Aberrant interchain disulfide bridge of tissue-nonspecific alkaline phosphatase with an Arg433 fi Cys substitution associated with severe hypophosphatasia Makiko Nasu 1 , Masahiro Ito 2 , Yoko Ishida 2 , Natsuko Numa 3 , Keiichi Komaru 4 , Shuichi Nomura 1 and Kimimitsu Oda 2,5 1 Division of Oral Health in Aging and Fixed Prosthodontics, 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 Pediatric Dentistry, Niigata University Graduate School of Medical and Dental Sciences, Japan 4 Kitasato Junior College of Health and Hygienic Sciences, Yamatomachi, Minami-Uonuma-shi, Niigata, Japan 5 Center for Transdisciplinary Research, Niigata University, Japan Keywords alkaline phosphatase; bone; disulfide bridge; hypophosphatasia; loss of function 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 0803 Tel: +81 25 227 2827 E-mail: oda@dent.niigata-u.ac.jp (Received 24 September 2006, accepted 23 October 2006) doi:10.1111/j.1742-4658.2006.05550.x Various mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene are responsible for hypophosphatasia characterized by defective bone and tooth mineralization; however, the underlying molecular mechanisms remain largely to be elucidated. Substitution of an arginine at position 433 with a histidine [TNSALP(R433H)] or a cysteine [TNSALP(R433C)] was reported in patients diagnosed with the mild or severe form of hypo- phosphatasia, respectively. To define the molecular phenotype of the two TNSALP mutants, we sought to examine them in transient (COS-1) and conditional (CHO-K1 Tet-On) heterologous expression systems. In contrast to an 80 kDa mature form of the wild-type and TNSALP(R433H), a unique disulfide-bonded 160 kDa molecular species appeared on the cell surface of the cells expressing TNSALP(R433C). Sucrose density gradient centri- fugation demonstrated that TNSALP(R433C) forms a disulfide-bonded dimer, instead of being noncovalently assembled like the wild-type. Of the five cysteine residues per subunit of the wild-type, only Cys102 is thought to be present in a free form. Replacement of Cys102 with serine did not affect the dimerization state of TNSALP(R433C), implying that TNSALP(R433C) forms a disulfide bridge between the cysteine residues at position 433 on each subunit. Although the cross-linking did not significantly interfere with the intracellular transport and cell surface expression of TNSALP(R433C), it strongly inhibited its alkaline phosphatase activity. This is in contrast to TNSALP(R433H), which shows enzyme activity comparable to that of the wild-type. Importantly, addition of dithiothreitol to the culture medium was found to partially reduce the amount of the cross-linked form in the cells expressing TNSALP(R433C), concomitantly with a significant increase in enzyme activity, suggesting that the cross-link between two subunits distorts the overall structure of the enzyme such that it no longer efficiently carries out its catalytic function. Increased susceptibility to proteases confirmed a Abbreviations Endo H, endo-b-N-acetylglucosaminidase H; ER, endoplasmic reticulum; GPI, glycosylphosphatidylinositol, PI-PLC, phosphatidylinositol- specific phospholipase C; TNSALP, tissue-nonspecific alkaline phosphatase; TNSALP(R433C), tissue-nonspecific alkaline phosphatase with an arginine to cysteine substitution at position 433; TNSALP(R433H), tissue-nonspecific alkaline phosphatase with an arginine to histidine substitution at position 433. 5612 FEBS Journal 273 (2006) 5612–5624 ª 2006 The Authors Journal compilation ª 2006 FEBS Hypophosphatasia is characterized by defective osteo- genesis with various degree of failure in mineralization of hard tissues such as bone and tooth [1–3]. Various mutations in the human tissue-nonspecific alkaline phosphatase (TNSALP, EC 3.1.3.1) gene are thought to be responsible for hypophosphatasia [1–5]. Hypo- phosphatasia is customarily divided into: (a) perinatal hypophosphatasia; (b) infantile hypophosphatasia; (c) childhood hypophosphatasia; (d) adult hypophospha- tasia; and (e) odonto-type hypophosphatasia. The bio- chemical hallmark of the disease is reduction in serum alkaline phosphatase activity. Variation in clinical expression is known to correlate well with variable residual enzymatic activities in hypophosphatasia patients (6,7). In general, the lower the activity, the more severe the symptoms. As of 24 July 2006, 184 mutations had been reported in the TNSALP gene worldwide, and about 80% of them are missense muta- tions [7] (http://www.sesep.usvq.fr. ⁄ Database.html). Recently, using a computer-assisted, three-dimensional model of TNSALP, Mornet et al. have proposed the categorization of missense mutations into different functional domains, such as the active site, the homodimer interface and the crown domain [8]. It is now easier to predict, estimate and probably under- stand the effects of some of the missense mutations on the TNSALP molecule. However, the structural evidence in itself may not be sufficient to assess the effects of other mutations on TNSALP, especially if a particular amino acid plays an essential role in the adoption of the native structure other than its role in maintaining the structure and function of the fully folded enzyme. In this respect, we previously reported that several TNSALP mutant proteins, which were reported in severe