Molecular and cellular basis of hypophosphatasia.

BACKGROUND: Hypophosphatasia (HPP) is an inherited disorder characterized by defective mineralization of the bone and teeth that is also associated with a deficiency of serum alkaline phosphatase (ALP). Patients with HPP exhibit a broad range of symptoms including stillbirth with an unmineralized skeleton, premature exfoliation and dental caries in childhood, and pseudo-fractures in adulthood. The broad clinical spectrum of HPP is attributed to various mutations in the ALPL gene, which encodes tissue-nonspecific alkaline phosphatase (TNSALP). Nevertheless, the molecular mechanisms underlying the genotypic and phenotypic relationship of HPP remain unclear. HIGHLIGHT: The expression of HPP-related TNSALP mutants in mammalian cells allows us to determine for the effects of mutations on the properties of TNSALP, which could contribute to a better understanding of the relationship between structure and function of TNSALP. CONCLUSION: Molecular characterization of TNSALP mutants helps establish the etiology and onset of HPP.

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

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 N-glycans 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.

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

Hypophosphatasia (HPP) is a genetic disease characterized by defective calcification of hard tissues such as bone and teeth accompanying deficiency of serum alkaline phosphatase (ALP) activity. Its development results from various mutations in the ALPL gene encoding tissue-nonspecific ALP (TNSALP). HPP is known to be transmitted in an autosomal recessive or autosomal dominant manner. A point mutation (c.323C>T) in the ALPL gene leading to a proline to leucine substitution at position 108 of TNSALP was first reported in a patient diagnosed with odonto-HPP (M Herasse et al., J Med Genet 2003;40:605-609), although the effects of this mutation on the TNSALP molecule have not been elucidated. To understand the molecular basis of this dominantly transmitted HPP, we first characterized TNSALP (P108L) by expressing it in COS-1 cells transiently. In contrast to wild-type TNSALP (WT), TNSALP (P108L) showed virtually no ALP activity. When coexpressed with TNSALP (WT), TNSALP (P108L) significantly inhibited the enzyme activity of TNSALP (WT), confirming that this mutant TNSALP exerts a dominant negative effect on TNSALP (WT). Using immunofluorescence and digestion with phosphatidylinositol-specific phospholipase C, we demonstrated that TNSALP (P108L) was anchored to the cell surface via glycosylphosphatidylinositol-like TNSALP (WT) in a Tet-On CHO cell expression system. Consistent with this, TNSALP (P108L) acquired endo-ß-N-acetylglucosaminidase H resistance and sialic acids, as evidenced by glycosidase treatments. Importantly, TNSALP (WT) largely formed a functional dimeric structure, while TNSALP (P108L) was found to be present as a monomer in the cell. This indicates that the molecular structure of TNSALP is affected by a missense mutation at position 108, which is in contact with the active site, such that it no longer assembles into the functional dimeric form. Collectively, these results may explain why TNSALP (P108L) loses its ALP activity, even though it is able to gain access to the cell surface.