Molecular phenotype of tissue-nonspecific alkaline phosphatase with a proline (108) to leucine substitution associated with dominant odontohypophosphatasia

Numa-Kinjoh, Natsuko; Komaru, Keiichi; Ishida, Yasushi; Sohda, Miwa; Oda, Kimimitsu

doi:10.1016/j.ymgme.2015.05.006

Cited by 10 publications

(5 citation statements)

References 28 publications

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“…2A, bottom), suggesting that even TNSALP (N430Q) reached the cell surface. We previously reported that dimerization was not obligatory for TNSALP to reach the cell surface and the mature monomeric form of TNSALP was able to access the cell surface [11][12][13]. Thus, it is highly likely that the mature monomeric form of TNSALP (N430Q) appeared on the cell surface.…”

Section: Resultsmentioning

confidence: 99%

“…The individual N ‐glycan deletion mutants of TNSALP were then analyzed by SDS/PAGE. Throughout the study, we used two types of SDS/PAGE: (a) conventional SDS/PAGE, in which TNSALP (WT) migrates as a dissociated monomer, and (b) recently developed SDS/PAGE specifically for ALPs , in which TNSALP (WT) retains its enzyme activity as a functional dimer in the presence of zinc ions. The immature and mature forms of TNSALP (WT) were both detected by conventional SDS/PAGE, as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Protein samples (5 μg of each homogenate) were reduced by heating at 95 °C for 5 min in the presence of 1% (v/v) 2‐mercaptoethanol (for conventional SDS/PAGE) or incubated with 0.5% SDS at pH 8.8 at 37 °C for 30 min prior to modified SDS/PAGE in the presence of zinc ions as described previously . Proteins were analyzed on a 7.5% (conventional) or 10% (modified) SDS/polyacrylamide gel, followed by electric transfer and western blotting using anti‐TNSALP antibody (dilution 1 : 4000) and peroxidase‐conjugated sheep anti‐(rabbit IgG) (dilution 1 : 4000) .…”

Section: Methodsmentioning

confidence: 99%

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Glycosylation‐deficient mutations in tissue‐nonspecific alkaline phosphatase impair its structure and function and are linked to infantile hypophosphatasia

Komaru¹,

Satou

Al-Shawafi

et al. 2016

The FEBS Journal

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Tissue-nonspecific alkaline phosphatase (TNSALP) is a membrane glycoprotein with a proposed role in bone mineralization. Indeed, mutations in TNSALP have been identified in patients with hypophosphatasia (HPP), a genetic disease characterized by hypomineralization of bone and teeth and a deficiency in serum ALP activity. TNSALP has five potential N-glycosylation sites at N140, N230, N271, N303 and N430 by standard nomenclature. A mutation at one of these sites, N430, was recently detected in a patient with infantile HPP. Using site-directed mutagenesis, we demonstrated that TNSALP has five N-glycans in transfected COS-1 cells and that individual single N-glycan deletion mutants of TNSALP retain the dimeric structure required for ALP activity, excluding the possibility that any single N-glycan plays a vital role in the structure and function of TNSALP. However, we found that TNSALP (N430Q) and TNSALP (N430E) mutants, but not a TNSALP (N430D) mutant, failed to form dimers. The TNSALP (N430S) mutant linked to infantile HPP was glycosylation-defective and unable to dimerise, similar to TNSALP (N430Q) and TNSALP (N430E) mutants; therefore, TNSALP (N430S) was established as a severe allele without strong ALP activity. By contrast to individual single N-glycan deletion mutants, TNSALP devoid of all five Nglycans was present to a much lesser extent than wild-type TNSALP in transfected cells, possibly reflecting its instability. A comprehensive analysis of a series of multiple N-glycan depletion mutants in TNSALP revealed that three N-glycans on N230, N271 and N303 were the minimal requirement for the structure and function of TNSALP and a prerequisite for its stable expression in a cell.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Glycosylation‐deficient mutations in tissue‐nonspecific alkaline phosphatase impair its structure and function and are linked to infantile hypophosphatasia

Komaru¹,

Satou

Al-Shawafi

et al. 2016

The FEBS Journal

Self Cite

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“…In the present study, after locating the identified mutations in 3D structure of the TNSALP, whose crystallographic co-ordinates have been determined by Mornet et al [ 19 ] based on human placental ALP, we found that the single heterozygous mutations R136H, Y388H, and V459A were located in the active site, the crown domain, and the homodimer interface, respectively ( Figure 3 ). C201 is one of the cysteine residues (C139–C201, C489–C497), which is essential for proper folding of monomeric TNSALP [ 21 ]. Mornet et al [ 19 ] revealed that cysteine residues at position 201 covalently link to 139 to form disulphide bond (C-139–C-201).…”

