Molecular basis of perinatal hypophosphatasia with
tissue-nonspecific alkaline phosphatase bearing
a conservative replacement of valine by alanine at
position 406
Structural importance of the crown domain
Natsuko Numa
1
, Yoko Ishida
2
, Makiko Nasu
3
, Miwa Sohda
2
, Yoshio Misumi
4
, Tadashi Noda
1
and Kimimitsu Oda
2,5
1 Division of Pediatric Dentistry, Niigata University Graduate School of Medical and Dental Sciences, Japan
2 Division of Oral Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Japan
3 Division of Oral Health in Aging and Fixed Prosthodontics, Niigata University Graduate School of Medical and Dental Sciences, Japan
4 Department of Cell Biology, Fukuoka University School of Medicine, Japan
5 Center for Transdisciplinary Research, Niigata University, Japan
Keywords
crown domain; glycosylphosphatidylinositol;
hypophosphatasia; raft; tissue-nonspecific
alkaline phosphatase
Correspondence
K. Oda, Division of Oral Biochemistry,
Niigata University Graduate School of

value compared
with that of the wild-type TNSALP. Interestingly, leucine and isoleucine,
but not phenylalanine, were able to substitute for valine, pointing to the
indispensable role of residues with a longer aliphatic side chain at position
406 of TNSALP. Taken together, this particular mutation highlights the
structural importance of the crown domain with respect to the catalytic
function of TNSALP.
Abbreviations
Endo H, endo-b-N-acetylglucosaminidase H; GPI, glycosylphosphatidylinositol; TNSALP (V406A), TNSALP with a valine to alanine substitution
at position 406; TNSALP, tissue-nonspecific alkaline phosphatase.
FEBS Journal 275 (2008) 2727–2737 ª 2008 The Authors Journal compilation ª 2008 FEBS 2727
Hypophosphatasia is caused by various mutations of
the tissue-nonspecific alkaline phosphatase (TNSALP)
gene (EC 3.1.3.1) [1–6]. To date a total of 191 distinct
mutations have been reported worldwide, and about
80% of these mutations are missense (http://www.
sesep.uvsq.fr./Database.html). Hypophosphatasia is
characterized by reduced levels of serum alkaline phos-
phatase activity and defective mineralization in bone
and tooth, and clinical severity is inversely correlated
to serum alkaline phosphatase levels [1,2,7]. Patients
suffering from severe hypophosphatasia, such as the
perinatal or infantile forms, develop severe defects in
skeletal bone mineralization, unequivocally demon-
strating that TNSALP is physiologically involved in
the mineralization process of bone. Consistent with
this concept, TNSALP-deficient mice are reported to
develop rickets and osteomalacia [8–10].
During the course of our study on several TNSALP
mutant proteins associated with the severe form of

crown domain is responsible for interacting with
extracellular matrix proteins, including collagen [4,21–
24]. Here we demonstrate that, in contrast to other
missense mutations associated with severe hypophos-
phatasia, the majority of TNSALP (V406A) molecules
are capable of reaching the cell surface at a rate simi-
lar to that of the wild-type enzyme, thus excluding
the possibility that transport incompetence is a major
molecular defect of TNSALP (V406A). Rather, it is
likely that this particular mutation affects the active
site of TNSALP through imposing a subtle change
on the crown domain, rendering TNSALP (V406A)
less efficient for its catalytic function.
Results
Transient expression of TNSALP (V406A)
When expressed transiently, TNSALP (V406A) pro-
duced only a weak cytochemical reaction product
compared with the wild-type enzyme (Fig. 1A). In
agreement with these staining patterns, the specific
alkaline phosphatase activity of the cell homogenate
expressing the mutant protein was less than one-quar-
ter of that of the cell homogenate expressing the
wild-type enzyme (Fig. 1B). Immunoblotting con-
firmed that both the wild-type protein and the
TNSALP (V406A) mutant consisted of a 66-kDa and
an 80-kDa molecular species (Fig. 1C), which repre-
sent an immature form bearing high mannose-type
N-linked oligosaccharides and a mature form bearing
complex-type oligosaccharides, respectively [11].
Besides, the amount of TNSALP (V406A) mutant

