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Original Studies |
Department of Environmental Medicine (G.C., T.M., M.Y., S.M., K.O.), Pediatrics (K.S.), Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Osaka 594-1101, Japan; and Department of Pediatrics (G.C., T.Y., M.S., S.N., S.O.) and Orthopedics (N.Y.), Osaka University Medical School, Suita, Osaka 565-0871, Japan
Address all correspondence and requests for reprints to: Keiichi Ozono, M.D., Department of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1101, Japan. E-mail: j61642{at}center.osaka-u.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The human TNSALP gene consists of 12 exons, including an alternative exon 1A or 1B and the coding exons 2–12 (5, 6). Eighteen point mutations (missense, deletion of codon and deletion of a nucleotide with frame shift) of TNSALP complementary DNA (cDNA) have been identified (2, 7, 8, 9, 10, 11, 12, 13, 14). The reported mutations associated with hypophosphatasia are scattered in exons 3, 4, 5, 6, 9, 10, 11, and 12. The elucidation of the molecular heterogeneity underlying hypophosphatasia may contribute to our understanding of the clinical heterogeneity observed in this disorder. For example, mutations at nucleotides 747, 1057, and 1309 are associated with mild (childhood and adult-form) hypophosphatasia (7) (the nucleotide number is designated relative to the initiation of cDNA; Ref. 15). Although the mutations responsible for mild hypophosphatasia may not cause a complete loss of ALP function, only a few mutagenesis experiments that examined mutated ALP activity have been reported (2, 7, 12).
The most severe form, the perinatal form, is almost always lethal. The precise mechanism underlying the lack of survival of such patients outside the uterus is not clear. However, the underdevelopment of the lungs associated with hypomineralization of bone is believed to be responsible for the deaths in the perinatal period in the severe form of hypophosphatasia (1). In contrast, we previously reported a neonatal patient with characteristic bone deformity and low serum levels of ALP activity who had survived without respiratory failure (12). The absence of any apparent hypomineralized bones observed in this patient supports the hypothesis that the maturation of the lungs that is critical for survival is related to bone development.
The mechanism whereby the lack of ALP activity causes the failure of mineralization of hard tissue also remains to be elucidated (16). Three major hypotheses have been postulated to explain how hypomineralization is caused by impaired ALP activity: an inability to concentrate organic phosphate, an accumulation of inorganic pyrophosphate, and a loss of bridging between collagen fibers and matrix vesicles (17). The initial site of mineralization is reported to be ALP-enriched matrix vesicles. ALP is attached to the outer surface of the matrix vesicle membrane, and it plays a critical role in mineralization. Therefore, the localization and function of the wild-type and TNSALP mutants associated with various forms of hypophosphatasia are important issues to be examined.
Concerning the relationship between structure and enzymatic activity, small regions of the protein have been found to be responsible for important functions of TNSALP, such as metal binding and membrane binding, partly on the basis of analogies to Escherichia coli ALP (15, 18). ALP is anchored in the plasma membrane by means of a covalent linkage to glycophosphatidylinositol (GPI) (19). More than a hundred proteins belong to the GPI-anchored protein family, and both the hydrophobicity of 20–30 carboxyl (C)-terminal amino acids and 2 amino acids with small side chains at the breaking point adjacent to the hydrophobic peptides are important for GPI anchoring (20, 21). The introduction of a mutation that generates an uncleaved signal for GPI anchoring leads to the retention in the endoplasmic reticulum of the mutated protein (21).
Green fluorescent protein (GFP) is used to visualize the transport and localization of various proteins in living cells (22). However, only a few proteins have been reported to localize on the cell surface by using this technique, and this method has not been used to detect the subcellular localization of any type of ALP (23). Several lines of evidence show that TNSALP is linked to the cell membrane by GPI anchoring, and the localization of TNSALP seems to be one of the critical factors for the mineralization of bones (16). Hence, we have decided to take advantage of GFP fusion with TNSALP to examine the subcellular localization of wild-type and mutated TNSALP.
