Mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene cause hypophosphatasia (HPP), an inborn error of metabolism characterized by defects in bone and teeth mineralization accompanying subnormal levels of serum alkaline phosphatase activity. Missense mutations at position 420 of TNSALP (standard nomenclature), which convert glycine to serine [TNSALP (G420S)] or alanine [TNSALP (G420A)], have been reported in perinatal and childhood HPP, respectively. When expressed in COS-1 cells, both TNSALP mutants were indistinguishable from wild-type TNSALP [TNSALP (W)] as evidenced by immunofluorescence and western blotting. Nevertheless, the two TNSALP mutants did not show substantial alkaline phosphatase activity. In agreement with transiently transfected cells, TNSALP (G420S) expressed in a Tet-On inducible expression system lacked its alkaline phosphatase activity, although this mutant was anchored to the cell surface lipid bilayers by glycosylphosphatidylinositol as an 80 kDa mature form bearing complex-type oligosaccharides like TNSALP (W). Importantly, TNSALP (G420S) was found to largely fail to assemble into the homodimer in contrast to TNSALP (W). Taken together, these results demonstrate that the glycine residue at position 420 is crucial for the subunit interaction of TNSALP and hence its catalytic function without affecting trafficking of monomeric TNSALP. We conclude that the dimerization defect is the molecular basis for perinatal HPP associated with the genotype G420S/G420S.
peptide N-glycosidase F (Flavobacterium meningosepticum)
TNSALP with a glycine to alanine substitution at position 420
TNSALP with a glycine to serine substitution at position 420
tissue-nonspecific alkaline phosphatase
There are four alkaline phosphatase (ALP) isozymes (EC 184.108.40.206) in humans: tissue-nonspecific, intestinal, placental and germ cell. Hypophosphatasia (HPP) is a rare inherited disorder resulting from loss-of-function mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene [1-4]. Clinical manifestations are variable, from stillbirth without mineralized bone to dental defects and pseudofractures developing late in adulthood, and are associated with decreased levels of serum ALP [1, 2]. It is customary to classify the patients into five main forms depending on the age at diagnosis: perinatal, infantile, childhood, adult and odonto. Generally, severe HPP (perinatal and infantile forms) is inherited in an autosomal recessive pattern, while the inheritance patterns for mild HPP (childhood, adult and odonto forms) are regarded as autosomal recessive or dominant conditions [1-3].
It has been well established that inorganic pyrophosphate, phosphoethanolamine and pyridoxal-5′-phosphate are natural substrates of TNSALP [1-4], as they are known to increase in the serum and/or urine of HPP patients. Elevated levels of three phospho compounds have been reported in TNSALP-deficient mice [5-7]. Mutations in the TNSALP gene could decrease the amount of the TNSALP molecule and even change the catalytic property of the TNSALP molecule on the matrix vesicle such that an accumulation of inorganic pyrophosphate, a crystal poison of hydroxyapatite formation, hampers calcification in bone matrix, leading to the hypomineralization seen in HPP patients [1-3, 8].
During co-translational translocation across the endoplasmic reticulum (ER) membrane by virtue of a signal sequence 17 amino acids in length, TNSALP properly folds and correctly assembles into the functional homodimer in the ER . Simultaneously, TNSALP is modified by N-linked oligosaccharides and glycosylphosphatidylinositol (GPI) at its carboxyl terminus and becomes a GPI-anchored membrane glycoprotein . On the way to the cell surface by way of the Golgi apparatus, TNSALP undergoes oligosaccharide processing to acquire endo-β-N-acetylglucosaminidase H resistance (complex-type oligosaccharides) and eventually is exposed on the cell membrane [9, 10].
In all, 261 TNSALP mutations have been reported worldwide as of July 2012 and the most prevailing mutation type is a missense mutation (76% of all mutations) (http://www.sesep.uvsq.fr/03_hypo_mutations.php). Mutations in HPP can affect any steps in protein folding, modifications and trafficking of TNSALP. TNSALP is converted to a GPI-anchorless secretory form with an extension at the carboxyl terminus in a frameshift TNSALP mutant (L520RfsX86) . Trafficking to the cell surface was perturbed to various degrees [12-17], with some severe mutations resulting in ER associated degradation . TNSALP (N170D) was found to accumulate at the cis-Golgi, instead of the ER, before degradation . Despite their cell surface localization, TNSALP (V423A) and TNSALP (R450C) showed reduced catalytic activity probably due to local structural hindrances of the crown domain caused by missense mutations [9, 10]. Interestingly, the association with wild-type TNSALP [TNSALP (W)] results in the retention of TNSALP (G249V), but not TNSALP (A116T), at the Golgi in the cases of dominantly transmitted mutations [18, 19].
