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

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 h

tissue-nonspecific alkaline phosphatase bearing

a conservative replacement of valine by alanine at

position 406

Structural importance of the crown domain

Natsuko Numa1, Yoko Ishida2, Makiko Nasu3, Miwa Sohda2, Yoshio Misumi4, Tadashi Noda1 and Kimimitsu Oda2,5

1 Division of Pediatric Dentistry, Niigata University Graduate School of Medical and Dental Sciences, Japan

2 Division of Oral Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Japan

3 Division of Oral Health in Aging and Fixed Prosthodontics, Niigata University Graduate School of Medical and Dental Sciences, Japan

4 Department of Cell Biology, Fukuoka University School of Medicine, Japan

5 Center for Transdisciplinary Research, Niigata University, Japan

Keywords

crown domain; glycosylphosphatidylinositol;

hypophosphatasia; raft; tissue-nonspecific

alkaline phosphatase

Correspondence

K Oda, Division of Oral Biochemistry,

Niigata University Graduate School of

Medical and Dental Sciences, 2-5274,

Gakkocho-dori, Niigata 951-8514, Japan

Fax: +81 25 227 0805

Tel: +81 25 227 2827

E-mail: oda@dent.niigata-u.ac.jp

(Received 30 November 2007, revised 18

January 2008, accepted 19 March 2008)

doi:10.1111/j.1742-4658.2008.06414.x

Hypophosphatasia, a congenital metabolic disease related to the tissue-non-specific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tis-sues A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophos-phatasia To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently trans-fected COS-1 cells and in stable CHO-K1 Tet-On cells Compared with the wild-type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum,

as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble frac-tion (raft) at a rate similar to that of the wild-type TNSALP TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K When purified as a glycosylphosphatidylinositol-anchorless soluble form, the mutant protein exhibited a remarkably lower Kcat⁄ Km 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.

Hypophosphatasia is caused by various mutations of

the tissue-nonspecific alkaline phosphatase (TNSALP)

gene (EC 3.1.3.1) [1–6] To date a total of 191 distinct

mutations have been reported worldwide, and about

80% of these mutations are missense (http://www

sesep.uvsq.fr./Database.html) Hypophosphatasia is

characterized by reduced levels of serum alkaline

phos-phatase activity and defective mineralization in bone

and tooth, and clinical severity is inversely correlated

to serum alkaline phosphatase levels [1,2,7] Patients

suffering from severe hypophosphatasia, such as the

perinatal or infantile forms, develop severe defects in

skeletal bone mineralization, unequivocally

demon-strating that TNSALP is physiologically involved in

the mineralization process of bone Consistent with

this concept, TNSALP-deficient mice are reported to

develop rickets and osteomalacia [8–10]

During the course of our study on several TNSALP

mutant proteins associated with the severe form of

hypophosphatasia, we found that the cell-surface

expression of the TNSALP mutants is remarkably

reduced The mutant proteins often fail to undergo

proper folding and correct assembly, resulting in

accu-mulation in the early stage of the secretory pathway

and eventual degradation in the endoplasmic reticulum

[11–15] However, the extent to which each TNSALP

mutant protein reaches the cell surface varies from one

mutation to another, depending on the position of the

mutation in the gene and the amino acid residue that

is replaced Fleisch et al proposed that TNSALP

regu-lates mineralization by hydrolyzing inorganic

pyro-phosphate, a poison of hydroxyapatite crystal [16,17],

at the site of biomineralization According to this

pro-posal, it is likely that defective bone formation

occur-ring in severe hypophosphatasia is closely related to

the number of cell-surface TNSALP mutant molecules

and their residual pyrophosphate-cleaving activity

[4,18,19]