of the wild-type TNSALP forms the aggregate in
transfected COS-1 cells [25]. To circumvent this draw-
back of transient expression, we established CHO-K1
Tet-On cells harboring a plasmid encoding TNSALP
(V406A). When incubated with doxycycline (an ana-
logue of tetracycline) TNSALP (V406A) appeared on
the cell surface (Fig. 2A, panel a) and exhibited weak
enzyme activity (Fig. 2A, panel c). The protein was
induced only with doxycycline, and no disulfide-
bonded aggregate was found on the top of the gel
(Fig. 2B). Note that most of the cellular TNSALP
(V406A) was present as the 80-kDa mature form. This
is in marked contrast to the transiently transfected
cells, where the 66-kDa form was a predominant
molecular species (Fig. 1C). This immunoblotting pat-
tern of the CHO-K1 Tet-On cells resembles that of
Saos-2 cells [14] – osteosarcoma producing a large
amount of TNSALP. Figure 3A shows pulse–chase
labeling experiments in combination with endo-b-N-
glucosaminidase H (Endo H) digestion. The wild-type
enzyme was synthesized as the 66-kDa Endo H-sensi-
tive form, which quickly became the 80-kDa Endo H-
resistant form. The processing of the newly synthesized
wild-type enzyme was complete by the end of the 2-h
chase period. This was also the case for TNSALP
(V406A) with only a small fraction being sensitive,
even at the end of the 2-h chase. Compatible with this
result, both the wild-type protein and the mutant pro-
tein were partitioned into a cold Triton X-100-insolu-
ble fraction (the raft) at a similar rate (Fig. 3B),

)1
of doxycycline for 14 h, were pulse-
labeled with [
35
S]methionine for 30 min and then the cells were collected at the indicated chase periods. The cells were lysed in the lysis
buffer and subjected to immunoprecipitation in (A). The immunoprecipitates on beads were incubated with or without Endo H prior to analy-
sis by SDS-PAGE (reducing condition) ⁄ fluorography. Some degradation products were observed in the samples during incubation with the
glycosidase. Left lane:
14
C-methylated protein markers of 200, 97.5, 66, 46 and 30 kDa, from the top of the gel. In panel B, the metabolically
labeled cells were lysed in cold 1% Triton X-100 and centrifuged at 15 000 g for 10 min. Triton X-100 soluble (S) and insoluble (I) fractions
were separated. The latter was further lysed in the lysis buffer and incubated at 37 °C for 20 min to extract TNSALP from the raft. Both sol-
uble and insoluble fractions were subjected to immunoprecipitation, followed by SDS-PAGE (reducing condition) ⁄ fluorography. Left lane:
14
C-methylated protein markers of 200, 97.5, 66, 46 and 30 kDa, from the top of the gel.
80 kD
a
DOX + – + –
Red
BA
ab
cd
Nonred
Fig. 2. Expression of TNSALP (V406A) in a CHO-K1 Tet-On cell line. (A) Established CHO-K1 Tet-On cells harboring a plasmid encoding
TNSALP (V406A) were cultured for 24 h in the absence (b, d) or presence (a, c) of doxycycline (1 lgÆmL
)1
) and stained for immunofluores-
cence using anti-TNSALP serum (a, b) or stained for alkaline phosphatase activity (c, d). (B) CHO-K1 Tet-On cells were cultured for 24 h in
the absence or presence of doxycycline (DOX) and analyzed by SDS-PAGE in the absence (Nonred) or presence (Red) of 2-mercaptoethanol,
followed by immunoblotting. An arrowhead indicates the top of the gel.

a K
cat
⁄ K
m
value that was < 10% of that of the wild-
type enzyme (Table 1), indicating that the conversion
of valine to alanine at position 406 in the crown
domain compromises the catalytic function of
TNSALP.
Protease sensitivity of TNSALP (V406A)
As shown in Fig. 5A, both the wild-type protein and
the mutant protein migrated at exactly the same posi-
tion, as judged by sucrose-density-gradient centrifuga-
tion, demonstrating that both the mutant protein and
the wild-type protein form a homodimer. However,
TNSALP (V406A) was found to be much more suscep-
tible to trypsin or proteinase K than the wild-type pro-
tein (Fig. 5B), suggesting that the conformation of the
crown domain of TNSALP (V406A) may be altered so
that each protease degrades the mutant protein more
easily, although its overall structure is not markedly
different from the wild-type protein.
Mutation analysis of the residue at position 406
Valine and alanine are usually classified into the same
amino acid group with a hydrophobic side chain.
Therefore, we hypothesized that not only hydrophobic-
ity, but also the length of the alkyl side chain of the
amino acid at position 406, is crucial to the catalytic
efficiency of TNSALP. This was the case. Leucine and
isoleucine, but not phenylalanine, successfully substi-