Section: Discussionmentioning

confidence: 99%

Four novel mutations in the ALPL gene in Chinese patients with odonto, childhood, and adult hypophosphatasia

Pang

Jiang

et al. 2018

Bioscience Reports

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Hypophosphatasia (HPP) is a rare inherited disorder characterized by defective bone and/or dental mineralization, and decreased serum alkaline phosphatase (ALP) activity. ALPL, the only gene related with HPP, encodes tissue non-specific ALP (TNSALP). Few studies were carried out in ALPL gene mutations in the Chinese population with HPP. The purpose of the present study is to elucidate the clinical and genetic characteristics of HPP in five unrelated Chinese families and two sporadic patients. Ten clinically diagnosed HPP patients from five unrelated Chinese families and two sporadic patients and fifty healthy controls were genetically investigated. All 12 exons and exon–intron boundaries of the ALPL gene were amplified by PCR and directly sequenced. The laboratory and radiological investigations were conducted simultaneously in these HPP ten patients. A 3D model of the TNSALP was used to predict the dominant negative effect of identified missense mutations. Three odonto, three childhood, and four adult types of HPP were clinically diagnosed. Ten mutations were identified in five unrelated Chinese families and two sporadic patients, including eight missense mutations and two frameshift mutations. Of which, four were novel: one frameshift mutation (p.R138Pfsx45); three missense mutations (p.C201R, p.V459A, p.C497S). No identical mutations and any other new ALPL mutations were found in unrelated 50 healthy controls. Our study demonstrated that the ALPL gene mutations are responsible for HPP in these Chinese families. These findings will be useful for clinicians to improve understanding of this heritable bone disorder.

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“…The human ALP peptide is synthesized as a native protein with a molecular weight of 57 kDa and it is then modified in the endoplasmic reticulum and in the Golgi apparatus, with the addition of sugar chains, until it reaches the mature form of about 80 kDa. The functional ALP enzyme is assumed to exist as a homodimer with a molecular weight of about 165-200 kDa [29][30][31]. Similarly, osteonectin is detectable as two different fragments, where the 45 kDa fragment represents the active form [32].…”

Section: Arbutin Cytocompatibility and Antioxidant Activitymentioning

confidence: 99%

Insights into Arbutin Effects on Bone Cells: Towards the Development of Antioxidant Titanium Implants

et al. 2020

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Arbutin is a plant-derived glycosylated hydroquinone with antioxidant features, exploited to combat cell damage induced by oxidative stress. The latter hinders the osseointegration of bone prostheses, leading to implant failure. Little is known about arbutin antioxidant effects on human osteoblasts, therefore, this study explores the in vitro protective role of arbutin on osteoblast-like cells (Saos-2) and periosteum-derived progenitor cells (PDPCs). Interestingly, cells exposed to oxidative stress were protected by arbutin, which preserved cell viability and differentiation. Starting from these encouraging results, an antioxidant coating loaded with arbutin was electrosynthesized on titanium. Therefore, for the first time, a polyacrylate-based system was designed to release the effective concentration of arbutin in situ. The innovative coating was characterized from the physico-chemical and morphological point of view to achieve an optimized system, which was in vitro tested with cells. Morpho-functional evaluations highlighted the high viability and good compatibility of the arbutin-loaded coating, which also promoted the expression of PDPC differentiation markers, even under oxidative stress. These results agreed with the coatings’ in vitro antioxidant activity, which showed a powerful scavenging effect against DPPH radicals. Taken together, the obtained results open intriguing opportunities for the further development of natural bioactive coatings for orthopedic titanium implants.

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Molecular phenotype of tissue-nonspecific alkaline phosphatase with a proline (108) to leucine substitution associated with dominant odontohypophosphatasia

Cited by 10 publications

References 28 publications

Glycosylation‐deficient mutations in tissue‐nonspecific alkaline phosphatase impair its structure and function and are linked to infantile hypophosphatasia

Glycosylation‐deficient mutations in tissue‐nonspecific alkaline phosphatase impair its structure and function and are linked to infantile hypophosphatasia

Four novel mutations in the ALPL gene in Chinese patients with odonto, childhood, and adult hypophosphatasia

Insights into Arbutin Effects on Bone Cells: Towards the Development of Antioxidant Titanium Implants

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