In the present report, we describe a neonatal patient with hypophosphatasia who had no respiratory complications and no apparent hypomineralization. Two mutations (Phe310Leu and delT1735, the amino acid number is designated relative to the initiation of mature TNSALP; Ref. 15) of the TNSALP gene were found, and mutated proteins were analyzed, in terms of their enzymatic activity and subcellular localization, using GFP chimeras. We also describe the recent profile of the female patient (mentioned above) with a relatively mild form of hypophosphatasia, whose onset was at birth (12). Both patients were compound heterozygotes for the TNSALP gene mutation; the common mutation was F310L.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The patient was a male baby born at the 40th gestational week.
He was the first baby of unrelated patients. The ultrasound examination
at the 37th gestational week had revealed a deformity of the
extremities. He had no respiratory problems, but the deformity of the
extremities was confirmed by roentgenogram. He was suspected to have
skeletal dysplasia and was referred to the Osaka University Hospital at
the age of 1 month. He had dimple skin on the lower legs, and the
roentgenogram findings resembled those of patient 2, affected with
hypophosphatasia except for the absence of bone spurs (Fig. 1
). Patient 1 was diagnosed as having
hypophosphatasia, on the basis of a low level of serum ALP activity (56
IU/L: reference range, 490 ± 215.5) and characteristic bone
findings. In addition, both parents had low ALP activity levels
(father, 32 IU/L; mother, 68 IU/L: reference range, 145 ± 32 and
161 ± 60.5, respectively). At the age of 1 yr, the patient
underwent an operation to correct the bilateral foot deformity, to
promote walking alone. He is surviving and has reached the age of 18
months without any other complications.
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The profile of this patient was described previously (12). The patient was a female with deformity of the long bones and the characteristic bone spurs in the fibulas, noted at birth (24). She was found to be heterozygous for F310L and G439R mutations (12). At the age of 1 yr, the patient underwent an operation to correct the bilateral foot deformity and resect the bone spurs. She has reached the age of 4 without any other complications, and the deformity of long bones improved. She is surviving; however, her height is below average (90.9 cm at the age 4, less than 3 percentile level of normal height), with bilateral, relatively short fibulas.
Sequence analysis
The sequence analysis of the TNSALP gene was performed after the extraction of genomic DNA and total RNA from the peripheral mononuclear cells of patient 1 and his parents. The primers described in the previous report were used for the PCR (12). cDNA was synthesized from the total RNA using random hexamer and reverse polymerase (Super Script II; Gibco BRL, Grand Island, NY). The fragments amplified by PCR or RT-PCR were subcloned into pT7-Blue T-vector (Novagen, Madison, WI). The fluorescence-based dideoxy sequencing was performed using a Model 373A sequencer (Perkin-Elmer Corp. , Norwalk, CT).
Mutation analysis of the TNSALP gene by RT-PCR and PCR with restriction enzyme digestion
The substitution of C for T at codon 310 generates the new StuI site (AGGCCT). Thus, we investigated whether the restriction enzyme StuI could digest the PCR products using primers corresponding to introns 8 and 9, as reported previously (12).
The deletion of nucleotide T at 1735 abolishes the DdeI site (CTNAG). Therefore, we investigated whether the restriction enzyme DdeI could digest the products amplified by PCR using forward and reverse primers corresponding to exon 12 (5'-CCCCCACGTGATGG CGTATGCAGCC, 5'-GTGCCCGGGCCCTGGGCCCTTCGAACAG). Because exon 12 contains another DdeI site at nucleotide 1748, the reverse primer, which contains two base-pair mismatches to eliminate the DdeI site other than the site of the mutation in the patient, was used in the PCR reaction.
Mutagenesis and expression
Mutagenesis of the deletion of T at 1735 was achieved by PCR-mediated nucleotide changes. The human TNSALP expression vector (pSV2Aalp) was generously provided by Dr. P. S. Henthorn (15). The TNSALP cDNA carrying the F310L mutation was generated in the previous study (12). The 3'-untranslated region (3'-UTR) sequence of pSV2Aalp, reported by Weiss et al. (GenBank accession number M24439) is different from that reported by Kishi et al. (GenBank accession number X14174) (25). The nucleotide sequence of the 3'-UTR of TNSALP cDNA in the patient was the same as the latter sequence. Therefore, the mutated TNSALP cDNA including the 3'-UTR found in the patient was substituted for the normal TNSALP cDNA using PmaI and PstI sites. As a result, the 5 amino acids at the extreme C-terminal of wild-type ALP was substituted, and 80 amino acids were added at the C-terminus in the delT1735 mutant. The introduction of the mutation was confirmed by the sequencing, as described above. The expression of each plasmid containing wild-type or mutant cDNA of TNSALP was confirmed by Northern blot analysis using 20 µg total RNA and the fragments of TNSALP cDNA released by the digestion with Pma C1 and Nae I as a probe (26).