hypophosphatasia patients, tend to form a high molecular mass aggregate in the endoplas- mic reticulum (ER), resulting in decreased cell surface appearance of the TNSALP mutants, suggesting impairment of the folding and assembly process for TNSALP [9–13]. Furthermore, some mutant proteins undergo proteasomal degradation [11–13]. Obviously, an ER exit defect could be an important factor in the etiology of severe forms of hypophosphatasia, irrespective of whether mutant enzymes exhibit vari- able residual enzyme activity [3]. TNSALP is an ectoenzyme anchored to the plasma membrane via glycosylphosphatidylinositol (GPI), and is believed to regulate biomineralization by hydrolyzing inorganic pyrophosphate, the extracellular matrix mineralization inhibitor, on the surface of osteoblasts, chondrocytes and matrix vesicles derived from them [3,14]. TNSALP(R433H arginine to histidine substitution) was found in a compound heterozygote (R433H ⁄ D389G) diagnosed with odontohypophosphatasia [15], whereas TNSALP(R433C arginine to cysteine substitu- tion) was found in two independent homozygous patients with infantile hypophosphatasia [16]. The three-dimensional structure of human TNSALP predicts that an arginine residue at position 433 is unique to TNSALP and is located at the entrance of the active site pocket, raising the possibility of its involvement in sub- strate positioning [8]. Because of its conservative nature, the replacement of arginine with histidine was assumed to affect the catalytic function of TNSALP less severely than replacement with cysteine. Here, we report that both TNSALP(R433H) and TNSALP(R433C) are anchored to the plasma membrane via GPI, like the wild-type. Nonetheless, in contrast to the wild-type and TNSALP(R433H), TNSALP(R433C) forms a covalent- ly cross-linked dimer with low catalytic efficiency, pre- sumably explaining the severity of the disease when this particular mutation is present in a homozygous state. Results Transient expression of TNSALP mutants in COS-1 cells Human TNSALP folds and assembles as a noncova- lently associated homodimer in the ER and then pro- ceeds through the secretory pathway to the plasma membrane, where it is anchored via GPI [9,10]. Of five potential N-glycosylation sites of TNSALP, three sites are attached by oligosaccharide chains when the pro- tein is expressed in COS-1 cells [9]. TNSALP is syn- thesized as a 66 kDa endo-b-N-glucosaminidase H (Endo H)-sensitive form, is processed to a mature 80 kDa Endo H-resistant form, and finally appears on the cell surface. To examine whether the two missense mutations at position 433 of TNSALP affect the biosynthesis of TNSALP, we transfected COS-1 cells with a plasmid encoding TNSALP(R433C) or gross conformational change of TNSALP(R433C) compared with the wild- type. Thus, loss of function resulting from the interchain disulfide bridge is the molecular basis for the lethal hypophosphatasia associated with TNS- ALP(R433C). M. Nasu et al. Aberrant interchain disulfide bridge FEBS Journal 273 (2006) 5612–5624 ª 2006 The Authors Journal compilation ª 2006 FEBS 5613 TNSALP(R433H). The cells were metabolically labeled with [ 35 S]methionine ⁄ cysteine for 3 h and subjected to immunoprecipitation using anti-TNSALP serum, fol- lowed by SDS ⁄ PAGE⁄ fluorography as shown in Fig. 1. Under reducing conditions, the wild-type and the two TNSALP mutants gave a similar electropho- retic pattern, consisting of the 66 kDa and 80 kDa forms. However, strikingly, a distinct pattern was obtained under nonreducing conditions. In addition to the two molecular forms, a 160 kDa and a 130 kDa form were found only in the cells expressing TNS- ALP(R433C) (Fig. 1, lanes 2 and 6), indicating that a considerable portion of newly synthesized TNS- ALP(R433C) is covalently cross-linked via a disulfide bond. As reported previously [13], TNSALP(D289V) is not processed to the 80 kDa form, as this mutant is transport-incompetent (Fig. 1, lanes 4 and 8). Instead of being conveyed to the Golgi apparatus, it accumu- lates in the ER, and is eventually degraded in the ubiquitin–proteasome pathway [13]. We consistently observed a high molecular mass aggregate even in the cells expressing the wild-type under nonreducing conditions (see the top of the gel, Fig. 1, lanes 5–8). Previously, we reported that a proportion of the newly synthesized TNSALP fails to be modified with GPI, and resultant GPI-anchorless TNSALP molecules form the aggregate in transfected cells [17]. This probably reflects a shortage of a GPI precursor pool in the ER of COS-1 cells where TNSALP is overexpressed ectopi- cally. The two TNSALP mutants appear on the cell surface Next, we investigated whether the TNSALP mutants gain access to the cell surface like the wild-type. The cells that expressed each TNSALP mutant were meta- bolically labeled and further incubated with phosphati- dylinositol-specific phospholipase C (PI-PLC). Upon digestion, the 80 kDa form was the only form in the culture media of the cells that expressed the wild-type or TNSALP(R433H) (Fig. 2, lanes 4 and 12). However, the 160 kDa form as well as the 80 kDa form were released into the medium from the cells expressing TNSALP(R433C) (Fig. 2, lane 8), indicating that the dimerization via a disulfide bridge does not severely affect the cell surface appearance of