Replacement of valine at position of 406 with

alanine was reported in a patient diagnosed with

peri-natal hypophosphatasia who was a compound

hetero-zygote for this mutation and A99T [20] The valine

residue at position 406 is located in a unique domain

called the crown domain [21] This domain shows the

lowest degree of homology among alkaline

phospha-tase isoenzymes, and isoenzyme-specific properties,

such as uncompetitive inhibition, heat stability and

allosteric behavior, are attributed to residues located

in this domain of each isoenzyme [4,22] Besides, this

crown domain is responsible for interacting with

extracellular matrix proteins, including collagen [4,21–

24] Here we demonstrate that, in contrast to other

missense mutations associated with severe

hypophos-phatasia, the majority of TNSALP (V406A) molecules are capable of reaching the cell surface at a rate simi-lar to that of the wild-type enzyme, thus excluding the possibility that transport incompetence is a major molecular defect of TNSALP (V406A) Rather, it is likely that this particular mutation affects the active site of TNSALP through imposing a subtle change

on the crown domain, rendering TNSALP (V406A) less efficient for its catalytic function

Results

Transient expression of TNSALP (V406A) When expressed transiently, TNSALP (V406A) pro-duced only a weak cytochemical reaction product compared with the wild-type enzyme (Fig 1A) In agreement with these staining patterns, the specific alkaline phosphatase activity of the cell homogenate expressing the mutant protein was less than one-quar-ter of that of the cell homogenate expressing the wild-type enzyme (Fig 1B) Immunoblotting con-firmed that both the wild-type protein and the TNSALP (V406A) mutant consisted of a 66-kDa and

an 80-kDa molecular species (Fig 1C), which repre-sent an immature form bearing high mannose-type N-linked oligosaccharides and a mature form bearing complex-type oligosaccharides, respectively [11] Besides, the amount of TNSALP (V406A) mutant was similar to that of the wild-type protein in trans-fected cells, indicating that the lower expression level does not account for the low specific enzyme activity

of the former Upon incubation with phosphatidylino-sitol-specific phospholipase C, both the wild-type pro-tein and the TNSALP (V406A) mutant were released into the medium (Fig 1D, lanes 4 and 8), confirming that they are anchored to the cell surface via glyco-sylphosphatidylinositol (GPI) By contrast, the 66-kDa form was the only molecular species observed within the cells expressing TNSALP (D289V) (Fig 1C), which is arrested and forms the disulfide-bonded aggregate in the endoplasmic reticulum [15], and is not able to gain access to the cell surface (Fig 1D, lane 12)

Expression of TNSALP (V406A) in a stable cell line

In the transient expression system, even the wild-type enzyme formed a disulfide-bonded aggregate, probably

as a result of the synthesis of an excess amount of TNSALP (Fig 1C, lanes 4–6, see the top of gel) In the present experiment, TNSALPs were expressed

under control of the CMV I.E Enhancer⁄ Promoter.

We observed the same aggregate also in a previous

experiment using a different expression vector

contain-ing the SV40 early promoter [11] Shortage of a

pre-cursor of GPI in the endoplasmic reticulum may be

one of the reasons why a small but significant fraction

of the wild-type TNSALP forms the aggregate in

transfected COS-1 cells [25] To circumvent this

draw-back of transient expression, we established CHO-K1

Tet-On cells harboring a plasmid encoding TNSALP

(V406A) When incubated with doxycycline (an

ana-logue of tetracycline) TNSALP (V406A) appeared on

the cell surface (Fig 2A, panel a) and exhibited weak

enzyme activity (Fig 2A, panel c) The protein was

induced only with doxycycline, and no

disulfide-bonded aggregate was found on the top of the gel

(Fig 2B) Note that most of the cellular TNSALP

(V406A) was present as the 80-kDa mature form This

is in marked contrast to the transiently transfected

cells, where the 66-kDa form was a predominant

molecular species (Fig 1C) This immunoblotting

pat-tern of the CHO-K1 Tet-On cells resembles that of

Saos-2 cells [14] – osteosarcoma producing a large

amount of TNSALP Figure 3A shows pulse–chase

labeling experiments in combination with endo-b-N-glucosaminidase H (Endo H) digestion The wild-type enzyme was synthesized as the 66-kDa Endo sensi-tive form, which quickly became the 80-kDa Endo H-resistant form The processing of the newly synthesized wild-type enzyme was complete by the end of the 2-h chase period This was also the case for TNSALP (V406A) with only a small fraction being sensitive, even at the end of the 2-h chase Compatible with this result, both the wild-type protein and the mutant pro-tein were partitioned into a cold Triton X-100-insolu-ble fraction (the raft) at a similar rate (Fig 3B), further supporting that the folding and assembly pro-cess and subsequent intracellular trafficking of TNSALP (V406A) are largely normal in the stable cell line

