Báo cáo khoa học: Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position 406 Structural importance of the crown domain potx

Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position 406 Structural importance of the crown domain Natsuko Numa 1 , Yoko Ishida 2 , Makiko Nasu 3 , Miwa Sohda 2 , Yoshio Misumi 4 , Tadashi Noda 1 and Kimimitsu Oda 2,5 1 Division of Pediatric Dentistry, Niigata University Graduate School of Medical and Dental Sciences, Japan 2 Division of Oral Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Japan 3 Division of Oral Health in Aging and Fixed Prosthodontics, Niigata University Graduate School of Medical and Dental Sciences, Japan 4 Department of Cell Biology, Fukuoka University School of Medicine, Japan 5 Center for Transdisciplinary Research, Niigata University, Japan Keywords crown domain; glycosylphosphatidylinositol; hypophosphatasia; raft; tissue-nonspecific alkaline phosphatase Correspondence K. Oda, Division of Oral Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, 2-5274, Gakkocho-dori, Niigata 951-8514, Japan Fax: +81 25 227 0805 Tel: +81 25 227 2827 E-mail: oda@dent.niigata-u.ac.jp (Received 30 November 2007, revised 18 January 2008, accepted 19 March 2008) doi:10.1111/j.1742-4658.2008.06414.x Hypophosphatasia, a congenital metabolic disease related to the tissue-non- specific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tis- sues. A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophos- phatasia. To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently trans- fected COS-1 cells and in stable CHO-K1 Tet-On cells. Compared with the wild-type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity. This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum, as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble frac- tion (raft) at a rate similar to that of the wild-type TNSALP. TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K. When purified as a glycosylphosphatidylinositol-anchorless soluble form, the mutant protein exhibited a remarkably lower K cat ⁄ K m value compared with that of the wild-type TNSALP. Interestingly, leucine and isoleucine, but not phenylalanine, were able to substitute for valine, pointing to the indispensable role of residues with a longer aliphatic side chain at position 406 of TNSALP. Taken together, this particular mutation highlights the structural importance of the crown domain with respect to the catalytic function of TNSALP. Abbreviations Endo H, endo-b-N-acetylglucosaminidase H; GPI, glycosylphosphatidylinositol; TNSALP (V406A), TNSALP with a valine to alanine substitution at position 406; TNSALP, tissue-nonspecific alkaline phosphatase. FEBS Journal 275 (2008) 2727–2737 ª 2008 The Authors Journal compilation ª 2008 FEBS 2727 Hypophosphatasia is caused by various mutations of the tissue-nonspecific alkaline phosphatase (TNSALP) gene (EC 3.1.3.1) [1–6]. To date a total of 191 distinct mutations have been reported worldwide, and about 80% of these mutations are missense (http://www. sesep.uvsq.fr./Database.html). Hypophosphatasia is characterized by reduced levels of serum alkaline phos- phatase activity and defective mineralization in bone and tooth, and clinical severity is inversely correlated to serum alkaline phosphatase levels [1,2,7]. Patients suffering from severe hypophosphatasia, such as the perinatal or infantile forms, develop severe defects in skeletal bone mineralization, unequivocally demon- strating that TNSALP is physiologically involved in the mineralization process of bone. Consistent with this concept, TNSALP-deficient mice are reported to develop rickets and osteomalacia [8–10]. During the course of our study on several TNSALP mutant proteins associated with the severe form of hypophosphatasia, we found that the cell-surface expression of the TNSALP mutants is remarkably reduced. The mutant proteins often fail to undergo proper folding and correct assembly, resulting in accu- mulation in the early stage of the secretory pathway and eventual degradation in the endoplasmic reticulum [11–15]. However, the extent to which each TNSALP mutant protein reaches the cell surface varies from one mutation to another, depending on