M 2-
amino-2-methyl-1,3-propanediol ⁄ HCl buffer (pH 10.5) containing
5m
M MgCl
2
and 0.1% Triton X-100.
K
m
K
cat
(s
)1
)
K
cat
⁄ K
m
· 10
3
(M
)1
s
)1
)
Wild-type TNSALP 0.21 971 4624
TNSALP (V406A)
mutant
0.09 34 377
N. Numa et al. Molecular basis of perinatal form of hypophosphatasia
FEBS Journal 275 (2008) 2727–2737 ª 2008 The Authors Journal compilation ª 2008 FEBS 2731

surface [12–15], whereas TNSALP (A162T) and
TNSALP (D277A) were present at the cell surface)
[11,12]. Residual activities of the latter mutant enzymes
may contribute to a highly variable clinical expressivity
of hypophosphatasia [4]. Improper folding and resul-
tant delayed trafficking are also molecular phenotypes
of TNSALP having missense mutations such as E174K,
G438S, I473F, G232V, I201T and F310L [27,28].
Recently we have characterized a unique mutation
associated with infantile hypophosphatasia that appar-
ently does not impair the trafficking of TNSALP [29].
Fig. 5. Molecular properties of the TNSALP
(V406A) mutant. Established CHO-K1 Tet-
On cells harboring a plasmid encoding the
wild-type TNSALP or the TNSALP (V406A)
mutant were cultured in the presence of
1 lgÆmL
)1
of doxycycline for 24 h. (A) Cells
were lysed and directly applied to a sucrose
density gradient. After centrifugation, 13
fractions were collected and assayed for
alkaline phosphatase activity. The figure
combines the results from two gradients:
black bar, wild-type TNSALP; white bar,
TNSALP (V406A) mutant. Abscissa: units of
enzyme activityÆmL
)1
of each fraction. b, a
and c denote bovine albumin (68 kDa), alco-

[30]. In this report we focused on the V406A missense
mutation. Figure 7 is a proposed 3D structural model
of human TNSALP, based on the crystallographic
analysis of human placental alkaline phosphatase, in
which both Val406 and Arg433 residues are high-
lighted. Valine at position 406 is located in the crown
domain consisting of 65 residues [4,21]. We have dem-
onstrated that TNSALP (V406A) is another allele with
severe effects, but does not show defective trafficking
like TNSALP (R433C). The rate of the intracellular
transport of the mutant protein was similar to that of
the wild-type protein, as assessed by the acquisition of
Endo H resistance. Besides, the mutant protein was
found to be incorporated into the raft at a kinetic rate
similar to that of the wild-type enzyme. GPI-anchored
protein is well known to be incorporated into the raft
in the Golgi apparatus [31], thus being ferried to the
apical surface of differentiated epithelial cells [32].
These findings strongly suggest that the cause of this
severe hypophosphatasia is not a defect in transport,
but the decreased catalytic activity of TNSALP
(V406A) itself. This was indeed confirmed by the
kinetic analysis of a purified GPI-anchorless soluble
version of the mutant protein. The K
cat
⁄ K
m
value of
the mutant TNSALP was less than one-tenth of the
K

Active site
Active site
Fig. 7. 3D model of human TNSALP. Valine at position 406 and
arginine at position 433 in the crown domain are highlighted.
Fig. 6. Expression of TNSALP (V406L), TNSALP (V406I) and
TNSALP (V406F) in COS-1 cells. COS-1 cells were transfected with
the plasmid encoding the wild-type TNSALP or the TNSALP
(V406A), TNSALP (V406L), TNSALP (V406I) or TNSALP (V406F)
mutants, for 24 h. Cell homogenates were assayed for alkaline
phosphatase activity (abscissa: unit activityÆmg of protein
)1
) (upper
panel). The same samples were analyzed by SDS-PAGE (under
non-reducing conditions), followed by immunoblotting using anti-
TNSALP serum (lower panel).
N. Numa et al. Molecular basis of perinatal form of hypophosphatasia
FEBS Journal 275 (2008) 2727–2737 ª 2008 The Authors Journal compilation ª 2008 FEBS 2733
the valine residue at position 406 on one subunit may
interact with the counterpart on the other subunit
through their long aliphatic hydrocarbon chains, thus
contributing to assume a proper conformation of the
crown domain, which is presumably indispensable for
the efficient catalytic function of TNSALP. In support
of our hypothesis, the cysteine residue at position 433
in one subunit of TNSALP (R433C) becomes cross-
linked to the counterpart of the other subunit [29],
implying that the two cysteine residues are sufficiently
close to stretch out to form a covalent linkage in the
crown domain. However, our hypothesis is not com-
patible with the current TNSALP structural model