Kinetics of the soluble form of TNSALP (V406A) Consistent with the biosynthetic studies in Fig 3, the expression level in the CHO-K1 Tet-On cell of TNSALP (V406A) was similar to that of the wild-type protein, as shown in Fig 4A,B However, the CHO-K1 Tet-On cells expressing TNSALP (V406A)

Fig 1 Transient expression of TNSALP mutant proteins in COS-1 cells (A) COS-1 cells expressing the wild-type TNSALP or the TNSALP (V406A) mutant were stained for alkaline phosphatase activity Each dish was incubated in a reaction mixture for 10 min at room tempera-ture In panels B and C, cell homogenates prepared from the transfected COS-1 cells were assayed for alkaline phosphatase (abscissa enzyme activity expressed in unitsÆmg)1of protein) or analyzed by SDS-PAGE under reducing (Red) or non-reducing (Nonred) conditions, fol-lowed by immunoblotting using anti-TNSALP serum The arrowhead indicates the top of the resolving gel The values are the means of two independent experiments (D) COS-1 cells expressing the wild-type TNSALP, or the TNSALP (V406A) or TNSALP (D289V) mutants were labeled with [ 35 S]methionine and incubated further in the absence (lanes 1, 2, 5, 6, 9 and 10) or presence (lanes 3, 4, 7, 8, 11 and 12) of phosphatidylinositol-specific phospholipase C Both cell lysates (C) and media (M) were subjected to immunoprecipitation, followed by SDS-PAGE (under reducing condition) ⁄ fluorography Left lane: 14

C-methylated protein markers of 200, 97.5, 66, 46 and 30 kDa, from the top of the gel.

Fig 3 Biosynthesis of TNSALP (V406A) in CHO-K1 Tet-On cells Established CHO-K1 Tet-On cells harboring a plasmid encoding the wild-type TNSALP or the TNSALP (V406A) mutant, which had been cultured in the presence of 0.5 lgÆmL)1of 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 insolsol-uble 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 kDa

Red

B A

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.

showed remarkably lower enzyme activity than those

expressing the wild-type enzyme, indicating that

TNSALP (V406A) has a compromised enzyme activity

Next, in an attempt to compare enzymatic properties

of the wild-type and TNSALP (V406A) proteins in

detail, we purified both the enzymes as GPI-anchorless

soluble forms We engineered the codons in TNSALP

cDNA to obtain a consecutive region of six histidine

residues and a premature stop codon upstream of a

putative C-terminal GPI-anchor signal sequence, as

described previously [26] Both wild-type and mutant

proteins were secreted into the medium from

transfect-ed COS-1 cells, and the proteins were applitransfect-ed to a

Ni-chelate column Eluted protein bands were

appar-ently homogeneous with a molecular mass of 70

(Fig 4C) and no disulfide-bonded aggregate was found

in this secreted form (data not shown) Km and Vmax

values were determined graphically using the direct lin-ear plot of Eisenthal & Cornish-Bowden TNSALP (V406A) showed a reduced Km value in association with a marked reduction in the Kcatvalue, resulting in

a Kcat⁄ Km value that was < 10% of that of the wild-type enzyme (Table 1), indicating that the conversion

of valine to alanine at position 406 in the crown domain compromises the catalytic function of TNSALP

Protease sensitivity of TNSALP (V406A)

