Disulfide bonds are critical for tissue-nonspecific alkaline phosphatase function revealed by analysis of mutant proteins bearing a C201-Y or C489-S substitution associated with severe hypophosphatasia

Abstract

Hypophosphatasia (HPP), a rare genetic disease characterized by reduced serum alkaline phosphatase (ALP) activity and failure in bone and tooth mineralization, is caused by mutations in tissue-nonspecific ALP (TNSALP) gene. Two missense mutations (C201Y and C489S, standardized nomenclature) of TNSALP, involved in intra-chain disulfide bonds, were reported in patients diagnosed with perinatal HPP (Taillandier A. et al. Hum. Mutat. 13 (1999) 171-172, Hum. Mutat. 15 (2000) 293). To investigate the role of the disulfide bond in TNSALP, we expressed TNSALP (C201Y) and TNSALP (C489S) in COS-1 cells transiently. Compared with the wild-type enzyme [TNSALP (W)], both the TNSALP mutants exhibited a diminished ALP activity in the cells, where a 66kDa immature form was predominant with a marginal amount of a 80kDa mature form of TNSALP. Detailed studies on Tet-On CHO established cell line expressing TNSALP (W) or TNSALP (C201Y) showed that the 66kDa form of TNSALP (C201Y) exists as a monomer in contrast to a dimer of TNSALP (W). Only a small fraction of the TNSALP (C201Y) reached cell surface as the 80kDa mature form, though most of the 66kDa form was found to be endo-β-N-acetylglucosaminidase H sensitive and rapidly degraded in proteasome following polyubiquitination. Collectively, these results indicate not only that the intra-subunit disulfide bonds are crucial for TNSALP to properly fold and assemble into the dimeric enzyme, but also that the development of HPP associated with TNSALP (C201Y) or TNSALP (C489S) is attributed to decreased cell surface appearance of the functional enzyme.

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

“…Attempts have been made to understand the molecular mechanism by which loss-of-function mutations in the ALPL gene culminate in the development of HPP by expressing and examining various TNSALP mutant proteins produced in vitro in cultured cells [7][8][9][10][11][12][13][14][15][16][17]. However, even though there are an increasing number of dominantly inherited TNSALP mutations, especially in milder HPP [6,14,18], their molecular study has been limited to a small number of cases to date [19][20][21][22].…”

“…However, high-molecular-mass disulfide-bonded aggregates were detected in the cells expressing TNSALP (A116T) under non-reducing conditions (Fig. 4A), indicating that two intrasubunit disulfide crosslinks, which are required for proper folding for the subunit of TNSALP [13], were perturbed, albeit to a lesser amount. These aggregates were completely absent from the cells producing TNSALP (P108L) as well as TNSALP (WT).…”

“…In theory, when the transcription of the ALPL gene is not impaired, these loss-of-function mutations could affect any biosynthetic step of TNSALP from the ER to the cell membrane, resulting in diminished and/or altered cell surface ALP activity. Studies in vitro in cultured cells have indicated that the cell surface localization of TNSALP mutant proteins is retarded to various degrees due to improper folding and resultant perturbed protein trafficking [7][8][9][10][11][12][13][14]. Some TNSALP mutant proteins are retained in the ER or cis-Golgi and eventually subjected to ER-associated degradation [10,11,13].…”

“…Studies in vitro in cultured cells have indicated that the cell surface localization of TNSALP mutant proteins is retarded to various degrees due to improper folding and resultant perturbed protein trafficking [7][8][9][10][11][12][13][14]. Some TNSALP mutant proteins are retained in the ER or cis-Golgi and eventually subjected to ER-associated degradation [10,11,13]. Even though some TNSALP mutants are able to reach the cell membrane like TNSALP (WT), it is likely that they exhibit diminished ALP activity and/or altered catalytic properties [15,16].…”

“…Unless otherwise stated, 5 μg of the cell homogenate was applied to western blotting. Cytochemical staining [10], ALP assay [7,11], immunofluorescence [11], western blotting [10,13], enzyme digestions using phosphatidylinositol-specific phospholipase C (PI-PLC) [20,21], or glycosidase such as endo-β-N-acetylglucosaminidase H (End H), neuraminidase (N), and peptide-N-glycosidase F (PNGase F) [13,21] were carried out as described previously. Antibody against human TNSALP was raised in rabbits [24].…”