the position of the mutation in the gene and the amino acid residue that is replaced. Fleisch et al. proposed that TNSALP regu- lates mineralization by hydrolyzing inorganic pyro- phosphate, a poison of hydroxyapatite crystal [16,17], at the site of biomineralization. According to this pro- posal, it is likely that defective bone formation occur- ring in severe hypophosphatasia is closely related to the number of cell-surface TNSALP mutant molecules and their residual pyrophosphate-cleaving activity [4,18,19]. Replacement of valine at position of 406 with alanine was reported in a patient diagnosed with peri- natal hypophosphatasia who was a compound hetero- zygote for this mutation and A99T [20]. The valine residue at position 406 is located in a unique domain called the crown domain [21]. This domain shows the lowest degree of homology among alkaline phospha- tase isoenzymes, and isoenzyme-specific properties, such as uncompetitive inhibition, heat stability and allosteric behavior, are attributed to residues located in this domain of each isoenzyme [4,22]. Besides, this crown domain is responsible for interacting with extracellular matrix proteins, including collagen [4,21– 24]. Here we demonstrate that, in contrast to other missense mutations associated with severe hypophos- phatasia, the majority of TNSALP (V406A) molecules are capable of reaching the cell surface at a rate simi- lar to that of the wild-type enzyme, thus excluding the possibility that transport incompetence is a major molecular defect of TNSALP (V406A). Rather, it is likely that this particular mutation affects the active site of TNSALP through imposing a subtle change on the crown domain, rendering TNSALP (V406A) less efficient for its catalytic function. Results Transient expression of TNSALP (V406A) When expressed transiently, TNSALP (V406A) pro- duced only a weak cytochemical reaction product compared with the wild-type enzyme (Fig. 1A). In agreement with these staining patterns, the specific alkaline phosphatase activity of the cell homogenate expressing the mutant protein was less than one-quar- ter of that of the cell homogenate expressing the wild-type enzyme (Fig. 1B). Immunoblotting con- firmed that both the wild-type protein and the TNSALP (V406A) mutant consisted of a 66-kDa and an 80-kDa molecular species (Fig. 1C), which repre- sent an immature form bearing high mannose-type N-linked oligosaccharides and a mature form bearing complex-type oligosaccharides, respectively [11]. Besides, the amount of TNSALP (V406A) mutant was similar to that of the wild-type protein in trans- fected cells, indicating that the lower expression level does not account for the low specific enzyme activity of the former. Upon incubation with phosphatidylino- sitol-specific phospholipase C, both the wild-type pro- tein and the TNSALP (V406A) mutant were released into the medium (Fig. 1D, lanes 4 and 8), confirming that they are anchored to the cell surface via glyco- sylphosphatidylinositol (GPI). By contrast, the 66-kDa form was the only molecular species observed within the cells expressing TNSALP (D289V) (Fig. 1C), which is arrested and forms the disulfide-bonded aggregate in the endoplasmic reticulum [15], and is not able to gain access to the cell surface (Fig. 1D, lane 12). Expression of TNSALP (V406A) in a stable cell line In the transient expression system, even the wild-type enzyme formed a disulfide-bonded aggregate, probably as a result of the synthesis of an excess amount of TNSALP (Fig. 1C, lanes 4–6, see the top of gel). In the present experiment, TNSALPs were expressed Molecular basis of perinatal form of hypophosphatasia N. Numa et al. 2728 FEBS Journal 275 (2008) 2727–2737 ª 2008 The Authors Journal compilation ª 2008 FEBS under control of the CMV I.E. Enhancer ⁄ Promoter. We observed the same aggregate also in a previous experiment using a different expression vector contain- ing the SV40 early promoter [11]. Shortage of a pre- cursor of GPI in the endoplasmic reticulum may be one of the reasons why a small but significant fraction of the wild-type TNSALP forms the aggregate in transfected COS-1 cells [25]. To circumvent this draw- back of transient expression, we