As shown in Fig 5A, both the wild-type protein and the mutant protein migrated at exactly the same posi-tion, as judged by sucrose-density-gradient centrifuga-tion, demonstrating that both the mutant protein and the wild-type protein form a homodimer However, TNSALP (V406A) was found to be much more suscep-tible to trypsin or proteinase K than the wild-type pro-tein (Fig 5B), suggesting that the conformation of the crown domain of TNSALP (V406A) may be altered so that each protease degrades the mutant protein more easily, although its overall structure is not markedly different from the wild-type protein

Mutation analysis of the residue at position 406 Valine and alanine are usually classified into the same amino acid group with a hydrophobic side chain Therefore, we hypothesized that not only hydrophobic-ity, but also the length of the alkyl side chain of the amino acid at position 406, is crucial to the catalytic efficiency of TNSALP This was the case Leucine and isoleucine, but not phenylalanine, successfully substi-tuted for the valine residue (Fig 6A) Replacement with phenylalanine resulted in a low enzyme activity, even though TNSALP (V406F) was processed to the 80-kDa mature form similarly to TNSALP (V406A) and appeared on the cell surface like the wild-type protein (data not shown)

Fig 4 Enzyme activity of wild-type TNSALP and the TNSALP

(V406A) mutant Established CHO-K1 Tet-On cells harboring a

plas-mid encoding the wild-type TNSALP or the TNSALP (V406A) mutant

were cultured in the presence of 1 lgÆmL)1of doxycycline After

24 h, the cells were homogenized and subjected to the alkaline

phos-phatase assay (abscissa: enzyme activity expressed in unitsÆmg)1of

protein; the values are the means of two independent experiments)

(A) or analyzed by SDS-PAGE (under reducing conditions), followed

by immunoblotting (5 lg each loaded) (B) (C) COS-1 cells were

transfected with the plasmid encoding the soluble form of the

wild-type TNSALP or the TNSALP (V406A) mutant After 48 h, the media

were collected and applied to the Ni-nitrilotriacetic acid column.

TNSALP was eluted with 250 m M imidazole Each eluate was

ana-lyzed by SDS-PAGE (reducing condition), followed by silver staining

(100 or 200 ng of protein loaded) Left lane: molecular mass markers

(200, 6, 46 and 30 kDa, from the top of the gel).

Table 1 Kinetic parameters of soluble forms of the wild-type TNSALP and the TNSALP (V406A) mutant The assay was carried out using p-nitrophenylphosphate as the substrate in 0.1 M 2-amino-2-methyl-1,3-propanediol ⁄ HCl buffer (pH 10.5) containing

5 m M MgCl 2 and 0.1% Triton X-100.

Km Kcat(s)1)

K cat ⁄ K m · 10 3

( M )1s)1)

Wild-type TNSALP 0.21 971 4624 TNSALP (V406A)

mutant

0.09 34 377

Hypophosphatasia is an inborn error of metabolism

related to bone and tooth The disease is caused by

various mutations on the TNSALP gene [1–6], which

is located on chromosome 1 (p34-p36.1) Reduction in

serum alkaline phosphatase levels is a biochemical

hall-mark of mutations in TNSALP, and patients develop

a variable degree of defective bone and tooth

minerali-zation The disease is categorized into five groups: (a)

perinatal hypophosphatasia, (b) infantile

hypophos-phatasia, (c) childhood hypophoshypophos-phatasia, (d) adult

hypophosphatasia and (e) odonto hypophosphatasia

[1–4] Perinatal and infantile forms of

hypophosphata-sia are severe and are usually transmitted as a recessive

trait, whereas the other three forms of

hypophosphata-sia are mild and are transmitted recessively or

domi-nantly So far, we have found that several missense

mutations, which were reported in patients with severe

hypophosphatasia, affect the folding and assembly

process of the TNSALP molecule As a result, these

TNSALP mutant proteins fail to acquire transport competence and accumulate in the early stages of the secretory pathway, followed by degradation in an ubiquitin⁄ proteasomal pathway [13–15] This leads to decreased levels of expression of TNSALP mutants on the cell surface, although the degree by which TNSALP mutants reach the cell surface differs from one mutation to another: TNSALP (R54C), TNSALP (N153D), TNSALP (E218G), TNSALP (D289V) and TNSALP (G317A) were totally absent from the cell surface [12–15], whereas TNSALP (A162T) and TNSALP (D277A) were present at the cell surface) [11,12] Residual activities of the latter mutant enzymes may contribute to a highly variable clinical expressivity