TNSALP(R433C). As a negative control, no TNSALP(D289V) was released into the medium by digestion with PI-PLC, because this mutant fails to exit from the ER. Immuno- fluorescence studies also confirmed the cell surface appearance of the wild-type, TNSALP(R433C) and TNSALP(R433H), but not TNSALP(D289V) (data not shown). Catalytic activity of TNSALP mutants An immunoblotting method showed essentially the same result for steady-state expression of the TNSALP mutants as the biosynthetic experiments (Fig. 3A, lanes 5 and 6), confirming that TNSALP(R433C) tends to become a disulfide-bonded form that is clearly differ- ent from the noncovalently associated forms of the wild-type, which migrates on the SDS gel as the 66 kDa or 80 kDa form. To address the question of whether the replacement of arginine at position 433 affects the catalytic function of TNSALP, the cells expressing the two mutants were assayed for alkaline phosphatase activity using p-nitro- phenylphosphate as a substrate (Fig. 3B). Conservative replacement of arginine with histidine was expected not to greatly change the catalytic function of TNSALP(R433H), although we consistently detected higher specific enzyme activity in the cells expres- sing TNSALP(R433H) than in those expressing the a Dk061 a Dk031 a Dk08 a Dk66 87654321 dernonder Fig. 1. Biosynthesis of TNSALP mutants in COS-1 cells. COS-1 cells, which had been transfected for 24 h with a plasmid enco- ding the wild-type (lanes 1 and 5), TNSALP(R433C) (lanes 2 and 6), TNSALP(R433H) (lanes 3 and 7) or TNSALP(D289V) (lanes 4 and 8), were labeled with [ 35 S]methionine ⁄ cysteine for 3 h. The cell lysates were immunoprecipitated with anti-TNSALP, and the immune com- plexes were then analyzed by SDS ⁄ PAGE ⁄ fluorography under redu- cing (lanes 1–4) or nonreducing (lanes 5–8) conditions. Double and single arrowheads indicate the tops of the stacking and resolving gels, respectively. Left lane: 14 C-methylated protein markers of 200, 97.4, 66, 46 and 30 kDa, from the top of the gel. Aberrant interchain disulfide bridge M. Nasu et al. 5614 FEBS Journal 273 (2006) 5612–5624 ª 2006 The Authors Journal compilation ª 2006 FEBS wild-type. In contrast to this, the cell homogenate of the cells that expressed TNSALP(R433C) showed a much reduced level of activity as compared with the wild-type. As a negative control, TNSALP(D289V) did not exhibit any enzyme activity, in agreement with a previous report [13]. K m (V max ) values for the wild-type, TNSALP(R433C) and TNSALP(R433H), which were determined using Lineweaver–Burk plots, were 0.23 mm (2.57 lmolÆmin )1 ), 0.50 mm (1.05 lmolÆmin )1 ) and 0.34 mm (3.69 lmolÆmin )1 ), respectively. As the expression level of TNSALP(R433H) was higher than that of the wild-type in the COS-1 cells, based on the immunoblotting results (Fig. 3A, lanes 1 and 3), it seems reasonable to assume that replacement of argin- ine with histidine at position 433 does not have much affect on the catalytic function of TNSALP, although a definite conclusion awaits its purification. In the case of TNSALP(R433C), however, we were uncertain whether the decrease in specific enzyme activity could be attrib- uted to disulfide bond formation, as a significant amount of the noncross-linked molecular species was also present in the cell homogenate (Fig. 3A, lane 6). Expression of TNSALP(R433C) in CHO-K1 Tet-On cells As it was difficult to separate the noncross-linked and the cross-linked form of TNSALP(R433C) from each other in the native state by means of biochemical methods such as gel filtration and electrophoresis, we turned to another strategy. We reasoned that if expres- sion levels of TNSALP(R433C) are kept at a relatively low level compared with transient expression, most of the newly synthesized TNSALP(R433C) molecules might be oxidized to become disulfide-bonded in the CLP-I P ++ ++ ++ + + MCMCMCMCMCMCMCMC C334Rd-typeliWV982DH334R aDk061 aDk031 aDk08 aDk66 16 15141312 11 1098 7 6 5 43 21 Fig. 2. Cell surface appearance of TNSALP mutants in COS-1 cells. COS-1 cells, which had been transfected with a plasmid enco- ding the wild-type, TNSALP(R433C), TNS- ALP(R433H) or TNSALP(D289V) for 24 h, were labeled with [ 35 S]methionine ⁄ cysteine for 3 h and chased for 2 h. The cells were then further incubated in the absence or presence of PI-PLC. The cell lysates (C) and media (M) were immunoprecipitated with anti-TNSALP, and the immune complexes were analysed by SDS ⁄ PAGE (nonreduc- ing) ⁄ fluorography. The single arrowhead indicates the top of the resolving gels. Left lane: 14 C-methylated protein markers as in Fig. 1. 