Novel aggregate formation of a frame-shift mutant protein of tissue-nonspecific alkaline phosphatase is ascribed to three cysteine residues in the C-terminal extension Retarded secretion and proteasomal degradation Keiichi Komaru1,2, Yoko Ishida1, Yoshihiro Amaya1, Masae Goseki-Sone3, Hideo Orimo4 and Kimimitsu Oda1,5 Division of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Gakkocho-dori, Niigata, Japan Kitasato Junior College of Health and Hygienic Sciences, Yamatomachi, Minami-Uonuma-shi, Niigata, Japan Department of Food and Nutrition, Japan Women’s University, Mejirodai, Bunkyo-ku, Tokyo, Japan Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan Center for Transdisciplinary Research, Niigata University, Japan Keywords aggregation; alkaline phosphatase; degradation; hypophosphatasia; proteasome; ubiquitin Correspondence K Oda, Division of Biochemistry, Course for Oral Life Science, Niigata University, Graduate School of Medical and Dental Sciences, 2–5274, Gakkocho-dori, Niigata, 951–8514, Japan Fax: +81 25 227 2831 Tel: +81 25 227 2827 E-mail: oda@dent.niigata-u.ac.jp (Received 21 December 2004, revised 30 January 2005, accepted February 2005) doi:10.1111/j.1742-4658.2005.04597.x In the majority of hypophosphatasia patients, reductions in the serum levels of alkaline phosphatase activity are caused by various missense mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene A unique frame-shift mutation due to a deletion of T at cDNA number 1559 [TNSALP (1559delT)] has been reported only in Japanese patients with high allele frequency In this study, we examined the molecular phenotype of TNSALP (1559delT) using in vitro translation ⁄ translocation system and COS-1 cells transiently expressing this mutant protein We showed that the mutant protein not only has a larger molecular size than the wild type enzyme by  12 kDa, reflecting an 80 amino acid-long extension at its C-terminus, but that it also lacks a glycosylphosphatidylinositol anchor In support of this, alkaline phosphatase activity of the cells expressing TNSALP (1559delT) was localized at the juxtanucleus position, but not on the cell surface However, only a limited amount of the newly synthesized protein was released into the medium and the rest was polyubiquitinated, followed by degradation in the proteasome SDS ⁄ PAGE and analysis by sucrose-density-gradient analysis indicated that TNSALP (1559delT) forms a disulfide-bonded high-molecular-mass aggregate Interestingly, the aggregate form of TNSALP (1559delT) exhibited a significant enzyme activity When all three cysteines at positions of 506, 521 and 577 of TNSALP (1559delT) were replaced with serines, the aggregation disappeared and instead this modified mutant protein formed a noncovalently associated dimer, strongly indicating that these cysteine residues in the C-terminal region are solely responsible for aggregate formation by cross-linking the catalytically active dimers Thus, complete absence of TNSALP on cell surfaces provides a plausible explanation for a severe lethal phenotype of a homozygote hypophosphatasia patient carrying TNSALP (1559delT) Abbreviations Bz-Asn-Gly-Thr-NH2, benzoyl-asparagine-glycine-threonine-amide; DMEM, Dulbecco’s modified Eagle’s medium; ER, endoplasmic reticulum; ECL, enhanced chemiluminescence; GPI, glycosylphosphatidylinositol; LLnL, N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal); LLM, N-acetyl-Lleucinyl-L-leucinyl-L-methional); MG-132, benzyloxycarbonyl-L-leucinyl-L-leucinyl-L-leucinal; PI-PLC, phosphatidylinositol-specific phospholipase C; PNGase F, peptide:N-glycosidase F; MEM, minimum essential medium; TNSALP, tissue-nonspecific alkaline phosphatase; sTNSALP, soluble form of TNSALP 1704 FEBS Journal 272 (2005) 1704–1717 ª 2005 FEBS K Komaru et al Novel aggregate formation of an alkaline phosphatase frame-shift mutant Hypophosphatasia is an inborn error of metabolism characterized by defective mineralization of hard tissues and reduced levels of tissue nonspecific alkaline phosphatase (TNSALP, EC 3.1.3.1) [1–3] Hypophosphatasia is classified into at least five categories depending on age of onset and severity: perinatal, infantile, childhood, adult and odontohypophosphatasia The disease is caused by various mutations in the TNSALP gene, which is located on chromosome 1p-36.1–34, and is transmitted in an autosomal recessive or a dominant manner The severity of the disease is inversely related to serum levels of alkaline phosphatase activity, therefore indicating that reduction of enzyme activity caused by the defective TNSALP genes are responsible for poor mineralization of bone and tooth TNSALP-deficient mice develop rickets and osteopenia postnatally and many die of seizure [4–6], recapitulating infantile hypophosphatasia Additionally, as with hypophosphatasia patients, elevated levels of inorganic pyrophosphate, phosphoethanolamine and pyridoxal-5¢-phosphate have been reported in the serum and urine of the knockout mice This raises the possibility that these phosphocompounds are natural substrates for TNSALP Recently Hessle et al [7] have postulated that the concerted action of nucleoside triphosphate pyrophosphohydolase and TNSALP regulates local concentration of inorganic pyrophosphate at the site of mineralization, which, as a poison of hydroxyapatite growth, in turn controls mineralization According to this idea, increased levels of inorganic pyrophosphate resulting from the defect of TNSALP are thought to be the main cause of hypomineralization Since Weiss et al [8] first identified a missense mutation (Ala162Thr) in the TNSALP gene of a patient diagnosed with infantile hypophosphatasia, more than 161 mutations have been reported and of all mutations about 80% of them are missense [3,9,(http://www.sesep uvsq.fr/Database.html)] To date, biochemical characterization of TNSALP mutant proteins have been limited to a small number of cases Nevertheless, some missense mutations, in particular associated with severe forms