The advent of a 3D structural model of TNSALP , which was constructed on the basis of X-ray crystallography of human placental ALP , has helped us to understand the effects of mutations on the TNSALP molecule by allowing us to map each missense mutation to different structural domains (active site or active site vicinity, active site valley, homodimer interface, crown domain, calcium site or calcium site vicinity, and others). Two missense mutations [TNSALP (G420S), TNSALP (G420A)] have been reported in a homozygous patient (G420S) and a compound heterozygote (G420A/E191K), who were diagnosed with perinatal and childhood HPP, respectively (http://www.sesep.uvsq.fr/03_hypo_mutations.php) . As glycine at position 420 [glycine (420)] of TNSALP is localized in the overlapping zone of the interface and crown domains (interfacial crown domain), it is of interest to see the effects of the replacement of glycine (420) with serine or alanine on the structure and function of TNSALP. However, little research has been conducted to investigate how missense mutations in this particular domain affect the TNSALP molecule. In this report, we show that both TNSALP (G420S) and TNSALP (G420A) are capable of reaching the cell membrane of transiently transfected cells like TNSALP (W), although both TNSALP mutants failed to exhibit substantial ALP activity. Moreover, a detailed study using Tet-On inducible CHO cells expressing TNSALP (G420S) demonstrated that this mutant was present as a monomer lacking ALP activity in contrast to the functional dimeric structure of TNSALP (W).
Expression of TNSALP (G420S) and TNSALP (G420A) in COS-1 cells
TNSALP (G420S) and TNSALP (G420A) were reported in patients diagnosed with perinatal HPP and childhood HPP, respectively (http://www.sesep.uvsq.fr/03_hypo_mutations.php) . To examine the effects of missense mutations at position 420 of TNSALP, we transfected COS-1 cells with pSG5 vector encoding TNSALP (G420S) or TNSALP (G420A). After 24 h, cells were processed for cytochemical staining for ALP activity as shown in Fig. 1A. In contrast to TNSALP (W), the TNSALP mutants developed no significant reaction products. Compatible with this observation, an ALP assay confirmed that both TNSALP mutants displayed only very low activity (Fig. 1B). These results strongly indicate that the replacement of glycine (420) abrogates the catalytic function of TNSALP. Figure 1C shows the results of coexpression experiments where TNSALP (W) was transfected either with TNSALP (G420S) or TNSALP (G420A) in varying ratios. As reported previously , TNSALP (G420A) showed a strong dominant negative effect. Interestingly, TNSALP (G420S) was also found to significantly suppress the enzyme activity of TNSALP (W), albeit to a lesser degree.
TNSALP is a GPI-anchored glycoprotein. Previous studies showed that TNSALP is synthesized as a 66 kDa form with high-mannose-type oligosaccharides in the ER and undergoes terminal glycosylation to become an 80 kDa form with complex-type oligosaccharide chains during passage across the Golgi apparatus [9, 10]. We and other groups have reported that several TNSALP mutants are partially or completely transport incompetent [12-17, 23]. At first, we speculated about the possibility that both TNSALP (G420S) and TNSALP (G420A) may fail to exit the ER. Figure 2A shows the immunofluorescence of transiently transfected cells. Cell surface localization of TNSALP (G420S) and TNSALP (G420A) was evident and indistinguishable from TNSALP (W), although both mutants showed quite low ALP activity. Consistent with this result, both the 66 kDa immature and the 80 kDa mature forms were detected in transfected cells (Fig. 2B), corroborating that the replacement of glycine does not profoundly affect intracellular trafficking and access to the cell surface of TNSALP.