Ò
) western blotting detection
reagent, peroxidase-conjugated donkey anti-(rabbit IgG)
and protein A–Sepharose CL-4B were obtained from Amer-
sham Pharmacia Biotech (Arlington Heights, IL, USA);
pALTER
Ò
-MAX, Altered sites
Ò
II mammalian mutagenesis
system was obtained from Promega (Madison, WI, USA);
QuikChange II Site-Directed Mutagenesis kit was obtained
from Stratagene (La Jolla, CA, USA); G418 and pansorbin
were obtained from Calbiochem (La Jolla CA, USA); Lipo-
fectamine Plus Reagent was obtained from Invitrogen
(Carlsbad, CA, USA); phosphatidylinositol-specific phos-
pholipase C was obtained from BIOMOL International,
L.P. (Plymouth Meeting, PA, USA); aprotinin, doxycycline
and saponin (Quillaja Bark) and l-1-tosylamide-2-phenyl-
ethyl-chloromethylketone-treated bovine pancreas trypsin
were obtained from Sigma Chemical Co. (St Louis, MO,
USA); proteinase K was obtained from Roche Diagnostics
(London, UK); Ni-nitrilotriacetic acid resin and the plas-
mid Midi-kit were obtained from Qiagen (Hilden, Ger-
many); antipain, chymostatin, elastatinal, leupeptin and
pepstatin A were obtained from Protein Research Founda-
tion (Osaka, Japan); and hygromycin B and (p-amidinophe-
nyl) methanesulfonylfluoride were obtained from Wako
Pure Chemicals (Tokyo, Japan). Antiserum against recom-
binant human TNSALP was raised in rabbits as described

(V406A) was further subcloned into pTRE2 to establish
stable cell lines. Transfection and screening of stable cell
lines were performed essentially according to the manufac-
turer’s protocol. CHO-K1 Tet-On cells, which successfully
produced the mutant TNSALP in the presence of doxy-
cycline, but not in its absence, were identified using
immunofluorescence. Establishment and characterization of
CHO-K1 Tet-On cells expressing the wild-type TNSALP
will be published elsewhere. Established CHO-K1 Tet-On
cells were cultured and passaged in the absence of doxycy-
cline until they were used for experiments. For immuno-
blotting or immunofluorescence studies, the cells were
cultured in the presence of 1 lgÆmL
)1
of doxycycline for
24 h before use. Alternatively, cells were cultured in
0.2–0.5 lgÆmL
)1
of doxycycline for 14 h before biosynthetic
experiments. For transient expression, COS-1 cells
(1.0–1.3 · 10
5
cells per 35-mm dish) were transfected with
0.5–0.8 lg of each plasmid using Lipofectamine Plus,
according to the manufacturer’s protocol, as described
Molecular basis of perinatal form of hypophosphatasia N. Numa et al.
2734 FEBS Journal 275 (2008) 2727–2737 ª 2008 The Authors Journal compilation ª 2008 FEBS
previously [14,15], and the transfected cells were incubated
for 24 h in a 5% CO
2

noisolation, as described previously [11,12]. The immune
complexes ⁄ Protein A beads were boiled in the absence or
presence of 1% (v ⁄ v) 2-mercaptoethanol and were then
analyzed by SDS ⁄ PAGE [9% (w ⁄ v) gels], followed by fluo-
rography [11].
Enzyme digestion
Endo H digestion and protease digestion using trypsin or
proteinase K were carried out as described previously
[11,12,29].
3D structure of TNSALP
A 3D model based on the crystal structure of human
placental alkaline phosphatase (./
Database.html) was downloaded and the program pymol
(http://pymol sourceforge.net/) was used to generate the
figure.
Miscellaneous procedures
Immunofluorescence for alkaline phosphatase was per-
formed as described previously [12,15]. Sucrose-density-gra-
dient centrifugation was performed as described previously
[14,25,29]. Transfer of proteins and subsequent procedures
were as described previously [25,29]. Proteins on mem-
branes were detected using ECL
Ò
western blotting detection
reagents. Purification of the soluble forms of the wild-type
TNSALP and TNSALP (V406A) was carried out essentially
as described previously [26]. Protein and alkaline phospha-
tase assays were performed as described previously [11,12].
One unit of alkaline phosphatase activity is defined as nmol
of p-nitrophenylphosphate hydrolyzed per min at 37 °C.

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