established CHO-K1 Tet-On cells harboring a plasmid encoding TNSALP (V406A). When incubated with doxycycline (an ana- logue of tetracycline) TNSALP (V406A) appeared on the cell surface (Fig. 2A, panel a) and exhibited weak enzyme activity (Fig. 2A, panel c). The protein was induced only with doxycycline, and no disulfide- bonded aggregate was found on the top of the gel (Fig. 2B). Note that most of the cellular TNSALP (V406A) was present as the 80-kDa mature form. This is in marked contrast to the transiently transfected cells, where the 66-kDa form was a predominant molecular species (Fig. 1C). This immunoblotting pat- tern of the CHO-K1 Tet-On cells resembles that of Saos-2 cells [14] – osteosarcoma producing a large amount of TNSALP. Figure 3A shows pulse–chase labeling experiments in combination with endo-b-N- glucosaminidase H (Endo H) digestion. The wild-type enzyme was synthesized as the 66-kDa Endo H-sensi- tive form, which quickly became the 80-kDa Endo H- resistant form. The processing of the newly synthesized wild-type enzyme was complete by the end of the 2-h chase period. This was also the case for TNSALP (V406A) with only a small fraction being sensitive, even at the end of the 2-h chase. Compatible with this result, both the wild-type protein and the mutant pro- tein were partitioned into a cold Triton X-100-insolu- ble fraction (the raft) at a similar rate (Fig. 3B), further supporting that the folding and assembly pro- cess and subsequent intracellular trafficking of TNSALP (V406A) are largely normal in the stable cell line. Kinetics of the soluble form of TNSALP (V406A) Consistent with the biosynthetic studies in Fig. 3, the expression level in the CHO-K1 Tet-On cell of TNSALP (V406A) was similar to that of the wild-type protein, as shown in Fig. 4A,B. However, the CHO-K1 Tet-On cells expressing TNSALP (V406A) Fig. 1. Transient expression of TNSALP mutant proteins in COS-1 cells. (A) COS-1 cells expressing the wild-type TNSALP or the TNSALP (V406A) mutant were stained for alkaline phosphatase activity. Each dish was incubated in a reaction mixture for 10 min at room tempera- ture. In panels B and C, cell homogenates prepared from the transfected COS-1 cells were assayed for alkaline phosphatase (abscissa enzyme activity expressed in unitsÆmg )1 of protein) or analyzed by SDS-PAGE under reducing (Red) or non-reducing (Nonred) conditions, fol- lowed by immunoblotting using anti-TNSALP serum. The arrowhead indicates the top of the resolving gel. The values are the means of two independent experiments. (D) COS-1 cells expressing the wild-type TNSALP, or the TNSALP (V406A) or TNSALP (D289V) mutants were labeled with [ 35 S]methionine and incubated further in the absence (lanes 1, 2, 5, 6, 9 and 10) or presence (lanes 3, 4, 7, 8, 11 and 12) of phosphatidylinositol-specific phospholipase C. Both cell lysates (C) and media (M) were subjected to immunoprecipitation, followed by SDS- PAGE (under reducing condition) ⁄ fluorography. Left lane: 14 C-methylated protein markers of 200, 97.5, 66, 46 and 30 kDa, from the top of the gel. N. Numa et al. Molecular basis of perinatal form of hypophosphatasia FEBS Journal 275 (2008) 2727–2737 ª 2008 The Authors Journal compilation ª 2008 FEBS 2729 Fig. 3. Biosynthesis of TNSALP (V406A) in CHO-K1 Tet-On cells. Established CHO-K1 Tet-On cells harboring a plasmid encoding the wild- type TNSALP or the TNSALP (V406A) mutant, which had been cultured in the presence of 0.5 lgÆmL )1 of doxycycline for 14 h, were pulse- labeled with [ 35 S]methionine for 30 min and then the cells were collected at the indicated chase periods. The cells were lysed in the lysis buffer and subjected to immunoprecipitation in (A). The immunoprecipitates on beads were incubated with or without Endo H prior to analy- sis by SDS-PAGE (reducing condition) ⁄ fluorography. Some degradation products were observed in the samples during incubation with the glycosidase. Left lane: 14 C-methylated protein markers of 200, 97.5, 66, 46 and 30 kDa, from the top of the gel. In panel B, the metabolically labeled cells were lysed in cold 1% Triton X-100 and centrifuged at 15 000 g for 10 min. Triton X-100 soluble (S) and insoluble (I) fractions