Expression of TNSALP (G420S) in Tet-On inducible CHO cells
Even TNSALP (W) tends to form a disulfide-bonded high-molecular-mass aggregate in transiently transfected cells, presumably due to the production of an excess amount of TNSALP in the ER [14, 15]. Figure 2B shows the TNSALP aggregate, which was revealed at the top of the gel only under non-reducing conditions. Next, we tried to establish a Tet-On CHO cell line expressing TNSALP (G420S) only in response to doxycycline (tetracycline analogue). We previously showed that no aggregates were formed in this inducible expression system and furthermore that the intracellular transport of TNSALP (W) to the cell surface was not delayed in contrast to transient expression [9, 10, 14, 15]. Expression was tightly regulated by doxycycline (Fig. 3A, lanes 1, 3, 5, 7 versus 2, 4, 6, 8) and the major molecular species of both TNSALP (W) and TNSALP (G420S) was the 80 kDa with a faint band of the 66 kDa immature form (Fig. 3A). No aggregates were observed under reducing or non-reducing conditions. In comparison with this result, it is evident that processing of the 66 kDa immature form to the 80 kDa mature form is disturbed in transiently transfected cells (Fig. 2B). Upon peptide N-glycosidase F (Flavobacterium meningosepticum) (PNGase F) digestion, both TNSALP (W) and the mutant migrated to the same position, suggesting that they were N-glycosylated to the same degree (Fig. 3B). Glycosidase digestion further confirmed that this 80 kDa form of TNSALP (G420S) was resistant to endo-β-N-acetyl-glucosaminidase H, but sensitive to neuraminidase like that of TNSALP (W) (Fig. 3B), supporting the notion that this mutant protein undergoes intracellular processing in the ER and Golgi apparatus. Like TNSALP (W), cell surface localization of TNSALP (G420S) was shown by immunofluorescence (Fig. 4A). Furthermore, the 80 kDa form, but not the 66 kDa form, was released into the medium from the cell upon digestion with phosphatidylinositol-specific phospholipase C (PI-PLC) (Fig. 4B), confirming that the 80 kDa form of TNSALP (G420S) is anchored to the cell membrane via the phosphatidylinositol moiety of GPI like the counterpart of TNSALP (W). However, despite this resemblance between the molecular phenotypes of TNSALP (W) and TNSALP (G420S), the latter completely lacked its ALP activity as shown in Fig. 4C.
Functional TNSALP comprises two identical subunits [20, 23]. Missense mutations are known to variously affect the assembly and aggregation state of the TNSALP molecule [15, 19, 23]. As SDS disassembles the dimeric form of TNSALP by disrupting non-covalent bonds, we investigated the molecular status of TNSALP (G420S) using sucrose-density-gradient centrifugation (Fig. 5). TNSALP (W) was found in fractions 7 and 8, where alcohol dehydrogenase (150 kDa) appeared, compatible with the dimeric status of TNSALP (W). On the other hand, TNSALP (G420S) co-migrated with bovine serum albumin (68 kDa), demonstrating that TNSALP (G420S) mostly exists as a monomer. Even though we did not directly analyze the TNSALP molecule on the cell surface by sucrose-density-gradient centrifugation, it appears highly likely that the 80 kDa form of TNSALP (G420S) exists as a monomer on the cell surface. Two lines of evidence support this. First, TNSALP (G420S) as well as TNSALP (W) are mostly present as the 80 kDa mature form in cells (Fig. 3A). Second, this mature form is released by PI-PLC digestion (Fig. 4B), therefore indicating that most of the 80 kDa form of TNSALP (G420S) and TNSALP (W) localizes on the cell surface in Tet-On CHO cells.
Protease digestion of TNSALP (G420S)
We have reported that missense mutations tend to render the TNSALP molecule more susceptible to proteases such as trypsin and proteinase K [9, 10], probably reflecting local conformational changes in TNSALP. As TNSALP (G420S) is present as a monomeric subunit, we assumed that this TNSALP mutant exhibits quite different sensitivity to proteases, as was the case. We homogenized CHO cells expressing TNSALP (W) or TNSALP (G420S) and incubated each homogenate with various concentrations of trypsin or proteinase K (Fig. 6). TNSALP (G420S) was found to be more sensitive to trypsin than TNSALP (W). Proteinase K also degraded TNSALP (G420S) more efficiently compared with TNSALP (W) (Fig. 6). These results suggest that protease sensitive peptide bonds are exposed in monomeric TNSALP (G420A).