of hypophosphatasia [4] Improper folding and resul-tant delayed trafficking are also molecular phenotypes

of TNSALP having missense mutations such as E174K, G438S, I473F, G232V, I201T and F310L [27,28] Recently we have characterized a unique mutation associated with infantile hypophosphatasia that appar-ently does not impair the trafficking of TNSALP [29]

Fig 5 Molecular properties of the TNSALP (V406A) mutant Established CHO-K1

Tet-On cells harboring a plasmid encoding the wild-type TNSALP or the TNSALP (V406A) mutant were cultured in the presence of

1 lgÆmL)1of 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)1of each fraction b, a and c denote bovine albumin (68 kDa), alco-hol dehydrogenase (141 kDa) and catalase (250 kDa), respectively, which were centri-fuged separately (B) The cells were col-lected and homogenized in 10 m M Tris-HCl (pH 8.0) using a sonicator The homogen-ates were incubated with increasing concen-trations (lgÆmL)1) of trypsin or proteinase K

in an ice ⁄ water bath for 30 min Each sam-ple was analyzed by SDS-PAGE (under reducing conditions), followed by immuno-blotting using anti-TNSALP serum.

TNSALP (R433C) forms a disulfide-bridged dimer

instead of a non-covalently assembled dimer like the

wild-type enzyme Although this mutant appears on

the cell surface at similar kinetics to the wild-type

enzyme, this novel covalent cross-linkage, but not the

replacement of the amino acid residue per se, is the

cause of the decreased enzyme activity of TNSALP

(R433C)

TNSALP (V406A) was reported in a patient

diag-nosed with perinatal hypophosphatasia, who is a

com-pound heterozygote carrying V406A and A99T [20]

A99T was also found in milder forms of the disease,

such as adult hypophosphatasia and odonto

hypophos-phatasia, and is known to be transmitted dominantly

[30] In this report we focused on the V406A missense

mutation Figure 7 is a proposed 3D structural model

of human TNSALP, based on the crystallographic

analysis of human placental alkaline phosphatase, in

which both Val406 and Arg433 residues are

high-lighted Valine at position 406 is located in the crown

domain consisting of 65 residues [4,21] We have

dem-onstrated that TNSALP (V406A) is another allele with

severe effects, but does not show defective trafficking

like TNSALP (R433C) The rate of the intracellular

transport of the mutant protein was similar to that of

the wild-type protein, as assessed by the acquisition of

Endo H resistance Besides, the mutant protein was

found to be incorporated into the raft at a kinetic rate

similar to that of the wild-type enzyme GPI-anchored protein is well known to be incorporated into the raft

in the Golgi apparatus [31], thus being ferried to the apical surface of differentiated epithelial cells [32] These findings strongly suggest that the cause of this severe hypophosphatasia is not a defect in transport, but the decreased catalytic activity of TNSALP (V406A) itself This was indeed confirmed by the kinetic analysis of a purified GPI-anchorless soluble version of the mutant protein The Kcat⁄ Km value of the mutant TNSALP was less than one-tenth of the