were separated. The latter was further lysed in the lysis buffer and incubated at 37 °C for 20 min to extract TNSALP from the raft. Both sol- uble and insoluble fractions were subjected to immunoprecipitation, followed by SDS-PAGE (reducing condition) ⁄ fluorography. Left lane: 14 C-methylated protein markers of 200, 97.5, 66, 46 and 30 kDa, from the top of the gel. 80 kD a DOX + – + – Red BA ab cd Nonred Fig. 2. Expression of TNSALP (V406A) in a CHO-K1 Tet-On cell line. (A) Established CHO-K1 Tet-On cells harboring a plasmid encoding TNSALP (V406A) were cultured for 24 h in the absence (b, d) or presence (a, c) of doxycycline (1 lgÆmL )1 ) and stained for immunofluores- cence using anti-TNSALP serum (a, b) or stained for alkaline phosphatase activity (c, d). (B) CHO-K1 Tet-On cells were cultured for 24 h in the absence or presence of doxycycline (DOX) and analyzed by SDS-PAGE in the absence (Nonred) or presence (Red) of 2-mercaptoethanol, followed by immunoblotting. An arrowhead indicates the top of the gel. Molecular basis of perinatal form of hypophosphatasia N. Numa et al. 2730 FEBS Journal 275 (2008) 2727–2737 ª 2008 The Authors Journal compilation ª 2008 FEBS showed remarkably lower enzyme activity than those expressing the wild-type enzyme, indicating that TNSALP (V406A) has a compromised enzyme activity. Next, in an attempt to compare enzymatic properties of the wild-type and TNSALP (V406A) proteins in detail, we purified both the enzymes as GPI-anchorless soluble forms. We engineered the codons in TNSALP cDNA to obtain a consecutive region of six histidine residues and a premature stop codon upstream of a putative C-terminal GPI-anchor signal sequence, as described previously [26]. Both wild-type and mutant proteins were secreted into the medium from transfect- ed COS-1 cells, and the proteins were applied to a Ni-chelate column. Eluted protein bands were appar- ently homogeneous with a molecular mass of 70 (Fig. 4C) and no disulfide-bonded aggregate was found in this secreted form (data not shown).K m and V max values were determined graphically using the direct lin- ear plot of Eisenthal & Cornish-Bowden. TNSALP (V406A) showed a reduced K m value in association with a marked reduction in the K cat value, resulting in a K cat ⁄ K m value that was < 10% of that of the wild- type enzyme (Table 1), indicating that the conversion of valine to alanine at position 406 in the crown domain compromises the catalytic function of TNSALP. Protease sensitivity of TNSALP (V406A) As shown in Fig. 5A, both the wild-type protein and the mutant protein migrated at exactly the same posi- tion, as judged by sucrose-density-gradient centrifuga- tion, demonstrating that both the mutant protein and the wild-type protein form a homodimer. However, TNSALP (V406A) was found to be much more suscep- tible to trypsin or proteinase K than the wild-type pro- tein (Fig. 5B), suggesting that the conformation of the crown domain of TNSALP (V406A) may be altered so that each protease degrades the mutant protein more easily, although its overall structure is not markedly different from the wild-type protein. Mutation analysis of the residue at position 406 Valine and alanine are usually classified into the same amino acid group with a hydrophobic side chain. Therefore, we hypothesized that not only hydrophobic- ity, but also the length of the alkyl side chain of the amino acid at position 406, is crucial to the catalytic efficiency of TNSALP. This was the case. Leucine and isoleucine, but not phenylalanine, successfully substi- tuted for the valine residue (Fig. 6A). Replacement with phenylalanine resulted in a low enzyme activity, even though TNSALP (V406F) was processed to the 80-kDa mature form similarly to TNSALP (V406A) and appeared on the cell surface like the wild-type protein (data not shown). Fig. 4. Enzyme activity of wild-type TNSALP and the TNSALP (V406A) mutant. Established CHO-K1 Tet-On cells harboring a plas- mid encoding the wild-type TNSALP or the TNSALP (V406A) mutant were cultured in the presence of 1 lgÆmL )1 of doxycycline. After 24 h, the cells were homogenized and subjected to the alkaline phos- phatase assay (abscissa: enzyme activity expressed in