TNSALP is 57% identical with and 74% homologous to placental ALP with four one-residue insertions, one three-residue insertion and one two-residue deletion in TNSALP [20, 24]. Mornet and colleagues have created a 3D model of human TNSALP by computer-simulating X-ray crystallographic data from human placental ALP . They successfully correlated the locations of amino acid substitutions with the degree of severity of the mutations by assigning various mutations to different functional domains of the TNSALP molecule. The crown domain comprises two flexible loop structures from each subunit and is a unique domain specific to mammalian ALPs that was observed for the first time in placental ALP, in which loop 423–452 is known to contribute isozyme-specific properties such as uncompetitive inhibition, heat stability and allosteric behavior in addition to providing a collagen binding site . The homodimer interface is a domain involved in the subunit interaction of TNSALP. As of 2001, 15 of 73 different missense mutations that cluster in the homodimer interface region have been found to be severe, suggestive of the structural importance of this domain . However, little study has been performed on the missense mutations located at the overlapping domain (interfacial crown domain) comprising the crown domain and homodimer interface.
Glycine (420) is well conserved among four human ALP isozymes  and resides in the interfacial crown domain. When expressed in mammalian cells, TNSALP (G420S) and TNSALP (G420A) almost lacked ALP activity as shown by qualitative (Fig. 1A) and quantitative (Fig. 1B) methods, indicating that glycine (420) is indispensable for ALP activity. However, unexpectedly, western blotting patterns of these TNSALP mutants were indistinguishable from that of TNSALP (W). Not only the 66 kDa immature molecular species but also the 80 kDa mature one were detected in transiently transfected cells (Fig. 2B), suggesting that terminal glycosylation concomitant with intracellular trafficking of these TNSALP mutants from the ER to the Golgi apparatus is not severely impaired. An immunofluorescence study further reinforced the notion that both TNSALP mutants have access to the cell membrane (Fig. 2A). A detailed study on TNSALP (G420S) using the Tet-On inducible expression system established that the 80 kDa form of this mutant underwent the trimming of mannose and acquired sialic acid, which takes place in the Golgi apparatus, as evidenced by digestion with endo-β-N-acetylglucosaminidase H and neuraminidase (Fig. 3A). Furthermore, the 80 kDa form, but not the 66 kDa form, was released upon digestion with PI-PLC (Fig. 4B), demonstrating that TNSALP (G420S) is anchored to the cell membrane via GPI like TNSALP (W). However, despite apparent similarities between TNSALP (W) and TNSALP (G420S), the latter completely lacked its catalytic activity (Fig. 4C). Remarkably, TNSALP (G420S) was found not to form the functional homodimer in contrast to TNSALP (W), as shown by sucrose-density-gradient centrifugation (Fig. 5), being compatible with the higher sensitivity of TNSALP (G420S) to proteases than TNSALP (W) (Fig. 6).
Thus, we demonstrated that glycine (420) plays a critical role in determining the homodimer assembly and catalytic function of TNSALP. Glycine (420) may be directly involved in monomer–monomer interactions or required for each subunit to acquire a proper structure competent for the homodimer. It is worth mentioning that monomeric TNSALP (G420S) gains access to the cell surface as the 80 kDa mature form (Fig. 4). In this regard, TNSALP (C201Y) lacking one intra-chain disulfide bond also failed to form the homodimer; however, in this case the resultant subunit was not capable of exiting from the ER . The development of perinatal HPP in a homozygote (G420S/G420S) can be accounted for by the complete loss of catalytic activity of TNSALP on the matrix vesicle, resulting in elevated levels of inorganic pyrophosphate, a negative regulator of hydroxyapatite formation, in the bone matrix. Correlations between genotype and phenotype are often difficult to establish because most HPP patients are compound heterozygous for missense mutations . The compound heterozygous patient (G420A/E191K) was diagnosed with childhood HPP . TNSALP (E191K), a moderate allele, can probably compensate TNSALP (G420A) lacking ALP activity like TNSALP (G420S) (Fig. 1), leading to moderate clinical expression. However, the situation is even more complex. Fauvert et al.  recently reported that TNSALP (G420A) strongly inhibits the activity of TNSALP (W) when they are co-transfected in a cell, raising the possibility that TNSALP (G420A) may interact with TNSALP (E191K) in the patient and affect its ALP activity. This strong dominant negative effect was confirmed in cells coexpressing TNSALP (W) and TNSALP (G420A) as shown in Fig. 1C. Moreover, we revealed that TNSALP (G420S) also exerts a weaker yet significant dominant negative effect on TNSALP (W) similar to TNSALP (A116T) [19, 22], suggesting that the monomeric TNSALP (G420S) molecule, which fails to assemble to each other, is capable of interacting with the subunit of TNSALP (W).
