title: Glycosylation-deficient mutations in tissue-nonspecific alkaline phosphatase impair its structure and function and are linked to infantile hypophosphatasia.
abstract: 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.
authors.affiliation: Kitasato Junior College of Health and Hygienic Sciences, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Komaru
authors.affiliation: Division of Oral Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Japan.
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Satou
authors.affiliation: Division of Oral Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Japan.
authors.forename: Hiba A
authors.initials: HA
authors.lastname: Al-Shawafi
authors.affiliation: Division of Oral Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Japan.
authors.forename: Natsuko
authors.initials: N
authors.lastname: Numa-Kinjoh
authors.affiliation: Division of Oral Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Japan.
authors.forename: Miwa
authors.initials: M
authors.lastname: Sohda
authors.affiliation: Division of Oral Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Japan.
authors.forename: Kimimitsu
authors.initials: K
authors.lastname: Oda
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D015971:Gene Expression Regulation, Enzymologic; D006031:Glycosylation; D006801:Humans; D007014:Hypophosphatasia / Q000201:enzymology* / Q000235:genetics*; D007223:Infant; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020125:Mutation, Missense; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; C548267:ALPL protein, human; D000469:Alkaline Phosphatase
keywords: N-glycan; dimerization; enzyme activity; hypophosphatasia; tissue-nonspecific alkaline phosphatase