Possible interference between tissue-non-specific alkaline phosphatase with an Arg54→Cys substitution and a counterpart with an Asp277→Ala substitution found in a compound heterozygote associated with severe hypophosphatasia

Abstract

Tissue-non-specific alkaline phosphatase (TNSALP) with an Arg(54)-->Cys (R54C) or an Asp(277)-->Ala (D277A)substitution was found in a patient with hypophosphatasia [Henthorn,Raducha, Fedde, Lafferty and Whyte (1992) Proc. Natl. Acad. Sci. U.S.A.89, 9924-9928]. To examine effects of these missense mutations onproperties of TNSALP, the TNSALP mutants were expressed ectopically inCOS-1 cells. The wild-type TNSALP was synthesized as a 66-kDa endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form, and processed to an 80-kDa mature form, which is anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). Although the mutant proteins were found to be modified by GPI, digestion with phosphatidylinositol-specific phospholipase C, cell-surface biotinylation and immunofluorescence observation demonstrated that the cell-surface appearance of TNSALP (R54C) and TNSALP (D277A) was either almost totally or partially retarded respectively. The 66-kDa Endo H-sensitive band was the only form, and was rapidly degraded in the cells expressing TNSALP (R54C). In contrast with cells expressing TNSALP(R54C), where alkaline phosphatase activity was negligible, significant enzyme activity was detected and, furthermore, the 80-kDa mature form appeared on the surface of the cells expressing TNSALP (D277A). Analysis by sedimentation on sucrose gradients showed that a considerable fraction of newly synthesized TNSALP (R54C) and TNSALP(D277A) formed large aggregates, indicating improper folding and incorrect oligomerization of the mutant enzymes. When co-expressed with TNSALP (R54C), the level of the 80-kDa mature form of TNSALP (D277A)was decreased dramatically, with a concomitant reduction in enzyme activity in the co-transfected cell. These findings suggest that TNSALP(R54C) interferes with folding and assembly of TNSALP (D277A) intrans when expressed in the same cell, thus probably explaining why a compound heterozygote for these mutant alleles developed severe hypophosphatasia.

Novel aggregate formation of a frame‐shift mutant protein of tissue‐nonspecific alkaline phosphatase is ascribed to three cysteine residues in the C‐terminal extension

“…We therefore concluded that TNSALP (1559delT-C506S ⁄ C521S ⁄ C577S) formed a noncovalently assembled dimer similarly to sTNSALP (Fig. 8B) and the wild type enzyme [12,13]. As expected, TNSALP (1559delT-C506S ⁄ C521S ⁄ C577S) was secreted threefold more than TNSALP (1559delT) (Fig.…”

“…8). At first glance this finding was quite puzzling as several missense TNSALP mutant proteins (R54C, N153D, E218G, D289V and G317D), which form similar highmolecular-mass aggregates in the transfected cells, exhibit no enzyme activity [10][11][12][13][14]. However, the substitution of cysteines for serines at position of 506, 521 and 577 of TNSALP (1559delT) provided a clue.…”

“…To date, biochemical characterization of TNSALP mutant proteins have been limited to a small number of cases. Nevertheless, some missense mutations, in particular associated with severe forms of hypophophatasia, were known to impair proper folding and correct assembly of TNSALP in the endoplasmic reticulum (ER), resulting in the loss of functional TNSALP from the cell surface [10][11][12][13][14]. Deletion of T at position 1559 of the cDNA [TNS-ALP (1559delT)] of the TNSALP gene was reported for the first time in hypophosphatasia patients by Orimo et al [15] and TNSALP (1559delT) is transmitted as a recessive trait.…”

Characterization of Six Missense Mutations in the Tissue-Nonspecific Alkaline Phosphatase (TNSALP) Gene in Chinese Children with Hypophosphatasia

“…It was reported that the mutations p.A115V, p.A162T, p.D277A and p.F310L had residual TNSALP enzyme activity and were properly anchored on the membrane [20,21,22,23]. However, other mutations, including p.R54C, p.N153D, p.E218G, p.D289V, p.G317D and c.1559delT, did not exhibit any residual ALP activity, had defects in membrane-anchoring and seemed to accumulate in the RE/Golgi apparatus before degradation in the proteasome [21,24,25,26,27,28]. Kiminitsu Oda et al demonstrated that the p.A116T protein failed to fold properly and forms disulfide-bonded aggregates, though it was indeed capable of interacting with the WT and reaching the cell surface [18].…”

Tissue Nonspecific Alkaline Phosphatase Is Activated via a Two-step Mechanism by Zinc Transport Complexes in the Early Secretory Pathway

“…Reduced activity of TNAP caused by missense mutations in the TNAP gene causes hypophosphatasia, a heritable form of rickets and osteomalacia (21,22). Moreover, partial reduction of TNAP activity and impairment of osteoblast maturation is observed in osteoblasts differentiated in vitro from precursor cells prepared from Znt5 Ϫ/Ϫ mice (23).…”