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 Nglycans 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.

Deglycosylation Differentially Regulates Weaned Porcine Gut Alkaline Phosphatase Isoform Functionality along the Longitudinal Axis

“…The N - and O -glycans of glycoproteins are participated in protein folding, trafficking, function and stability [ 32 , 33 ]. The N - and O -glycan chains of AP isoforms are synthesized with various glycosidases and glycosyltransferases by adding monosaccharides in the path from the endoplasmic reticulum (ER) and Golgi apparatus to the apical membrane anchored via glycosylphosphatidylinositol (GPI) [ 30 , 59 , 62 ].…”

“…The bone AP isoforms (B1, B2, and B/I) differed in N -linked glycans, which resulted in significantly different catalytic properties [ 32 ]. A study using site-directed mutagenesis shown that the dimeric structure required for TNAP activity remained in individual single N -glycan deletion mutants [ 33 ]. The infantile hypophosphatasia was linked to TNAP mutants (N430S) that were glycosylation-defective and unable to dimerize; and the N -glycans on N230, N271 and N303 were the minimal requirement for the structure, function and stable expression of TNAP in a cell [ 33 ].…”

“…A study using site-directed mutagenesis shown that the dimeric structure required for TNAP activity remained in individual single N -glycan deletion mutants [ 33 ]. The infantile hypophosphatasia was linked to TNAP mutants (N430S) that were glycosylation-defective and unable to dimerize; and the N -glycans on N230, N271 and N303 were the minimal requirement for the structure, function and stable expression of TNAP in a cell [ 33 ]. Thus, different glycosylation sites may impact the AP enzyme structure and functionality in many ways and different AP isoforms may respond to N - or O -glycosylation differentially.…”

“…Glycosylation variation in causing hypophosphatasia in chronic renal and bone diseases resulted from different catalytic properties of TNAP isoforms [ 32 ]. A study using site-directed mutagenesis indicated that human TNAP N -glycans at specific sites were critical in dimerization that was associated with infantile hypophosphatasia and stable expression of functional TNAP [ 33 ]. Bublitz et al [ 34 ] examined roles of N -glycosylation in calf enterocytic vesicle-mediated exocytotic export of AP into gut lumen; however, they did not find any link to AP functionality.…”

Biochemical and functional properties of mammalian bone alkaline phosphatase isoforms during osteogenesis

“…It has later been confirmed that all TNALPs are N-glycosylated and that bone and kidney ALP are O-glycosylated but not LALP [82,134]. The two sites in GCALP are occupied by N-glycans and deletion of either one, or two of these glycans, do not affect the enzymatic activity of GCALP [135].…”