of hypophophatasia, were known to impair proper folding and correct assembly of TNSALP in the endoplasmic reticulum (ER), resulting in the loss of functional TNSALP from the cell surface [10–14] Deletion of T at position 1559 of the cDNA [TNSALP (1559delT)] of the TNSALP gene was reported for the first time in hypophosphatasia patients by Orimo et al [15] and TNSALP (1559delT) is transmitted as a recessive trait TNSALP (1559delT) was originally referred to as TNSALP (1735delT) [16–18] According to the recommended nomenclature, the adenine of the FEBS Journal 272 (2005) 1704–1717 ª 2005 FEBS initiator ATG codon in the cDNA of TNSALP is denoted as nucleotide +1 instead of the first nucleotide of the cDNA clone originally isolated by Weiss et al [8] TNSALP (1559delT) is unique in that so far, this frameshift mutation has been reported only in the Japanese population [18] The patients are largely compound heterozygotes with different missense mutations, or in some cases with undetected mutations on the opposite allele, and exhibit clinical manifestations varying from infantile to odontohypophosphatasia [18] Quite recently, a case of a homozygous patient of TNSALP (1559delT) has been reported and classified as the prenatal lethal form, confirming that this mutation represents a severe allele [19] This frameshift mutation is assumed to eliminate the original translational stop codon and instead causes the extension consisting of 80 amino acid residues at the C-terminus of TNSALP Goseki et al detected a larger form of TNSALP in vivo in the serum of the patients carrying this mutation [16]; however, little is known about the molecular phenotype of TNSALP (1559delT) underlying the clinical symptoms In this report we have elucidated the biosynthesis of TNSALP (1559delT) in a heterologous expression system and in vitro translation ⁄ translocation system Our results shows that although TNSALP (1559delT) is synthesized as a secretory form lacking glycosylphosphatidylinositol (GPI), most of newly synthesized molecules form the aggregate and fail to exit from the ER Furthermore, the accumulated TNSALP (1559delT) was found to be polyubiquitinated under the condition where cellular proteasome activity was blocked, indicative of ubiquitin ⁄ proteasome pathway as part of an ER quality control mechanism We also have demonstrated that three cysteine residues in the C-terminal extension of this frameshift mutant protein are responsible for the formation of the novel aggregate retaining enzyme activity Results In vitro translation/translocation The deletion of T at cDNA number 1559 causes a frameshift downstream from leucine at position 503 of TNSALP, resulting in the elimination of an original translational stop codon Thus, the cDNA of TNSALP (1559delT) was predicted to encode a large sized TNSALP molecule with an additional 80 amino acidlong extension at the C-terminus (Fig 1) To confirm this prediction, we performed in vitro translation experiments as shown in Fig 2A The molecular mass of TNSALP (1559delT) was estimated to be 66 kDa 1705 Novel aggregate formation of an alkaline phosphatase frame-shift mutant TNSALP GPLLLALALYPLSVLF 506 521 1559delT GPLLLALALYP RASCSEGPGPGHPQARDRCQLPTRQPPSQGARWGPP LQLQERGPRKPKSAAHLAPLWNLPQGPNPLLASSLCSLPAALWPTG Fig Predicted amino acid sequence of TNSALP (1559delT) A single T deletion in the cDNA at nucleotide 1559 of tissue nonspecific alkaline phosphatase (TNSALP) changes the amino acid sequence at leucine 503 and downstream until the new stop codon appears Accordingly, this frame-shift mutation predicts that TNSALP (1559delT) is 80 amino acids longer than the wild type TNSALP Three cysteine residues at positions of 506, 521 and 577 are marked A 1559delT TNSALP 80 kDa 54 kDa B PNGase F TNSALP - + 1559delT - + 80 kDa 66 kDa 54 kDa Fig In vitro transcription ⁄ translation (A) Transcription-coupled translation of TNSALP or TNSALP (1559delT) were carried out in the absence (lanes and 4) or presence (lanes 2, 3, and 6) of canine pancreatic microsomes The N-glycosylation inhibitor Bz-Asn-Gly-Thr-NH2 was added at a final concentration of 0.5 mM (lanes and 6) Aliquots of the translation reactions were analysed by SDS ⁄ PAGE, followed by fluorography The leftmost lane shows 14 C-methylated protein markers of 200, 97.4, 66 and 46 kDa, from the top of the gel (B) The translation products (A, lanes and 5) were further incubated in the absence or presence of PNGase F, followed by SDS ⁄ PAGE ⁄ fluorography Left lane: 14C-methylated protein markers as in Fig 2A and larger than the wild type enzyme by  12 kDa This value is in close agreement with the calculated molecular mass (65 796) based on the amino acid 1706 sequence of TNSALP (1559delT) When translation was carried out in the presence of the canine microsome, TNSALP (1559delT) became a 80 kDa form; however, the appearance of the 80 kDa form was greatly diminished in the presence of Bz-Asn-Gly-ThrNH2, an inhibitor of N-glycosylation (Fig 2A, lane 6) Furthermore, upon incubation with PNGase F, which cleaves N-linked oligosaccharides between innermost N-acetylglucosamine and asparagine residue of glycoproteins, the 80 kDa form was completely converted to the 66 kDa form (Fig 2B), indicating that TNSALP (1559delT) is cotranslationally N-glycosylated to become the 80 kDa form in the microsome Thus, it is unlikely that the additional 80 amino acid residues at the C-terminus strongly affect the cotranslational translocation of TNSALP (1559delT) across the ER membrane Phase separation using Triton X-114 66 kDa K Komaru et al Given our finding that TNSALP (1559delT) is synthesized as a larger protein with a C-terminal extension, we considered the possibility that this frame-shift mutant protein fails to be attached by a GPI, because a putative GPI-anchor signal consisting of a stretch of hydrophobic amino acids is abrogated Previous studies have demonstrated that the wild type TNSALP expressed in the COS-1 cell is modified by GPI as shown by its