So far, the missense mutations associated with severe HPP can be grouped into four categories. First, perturbation of trafficking of the TNSALP molecule results from protein misfolding [12-17, 23]. Some mutants never appear on the cell surface [14, 15], while others have access to it to various degrees [12, 14-17, 23]. In addition, mutations may affect not only trafficking but also the kinetic properties of the residual activity of TNSALP mutants , explaining the variable expressivity of the disease. Disulfide linkage occurring in an oxidative milieu in the ER also plays a critical role for the protein folding of TNSALP. TNSALP (C201Y) and probably TNSALP (C489S) largely failed to assemble into a dimeric structure. Instead of being ferried to the cell surface, at least TNSALP (C201Y) lacking one of two intra-chain disulfide bonds needed for a proper tertiary structure resulted in retention in the ER, followed by polyubiquitination and degradation in the proteasome . Calcium binding is also important for TNSALP to acquire a proper structure as shown by the analyses of TNSALP (D306V) and TNSALP (E235G)  as these TNSALP mutants are essentially transport incompetent and are subjected to degradation in the ER. Both D306 and E235 are known to be involved in calcium coordination . Second, GPI addition is blocked and consequently GPI-less TNSALP becomes a soluble protein. One frameshift mutant (L520RfsX86) was secreted as a secretory protein with an additional 80 amino acid extension at the carboxyl end of TNSALP . Nascent TNSALP has a carboxyl-terminal propeptide, which includes a hydrophobic amino acid stretch and presumably serves as a GPI-anchor signal. Thus, this particular frameshift mutation abrogates the putative GPI-anchor signal by changing the reading frame at the carboxyl terminus. Third, substitutions in the crown domain of TNSALP do not grossly affect the trafficking of TNSALP (V423A) and TNSALP (R450C), but significantly suppress catalytic activity [9, 10]. Compared with TNSALP (G420S), both TNSALP (V423A) and TNSALP (R450C) were found to assemble into the dimer [9, 10]. It is likely that these missense mutations elicit local structural distortions of the crown domain, resulting in the decreased enzyme activity of TNSALP. Finally, as demonstrated in this study, the mutations involved in the interfacial crown domain blocked the monomer to monomer interaction of TNSALP.
There have been increasing attempts to develop a clinical treatment for HPP including ex vivo and in vivo gene therapy [27-38]. Among these, enzyme replacement therapy using a soluble form of recombinant TNSALP attached with a bone-targeted motif has been extensively studied in an animal model of HPP [33-36] and successfully applied to HPP patients [37, 38] (http://clinicaltrials.gov/).
In conclusion, our results demonstrate that the replacement of glycine (420) with serine located at the interfacial crown domain leads to the dysfunction of TNSALP by blocking its subunit assembly, thus contributing to the understanding of the molecular basis of the severity of this particular missense mutation. We also showed that, instead of being degraded in the ER, unassembled monomeric TNSALP (G420S) reaches the cell surface as the 80 kDa mature form and is anchored to the cell membrane via GPI, indicating that dimerization is not an obligatory step for trafficking of the TNSALP molecule to the cell surface.
Materials and methods
Our reagents were obtained from the following commercial sources: antipain, chymostatin, elastatinal, leupeptin and pepstatin A from Protein Institute Inc. (Osaka, Japan); aprotinin, baker's yeast alcohol dehydrogenase, bovine serum albumin, phosphatidylinositol-specific phospholipase C (Bacillus cereus, PI-PLC), l-1-tosylamide-2-phenylethyl-chloromethyl ketone-treated bovine pancreas trypsin from Sigma Chemical Co. (St Louis, MO, USA); bovine liver catalase, hygromycin B, p-amidinophenyl-methanesulfonyl fluoride from Wako Pure Chemicals (Tokyo, Japan); endo-β-N-acetylglucosaminidase H (Streptomyces griseus) from Seikagaku Kogyo (Kyoto, Japan); Enhanced Chemiluminescence Plus (ECL Plus) western blotting detection reagents, peroxidase-conjugated donkey anti-(rabbit IgG) from Amersham Pharmacia Biotech (Arlington Heights, IL, USA); fetal bovine serum, Lipofectamine Plus Reagent and Opti-MEM from Invitrogen (Carlsbad, CA, USA); G418 from Calbiochem (La Jolla, CA, USA); High Speed Plasmid Midi-kit from Qiagen (Hilden, Germany); neuraminidase (Arthrobacter ureafaciens) and proteinase K from Roche (Indianapolis, IN, USA); peptide N-glycosidase F (Flavobacterium meningosepticum, PNGase F) from New England Biolabs (Beverly, MA, USA); pTRE2-hygro and BD® CHO-K1 Tet (tetracycline)-On cell line and Tet system approved fetal bovine serum from Clontech (Palo Alto, CA, USA); QuikChange Lightning Site-Directed Mutagenesis Kit from Stratagene (La Jolla, CA, USA); rhodamine-conjugated sheep anti-(rabbit IgG) from Cappel (Solon, OH, USA). Antibody to human TNSALP was raised in rabbits as described previously .