Measurement of ALP activity
The expression vectors of the normal and mutated ALP cDNA were transfected into COS-7 cells by the diethylaminoethyl-dextran method. Two days after the transfection, the lysates of whole cells, harvested in 10 mmol/L Tris-HCl (pH 7.4) and 0.05% Triton X-100, were used for the measurement of ALP activity after the sonication. The ALP activity was measured by the Lowry method using p-nitrophenylphosphate as a substrate in glycine alkaline buffer containing 10 mmol/L MgCl2 (27).
The ALP activity of the fusion proteins of ALP to GFP, described in detail below, was also measured. The Saos-2 cells stably transfected with the expression vectors of the fusion proteins of wild-type (GFP-ALP) or mutated ALP (GFP-ALPdelT1735 and GFP-ALPF310L) were harvested in RIPA buffer [1% Triton X-100, 1% Na deoxycholate, 0.1% SDS, 150 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 7.4), 5 mmol/L EDTA, and protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany)] 2 days after the transfection. The fusion proteins were immunoprecipitated by monoclonal anti-GFP antibody (Boehringer Mannheim) at 4 C for 3 h, followed by the incubation with protein A/G-conjugated agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 C overnight. After the agarose was washed with RIPA buffer, the agarose containing the fusion protein was used for the ALP assay.
Constructs of the fusion proteins of ALP to GFP
The expression vectors of fusion proteins (GFP-ALP) were
reconstructed in pcDNA3.1 vector (Invitrogen, Carlsbad,
CA) using synthesized nucleotides coding the signal peptide of TNSALP,
cDNA of GFP (238 amino acids) obtained by the digestion of pGreen
Lantern (Gibco BRL) with NotI, and cDNA of
mature TNSALP (507 amino acids in wild-type and F310L, 587 amino acids
in delT1735), obtained from pSV2Aalp with a 3'-UTR sequence
corresponding to that reported by Kishi et al. (see Fig. 4A)
(25). As a result, GFP was inserted between the signal peptide and the
mature peptides of the TNSALP. Nine amino acids (SSTVAAAAT) and seven
amino acids (LGGRSST) were generated between the signal peptide and GFP
and between GFP and the mature TNSALP, respectively, in the process of
constructing the expression plasmids. In the transient transfection
experiments, the expression vectors of the fusion proteins of wild-type
(GFP-ALP) or mutated ALPs (GFP-ALPdelT1735 and GFP-ALPF310L) were
transfected into cells of the human osteosarcoma-derived osteoblastic
cell line Saos-2 and MG63 using lipofectamine (Gibco BRL)
and TransFast (Promega Corp., Madison, WI), respectively.
Twenty-four hours after the transfection, the living cells were
subjected to observation with fluorescent microscopy (BH-2,
Olympus Corp., Tokyo, Japan).
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Saos-2 cells were transfected with the expression vectors containing the cDNA of GFP-ALP, GFP-ALPdelT1735, and GFP-ALPF310L, using lipofectamine. Cells were selected by neomycin resistance (G418, Gibco BRL), and several clones were obtained. The expression of the ALP proteins was confirmed by the visualization of GFP by fluorescent microscopy and Western blotting using anti-GFP antibody.
Analysis of chimera proteins by Western blotting
Whole-cell lysates of Saos-2 cells, transiently or stably transfected with the expression vector of fusion proteins grown in a 60-mm dish, were obtained in 200 µL RIPA buffer. The lysates, containing 40 µg protein, were boiled and applied on a 7.5% polyacrylamide gel containing 0.1% SDS. After the electrophoresis in Tris-glycine buffer containing 0.1% SDS, the proteins were transblotted to a PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA). After blocking with Block Ace (Dainippon Pharmaceuticals, Osaka, Japan), the membrane was incubated with a mouse monoclonal anti-GFP antibody (Boehringer Mannheim), followed by incubation with peroxidase-labeled antimouse IgG antibody (Amersham, Buckinghamshire, England). The immunocomplex was visualized by enhanced chemiluminescence (ECL; Amersham).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A missense mutation (T-to-C conversion, resulting in a Phe to Leu conversion at codon 310: F310L) was detected in exon 9. The deletion of T at position 1735 (leading to a frame shift, resulting in the substitution of 5 amino acids at the C-terminus of wild-type ALP and the addition of 80 amino acids: delT1735) was found in exon 12 in patient 1 (data not shown).