sensitivity to phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves between phosphatidylglycerol and phosphoinositol of GPI, and metabolic labeling using [3H]ethanolamine, a component of GPI [10–13] To examine if TNSALP (1559delT) is modified by GPI, we exploited a phase separation method by Bordier [20] After metabolic labeling, the cells expressing either the wild type or TNSALP (1559delT) were lysed in a buffer containing TX-114 on ice, then warmed at 25 °C A detergent phase was separated from an aqueous phase by centrifugation and both phases were subjected to immunoprecipitation Newly synthesized wild type enzyme was largely partitioned into the detergent phase as shown in Fig (lanes & 2) The band in the aqueous phase probably represents GPI-anchor-less molecules due to overexpression of the enzyme in the transiently transfected cells It is noteworthy that a 66 kDa form, but not an 80 kDa form was partitioned into the aqueous phase As the 66 kDa form of the wild type migrates to the Golgi complex and becomes the 80 kDa form as described previously [10,12], this result suggests that the GPI-less molecules fail to exit the ER However, this partition behavior of the wild type enzyme completely changed upon incubation with PI-PLC prior to phase separation All wild type FEBS Journal 272 (2005) 1704–1717 ª 2005 FEBS K Komaru et al Novel aggregate formation of an alkaline phosphatase frame-shift mutant TNSALP sTNSALP 1559delT - - + + - - - det PI-PLC aq det aq det aq det aq - 80 kDa 66 kDa Fig Phase separation COS-1 cells expressing the wild type TNSALP (lanes 1–4), sTNSALP (lanes and 6) or TNSALP (1559delT) (lanes and 8) were labeled with [35S]methionine ⁄ cysteine for h The cells were lysed in buffer containing Triton X-114 and partitioned into detergent (det) and aqueous (aq) phases before (lanes 1, 2, 5–8) or after (lanes and 4) PI-PLC treatment Each phase was subjected to immunoprecipitation The immune complexes were analysed by SDS ⁄ PAGE, followed by fluorography Left lane: 14Cmethylated protein markers of 97.4, 66 and 46 kDa TNSALP molecules were now partitioned into the aqueous phase (Fig 3, lanes & 4), indicating that the wild type TNSALP molecule in the detergent phase represents a GPI-anchored membrane form In contrast to the wild type, TNSALP (1559delT) was exclusively found in the aqueous phase even without PI-PLC digestion (Fig 3, lanes & 8), strongly arguing that TNSALP (1559delT) lacks a GPI For comparison, we also expressed and analyzed a soluble truncated form of TNSALP (sTNSALP), which lacks the C-terminal 23 amino acids including a putative GPI-anchor signal sequence [21] As expected, sTNSALP was found to be recovered only in the aqueous phase like TNSALP (1559delT) (Fig 3, lanes and 6), further supporting that TNSALP (1559delT) is not modified by a GPI Biosynthesis of TNSALP (1559delT) If TNSALP (1559delT) is not attached by a GPIanchor, a prediction is that this mutant is no longer embedded into the lipid bilayer via GPI, which helps anchor TNSALP to the plasma membrane, but is secreted out of the cell To investigate whether this mutant protein is secreted, we labeled the transfected cells with [35S]methionine ⁄ cysteine and followed the kinetics of TNSALP secretion As reported previously [10,12], the wild type TNSALP was synthesized as the 66 kDa Endo H-sensitive form and underwent processing of N-linked oligosaccharides to become the 80 kDa Endo H-resistant mature species Both the 66 kDa and FEBS Journal 272 (2005) 1704–1717 ª 2005 FEBS 80 kDa forms of TNSALP were detected in the transfected cells, though the conversion of the precursor to the 80 kDa form obviously is not efficient in our transient expression system (Fig 4A, lanes 1–3) In contrast, in the cells expressing TNSALP (1559delT), an 80 kDa form – which corresponds in molecular mass to the in vitro 80 kDa translational product (Fig 2) – was the only molecular species throughout the chase time (Fig 4A, lane 6) The intensity of the 80 kDa form of TNSALP (1559delT) rapidly decreased as the chase time elapsed However, this decline is not simply accounted for by the secretion of the mutant protein into the medium, as no band was detectable even in the h chase culture medium (Fig 4A, lane 8) Only after a prolonged exposure, however, a 90 kDa form of TNSALP (1559delT) was found in the medium (results not shown) To confirm that TNSALP (1559delT) is indeed secreted into the medium, albeit in a lesser amount, culture media were collected from continuously radiolabeled transfected cells and any secreted TNSALP (1559delT) was immunoprecipitated A 90 kDa form of TNSALP (1559delT) became evident in the medium (Fig 4B, lane 4), suggesting that the 80 kDa form was processed to the 90 kDa form in the Golgi apparatus before being released into the medium In support of this, this secretory form was found to be sensitive to PNGase F but resistant to Endo H (Fig 4B, lanes & 6) On the other hand, the GPI-anchor-less sTNSALP was efficiently secreted out of the cells even after 0.5 h chase (Fig 4C), indicating that sTNSALP behaves like a genuine secretory protein Thus, we conclude that TNSALP (1559delT) is newly synthesized as the 80 kDa soluble form and mostly undergoes degradation, resulting in only a portion of it being secreted as the 90 kDa Endo H-resistant form Degradation and ubiquitination of TNSALP (1559delT) We next examined the effect of several protease inhibitors on the degradation of TNSALP (1559delT) Inhibitors of proteasome function, such as LLnL and MG-132, but not a calpain inhibitor (LLM) remarkably blocked the degradation of TNSALP (1559delT) (Fig 5A,B), indicative of involvement of the proteasome Consistent with this observation, leupeptin and pepstatin A (inhibitors of lysosomal proteases) had no effect on the degradation (results not shown) Quite recently we have reported that TNSALP (D289V), which is associated with perinatal hypophosphatasia, undergoes polyubiquitination prior to the degradation in the