unitsÆmg )1 of protein; the values are the means of two independent experiments) (A) or analyzed by SDS-PAGE (under reducing conditions), followed by immunoblotting (5 lg each loaded) (B). (C) COS-1 cells were transfected with the plasmid encoding the soluble form of the wild- type TNSALP or the TNSALP (V406A) mutant. After 48 h, the media were collected and applied to the Ni-nitrilotriacetic acid column. TNSALP was eluted with 250 m M imidazole. Each eluate was ana- lyzed by SDS-PAGE (reducing condition), followed by silver staining (100 or 200 ng of protein loaded). Left lane: molecular mass markers (200, 6, 46 and 30 kDa, from the top of the gel). Table 1. Kinetic parameters of soluble forms of the wild-type TNSALP and the TNSALP (V406A) mutant. The assay was carried out using p-nitrophenylphosphate as the substrate in 0.1 M 2- amino-2-methyl-1,3-propanediol ⁄ HCl buffer (pH 10.5) containing 5m M MgCl 2 and 0.1% Triton X-100. K m K cat (s )1 ) K cat ⁄ K m · 10 3 (M )1 s )1 ) Wild-type TNSALP 0.21 971 4624 TNSALP (V406A) mutant 0.09 34 377 N. Numa et al. Molecular basis of perinatal form of hypophosphatasia FEBS Journal 275 (2008) 2727–2737 ª 2008 The Authors Journal compilation ª 2008 FEBS 2731 Discussion Hypophosphatasia is an inborn error of metabolism related to bone and tooth. The disease is caused by various mutations on the TNSALP gene [1–6], which is located on chromosome 1 (p34-p36.1). Reduction in serum alkaline phosphatase levels is a biochemical hall- mark of mutations in TNSALP, and patients develop a variable degree of defective bone and tooth minerali- zation. The disease is categorized into five groups: (a) perinatal hypophosphatasia, (b) infantile hypophos- phatasia, (c) childhood hypophosphatasia, (d) adult hypophosphatasia and (e) odonto hypophosphatasia [1–4]. Perinatal and infantile forms of hypophosphata- sia are severe and are usually transmitted as a recessive trait, whereas the other three forms of hypophosphata- sia are mild and are transmitted recessively or domi- nantly. So far, we have found that several missense mutations, which were reported in patients with severe hypophosphatasia, affect the folding and assembly process of the TNSALP molecule. As a result, these TNSALP mutant proteins fail to acquire transport competence and accumulate in the early stages of the secretory pathway, followed by degradation in an ubiquitin ⁄ proteasomal pathway [13–15]. This leads to decreased levels of expression of TNSALP mutants on the cell surface, although the degree by which TNSALP mutants reach the cell surface differs from one mutation to another: TNSALP (R54C), TNSALP (N153D), TNSALP (E218G), TNSALP (D289V) and TNSALP (G317A) were totally absent from the cell surface [12–15], whereas TNSALP (A162T) and TNSALP (D277A) were present at the cell surface) [11,12]. Residual activities of the latter mutant enzymes may contribute to a highly variable clinical expressivity of hypophosphatasia [4]. Improper folding and resul- tant delayed trafficking are also molecular phenotypes of TNSALP having missense mutations such as E174K, G438S, I473F, G232V, I201T and F310L [27,28]. Recently we have characterized a unique mutation associated with infantile hypophosphatasia that appar- ently does not impair the trafficking of TNSALP [29]. Fig. 5. Molecular properties of the TNSALP (V406A) mutant. Established CHO-K1 Tet- On cells harboring a plasmid encoding the wild-type TNSALP or the TNSALP (V406A) mutant were cultured in the presence of 1 lgÆmL )1 of doxycycline for 24 h. (A) Cells were lysed and directly applied to a sucrose density gradient. After centrifugation, 13 fractions were collected and assayed for alkaline phosphatase activity. The figure combines the results from two gradients: black bar, wild-type TNSALP; white bar, TNSALP (V406A) mutant. Abscissa: units of enzyme activityÆmL )1 of each fraction. b, a and c denote bovine albumin (68 kDa), alco- hol dehydrogenase (141 kDa) and catalase (250 kDa), respectively, which were centri- fuged separately. (B) The cells were col- lected and homogenized in 10 m M Tris-HCl (pH 8.0) using a sonicator. The homogen- ates were incubated with increasing concen- trations (lgÆmL )1 ) of trypsin