The former mutation was also detected in one allele of the mother of
patient 1, and the latter was detected in one allele of his father. No
other mutation was observed in the entire coding region of the TNSALP
cDNA. The former mutation (F310L) was confirmed by the digestion with
the restriction enzyme StuI, and the delT1735 mutation was
confirmed by the loss of digestion with another restriction enzyme,
DdeI (Fig. 2, A and B).
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The activity of the mutated ALP enzyme was measured using the
transient expression system. The COS-7 cells transfected with the
mutated plasmid at nucleotide 1735 (delT1735) exhibited low ALP
activity, corresponding to the COS-7 cells transfected with the mock
expression vector (15 ± 9% and 13 ± 4%, respectively). In
contrast, the COS-7 cells transfected with the mutated plasmid at codon
310 (F310L) exhibited a level 72 ± 14% of the wild-type ALP
control level (Fig. 3A). The expression
of these plasmids was almost equal, as demonstrated by the Northern
blot study (Fig. 3, B and C).
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Two previously described polymorphisms in the TNSALP gene were also detected in the parents of patient 1 (7). The bases of the polymorphic site in exon 7 and exon 9 were T/T and A/A in the alleles of the patient (963T/T, 1052A/A), in those of his father (963T/T, 1052A/G), and in those of his mother (963T/C, 1052A/G). One allele of each parent that possesses T and A at nucleotides 963 and 1052, respectively, had a mutation (father: delT1735, mother: F310L), and these mutations were transferred to the proband.
Localization of GFP-ALP, GFP-ALPF310L, and GFP-ALPdelT1735
As shown in Fig. 4B
, GFP-ALP and
GFP-ALPF310L seemed to localize on the cell surface in the cells stably
transfected with the expression plasmids of corresponding cDNA. In most
cells, GFP-ALP was also observed in the cytoplasm but never in the
nucleus. These findings were clearly different from the distribution of
GFP alone, which existed in both cytoplasm and nucleus, suggesting that
the localization of GFP-ALP on the cell surface is dependent on the
character of ALP. In contrast, GFP-ALPdelT1735 did not localize on cell
surface and was located only in the cytoplasm. These data were
essentially the same as those obtained in transient transfection
experiments using both Saos-2 and MG63 cells (data not shown). The
expression level and the size of chimera proteins were examined by a
Western blot analysis using anti-GFP antibody (Fig. 4C). The
GFP-ALPdelT1735 protein was slightly larger than the GFP-ALP and
GFP-ALPF310L proteins, indicating the addition of 80 amino acids in
GFP-ALPdelT1735. In addition, no apparent degradation products were
observed, indicating that GFP was fused to ALP in the transfected
cells.
The enzymatic activity of these chimera proteins was estimated after
the separation of the chimera proteins from endogenous ALP by
immunoprecipitation. The activity of these chimera proteins
corresponded to the ALP activity of cell lysates transfected with ALP
expression vectors shown in Fig. 3A; 100% in GFP-ALP, 60 ± 7%
in GFP-ALPF310L, and 9 ± 1% in GFP-ALPdelT1735 (mean ±
SD, Fig. 4D).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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To date, eighteen mutations of the TNSALP gene have been reported in patients with hypophosphatasia (2, 7, 8, 9, 10, 11, 12, 13, 14). The association between certain mutations and clinical subforms is obscure, although mutations at nucleotides 747, 1057, and 1309 seem to be associated with childhood and adult-form hypophosphatasia (7). Our patients were found to be heterozygous for F310L and delT1735 (patient 1) and F310L and G439R (patient 2). The clinical features of the patients and the results of the reconstruction (mutagenesis) experiments (72% activity of ALPF310L, compared with 100% of wild-type ALP) support the idea that F310L is associated with the relatively mild (nonlethal) form of hypophosphatasia manifested at birth. It is likely that residual ALP activity of the F310L mutant contributed to the mineralization in bones and to long survival of these patients.