proteasome in the transfected COS-1 cells This 1707 Novel aggregate formation of an alkaline phosphatase frame-shift mutant A A cell medium TNSALP 1559delT K Komaru et al Control LLM LLnL MG132 TNSALP 1559delT 80 kDa 80 kDa 66 kDa B B cell 1559delT -LLnL +LLnL medium 90 kDa 80 kDa 80 kDa 54 kDa C cell sTNSALP C HA-Ub LLnL - medium - + + + - + - - + + + - + 72 kDa PolyUb Fig Pulse-chase experiment (A) Cells expressing TNSALP (lanes 1–3, 7) or TNSALP (1559delT) (lanes 4–6, 8) were pulse-labeled with [35S]methionine ⁄ cysteine for 30 (lanes and 4) and chased for h (lanes, and 5) or for h (lanes and 6) At h chase period, the media (lanes and 8) were removed and the cells were lysed for immunoprecipitation The immune complexes were analysed by SDS ⁄ PAGE and fluorography Left lane: 14 C-methylated protein markers of 97.4, 66 and 46 kDa (B) Cells expressing TNSALP (1559delT) were labeled for h with [35S]methionine ⁄ cysteine After h, the medium was removed (lanes 4–6) and the cells (lanes 1–3) were lysed for immunoprecipitation The immunoprecipitates were incubated in the absence (lanes and 4) or presence of PNGase F (lanes and 5) or Endo H (lanes and 6) prior to SDS ⁄ PAGE ⁄ fluorography Left lane: 14 C-methylated protein markers of 97.4, 66 and 46 kDa (C) Cells expressing sTNSALP were pulse-labeled with [35S]methionine ⁄ cysteine for 30 and chased for h (lane 1), for 0.5 h (lanes and 5), for h (lanes and 6) or h (lanes and 7) The media and cell lysates were subjected to immunoprecipitation and the immune complexes were analysed by SDS ⁄ PAGE ⁄ fluorography Left lane: 14 C-methylated protein markers of 200, 97.4, 66, 46 and 30 kDa 1708 anti-Ub anti-HA Fig Degradation and ubiquitination (A) Cells expressing TNSALP (1559delT) were pulse-labeled with [35S]methionine ⁄ cysteine for 30 and chased for h in the absence (lanes and 2) or presence of 50 lM LLM (lanes and 4), 50 lM LLnL (lanes and 6) or 50 lM MG-132 (lanes and 8) The cell lysates were subjected to immunoprecipitation and the immune complexes were analysed by SDS ⁄ PAGE and fluorography Left lane: 14C-methylated protein markers of 200, 97.4, 66, 46 and 30 kDa (B) Cells expressing TNSALP (1559delT) were pulse-labeled with [35S]methionine ⁄ cysteine for 30 and chased for h (lanes and 4), h (lanes and 5) or h (lanes and 6) in the absence (lanes 1–3) or presence of 50 lM LLnL (lanes 4–6) The immune complexes were analysed by SDS ⁄ PAGE and fluorography Left lane: 14C-methylated protein markers (Fig 4A) (C) Cells expressing TNSALP (1559delT) alone or TNSALP (1559delT) and HA-ubiquitin were incubated in the absence (–) or presence (+) of 50 lM LLnL for h Then the cells were lysed and subjected to immunoprecipitated with antiTNSALP After transfer, membranes were reacted with antiubiquitin (anti-Ub) or anti-influenza hemagglutinin epitope (anti-HA) Igs FEBS Journal 272 (2005) 1704–1717 ª 2005 FEBS K Komaru et al Novel aggregate formation of an alkaline phosphatase frame-shift mutant finding prompted us to determine if TNSALP (1559delT) also is ubiquitinated prior to degradation in the proteasome To this end we transfected the cells with the plasmid encoding TNSALP (1559delT) with or without the plasmid encoding ubiquitin bearing the N-terminal influenza HA epitope TNSALP (1559delT) was immnoprecipitated with anti-TNSALP Igs and subsequently the immunoprecipitates were subject to immunoblotting using either anti-ubiquitin or anti-HA Igs (Fig 5C) Not only did proteasome inhibitors did not affect ubiquitination, but also overall biosynthesis of the wild type enzyme (results not shown) [14] Remarkably, TNSALP (1559delT) was found to be heavily ubiquitinated in the presence of the inhibitor of proteasome function Furthermore, the extent of ubiquitination of TNSALP (1559delT) was further augmented in the cells expressing ubiquitin, strongly demonstrating that this mutant protein is degraded via ubiquitin ⁄ proteasome pathway mutant protein possesses catalytic activity and is concentrated in the Golgi apparatus on its way to being discharged In keeping with this morphological observation we found a low but significant enzyme activity in both the homogenate and culture medium of the cells expressing TNSALP (1559delT) (Fig 7A) However, an immunoblotting experiment demonstrated that the amount of TNSALP (1559delT) in the cell was less than one tenth of that of the wild type at steady state (Fig 7B, lanes and 6), probably reflecting its rapid degradation as shown in Fig Taking these values into consideration, the relative specific enzyme activity of the mutant protein was calculated to be about one third of that of the wild type (Fig 7C) In contrast to the culture medium of the cells expressing the wild type enzyme, very high enzyme activity was detected in that of the cells expressing sTNSALP (Fig 7A), consistent with a metabolic labeling study showing that sTNSALP is rapidly secreted out of the cell (Fig 4C) Catalytic activity of TNSALP (1559delT) Aggregation of TNSALP (1559delT) Figure shows cytohistochemistry for alkaline phosphatase In contrast to the wild type enzyme, virtually no alkaline phosphatase activity was detected on the cell surface of cells expressing TNSALP (1559delT) A faint staining might be attributed to secreted TNSALP (1559delT) trapped on the cell surface because of its aggregate nature (see below) These observations are compatible with the finding that the wild type, but not the mutant, protein is attached by GPI as shown in Fig Interestingly, we detected strong alkaline phosphatase activity at a juxtanucleus position in the cells expressing TNSALP (1559delT) as well as the wild type, suggesting that this Previously, we have reported that several TNSALP missense mutants tend to form a disulfide-bonded high-molecular-mass aggregate in transfected cells presumably due to defective folding and random association of mutant proteins [10–14] To investigate if this is also the case for TNSALP (1559delT), the newly synthesized mutant protein was