or proteinase K in an ice ⁄ water bath for 30 min. Each sam- ple was analyzed by SDS-PAGE (under reducing conditions), followed by immuno- blotting using anti-TNSALP serum. Molecular basis of perinatal form of hypophosphatasia N. Numa et al. 2732 FEBS Journal 275 (2008) 2727–2737 ª 2008 The Authors Journal compilation ª 2008 FEBS TNSALP (R433C) forms a disulfide-bridged dimer instead of a non-covalently assembled dimer like the wild-type enzyme. Although this mutant appears on the cell surface at similar kinetics to the wild-type enzyme, this novel covalent cross-linkage, but not the replacement of the amino acid residue per se, is the cause of the decreased enzyme activity of TNSALP (R433C). TNSALP (V406A) was reported in a patient diag- nosed with perinatal hypophosphatasia, who is a com- pound heterozygote carrying V406A and A99T [20]. A99T was also found in milder forms of the disease, such as adult hypophosphatasia and odonto hypophos- phatasia, and is known to be transmitted dominantly [30]. In this report we focused on the V406A missense mutation. Figure 7 is a proposed 3D structural model of human TNSALP, based on the crystallographic analysis of human placental alkaline phosphatase, in which both Val406 and Arg433 residues are high- lighted. Valine at position 406 is located in the crown domain consisting of 65 residues [4,21]. We have dem- onstrated that TNSALP (V406A) is another allele with severe effects, but does not show defective trafficking like TNSALP (R433C). The rate of the intracellular transport of the mutant protein was similar to that of the wild-type protein, as assessed by the acquisition of Endo H resistance. Besides, the mutant protein was found to be incorporated into the raft at a kinetic rate similar to that of the wild-type enzyme. GPI-anchored protein is well known to be incorporated into the raft in the Golgi apparatus [31], thus being ferried to the apical surface of differentiated epithelial cells [32]. These findings strongly suggest that the cause of this severe hypophosphatasia is not a defect in transport, but the decreased catalytic activity of TNSALP (V406A) itself. This was indeed confirmed by the kinetic analysis of a purified GPI-anchorless soluble version of the mutant protein. The K cat ⁄ K m value of the mutant TNSALP was less than one-tenth of the K cat ⁄ K m value of the soluble form of the wild-type enzyme, indicating that the replacement of valine with alanine at position 406 in the crown domain somehow strongly affects the catalytic efficiency of TNSALP. Considering that TNSALP (V406A) migrates at the same position as the wild-type enzyme, as judged by sucrose-density-gradient centrifugation, it is likely that the overall structure of the mutant protein is not grossly changed. Nevertheless, enhanced susceptibility to trypsin, or especially to proteinase K, suggests a subtle distortion of the crown domain of the mutant protein. Currently we do not have a definite answer as to how this missense mutation at position 406 in the crown domain results in a remarkable decrease in the catalytic efficiency of TNSALP. In this respect, it is worth pointing out that residues in the crown domain are closely related to the enzymatic properties of TNSALP [22]. Interestingly, when valine at posi- tion 406 is replaced with leucine or isoleucine, both of which have a longer aliphatic chain than alanine, these TNSALP mutant proteins were processed to the 80- kDa form and exhibited enzyme activity similar to that of the wild-type protein. This leads us to speculate that Crown domain V406 V406 R433R433 Active site Active site Fig. 7. 