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Glycosylation‐deficient mutations in tissue‐nonspecific alkaline phosphatase impair its structure and function and are linked to infantile hypophosphatasia
Author(s) -
Komaru Keiichi,
Satou Yasuhito,
AlShawafi Hiba A.,
NumaKinjoh Natsuko,
Sohda Miwa,
Oda Kimimitsu
Publication year - 2016
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13663
Subject(s) - hypophosphatasia , mutant , glycan , glycosylation , mutation , biology , alkaline phosphatase , mutagenesis , glycoprotein , biochemistry , microbiology and biotechnology , genetics , chemistry , gene , enzyme
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.