0 00 0 1 0002 00 0 3 0004 0 00 5 V982DH334RC334RTW Enzyme activity (U/mg protein) B 4321 8765 dernonder a Dk061 aDk031 aDk08 a D k6 6 A Fig. 3. Steady-state expression of TNSALP mutants in COS-1 cells. (A) COS-1 cells, which had been transfected with a plasmid enco- ding the wild-type (lanes 1 and 5), TNSALP(R433C) (lanes 2 and 6), TNSALP(R433H) (lanes 3 and 7) or TNSALP(D289V) (lanes 4 and 8) for 24 h, were homogenized, and 10 lg of each homogenate was directly separated by SDS ⁄ PAGE under reducing (lanes 1–4) or non- reducing (lanes 5–8) conditions and subjected to immunoblotting using anti-TNSALP. Double and single arrowheads indicate the tops of the stacking and resolving gels, respectively. (B) The same homogenates as described in (A) were assayed for alkaline phos- phatase activity and protein. Values are means of two independent experiments. M. Nasu et al. Aberrant interchain disulfide bridge FEBS Journal 273 (2006) 5612–5624 ª 2006 The Authors Journal compilation ª 2006 FEBS 5615 ER. This was the case. We succeeded in establishing a CHO-K1 Tet-On (Tet-On) cell line that expresses TNSALP(R433C) only in response to the addition of doxycycline (a tetracycline analog). In marked contrast to transient expression (Fig. 3), the 160 kDa disulfide- bonded form was the predominant molecular species in the Tet-On cells, with a trace amount of the 80 kDa noncross-linked form, over a wide range of expression conditions (Fig. 4). Induction of TNSALP(R433C) was found to be regulated tightly, as no band was observed in the absence of doxycycline (Fig. 4). Con- sistent with this, the alkaline phosphatase activity of Tet-On cells was negligible in the absence of the indu- cer (data not shown). When its synthesis was induced, TNSALP(R433C) was localized on the cell surface of the Tet-On cells, as judged by immunofluorescence (Fig. 5A) and PI-PLC digestion (Fig. 5B). Next, the detergent extracts of cells expressing the wild-type or TNSALP(R433C) were fractionated by sucrose density gradient centrifugation, and the distribution of TNSALP was analyzed by immunoprecipitation (Fig. 6). Both the wild-type and TNSALP(R433C) appeared at exactly the same position across the gradient, demonstrating that the disulfide-bonded 0.5 0.20DOX 1.00.50.201.0 nonredred a Dk061 a Dk031 aD k 0 8 a D k66 Fig. 4. Steady-state expression of TNS- ALP(R433C) in Tet-On cells.The established Tet-On cells harboring a plasmid encoding TNSALP(R433C) were cultured with differ- ent concentrations of doxycycline for 24 h. The cells were homogenized, and 5 lgof each homogenate was separated by SDS ⁄ PAGE under reducing (red) or non- reducing (nonred) conditions; this was fol- lowed by immunoblotting with anti-TNSALP. DOX, doxycycline. PCLP-I ++ MCMC aDk061 aDk031 a Dk08 a Dk66 AB Fig. 5. Cell surface appearance of TNSALP(R433C) in Tet-On cells. (A) Established Tet-On cells harboring a plasmid encoding TNS- ALP(R433C) were cultured with 0.5 lgÆmL )1 doxycycline for 24 h. After fixation, the cells were reacted with anti-TNSALP and then with anti-(rabbit IgG)–rhodamine. (B) The established Tet-On cells, which had been cultured with 1.0 lgÆmL )1 doxycycline for 14 h, were labeled with [ 35 S]methionine ⁄ cysteine for 0.5 h and chased for 1 h. The cells were further incubated in the absence or presence of PI-PLC. The cell lysates (C) and media (M) were immunoprecipitated with anti-TNSALP, and the immune complexes were analysed by SDS ⁄ PAGE (nonreduc- ing) ⁄ fluorography. Left lane: 14 C-methylated protein markers as in Fig. 1. Aberrant interchain disulfide bridge M. Nasu et al. 5616 FEBS Journal 273 (2006) 5612–5624 ª 2006 The Authors Journal compilation ª 2006 FEBS TNSALP(R433C) forms a dimer like the wild-type. The pulse-chase experiments demonstrated that the wild-type 66 kDa form was efficiently processed to the mature 80 kDa form, and this mature form was the only form found in the cell at 2 h chase time (Fig. 7A). Similarly, the majority of TNSALP(R433C) was 121110987654321 Wild-type C 334R aDk 08 a Dk061 a D k 0 8 c ab Fig. 6. Sucrose density gradient analysis of TNSALP(R433C). The established Tet-On cells harboring a plasmid encoding the wild-type or TNSALP(R433C) were cultured with 1.0 lgÆmL )1 doxycycline for 12 h. The cells were labeled with [ 35 S]methionine ⁄ cysteine for 1 h and fur- ther chased for 3 h. The cells were lysed, loaded on the top of the gradient [5–35% (w ⁄ w) sucrose], and centrifuged for 18 h at 4 °C. Each 400 lL fraction was collected from the top (fraction 1) of the gradient and immunoprecipitated. The immune complexes were separated by SDS ⁄ PAGE (nonreducing), followed by fluorography. The arrowhead indicates an unknown band. BSA (b, 68 kDa), alcohol dehydrogenase (a, 141 kDa) and catalase (c, 250 kDa) were applied on a separate gradient as size markers. Left lane: 14 C-methylated protein markers of 200, 97.4 and 66 kDa from the top of the gel. aDk08 aDk66 aDk 0 61 aDk0 31 a Dk66 0)h(esahC AB 215.0 epyt- d liW C33 4 R a Dk031 aDk66 +-HodnE Fig. 7. Biosynthesis of TNSALP(R433C) in Tet-On cells. (A) The established Tet-On cells harboring a plasmid encoding the wild-type or TNSALP(R433C) were cultured with 1.0 lgÆmL )1 doxycycline for 14 h, labeled with [ 35 S]methionine ⁄ cysteine for 0.5 h, and chased for up to 2 h. The cells were lysed and immunoprecipitated with anti-TNSALP, and the immune complexes were separated by SDS ⁄ PAGE (non- reducing), followed by fluorography. Left lane: 14 C-methylated protein markers of 200, 97.4 and 66 kDa from the top of the gel. (B) The established Tet-On cells harboring a plasmid encoding TNSALP(R433C) were cultured with 1.0 lgÆmL )1 doxycycline for 14 h. The cells were pulse-labeled with [ 35 S]methionine ⁄ cysteine for 0.5 h and immunoprecipitated for Endo H digestion. The immunoprecipitates were analyzed by SDS ⁄ PAGE (nonreducing) ⁄ fluorography. M. Nasu et al. Aberrant interchain disulfide bridge FEBS Journal 273 (2006) 5612–5624 ª 2006 The Authors Journal compilation ª 2006 FEBS 5617 efficiently converted to the 160 kDa form, although a