3D model of human TNSALP. Valine at position 406 and arginine at position 433 in the crown domain are highlighted. Fig. 6. Expression of TNSALP (V406L), TNSALP (V406I) and TNSALP (V406F) in COS-1 cells. COS-1 cells were transfected with the plasmid encoding the wild-type TNSALP or the TNSALP (V406A), TNSALP (V406L), TNSALP (V406I) or TNSALP (V406F) mutants, for 24 h. Cell homogenates were assayed for alkaline phosphatase activity (abscissa: unit activityÆmg of protein )1 ) (upper panel). The same samples were analyzed by SDS-PAGE (under non-reducing conditions), followed by immunoblotting using anti- TNSALP serum (lower panel). N. Numa et al. Molecular basis of perinatal form of hypophosphatasia FEBS Journal 275 (2008) 2727–2737 ª 2008 The Authors Journal compilation ª 2008 FEBS 2733 the valine residue at position 406 on one subunit may interact with the counterpart on the other subunit through their long aliphatic hydrocarbon chains, thus contributing to assume a proper conformation of the crown domain, which is presumably indispensable for the efficient catalytic function of TNSALP. In support of our hypothesis, the cysteine residue at position 433 in one subunit of TNSALP (R433C) becomes cross- linked to the counterpart of the other subunit [29], implying that the two cysteine residues are sufficiently close to stretch out to form a covalent linkage in the crown domain. However, our hypothesis is not com- patible with the current TNSALP structural model shown in Fig. 7. It seems that the side chains of two valine residues at position 406 are too far apart to interact with each other in the crown domain. Never- theless, it is worth noting that the overall homology of the crown domain between TNSALP and the placental isoenzyme is around 72% at the amino acid level, while that of the loop comprising residues 405–435 is only 50% [21]. Another possibility is that valine at position 406 is involved in a substrate–enzyme interac- tion instead of interacting with the counterpart of the other subunit. Obviously, the definite conclusion awaits the crystallographic analysis of human TNSALP, although our findings imply a close func- tional relationship between the active site and the crown domain of the TNSALP molecule, which may be physiologically relevant to biomineralization. Materials and methods Materials Express 35 S 35 S protein labeling mix (> 1000 CiÆmmol )1 ) was obtained from Dupont-New England Nuclear (Boston, MA, USA); and 14 C-methylated proteins and the enhanced chemiluminescence (ECL Ò ) western blotting detection reagent, peroxidase-conjugated donkey anti-(rabbit IgG) and protein A–Sepharose CL-4B were obtained from Amer- sham Pharmacia Biotech (Arlington Heights, IL, USA); pALTER Ò -MAX, Altered sites Ò II mammalian mutagenesis system was obtained from Promega (Madison, WI, USA); QuikChange II Site-Directed Mutagenesis kit was obtained from Stratagene (La Jolla, CA, USA); G418 and pansorbin were obtained from Calbiochem (La Jolla CA, USA); Lipo- fectamine Plus Reagent was obtained from Invitrogen (Carlsbad, CA, USA); phosphatidylinositol-specific phos- pholipase C was obtained from BIOMOL International, L.P. (Plymouth Meeting, PA, USA); aprotinin, doxycycline and saponin (Quillaja Bark) and l-1-tosylamide-2-phenyl- ethyl-chloromethylketone-treated bovine pancreas trypsin were obtained from Sigma Chemical Co. (St Louis, MO, USA); proteinase K was obtained from Roche Diagnostics (London, UK); Ni-nitrilotriacetic acid resin and the plas- mid Midi-kit were obtained from Qiagen (Hilden, Ger- many); antipain, chymostatin, elastatinal, leupeptin and pepstatin A were obtained from Protein Research Founda- tion (Osaka, Japan); and hygromycin B and (p-amidinophe- nyl) methanesulfonylfluoride were obtained from Wako Pure Chemicals (Tokyo, Japan). Antiserum against recom- binant human TNSALP was raised in rabbits as described previously [26]. The pTRE2 and BD Ò CHO-K1 Tet-On cell line and Tet systems approved fetal bovine serum from BD Biosciences Clontech (Palo Alto, CA, USA). Plasmids and transfection The pALTER-MAX Ò encoding the wild-type TNSALP was constructed as described previously [14]. Mutations were introduced at specific sites using the Altered sites Ò II mam- malian mutagenesis system, as described previously [14,15]. The oligonucleotides used were: TNSALP (V406A), 5¢-TTC ACC GCC CAC TGC CTT GTA GCC AGG-3¢ and a sol- uble form of TNSALP (V406A), 5¢-GCA GCA AGG CTG CCT GCC TAG TGA TGG TGA TGG TGA TGG CTG GCA GGA GCA CA-3¢. TNSALP (V406L), TNSALP (V406I) and TNSALP (V406F) were created using the QuikChange II Site-Directed Mutagenesis kit with the following primers: V406L, 5¢-CCT GGC TAC AAG CTG GTG GGC GGT G-3¢ and 5¢-CAC CGC CCA CCA GCT TGT AGCCAG G-3¢; V406I, 5¢-CCT GGC TAC AAG ATA GTG GGC GGT GAA-3¢ and 5¢-TTC ACC