immunoprecipitated and analysed by SDS ⁄ PAGE under reducing or nonreducing condition TNSALP (1559delT) formed a large aggregate bonded by multiple disulfide-bonds at the top of the resolving gel (Fig 8A, lanes & 4) In contrast, only a small amount of the wild type enzyme formed the aggregate (lanes and 3) The aggregate saponin - saponin + TNSALP 1559delT Fig Cytohistochemical staining for alkaline phosphatase Cells expressing TNSALP or TNSALP (1559delT) were stained for alkaline phosphate activity in the absence or presence of saponin FEBS Journal 272 (2005) 1704–1717 ª 2005 FEBS 1709 Novel aggregate formation of an alkaline phosphatase frame-shift mutant A K Komaru et al C 3000 2500 Alkaline phosphatase activity Alkaline phosphatase (unit/mg protein or ml) 3000 2000 1500 1000 2500 2000 1500 1000 500 500 0 TNSALP B sTNSALP TNSALP 2.5 TNSALP 1559deT 1559delT sTNSALP 1559delT 10 10 10 10 80 kDa 66 kDa thus found in the cells expressing the wild type could be GPI-anchor-less molecules, which are retained in the ER (Fig 3) Note that the secreted TNSALP (1559delT) also formed large aggregates (Fig 8A, lanes & 8) Addition of dithiothreitol in the culture medium did not enhance the secretion of the mutant, but rather inhibited it (results not shown) In good agreement with the SDS ⁄ PAGE, sucrose gradient centrifugation further demonstrated that TNSALP (1559delT) tends to form large aggregates Considerable amount of cellular activity and most of secreted activity was recovered in the bottom three fractions (Fig 8B) In contrast, sTNSALP peaked at fraction (Fig 8B) Because the wild type enzyme also appeared in fractions and in a similar analysis [12,13], this result indicates that sTNSALP forms a dimer Importantly, Km values estimated by Lineweaver–Burk plots were 4.3 · 10)4 m (wild type, cell homogenate), 1.9 · 10)4 m [TNSALP (1559delT), fractions 10–12 of the medium] and 5.5 · 10)4 m (sTNSALP, medium) This finding indicates that the C-terminal extension of 1710 Fig Enzyme activity of TNSALP (1559delT) (A) COS-1 cells, which had been transfected with the plasmids encoding the wild type TNSALP, sTNSALP or TNSALP (1559delT), were cultured for 24 h and then homogenized in the 50 mM Tris ⁄ HCl (pH 7.5) The cell homogenates (white bars) and media (black bars) were assayed for alkaline phosphatase and expressed in unit per mg protein (cell) or unit per mL culture medium, respectively (B) In addition to the cell homogenates (lanes 1–6) prepared as described in (A), cells expressing TNSALP (1559delT) were incubated in the presence of of LLnL (10 lM) for 24 h and then homogenized (lane 7) The homogenates were analysed by immunoblotting with anti-TNSALP The numbers above the fluorogram shows the amounts (lg) of protein applied on SDS ⁄ PAGE (C) The relative specific enzyme activities of the cell homogenates prepared from cells expressing TNSALP or TNSALP (1559delT) described as in A were calculated based on the relative amount (10 : 1) of both proteins in the homogenates as described in B (ordinate, arbitrary unit) TNSALP (1559delT) does not significantly affect the substrate affinity of this mutant, thus differentiating TNSALP (1559delT) from other missense TNSALP mutants possessing no catalytic activity, such as TNSALP (R54C), TNSALP (N153D), TNSALP (E218G), TNSALP (D289V) and TNSALP (G317D) [10–14] Addition of dithiothreitol into the culture media and cell lysates of the cells expressing TNSALP (1559delT) did not enhance the enzyme activity (results not shown) Replacement of three cysteines with serine residues in the C-terminus region Despite its aggregation state, TNSALP (1559delT) shows catalytic activity comparable to that of the wild type and sTNSALP as described above We therefore speculated that TNSALP (1559delT) becomes correctly folded and assembled by the time that the cysteine residues in the C-terminal region emerge through the translocon of the ER, and that it eventually undergoes FEBS Journal 272 (2005) 1704–1717 ª 2005 FEBS K Komaru et al Novel aggregate formation of an alkaline phosphatase frame-shift mutant A cell red nonred medium red nonred 90 kDa 80 kDa 66 kDa sTNSALP (cell) 90 alkaline phosphatase (unit/ml) 80 70 60 50 40 30 20 10 4000 3500 3000 2500 2000 1500 1000 500 10 11 12 alkaline phosphatase (unit/ml) 25 2 10 11 12 1559delT (medium) a c b c 1559delT (cell) 20 15 10 0 multiple cross-linking reactions via the cysteine residues To address this possibility, three cysteines were substituted for serine residues in the C-terminal extension of TNSALP (1559delT) (Fig 1) Initially we attempted to simultaneously replace all three cysteine residues at positions 506, 521 and 577 However, only two plasmids were obtained in which two out of three cysteine residues were replaced [TNSALP (1559delTC506C ⁄ C521S ⁄ C577S), TNSALP (1559delT-C506S ⁄ FEBS Journal 272 (2005) 1704–1717 ª 2005 FEBS sTNSALP (medium) 4500 alkaline phosphatase (unit/mg protein) Fig Sucrose-density-gradient analysis of TNSALP (1559delT) (A) Cells expressing TNSALP (lanes 1, 3, and 7) or TNSALP (1559delT) (lanes 2, 4, and 8) were continuously labeled with [35S]methionine ⁄ cysteine for h The media and cell lysates were subjected to immunoprecipitation The immune complexes were boiled in the absence (nonreducing condition) or presence (reducing condition) of 2-mercaptoethanol and analysed by SDS ⁄ PAGE, followed by fluorography An arrowhead indicates the top position of the resolving gel Left lane: 14 C-methylated protein markers of 200, 97.4, 66 and 46 kDa (B) After 24 h posttransfection, the lysates and media prepared from cell cultures expressing sTNSALP or TNSALP (1559delT) were directly applied on the top of sucrose-density-gradient analysis (5–35%) After centrifugation, each 400 lL fraction was collected from the top of the gradient and assayed for alkaline phosphatase activity (ordinate, unit per mL fraction) BSA (b, 68 kDa), alcohol dehydrogenase (a, 141 kDa) and catalase (250 kDa) were loaded on to a separate gradient as