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The structural pathology for hypophosphatasia caused by malfunctional tissue non-specific alkaline phosphatase.

ABSTRACT: Hypophosphatasia (HPP) is a metabolic bone disease that manifests as developmental abnormalities in bone and dental tissues. HPP patients exhibit hypo-mineralization and osteopenia due to the deficiency or malfunction of tissue non-specific alkaline phosphatase (TNAP), which catalyzes the hydrolysis of phosphate-containing molecules outside the cells, promoting the deposition of hydroxyapatite in the extracellular matrix. Despite the identification of hundreds of pathogenic TNAP mutations, the detailed molecular pathology of HPP remains unclear. Here, to address this issue, we determine the crystal structures of human TNAP at near-atomic resolution and map the major pathogenic mutations onto the structure. Our study reveals an unexpected octameric architecture for TNAP, which is generated by the tetramerization of dimeric TNAPs, potentially stabilizing the TNAPs in the extracellular environments. Moreover, we use cryo-electron microscopy to demonstrate that the TNAP agonist antibody (JTALP001) forms a stable complex with TNAP by binding to the octameric interface. The administration of JTALP001 enhances osteoblast mineralization and promoted recombinant TNAP-rescued mineralization in TNAP knockout osteoblasts. Our findings elucidate the structural pathology of HPP and highlight the therapeutic potential of the TNAP agonist antibody for osteoblast-associated bone disorders.

SUBMITTER: Yu Y

PROVIDER: S-EPMC10329691 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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The structural pathology for hypophosphatasia caused by malfunctional tissue non-specific alkaline phosphatase.

Yu Yating Y Rong Kewei K Yao Deqiang D Zhang Qing Q Cao Xiankun X Rao Bing B Xia Ying Y Lu Yi Y Shen Yafeng Y Yao Ying Y Xu Hongtao H Ma Peixiang P Cao Yu Y Qin An A

Nature communications 20230708 1

Hypophosphatasia (HPP) is a metabolic bone disease that manifests as developmental abnormalities in bone and dental tissues. HPP patients exhibit hypo-mineralization and osteopenia due to the deficiency or malfunction of tissue non-specific alkaline phosphatase (TNAP), which catalyzes the hydrolysis of phosphate-containing molecules outside the cells, promoting the deposition of hydroxyapatite in the extracellular matrix. Despite the identification of hundreds of pathogenic TNAP mutations, the d ...[more]

PMID: 37422472

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Project description:Tissue-non-specific alkaline phosphatase (TNSALP) is an ectoenzyme anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). A TNSALP mutant with an Asn(153)-->Asp (N153D) substitution was reported in a foetus diagnosed with perinatal hypophosphatasia (Mornet, Taillandier, Peyramaure, Kaper, Muller, Brenner, Bussiere, Freisinger, Godard, Merrer et al. (1998) Eur. J. Hum. Genet. 6, 308-314). When expressed ectopically in COS-1 cells, the wild-type TNSALP formed active non-covalently associated dimers, whereas TNSALP (N153D) formed aberrant disulphide-bonded high-molecular-mass aggregates devoid of enzyme activity. Cell-surface biotinylation and digestion with phosphatidylinositol-specific phospholipase C showed that TNSALP (N153D) failed to reach the cell surface. Instead, double immunofluorescence demonstrated that TNSALP (N153D) partially co-localized with a cis-Golgi marker (GM-130) at the steady-state. Upon treatment with brefeldin A, TNSALP (N153D) was still co-localized with GM-130, further supporting the finding that this mutant is localized in the cis-Golgi. Consistent with morphological results, pulse-chase experiments showed that newly synthesized TNSALP (N153D) remained endo-beta-N-acetylglucosaminidase H-sensitive throughout the chase. Eventually, after a prolonged chase time, the mutant was found to be partly degraded in a proteasome-dependent manner. Since the mutant TNSALP was significantly labelled with [3H]ethanolamine, a component of GPI, comparable with the wild-type enzyme, it is unlikely that the abortive synthesis of the mutant is due to a defect in GPI-attachment. Interestingly, when asparagine was replaced by glutamine at position 153 (N153D), TNSALP (N153Q) was indistinguishable from the wild-type enzyme in terms of its molecular properties, suggesting the possible importance of amino acids with a polar amide group at position 153. Taken together, these findings indicate that replacing asparagine with aspartic acid at position 153 causes misfolding and incorrect assembly of TNSALP, which results in its retention at the cis-Golgi en route to the cell surface, followed by a delayed degradation, presumably as part of a quality-control process. We postulate that the molecular basis of the perinatal hypophosphatasia associated with TNSALP (N153D) is due to the absence of mature TNSALP at the cell surface.