GCC CAC TAT CTT GTA GCC AGG-3¢; and V406F, 5¢-CCT GGC TAC AAG TTC GTG GGC GGT G-3¢ and 5¢-CAC CGC CCA CGA ACT TGT AGC CAG G-3¢, respectively. The DNA sequence of the mutation sites was verified by DNA sequence analyses. The cDNA encoding TNSALP (V406A) was further subcloned into pTRE2 to establish stable cell lines. Transfection and screening of stable cell lines were performed essentially according to the manufac- turer’s protocol. CHO-K1 Tet-On cells, which successfully produced the mutant TNSALP in the presence of doxy- cycline, but not in its absence, were identified using immunofluorescence. Establishment and characterization of CHO-K1 Tet-On cells expressing the wild-type TNSALP will be published elsewhere. Established CHO-K1 Tet-On cells were cultured and passaged in the absence of doxycy- cline until they were used for experiments. For immuno- blotting or immunofluorescence studies, the cells were cultured in the presence of 1 lgÆmL )1 of doxycycline for 24 h before use. Alternatively, cells were cultured in 0.2–0.5 lgÆmL )1 of doxycycline for 14 h before biosynthetic experiments. For transient expression, COS-1 cells (1.0–1.3 · 10 5 cells per 35-mm dish) were transfected with 0.5–0.8 lg of each plasmid using Lipofectamine Plus, according to the manufacturer’s protocol, as described Molecular basis of perinatal form of hypophosphatasia N. Numa et al. 2734 FEBS Journal 275 (2008) 2727–2737 ª 2008 The Authors Journal compilation ª 2008 FEBS previously [14,15], and the transfected cells were incubated for 24 h in a 5% CO 2 ⁄ 95% air incubator before use. For purification of the soluble forms of enzymes, five 100-mm dishes were transfected and cultured for 48 h. COS-1 cells were cultured in DMEM supplemented with 10% fetal bovine serum [9]. Metabolic labeling and immunoprecipitation For pulse-chase experiments, cells were pre-incubated for 0.5–1 h in methionine ⁄ cysteine-free DMEM and labeled with 50–100 lCi of [ 35 S]methionine ⁄ cysteine for 0.5 h in the fresh methionine ⁄ cysteine-free MEM. After a pulse period, cells were washed and chased in the DMEM as described previously [15,29]. After metabolic labeling, the medium was removed and the cells were lysed in 0.5 mL of lysis buffer [1% (w ⁄ v) Triton X-100 ⁄ 0.5% (w ⁄ v) sodium deoxy- cholate ⁄ 0.05% (w ⁄ v) SDS in NaCl ⁄ P i ]. For separation of Triton X-100-soluble and Triton X-100-insoluble fractions, labeled cells were lysed in 1% Triton X-100 in NaCl ⁄ P i instead of in the lysis buffer and were centrifuged at 15 000 g for 10 min before lysing the insoluble fraction fur- ther in the lysis buffer for immunoprecipitation. A prote- ase-inhibitor cocktail (antipain, aprotinin, chymostatin, elastatinal, leupeptin, pepstatin A) was added to cell lysates and media (10 lg of each protease inhibitor per mL). The lysates were incubated for 20 min at 37 °C to extract TNSALP. The lysates and media were subjected to immu- noisolation, as described previously [11,12]. The immune complexes ⁄ Protein A beads were boiled in the absence or presence of 1% (v ⁄ v) 2-mercaptoethanol and were then analyzed by SDS ⁄ PAGE [9% (w ⁄ v) gels], followed by fluo- rography [11]. Enzyme digestion Endo H digestion and protease digestion using trypsin or proteinase K were carried out as described previously [11,12,29]. 3D structure of TNSALP A 3D model based on the crystal structure of human placental alkaline phosphatase (http://www.sesep.uvsq.fr./ Database.html) was downloaded and the program pymol (http://pymol sourceforge.net/) was used to generate the figure. Miscellaneous procedures Immunofluorescence for alkaline phosphatase was per- formed as described previously [12,15]. Sucrose-density-gra- dient centrifugation was performed as described previously [14,25,29]. Transfer of proteins and subsequent procedures were as described previously [25,29]. Proteins on mem- branes were detected using ECL Ò western blotting detection reagents. 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Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position. activity [4,18,19]. Replacement of valine at position of 406 with alanine was reported in a patient diagnosed with peri- natal hypophosphatasia who was a compound