to rickets, we hypothesized that TNAP also exerts anti-inflammatory effects relying on the hydrolysis of pro-inflammatory adenosine nucleotides into the anti-inflammatory adenosine. We explored this hypothesis in bone metaphyses of 7-day-old Alpl+/- mice (encoding TNAP), in mineralizing hypertrophic chondrocytes and osteoblasts, and non-mineralizing mesenchymal stem cells (MSCs) and neutrophils, which express TNAP and are present, or can be recruited in the metaphysis. Bone metaphyses of 7-day-old Alpl+/- mice had significantly increased levels of Il-1? and Il-6 and decreased levels of the anti-inflammatory Il-10 cytokine as compared with Alpl+/+ mice. In bone metaphyses, murine hypertrophic chondrocytes and osteoblasts, Alpl mRNA levels were much higher than those of the adenosine nucleotidases Npp1, Cd39 and Cd73. In hypertrophic chondrocytes, inhibition of TNAP with 25 ?M of MLS-0038949 decreased the hydrolysis of AMP and ATP. However, TNAP inhibition did not significantly modulate ATP- and adenosine-associated effects in these cells. We observed that part of TNAP proteins in hypertrophic chondrocytes was sent from the cell membrane to matrix vesicles, which may explain why TNAP participated in the hydrolysis of ATP but did not significantly modulate its autocrine pro-inflammatory effects. In MSCs, TNAP did not participate in ATP hydrolysis nor in secretion of inflammatory mediators. In contrast, in neutrophils, TNAP inhibition with MLS-0038949 significantly exacerbated ATP-associated activation and secretion of IL-1?, and extended cell survival. Collectively, these results demonstrate that TNAP is a nucleotidase in both hypertrophic chondrocytes and neutrophils, and that this nucleotidase function is associated with autocrine effects on inflammation only in neutrophils.

Project description:Tissue-nonspecific alkaline phosphatase (TNALP) is a glycoprotein expressed by osteoblasts that promotes bone mineralization. TNALP catalyzes the hydrolysis of the mineralization inhibitor inorganic pyrophosphate and ATP to provide inorganic phosphate, thus controlling the inorganic pyrophosphate/inorganic phosphate ratio to enable the growth of hydroxyapatite crystals. N-linked glycosylation of TNALP is essential for protein stability and enzymatic activity and is responsible for the presence of different bone isoforms of TNALP associated with functional and clinical differences. The site-specific glycosylation profiles of TNALP are, however, elusive. TNALP has 5 potential N-glycosylation sites located at the asparagine (N) residues 140, 230, 271, 303, and 430. The objective of this study was to reveal the presence and structure of site-specific glycosylation in TNALP expressed in osteoblasts. Calvarial osteoblasts derived from Alpl+/- expressing SV40 Large T antigen were transfected with soluble epitope-tagged human TNALP. Purified TNALP was analyzed with a lectin microarray, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and liquid chromatography with tandem mass spectrometry. The results showed that all sites (n = 5) were fully occupied predominantly with complex-type N-glycans. High abundance of galactosylated biantennary N-glycans with various degrees of sialylation was observed on all sites, as well as glycans with no terminal galactose and sialic acid. Furthermore, all sites had core fucosylation except site N271. Modelling of TNALP, with the protein structure prediction software ColabFold, showed possible steric hindrance by the adjacent side chain of W270, which could explain the absence of core fucosylation at N271. These novel findings provide evidence for N-linked glycosylation on all 5 sites of TNALP, as well as core fucosylation on 4 out of 5 sites. We anticipate that this new knowledge can aid in the development of functional and clinical assays specific for the TNALP bone isoforms.

Project description:We analyzed polymorphism of the ALPL gene in patients with low serum levels of tissue-nonspecific alkaline phosphatase (TNAP). The presence of three or more of the less frequent alleles of ALPL polymorphisms was associated with significantly lower TNAP serum level and higher frequencies of metatarsal fractures, which may help confirm a clinical suspicion of adult hypophosphatasia.IntroductionAlkaline phosphatases (ALPs) are membrane-bound enzymes that hydrolyze monophosphate esters at a high pH (pH 8-10). Inorganic pyrophosphate, pyridoxal 5-phosphate, the activated form of vitamin B6 (PLP), and phosphoethanolamine (PEA), are natural substrates of ALPs. Hypophosphatasia (HPP, OMIM 146300, 241500, 241510) is a heterogeneous rare metabolic bone disease caused by loss-of-function mutations in the tissue-nonspecific alkaline phosphatase gene (ALPL; MIM 171760) with a deficiency of TNAP. Clinical presentation of HPP in adults demonstrated a wide range of manifestations, many of which are nonspecific. In the present study, we screened the polymorphic genetic variants of ALPL in 56 subjects presenting low serum levels of TNAP and/or other clinical signs of adult HPP in order to evaluate a possible role of polymorphic variants in the diagnosis and management of HPP in adults.MethodsGenomic DNA was extracted from peripheral blood and ALPL gene was sequenced by PCR-based Sanger technique.ResultsFourteen different polymorphic variants were found in the study population. A lower serum level of TNAP and higher frequencies of metatarsal fractures were observed in patients bearing three or more of the minor frequency alleles (MFAs) of the ALPL polymorphic variants. The presence of some MFAs, mostly as a contemporary presence of three or more of them, was found to be mainly represented in patients having both a significantly lower level of TNAP and a higher level of vitamin B6.ConclusionThe genetic analysis and presence of some polymorphic variants may be an instrument to confirm clinical and biochemical data, consider adult HPP, and help clinicians be cautious in the administration of anti-reabsorption drugs.

Dataset Information

Molecular evolution of the tissue-nonspecific alkaline phosphatase allows prediction and validation of missense mutations responsible for hypophosphatasia.

Publications

Molecular evolution of the tissue-nonspecific alkaline phosphatase allows prediction and validation of missense mutations responsible for hypophosphatasia.

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