Project description:BackgroundTissue non-specific alkaline phosphatase (TNSALP; encoded by the ALPL gene) has a critical role in the postnatal regulation of phosphate homeostasis, yet how TNSALP activity and expression are regulated during pregnancy remain largely unknown. This study tested the hypothesis that progesterone (P4) and/or interferon tau (IFNT) regulate TNSALP activity during pregnancy in sheep.MethodsIn Exp. 1, ewes were bred and received daily intramuscular injections of either corn oil vehicle (CO) or 25 mg progesterone in CO (P4) for the first 8 days of pregnancy and were hysterectomized on either Day 9, 12, or 125 of gestation. In Exp. 2, ewes were fitted with intrauterine catheters on Day 7 of the estrous cycle and received daily intramuscular injections of 50 mg P4 in CO and/or 75 mg progesterone receptor antagonist (RU486) in CO from Days 8 to 15, and twice daily intrauterine injections of either control proteins (CX) or IFNT (25 µg/uterine horn/d) from Days 11 to 15 (treatment groups: P4 + CX; P4 + IFNT; RU486 + P4 + CX; and RU486 + P4 + IFNT) and were hysterectomized on Day 16.ResultsIn Exp. 1, endometria from ewes administered P4 had greater expression of ALPL mRNA than ewes administered CO on Day 12. TNSALP activity appeared greater in the epithelia, stratum compactum stroma, and endothelium of the blood vessels in the endometrium and myometrium from ewes administered P4 than ewes administered CO on Day 12. On Day 125, TNSALP activity localized to uterine epithelial and endothelial cells, independent of P4 treatment. TNSALP activity in placentomes appeared greater in P4 treated ewes and was detected in endothelial cells and caruncular tissue in P4 treated but not CO treated ewes. In Exp. 2, endometrial homogenates from ewes administered RU486 + P4 + CX had lower TNSALP activity those for P4 + CX and P4 + IFNT ewes. Immunoreactive TNSALP protein appeared greater in the mid- and deep-glandular epithelia in RU486 + P4 + CX treated ewes as compared to the other treatment groups. Enzymatic activity appeared greater on the apical surface of the deep glandular epithelia in endometria from ewes treated with RU486 + P4 + CX compared to the other treatment groups.ConclusionsThese results suggest that P4, but not IFNT, regulates the expression and activity of TNSALP in utero-placental tissues and has the potential to contribute to the regulation of phosphate availability that is critical for conceptus development during pregnancy.

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.

Project description:Tissue-non-specific alkaline phosphatase (TNSALP) with an Arg(54)-->Cys (R54C) or an Asp(277)-->Ala (D277A)substitution was found in a patient with hypophosphatasia [Henthorn,Raducha, Fedde, Lafferty and Whyte (1992) Proc. Natl. Acad. Sci. U.S.A.89, 9924-9928]. To examine effects of these missense mutations onproperties of TNSALP, the TNSALP mutants were expressed ectopically inCOS-1 cells. The wild-type TNSALP was synthesized as a 66-kDa endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form, and processed to an 80-kDa mature form, which is anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). Although the mutant proteins were found to be modified by GPI, digestion with phosphatidylinositol-specific phospholipase C, cell-surface biotinylation and immunofluorescence observation demonstrated that the cell-surface appearance of TNSALP (R54C) and TNSALP (D277A) was either almost totally or partially retarded respectively. The 66-kDa Endo H-sensitive band was the only form, and was rapidly degraded in the cells expressing TNSALP (R54C). In contrast with cells expressing TNSALP(R54C), where alkaline phosphatase activity was negligible, significant enzyme activity was detected and, furthermore, the 80-kDa mature form appeared on the surface of the cells expressing TNSALP (D277A). Analysis by sedimentation on sucrose gradients showed that a considerable fraction of newly synthesized TNSALP (R54C) and TNSALP(D277A) formed large aggregates, indicating improper folding and incorrect oligomerization of the mutant enzymes. When co-expressed with TNSALP (R54C), the level of the 80-kDa mature form of TNSALP (D277A)was decreased dramatically, with a concomitant reduction in enzyme activity in the co-transfected cell. These findings suggest that TNSALP(R54C) interferes with folding and assembly of TNSALP (D277A) intrans when expressed in the same cell, thus probably explaining why a compound heterozygote for these mutant alleles developed severe hypophosphatasia.

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The structural pathology for hypophosphatasia caused by malfunctional tissue non-specific alkaline phosphatase.

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The structural pathology for hypophosphatasia caused by malfunctional tissue non-specific alkaline phosphatase.

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