molecular mass markers alkaline phosphatase (unit/mg protein) B 10 11 12 10 11 12 C521C ⁄ C577S)] When these two proteins were expressed in COS-1 cells, the amount of the large aggregate was markedly reduced and instead the crosslinked dimer became prominent (Fig 9A, lanes 11–14) Note the decrease in the aggregate on the stacking gel Next, we introduced the third mutation into TNSALP (1559delT-C506S ⁄ C521C ⁄ C577S) The aggregation state was dramatically changed in the cells expressing TNSALP (1559delT-C506S ⁄ C521S ⁄ C577S) 1711 Novel aggregate formation of an alkaline phosphatase frame-shift mutant A red C M C M C K Komaru et al nonred M C M C M C M C M C M dimer 90 kDa 10 11 12 13 14 15 16 B 1559deT (serines) Alkaline phosphatase (nmol/min/ml medium) 1559delT c a b 120 350 100 300 250 80 200 60 150 40 100 20 50 0 10 11 12 10 11 12 Fig Replacement of the cysteine residues in the C-terminal extension (A) Cells were transfected with pALTERÒ-MAX encoding TNSALP (1559delT) (lanes 1, 2, and 10), pALTERÒ-MAX encoding TNSALP (1559delT-C506S ⁄ C521C ⁄ C577S) (lanes 3, 4, 11 and 12), pALTERÒMAX encoding TNSALP (1559delT-C506C ⁄ C521S ⁄ C577S) (lanes 5, 6, 13 and14) or pAltermax encoding TNSALP (1559delT-C506S ⁄ C521S ⁄ C577S) (lanes 7, 8, 15 and 16) After 24 h the cells were continuously labeled with [35S]methionine ⁄ cysteine After h, the media (M) and cell lysates (C) were subjected to immnoprecipitation Iodoacetoamide was added to both the cell lysates and media (final concentration of 25 mM) The immune complexes were analysed by SDS ⁄ PAGE in the absence (nonreducing condition) or presence (reducing condition) of 2-mercaptoethanol, followed by fluorography Double and single arrowheads indicate the top of the stacking and resolving gels, respectively Left lane: 14C-methylated protein markers of 200, 97.4, 66, 46 and 30 kDa (B) After 24 h post-transfection, the media were removed from the cell cultures expressing either TNSALP (1559delT) or TNSALP (1559delT-C506S ⁄ C521S ⁄ C577S) [1559delT (serines)] and directly applied on the top of the sucrose-density-gradient After centrifugation, each 400 lL fraction was collected from the top of the gradient and assayed for alkaline phosphatase activity (ordinate, unit per mL fraction) BSA (b, 68 kDa), alcohol dehydrogenase (a, 141 kDa) and catalase (c, 250 kDa) were loaded on to a separate gradient as molecular mass markers Not only the aggregate but also the covalently linked dimer almost disappeared (Fig 9A, lanes, 15 and 16) This modified TNSALP (1559delT) was found to sediment at a dimer position as judged by sucrose-density centrifugation (Fig 9B) We therefore concluded that TNSALP (1559delT-C506S ⁄ C521S ⁄ C577S) formed a noncovalently assembled dimer similarly to sTNSALP (Fig 8B) and the wild type enzyme [12,13] As expected, TNSALP (1559delT-C506S ⁄ C521S ⁄ C577S) was secreted threefold more than TNSALP (1559delT) (Fig 9B; compare ordinates) 1712 Discussion TNSALP (1559delT) is a large-sized secretory protein lacking GPI A growing number of genetic diseases have been related to defective post-translational folding and resultant degradation in the ER as part of the ER quality control system [22–24] TNSALP missense mutant proteins, in particular associated with severe form hypophosphatasia, fall into this category The missense FEBS Journal 272 (2005) 1704–1717 ª 2005 FEBS K Komaru et al Novel aggregate formation of an alkaline phosphatase frame-shift mutant mutations such as TNSALP (R54C), TNSALP (N153D), TNSALP (E218G), TNSALP (D289V) and TNSALP (G317D) are causes for severe molecular phenotypes and exhibit only negligible alkaline phosphatase activity when expressed in the cell ectopically These mutants were found to form disulfide-bonded high-molecular-mass aggregates and accumulate in the ER and ⁄ or cis-Golgi, followed by degradation via the proteasome In contrast to these missense mutants, TNSALP (1559delT) is unique in that it has the long C-terminal extension due to the frameshift mutation In vitro translation ⁄ translocation experiments demonstrated that the translational product (66 kDa) of the mutant protein is larger than that of the wild type by  12 kDa, compatible with an additional 80 amino acid residues at C-terminus (Fig 1) This 66 kDa product becomes the 80 kDa form in the presence of the microsome Probably the increase in molecular mass is solely due to the acquisition of N-linked oligosaccharides, as supported by two lines of evidence First, the molecular shift was remarkably diminished when translation ⁄ translocation experiments were carried out in the presence of an inhibitor of N-linked oligosaccharide attachment Second, the 80 kDa form was converted into the 66 kDa form by digestion with PNGase F Consistent with the in vitro translation, we observed the 80 kDa form immediately following a pulse-period in the cultured cells expressing TNSALP (1559delT) (Fig 4A, lane 4) Another feature of this mutant is its solubility In contrast with the missense mutants mentioned above, TNSALP (1559delT) is a soluble enzyme lacking a GPI-anchor This was examined by phase separation using Triton X-114 The wild type enzyme is largely partitioned into the detergent phase and moved into the aqueous phase only after PI-PLC digestion (Fig 3) TNSALP (1559delT) was exclusively partitioned into the aqueous phase without PI-PLC digestion As a control, a soluble truncated form of TNSALP (sTNSALP) was also partitioned into the aqueous phase, further supporting the hypothesis that TNSALP (1559delT) lacks GPI With respect to secretion, it is of interest that several missense TNSALP mutant proteins are reported to be secreted out of the cell, such as sTNSALP, when they are synthesized as soluble forms lacking GPI [25] TNSALP (1559delT) forms an aggregate and is degraded Although TNSALP (1559delT) is a soluble enzyme, its secretion was far less efficient than that of sTNSALP (Fig 4A,C) Nevertheless, a small portion of TNSALP FEBS Journal 272 (2005) 1704–1717 ª 2005 