small proportion of it remained unprocessed even after 2 h of chase. Thus we cannot exclude the possibility that this missense mutation also affects acquisition of the transport competence of TNSALP. The dimeri- zation of TNSALP(R433C) must occur at the ER, as the 130 kDa form appeared immediately after the pulse period (Fig. 7A). Furthermore, the 130 kDa disulfide-bonded TNSALP(R433C) was sensitive to Endo H digestion (Fig. 7B). The disulfide bridge suppresses the catalytic function of TNSALP(R433C) The predominance of the dimer form of TNS- ALP(R433C) in the Tet-On cells in response to doxy- cycline allowed us to unambiguously evaluate the enzyme activity of this disulfide-bonded TNSALP (R433C) (Fig. 8). The specific enzyme activity of the cells expressing TNSALP(R433C) was only one-twen- tieth of those expressing the wild-type enzyme. K m (and V max ) values obtained by kinetic studies are: 0.45 m m (6.75 lmolÆmin )1 ) for the wild-type and 0.66 mm (0.34 lmolÆmin )1 ) for the disulfide-bonded TNSALP (R433C). As the wild-type and TNSALP(R433C) in Tet-On cells were comparable in their expression levels as estimated by immunoblotting (Fig. 9, lanes 1 and 2), it is likely that the disulfide bond formation substan- tially suppresses the catalytic efficiency of TNS- ALP(R433C) without much affecting its substrate binding. Figure 9 shows the effect of dithiothreitol on the biosynthesis of TNSALP(R433C). Dithiothreitol is a membrane-permeable reducing agent and is known to render the lumen of the ER unfavorable for oxida- tion of sulfhydryl groups on cysteine residues [18]. The cells were incubated with doxycycline in the absence or presence of dithiothreitol for 12 h or 24 h. A small but significant amount of the 80 kDa form of TNSALP(R433C) was found to appear in the cells only with dithiothreitol (Fig. 9A, lanes 3 and 7). A concentration of 1 mm of dithiothreitol was optimal, and higher concentrations of dithiothrei- tol tended to inhibit the synthesis of TNSALP (R433C) induced by doxycycline. Importantly, we detected an increase in the enzyme activity of the cells concomitantly with the appearance of the 80 kDa form (Fig. 9B), suggesting that TNSALP (R433C) is capable of exhibiting its catalytic activity unless it is oxidized to form an interchain disulfide bond. However, we failed to increase the enzyme activity of the cell homogenate prepared from Tet-On cells expressing TNSALP(R433C) by incubating them with dithiothreitol or 2-mercaptoethanol under var- ious conditions. 0 0002 0004 0 00 6 0008 00001 00021 00041 C334RTW Enzyme activity (U/mg protein) Fig. 8. Alkaline phosphatase activity in the Tet-On cells expressing TNSALP(R433C). After the established Tet-On cells harboring a plasmid encoding the wild-type or TNSALP(R433C) had been cul- tured with 1 lgÆmL )1 doxycycline for 24 h, the cells were homo- genized and assayed for alkaline phosphatase and protein. The homogenates (5 lg each) were also used for immunoblotting (Fig. 9, lanes 1 and 2). Values are means of two independent experiments. Fig. 9. Effects of dithiothreitol on the expression of TNS- ALP(R433C). After the established Tet-On cells harboring a plasmid encoding the wild-type (lane 1) or TNSALP (R433C) (lanes 2–9) had been cultured with 1 lgÆmL )1 doxycycline for 12 h or 24 h in the presence of different concentrations of dithiothreitol (A), the cell homogenates (5 lg each) were used for immunoblotting (nonreduc- ing). (B) The same cell homogenates as described in (A) were investigated for alkaline phosphatase activity. The open bar and closed bar represent 24 h or 12 h of incubation with dithiothreitol, respectively. Values are means of two experiments. Aberrant interchain disulfide bridge M. Nasu et al. 5618 FEBS Journal 273 (2006) 5612–5624 ª 2006 The Authors Journal compilation ª 2006 FEBS Next, we compared protease susceptibility between the wild-type and TNSALP(R433C). As shown in Fig. 10, the wild-type enzyme was largely resistant to trypsin digestion at concentrations up to 50 lgÆmL )1 , whereas the mutant protein was found to be degraded at higher concentrations of trypsin. The same holds true for proteinase K digestion. The mutant protein completely disappeared even at 0.5 lgÆmL )1 (lane 7), but not the wild-type (lane 2). These results therefore suggest that the interchain disulfide bond markedly changes the tertiary structure of TNSALP such that TNSALP(R433C) becomes more susceptible to the proteases. An interchain disulfide bridge forms between two cysteines at position 433 Human TNSALPs have five cysteine residues (C102, C122, C184, C472 and C480) per subunit, and their positions are well conserved among four isoenzymes [3,19]. C122 and C472 are thought to bond to C184 and C480 in the same subunit, respectively, whereas C102 is in a free state, raising the possibility that C102 is involved in the interchain disulfide bridge of TNSALP(R433C). To address this question, we replaced C102 with serine and expressed TNSALP (C102S) in the COS-1 cells as shown in Fig. 11. TNSALP(C102S) consists of the 66 kDa immature and 80 kDa mature forms, and showed a similar specific enzyme activity to that of the wild-type. Also, a TNS- ALP double mutant (C102S ⁄ R433C) was found to be indistinguishable from TNSALP(R433C) as assessed by immunoblotting, as shown in Fig. 11A (lanes 7 and 