The F310L and the delT1735 mutations identified in these patients have been reported in two and three other Japanese patients with hypophosphatasia, respectively (10, 12, 14). These mutations thus seem to be rather common in Japanese patients. Because both mutations can be detected by the digestion of PCR fragments with restriction enzymes (StuI and DdeI), we recommend the performance of a digestion study to determine the mutation of the TNSALP gene in Japanese patients with hypophosphatasia.
A number of polymorphisms have been reported at the TNSALP locus (7). Two polymorphic sites of the coding region have been reported in Caucasian populations, and we found that they are also polymorphic in the Japanese population. The almost equal rates of nucleotides (T: 0.42, C: 0.58 in exon 7; and A: 0.42, G: 0.58 in exon 9) in 19 Japanese people (G. Cai, unpublished data) and nonlinks with each other indicate that these polymorphisms of the coding region are informative for family analyses. In our study, the polymorphic sites indicated that the allele carrying T in exon 7 and A in exon 9 was linked to the F310L mutation in both patients, suggesting that the F310L mutation may be derived from a distant common ancestor rather than through independent mutation.
ALP is thought to be essential for the mineralization of bone. The precise mechanism whereby the reduced activity of ALP leads to the hypomineralization of bone is still to be determined, but three major hypotheses have been postulated to explain how hypomineralization is caused by reduced ALP activity, as noted in the beginning section of this text: an inability to concentrate organic phosphate, an accumulation of inorganic pyrophosphate, and a loss of bridging between collagen fibers and matrix vesicles. In the perinatal form of hypophosphatasia, hypomineralization, as well as severely affected bone formation, is observed; but hypomineralization and vulnerability to bone fracture are observed, even in the adult form. The hypomineralization of bone is, therefore, a characteristic feature of hypophosphatasia associated with impaired TNSALP activity in humans; this feature seems to be in contrast to mice with a defect of the TNSALP gene, whose dominant feature is convulsion (29, 30).
GFP is used to visualize the transport and localization of various proteins (22). To our knowledge, this is the first report that showed the localization of ALP at the cell surface, using GFP chimeras. The results are consistent with the previous findings obtained with the immunostaining of ALP protein (1, 19). In addition, the loss of localization of GFP-ALPdelT1735 at the cell surface was detected. It is likely that the aberrant localization of ALPdelT1735 is caused by the change of hydrophobicity of the C-terminal amino acids associated with the frame shift (hydropathy plot; data not shown).
The ALPdelT1735 had no enzymatic activity (Figs. 3A and 4D). The membrane anchoring is thought not to be
essential for the enzymatic activity, because the soluble ALP without
membrane still possesses the enzymatic activity. In our experiment
using immunoprecipitation, GFP-ALPdelT1735 did not have enzymatic
activity, whereas wild-type and ALPF310L hydrolyzed the substrate.
These data suggest that the change of conformation caused by the
addition of amino acids at the C-terminus leads to the loss of both
membrane localization and the enzymatic activity. The patients
homozygous for the delT1735 mutation had severe hypomineralized bones
and died soon after birth (G. Cai, manuscript in preparation). Hence,
the ALPdelT1735 mutant causes hypomineralization, but it is not clear
whether the loss of enzymatic activity and/or the loss of membrane
localization contributes to the hypomineralization. It will be of
interest to determine whether the ALP mutant, which loses membrane
localization but still has enzymatic activity, causes the impairment of
mineralization in bones.
In conclusion, we reported the cases of two patients with a relatively mild form of hypophosphatasia whose onset was at birth. They were compound heterozygotes for the TNSALP gene mutation; the common mutation was F310L. Because the mutated protein (F310L) has residual ALP activity, it is thought to contribute to the mildness of the clinical manifestations in these nonlethal forms of neonatal hypophosphatasia. The mutated ALP produced by the other mutation (delT1735) found in patient 1 was shown, by the analysis of GFP chimera, to have lost the membrane localization.
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Acknowledgments |
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Footnotes |
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Received December 2, 1997.
Revised May 28, 1998.
Accepted July 20, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Asp mutation in ALPL causes the
perinatal (lethal) form of hypophosphatasia in Canadian Mennonites. Genomics. 17:215–217.[CrossRef][Medline]
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