FEBS (1559delT) progressed to the Golgi apparatus, acquired Endo H-resistance and was then released as the 90 kDa form into the medium (Fig 4B) Analyses by SDS ⁄ PAGE and sucrose-density-gradient analysis demonstrated that TNSALP (1559delT) formed a disulfide-bonded high-molecular-mass aggregate in the transfected cells (Fig 8), implying that this aggregation state is probably a cause of impaired secretion of TNSALP (1559delT) The aggregation may lower the probability of TNSALP (1559delT) being segregated into COP II vesicles at the exit site from ER and therefore the mutant protein remains longer in the lumen of the ER and finally is diverted to the degradation pathway Because the degradation is blocked by inhibitors of proteasome function (Fig 5A,B), it is likely that TNSALP (1559delT) is eventually degraded in the proteasome in the cytoplasm We also found that TNSALP (1559delT) is polyubiquitinated before being destroyed in the proteasome (Fig 5C) TNSALP (1559delT) is not the only TNSALP mutant protein that is degraded via the ubiquitin ⁄ proteasome pathway TNSALP (D289V), which is associated perinatal hypophosphatasia, is another example [14] These findings suggest that the biosynthesis of TNSALP is under scrutiny of the ER quality control system Improperly folded and incorrectly assembled molecules are moved into cytoplasm in the early stage of the secretory pathway [26–28] However, much remains to be learned regarding the molecular mechanism leading to degradation from the ER How are mutant forms of TNSALP but not the wild type recognized and retrotranslocated into the cytoplasm? What type of ubiquitin ligase(s) is involved in the ubiquitination of TNSALP mutant proteins prior to destruction in the proteasome? Furthermore, as both TNSALP (1559delT) and TNSALP (D289V) are present in aggregate state, is it an obligatory process to reduce the disulfide-bonded aggregate prior to translocation in an opposite direction? Two molecules have recently emerged as key components of the ER quality control system, namely a Man8GlcNAc2-binding lectin (EDEM) [29,30], and SCFFbs2 ubiquitin ligase complex, which specifically targets N-linked high-mannosetype oligosaccharide chains of glycoproteins [31] The involvement of EDEM and ⁄ or SCFFbs2 in the degradation of TNSALP mutant proteins is currently being investigated The aggregate form of TNSALP (1559delT) possesses enzyme activity TNSALP (1559delT) retains the catalytic function comparable to the wild type enzyme, even though it 1713 Novel aggregate formation of an alkaline phosphatase frame-shift mutant forms large aggregates in both the cell and the medium This is supported by several lines of evidence as follows: (a) cytohistochemistry for alkaline phosphatase activity (Fig 6); (b) enzyme assay of the cell homogenate and culture medium of the cells expressing TNSALP (1559delT) (Fig 7); (c) the Km value of TNSALP (1559delT); and (d) SDS ⁄ PAGE in conjunction with sucrose-density-gradient analysis (Fig 8) At first glance this finding was quite puzzling as several missense TNSALP mutant proteins (R54C, N153D, E218G, D289V and G317D), which form similar highmolecular-mass aggregates in the transfected cells, exhibit no enzyme activity [10–14] However, the substitution of cysteines for serines at position of 506, 521 and 577 of TNSALP (1559delT) provided a clue The disulfide-bonded aggregation almost disappeared in the cell lysate and the culture medium of the cells expressing TNSALP (1559delT-C506S ⁄ C521S ⁄ C577S) (Fig 9) Importantly, thus modified TNSALP (1559delT) formed a noncovalently assembled homodimer like sTNSALP (Figs and 9) and the wild type [12,13], as judged by sucrose-density-gradient analysis Collectively, our findings strongly indicate that as it emerges through the translocon into the ER lumen, TNSALP (1559delT) adopts its proper conformation and assembles into the dimer structure; however, this catalytically active dimer further undergoes multiple cross-linkings among the dimers via the three cysteine residues in the C-terminal region Molecular phenotype and disease So far TNSALP (1559delT) has been reported only in the Japanese population In addition, this mutation has been found in about 71% of the Japanese hypophosphatasia patients with an allele frequency of 36% [18] However, it is unlikely that this specific mutation derives from a single founder, based on haplotype analysis [18] For Caucasians, TNSALP (E174K) has been repeatedly reported [32] Our results raise the possibility that TNSALP (1559delT) may be secreted into the circulation of patients carrying this mutation, albeit in a limited amount, as an aggregate form still possessing enzyme activity With regard to this, it is of interest that a large-sized TNSALP was detected in the sera of patients carrying this frame-shift mutation [16] However, this soluble form of TNSALP (1559delT) may quickly lose its catalytic activity during circulation, as the serum level of alkaline phosphatase activity in a homozygous patient was reported to be quite low [19] TNSALP on cell surfaces, but not circulating alkaline phosphatase is physiologically important Intravenous infusions of plasma from Paget disease or purified 1714 K Komaru et al alkaline phosphatase to hypophosphatasia patients failed to improve clinical conditions [33] Experimental procedures Materials Express 35S35S protein labeling mix (> 1000 CiỈmmol)1) was obtained from Dupont-New England Nuclear (Boston, MA, USA) 14C-methylated proteins and enhanced chemiluminescence western blotting detection reagent, peroxidaseconjugated donkey anti-(rabbit IgG) Ig and Protein A-Sepharose CL-4B from Amersham Pharmacia Biotech (Arlington Heights, IL, USA) pALTERÒ-MAX, Altered sitesÒ II mammalian mutagenesis system, TNTÒT7 coupled reticulocyte lysate system, T7 polymerase, Flexi rabbit reticulocyte lysate and canine pancreas microsome were from Promega (Madison, WI, USA); benzoyl-asparagine-glycinethreonine-amide (Bz-Asn-Gly-Thr-NH2), from