Plasmids and transfection
The construction of pSG5 vector encoding TNSALP (W) was described previously . Specific mutations were introduced into the plasmid using the QuikChange Lightning Site-Directed Mutagenesis Kit essentially according to the manufacturer's protocol. Oligonucleotides used were as follows: TNSALP (G420S), 5′-TATGGCAATGGGCCTAGCTACAAGGTGGTGG-3′ and 5′-CCACCACCTTGTAGCTAGGCCCATTGCCATA-3′; TNSALP (G403A), 5′-TATGGCAATGGGCCTGCCTACAAGGTGGTGG-3′ and 5′-CCACCACCTTGTAGGCAGGCCCATTGCCATA-3′.
Sequencing of the cDNA of each TNSALP mutant confirmed the mutation. To established stable cell lines, cDNA encoding TNSALP (G420S) was further inserted into pTRE2-hygro. Transfection and screening of stable cell lines were carried out as described previously [19, 40]. Tet-On CHO K1 cells expressing TNSALP (W) were established previously . For the induction of TNSALP, the cells were cultured in the presence of 1 µg·mL−1 doxycycline for 24 h before being used. Transient expression in the COS-1 cell was performed as described previously [19, 40].
Cell pellets, which were collected by scraping, were homogenized using a sonicator in 100–200 µL of 10 mm Tris/HCl (pH 7.2). Cell homogenates (5–10 µg) were directly analyzed by SDS/PAGE in the absence or presence of 1% (v/v) 2-mercaptoethanol. Electric transfer was carried out at 20 V for 30 min using the Bio-Rad Trans-Blot SD Semi-Dry Electric Transfer Cell (Hercules, CA, USA). The membrane was then blocked with 5% skim milk and further incubated with anti-TNSALP serum (1 : 4000), followed by peroxidase-conjugated sheep anti-(rabbit IgG) (1 : 4000). Protein bands on the membrane were detected using ECL Plus reagent as described previously .
The cells expressing TNSALP (W) or TNSALP (G420S) were homogenized and used for glycosidase digestions (PNGase F, neuraminidase and endo-β-N-acetylglucosaminidase H) essentially as described previously .
To estimate the sensitivity to trypsin or proteinase K, Tet-On cells expressing TNSALP (W) or TNSALP (G420S) were homogenized in 10 mm Tris/HCl (pH 8.0) using a sonicator. Each 5 µg of homogenate was incubated with increasing concentrations of trypsin or proteinase K at pH 8.0 in a total volume of 20 µL in an ice/water bath, essentially according to Akiyama and Ito . After 30 min, 1 µL of 100 mm p-amidinophenylethanesulfonyl fluoride was added to stop the reaction. Each sample was mixed with 10 µL of three-fold SDS sample buffer and heated at 95 °C for 5 min, followed by SDS/PAGE western blotting.
Anchoring of the TNSALP molecule to the cell membrane via GPI was determined using PI-PLC digestion [19, 23]. The molecular size of the TNSALP molecule was compared using sucrose-density-gradient centrifugation [19, 23]. Cytochemical staining for ALP activity was carried out as described previously [15, 19]. Cell surface appearance of the TNSALP molecule was assessed by immunofluorescence . Protein content and ALP activity were determined as described previously . One unit of ALP activity is expressed as nmoles of p-nitrophenylphosphate hydrolyzed per minute at 37 °C.
The authors thank Ms M. Okamura for her assistance. We would also like to thank the anonymous referees for their invaluable remarks and suggestions for the coexpression study. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports and Technology of Japan (to K.O.) and a Grant from the Ministry of Health, Labour and Welfare of Japan (to K.O.).