8), suggesting that a disulfide bond forms between C433 residues on two subunits of TNSALP(R433C). Discussion Hypophosphatasia and TNSALP mutants Inorganic pyrophosphate is believed to play a pivotal role in bone matrix mineralization [20,21]. At lower concentrations (0.01–0.1 mm), pyrophosphate enhances mineralization, whereas it inhibits the formation of hydroxyapatite at concentrations higher than 1 mm. TNSALP is thought to promote mineralization by hydrolyzing pyrophosphate into phosphate. Fine regu- lation of pyrophosphate levels at the site of mineral- ization also requires at least two other proteins: nucleoside triphosphate pyrophosphatase phospho- diesterase (or PC-1), which generates pyrophosphate from nucleoside triphosphate, and a channel protein ANK (ankylosis), which mediates transport of pyro- phosphate across the plasma membrane of osteoblasts [3,22,23]. Various mutations in the TNSALP gene cause a her- editary disease known as hypophosphatasia, which is characterized by defective osteogenesis, unequivocally pointing to the physiologic relevance of the enzyme in biomineralization [4,5,7]. In support of this, relevant knock-out mice develop rickets and osteomalacia, thus recapitulating infantile hypophosphatasia [24–26]. In TNSALP-deficient mice, the initiation of mineral crystallization occurs within matrix vesicles; however, 2101 9 87654321 019876543 C334Rd-typeliwC334Rd-typeliw nispyrtKesanietorp aDk08 aDk061 Fig. 10. Protease sensitivity. After the established Tet-On cells harboring a plasmid encoding the wild-type or TNSALP(R433C) had been cul- tured with 1 lgÆmL )1 doxycycline for 24 h, the cells were homogenized in 10 mM Tris ⁄ HCl (pH 8.0), using a sonicator. The homogenates were incubated with trypsin or proteinase K in an ice ⁄ water bath for 30 min at the indicated concentrations. For trypsin, the final concentra- tions were (lg ⁄ mL): lanes 1 and 6, 0; lanes 2 and 7, 5; lanes 3 and 8, 10; lanes 4 and 9, 20; lanes 5 and 10, 50. For proteinase K, the final concentrations were (lg ⁄ mL): lanes 1 and 6, 0; lanes 2 and 7, 0.5; lanes 3 and 8, 1.0; lanes 4 and 9, 5.0; lanes 5 and 10, 10. Lanes 1–5 and lanes 6–10 were analyzed by SDS ⁄ PAGE in the presence or absence of 2-mercaptoethanol, respectively. M. Nasu et al. Aberrant interchain disulfide bridge FEBS Journal 273 (2006) 5612–5624 ª 2006 The Authors Journal compilation ª 2006 FEBS 5619 the subsequent proliferation and growth stage of min- eralization is severely impaired, leading to an increase in noncalcified bone matrix (osteoid) [27], consistent with what is seen in hypophosphatasia patients [28]. Hypophosphatasia patients show wide-ranging clinical manifestations, from stillbirth with an almost unminer- alized skeleton to premature loss of deciduous teeth in childhood and pseudofracture first presenting in adult life [1,2]. The symptoms of hypophosphatasia are well known to correlate with the residual enzyme activities of affected patients [1,2,6,7]. During the course of our studies on the biosynthesis of several TNSALP mutants, we found that the missense mutations associ- ated with severe hypophosphatasia variously affect the efficiency with which TNSALP properly folds and cor- rectly assembles, depending upon the position of a missense mutation and the nature of a substituted amino acid. For example, TNSALP(A162T), found in a homozygous patient diagnosed with a lethal infantile form of hypophosphatasia [4], mainly formed a high molecular mass aggregate in the ER, and only a small proportion of newly synthesized TNSALP(A162T) reached its site of action, the cell surface [9,10]. Conse- quently, the cell surface expression of this TNSALP mutant is much reduced compared with that of the wild-type. More TNSALP(D277A) was found to gain access to the cell surface than TNSALP(A162T), albeit with a significant population in the ER [10]. Alternatively, TNSALP(R54C), TNSALP(N153D), TNSALP(E218G), TNSALP(D289V) and TNSALP (G317D) never appeared on the plasma membrane [9–13]. Interestingly, a recent study has shown that alkaline phosphatase acquires Zn 2+ , which is indis- pensable for its catalytic activity, in the Golgi apparatus on its way to the plasma membrane [29]. This leads to the speculation that TNSALP mutants, which are retained in the ER due to a folding defect, not only fail to appear on the cell surface, but also are not able to acquire Zn 2+ . Consistent with this, the TNSALP mutants with defective ER-to-Golgi transport did not show measurable alkaline phospha- tase activity when being expressed in COS-1 cells [10–13]. TNSALP(R433C) becomes cross-linked via a disulfide bridge Mammalian alkaline phosphatases have five cysteine residues per subunit, and their positions are well con- served [3,19,30]. C102 is believed to be present only in a free state, whereas C122 and C472 bind to C184 and C480, respectively, in the same subunit. Both TNSALP(C184Y) and TNSALP(C472S) have been reported in perinatal hypophosphatasia patients [15,31], implying that the two interchain disulfide bonds are necessary for the correct folding and assembly of TNSALP. TNSALP(R433C) was repor- ted in homozygous patients diagnosed with lethal infantile hypophosphatasia [16,32]. In contrast to the TNSALP mutants showing various degrees of folding defect, TNSALP(R433C) did not form a high molecu- lar