BACHEM AG (Bubendorf, Switzerland); Lipofectamine Plus Reagent from Invitrogen (Carlsbad, CA, USA); N-acetyl-l-leucinyll-leucinyl-l-norleucinal (LLnL), N-acetyl-l-leucinyl-l-leucinyl-l-methional (LLM), aprotinin and saponin (Quillaja Bark) from Sigma Chemical Co (St Louis, MO, USA); peptide:N-glycosidase F (PNGase F) from New England Biolabs, Inc (Beverly, MA, USA); anti-HA Igs from BAbCO (Richmond, CA, USA); anti-multiubiquitin Igs from MBL (Nagoya, Japan); peroxidase-conjugated goat anti(mouse IgG) from Molecular Probes, Inc (Eugene, OR, USA); antipain, chymostatin, elastatinal, leupeptin and MG-132 (benzyloxycarbonyl-l-leucinyl-l-leucinyl-l-leucinal) and pepstatin A from Protein Research Foundation (Osaka, Japan); phosphatidylinositol-specific phospholipase C (PI-PLC) from Funakoshi Co (Tokyo, Japan); Triton X-114 from Nacalai Tesque, Inc (Kyoto, Japan) Antiserum against recombinant human TNSALP was raised in rabbits as described previously [21] COS-1 cells were cultured in Dulbecco’s modified Eagle’s minimum essential medium (DMEM) supplemented with 10% (v ⁄ v) fetal bovine serum [10] MG-132, LLnL and LLM were dissolved in dimethylsulfoxide (50 mm stock solution) and stored at )20 °C Plasmids and transfection The plasmids encoding the wild type TNSALP, TNSALP (1559delT) or secretory form of TNSALP (sTNSALP) were constructed as described previously [10–12,21] For mutations, the cDNAs of wild type TNSALP and TNSALP (1559delT) were subcloned into pALTERÒMAX Mutations were introduced at specific sites to replace three cysteine residues with serines using Altered sitesÒ II mammalian mutagenesis system as described previously [13,14] Oligonucleotides used were: C506S, 5¢-CCCTCAGAACTG FEBS Journal 272 (2005) 1704–1717 ª 2005 FEBS K Komaru et al Novel aggregate formation of an alkaline phosphatase frame-shift mutant GACGCTC-3¢; C521S, 5¢-GTGTGGGAAGTTGAGAT CTGTCACGGG-3¢; C577S, 5¢-GGGAGGGAGCTAAGG CTGG-3¢ The mutations were verified by DNA sequencing A plasmid encoding influenza hemaggulutinin (HA)tagged ubiquitin was provided by D Bohmann (EMBL, Heidelberg, Germany) Cells (1.0–1.3 · 105 cells per 35 mm dish) were transfected with 0.8–1 lg of each plasmid using Lipofectamine Plus according to the manufacturer’s protocol as described previously [13,14] and the transfected cells were incubated for 24 h in 5% CO2 ⁄ 95% air (v ⁄ v) incubator before use In vitro transcription/translation Transcription-coupled translation was performed using the TNTÒT7 coupled reticulocyte lysate system essentially according to the manufacturer’s protocol Transcription ⁄ translation was carried out with [35S]methionine ⁄ cysteine at 30 °C for 90 in the absence or presence of canine pancreatic microsomal membrane as described previously [14] Metabolic labelling and immunoprecipitation For pulse-chase experiments, cells were preincubated for 0.5–1 h in the methionine ⁄ cysteine-free DMEM and labeled with 50–100 lCi of [35S]methionine ⁄ cysteine for 0.5 h in the fresh methionine ⁄ cysteine-free MEM After a pulse period, cells were washed and chased in DMEM as described previously [10,14] When protease inhibitors were included, inhibitors were added at the start of starvation and present throughout entire pulse ⁄ chase experiments 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 deoxycholate ⁄ 0.05% (w ⁄ v) SDS in NaCl ⁄ Pi] A protease inhibitors cocktail (antipain, aprotinin, chymostatin, elastatinal, leupeptin, pepstatin A) was added to cell lysates and media (10 lgỈmL)1 for each) Unless stated otherwise, iodoacetoamide was not added to the lysates and media The lysates were incubated for 20 at 37 °C to extract TNSALP The lysates and media were subjected to immunoisolation as described previously [10,14] The immune complexes ⁄ Protein A beads were boiled in the absence or presence of 1% (v ⁄ v) 2-mercaptoethanol, and then analyzed by SDS ⁄ PAGE [9% (w ⁄ v) gels], followed by fluorography [10] Phase separation using Triton X-114 Following metabolic labeling, cells were collected, sonicated in the 20 mm Tris ⁄ HCl buffer (pH 7.5) containing 150 mm NaCl and 0.1% (w ⁄ v) Triton X-114 and incubated in the absence or presence of PI-PLC (0.05 unit) for h at 37 °C Samples were then adjusted to a final concentration of 1% FEBS Journal 272 (2005) 1704–1717 ª 2005 FEBS (w ⁄ v) Triton X-114 and subjected to phase separation essentially according to Bordier [20] TNSALP molecules recovered in aqueous and detergent phases were immunoprecipitated Miscellaneous procedures Cytohistochemical staining for alkaline phosphatase was performed as described previously [12] Sucrose-densitygradient analysis was performed as described previously [12,13] Electric transfer of proteins and subsequent procedures were described as before [13,14] Proteins on membranes were detected with enhanced chemiluminescence western blotting detection reagents Digestion of [35S]TNSALP with PNGase F and Endo H was carried out as described previously [10], as were the protein and alkaline phosphatase assays [12] One unit of alkaline phosphatase activity is defined as nmol of p-nitrophenylphosphate hydrolyzed per at 37 °C Acknowledgements We would like to thank Dr Dirk Bohmann for sending plasmids 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 by a grant for the Promotion of Niigata University Research Project (to K.O.) 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Komaru et al Novel aggregate formation of an alkaline phosphatase frame-shift mutant Hypophosphatasia is an inborn error of metabolism characterized by defective mineralization of hard tissues and... 2005 FEBS 1709 Novel aggregate formation of an alkaline phosphatase frame-shift mutant A K Komaru et al C 3000 2500 Alkaline phosphatase activity Alkaline phosphatase (unit/mg protein or ml) 3000... hypophosphatasia, undergoes polyubiquitination prior to the degradation in the proteasome in the transfected COS-1 cells This 1707 Novel aggregate formation of an alkaline phosphatase frame-shift mutant