mass aggregate. Instead, it formed a covalently cross-linked homodimer, as evidenced by sucrose den- sity gradient centrifugation (Fig. 6). As replacement reducing AB nonreducing 123 4 5678 6000 5000 4000 160 kDa 80 kDa Enzyme activity (U/mg protein) 3000 2000 1000 0 WT C102S R433C C102S/R433C Fig. 11. Expression of a TNSALP double mutant (C102S ⁄ R433C) in COS-1 cells. COS-1 cells expressing wild-type enzyme (lanes 1 and 5), TNSALP(C102S) (lanes 2 and 6), TNSALP(R433C) (lanes 3 and 7) or TNSALP(C102S ⁄ R433C) (lanes 4 and 8) were homogenized. The homo- genates were analyzed by SDS ⁄ PAGE under reducing or nonreducing conditions, and this was followed by immunoblotting with anti- TNSALP (A) or assayed for alkaline phosphatase (B). Aberrant interchain disulfide bridge M. Nasu et al. 5620 FEBS Journal 273 (2006) 5612–5624 ª 2006 The Authors Journal compilation ª 2006 FEBS of C102 with serine did not affect the cross-linking of TNSALP(R433C) (Fig. 11), this result strongly indi- cates that a sulfhydryl group on the cysteine residue at position 433 of one subunit is oxidized to bond to the counterpart of the other subunit. This covalent cross-linkage of TNSALP(R433C) occurs in an early stage of the secretory pathway, as the cross-linked molecular species appeared in the cell immediately after a pulse-labeling period, and besides this, the 130 kDa form was sensitive to Endo H digestion. Also, the results of the pulse-chase experiments suggest that most newly synthesized TNSALP(R433C) migrated from the ER to the Golgi apparatus at a similar rate to the wild-type enzyme (Fig. 7). The cell surface appearance of TNSALP(R433C) was shown by immunofluorescence microscopy and PI-PLC digestion (Fig. 5), indicating that TNSALP(R433C) resides on the cell surface as a GPI-anchored ecto- enzyme, like the wild-type. Thus, it is likely that the cross-linkage between the subunits did not greatly affect the biosynthesis and intracellular transport of this mutant protein. However, the intersubunit cross- linkage did severely affect the catalytic activity of TNSALP(R433C). This is based on the findings in Tet-On cells, which predominantly express the cross- linked form of TNSALP(R433C) in response to doxy- cycline. Considering that the expression levels of TNSALP(R433C) and the wild-type in each Tet-On cell line are very similar (Fig. 9A), comparison of K m and V max values suggests that the catalytic efficiency of the mutant protein is dramatically reduced com- pared with that of the wild-type. Increased suscepti- bility of TNSALP(R433C) to proteases supports the notion that the disulfide bridge has a profound effect on the structure of TNSALP (Fig. 10). The effects of substitution of R433 either with alanine or aspartate on the catalytic properties of TNSALP were reported by Kozlenkov et al. [33]. Both TNSALP(R433A) and TNSALP(R433D) showed a noticeable decrease in k cat with a moderate increase in K m . One might argue that the substitution of arginine with cysteine itself, but not the disulfide bridge, decreases the catalytic activity of TNSALP(R433C). However, this is unli- kely, for the following reasons: first, the COS-1 cells, which express both noncross-linked and cross-linked TNSALP(R433C), showed considerable enzyme acti- vity (Fig. 3). Second, when the Tet-On (R433C) cells were cultured in the presence of doxycycline and di- thiothreitol, a significant amount of TNSALP(R433C) failed to become cross-linked, and concomitantly we detected an increase in enzyme activity in the cell homogenates. Taken together, these facts suggest that the diminished catalytic function of TNSALP(R433C) due to its disulfide-bonded linkage is a likely cause for the lethal hypophosphatasia resulting from the homozygous presence of this mutation. To our know- ledge, this is the first TNSALP missense mutation associated with severe hypophosphatasia that abro- gates the catalytic activity of TNSALP without signi- ficantly affecting its cell surface expression. TNSALP(R433H) was reported in a compound heterozygote (R433H ⁄ D389G) diagnosed with a mild form of hypophosphatasia [15]. Therefore, it is reason- able to assume that TNSALP(R433H) does not have a severe effect, unlike TNSALP(R433C). Also, as the substitution of arginine with histidine is a conservative replacement, it was expected that this mutation would not much affect TNSALP activity. When expressed in COS-1 cells, TNSALP(R433H) showed enzyme activity comparable to that of the wild-type. Also, its biosyn- thesis and cell surface appearance were not measurably disturbed (Figs 1–3), further highlighting the clinical importance of the substitution of arginine at position 433 with cysteine. Experimental procedures Materials Express 35 S 35 S protein labeling mix (> 1000 CiÆmmol )1 ) was obtained from Dupont-New England Nuclear (Boston, MA), and 14 C-methylated proteins and enhanced chemiluminescence western blotting detection reagent, per- oxidase-conjugated donkey anti-(rabbit IgG) and protein A–Sepharose CL-4B were obtained from Amersham Phar- macia Biotech (Arlington Heights, IL); the pALTER-MAX, Altered sites II mammalian mutagenesis system was obtained from Promega (Madison, WI); the QuikChange II Site-Directed Mutagenesis kit was obtained from Stratagene (La Jolla, CA); G418 and pansorbin were obtained from Calbiochem (La Jolla CA); Lipofectamine Plus Reagent was obtained from Invitrogen (Carlsbad, CA); PI-PLC was obtained from BIOMOL International, L.P. (Plymouth Meeting, PA); aprotinin, doxycycline and saponin (Quillaja Bark) and l-1-tosylamide-2-phenylethyl-chloromethyl ketone-treated bovine pancreas trypsin were obtained from Sigma Chemical Co. (St Louis, MO); proteinase K was obtained from Roche Diagnotics (London, UK); antipain, chymostatin, elastatinal, leupeptin and pepstatin A were obtained from the Protein Research Foundation (Osaka, Japan); hygromycin B and p-amidinophenylmethanesulfonyl fluoride were obtained from Wako Pure Chemicals (Tokyo, Japan); and serum against recombinant human TNSALP was raised in rabbits as described previously [34]. pTRE2 and the BD CHO-K1 Tet-On cell line and Tet system approved fetal bovine serum were obtaied from BD Biosciences Clontech (Palo Alto, CA). M. Nasu et al. Aberrant interchain disulfide bridge FEBS Journal 273 (2006) 5612–5624 ª 2006 The Authors Journal compilation ª 2006 FEBS 5621 [...]... intracellular transport of tissue-nonspecific alkaline phosphatase with an Ala162 fi Thr mutation associated with lethal hypophosphatasia J Biochem (Tokyo) 123, 968–977 10 Fukushi-Irie M, Ito M, Amaya Y, Amizuka N, Ozawa H, Omura S, Ikehara Y & Oda K (2000) Possible interference between tissue-non-specific alkaline phosphatase with an Arg54 fi Cys substitution and a counterpart with an Asp277 fi Ala substitution. .. attention and suggesting the protease sensitivity assay 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 KO) and by a grant for the Promotion of Niigata University Research Project (to KO) Aberrant interchain disulfide bridge 12 References 1 Harris H (1989) The human alkaline phosphatases: what we know and... Oda K (2002) Retention at the cis-Golgi and delayed degradation of tissuenon-specific alkaline phosphatase with an Asn153 fi Asp substitution, a cause of perinatal hypophosphatasia Biochem J 361, 473–480 Ishida Y, Komaru K, Ito M, Amaya Y, Kohno S & Oda K (2003) Tissue-nonspecific alkaline phosphatase with an Asp289 fi Val mutation fails to reach the cell surface and undergoes proteasome-mediated degradation... N461I, C472S) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in patients with hypophosphatasia Hum Mutat 15, 293 Mornet E, Taillandier A, Peyramaure S, Kaper F, Muller F, Brenner R, Bussiere P, Friesinger P, Godard J, Le Merrer M et al (1998) Identification of fifteen novel mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in European patients with severe hypophosphatasia. .. substitution found in a compound heterozygote associated with severe hypophosphatasia Biochem J 348, 633–642 11 Fukushi M, Amizuka N, Hoshi K, Ozawa H, Kumagai H, Omura S, Misumi Y, Ikehara Y & Oda K (1998) 13 14 15 16 17 18 19 20 21 Intracellular retention and degradation of tissue-nonspecific alkaline phosphatase with a Gly317 fi Asp substitution associated with lethal hypophosphatasia Biochem Biophys Res Commun... Protein and alkaline phosphatase assays were performed as described previously [9,10] One unit of alkaline phosphatase activity is defined as nmoles of p-nitrophenylphosphate hydrolyzed per min at 37 °C Acknowledgements We thank Dr Yoshio Misumi, Dr Miwa Sohda and Dr Tsuneo Imanaka for their advice on establishing TetOn cells expressing a TNSALP mutant We also thank anonymous reviewers for bringing Cys1 02... establishment of a model of infantile hypophosphatasia Devdyn 208, 432–446 Fedde KN, Blair L, Silverstein J, Coburn SP, Ryan ´ LM, Weinstein RS, Waymire K, Narisawa S, Millan JL, MacGregor GR et al (1999) Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia J Bone Mineral Res 14, 2015–2026 Anderson HC, Sipe JB, Hessle L, Dhanyamraju R, Atti... Characterization of eleven novel mutations (M45L, R119H, 544delG, G145V, H154Y, C184Y, D289V, 862+5A, 1172delC, R411X, E459K) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in patients with severe hypophosphatasia Hum Mutat 13, 171–172 Whyte MP, Essmyer K, Geimer M & Mumm S (2006) Homozygosity for TNSALP mutation 1348C>T (Arg43 3Cys) causes infantile hypophosphatasia manifesting transient disease... transporters, ZnT5 and ZnT7, are 5624 30 31 32 33 34 35 36 required for the activation of alkaline phosphatases, zinc-requiring enzymes that are glycosylphosphatidylinositol-anchored to the cytoplasmic membrane J Biol Chem 280, 637–643 Kozlenkov A, Manes T, Hoylaerts MF & Millan JL (2002) Function assignment to conserved residues in mammalian alkaline phosphatases J Biol Chem 277, 22992–22999 Taillandier... pyrophosphate and osteopontin by akp2, enpp1 and a; an integrated model of the pathogenesis of mineralization disorders Am J Pathol 164, 1199–2019 Anderson HC, Harmey D, Camacho NP, Garimella R, Sipe JB, Tague S, Bi X, Johnson K, Terkeltaub R & Millan JL (2005) Sustained osteomalacia of long bones despite major improvement in other hypophosphatasiarelated mineral deficits in tissue nonspecific alkaline phosphatase . Aberrant interchain disulfide bridge of tissue-nonspecific alkaline phosphatase with an Arg433 fi Cys substitution associated with severe hypophosphatasia Makiko. tissue-non-specific alkaline phosphatase with an Arg 54 fi Cys substitution and a counterpart with an Asp 277 fi Ala substitution found in a com- pound heterozygote associated