Kcat⁄ Km value of the soluble form of the wild-type enzyme, indicating that the replacement of valine with alanine at position 406 in the crown domain somehow strongly affects the catalytic efficiency of TNSALP Considering that TNSALP (V406A) migrates at the same position as the wild-type enzyme, as judged by sucrose-density-gradient centrifugation, it is likely that the overall structure of the mutant protein is not grossly changed Nevertheless, enhanced susceptibility

to trypsin, or especially to proteinase K, suggests a subtle distortion of the crown domain of the mutant protein Currently we do not have a definite answer as

to how this missense mutation at position 406 in the crown domain results in a remarkable decrease in the catalytic efficiency of TNSALP In this respect, it is worth pointing out that residues in the crown domain are closely related to the enzymatic properties of TNSALP [22] Interestingly, when valine at posi-tion 406 is replaced with leucine or isoleucine, both of which have a longer aliphatic chain than alanine, these TNSALP mutant proteins were processed to the 80-kDa form and exhibited enzyme activity similar to that

of the wild-type protein This leads us to speculate that

Crown domain

R433

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).

the valine residue at position 406 on one subunit may

interact with the counterpart on the other subunit

through their long aliphatic hydrocarbon chains, thus

contributing to assume a proper conformation of the

crown domain, which is presumably indispensable for

the efficient catalytic function of TNSALP In support

of our hypothesis, the cysteine residue at position 433

in one subunit of TNSALP (R433C) becomes

cross-linked to the counterpart of the other subunit [29],

implying that the two cysteine residues are sufficiently

close to stretch out to form a covalent linkage in the

crown domain However, our hypothesis is not

com-patible with the current TNSALP structural model

shown in Fig 7 It seems that the side chains of two

valine residues at position 406 are too far apart to

interact with each other in the crown domain

Never-theless, it is worth noting that the overall homology of

the crown domain between TNSALP and the placental

isoenzyme is around 72% at the amino acid level,

while that of the loop comprising residues 405–435 is

only 50% [21] Another possibility is that valine at

position 406 is involved in a substrate–enzyme

interac-tion instead of interacting with the counterpart of the

other subunit Obviously, the definite conclusion

awaits the crystallographic analysis of human

TNSALP, although our findings imply a close

func-tional relationship between the active site and the

crown domain of the TNSALP molecule, which may

be physiologically relevant to biomineralization

Materials and methods

Materials

Express35S35S protein labeling mix (> 1000 CiÆmmol)1)

was obtained from Dupont-New England Nuclear (Boston,

MA, USA); and14C-methylated proteins and the enhanced

chemiluminescence (ECL) 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-MAX, Altered sitesII mammalian mutagenesis

system was obtained from Promega (Madison, WI, USA);

QuikChange II Site-Directed Mutagenesis kit was obtained

from Stratagene (La Jolla, CA, USA); G418 and pansorbin

were obtained from Calbiochem (La Jolla CA, USA);

Lipo-fectamine Plus Reagent was obtained from Invitrogen

(Carlsbad, CA, USA); phosphatidylinositol-specific

phos-pholipase C was obtained from BIOMOL International,

L.P (Plymouth Meeting, PA, USA); aprotinin, doxycycline

and saponin (Quillaja Bark) and

l-1-tosylamide-2-phenyl-ethyl-chloromethylketone-treated bovine pancreas trypsin

were obtained from Sigma Chemical Co (St Louis, MO,

USA); proteinase K was obtained from Roche Diagnostics (London, UK); Ni-nitrilotriacetic acid resin and the plas-mid Midi-kit were obtained from Qiagen (Hilden, Ger-many); antipain, chymostatin, elastatinal, leupeptin and pepstatin A were obtained from Protein Research Founda-tion (Osaka, Japan); and hygromycin B and (p-amidinophe-nyl) methanesulfonylfluoride were obtained from Wako Pure Chemicals (Tokyo, Japan) Antiserum against recom-binant human TNSALP was raised in rabbits as described previously [26] The pTRE2 and BDCHO-K1 Tet-On cell line and Tet systems approved fetal bovine serum from BD Biosciences Clontech (Palo Alto, CA, USA)

Plasmids and transfection The pALTER-MAXencoding the wild-type TNSALP was constructed as described previously [14] Mutations were introduced at specific sites using the Altered sitesII mam-malian mutagenesis system, as described previously [14,15] The oligonucleotides used were: TNSALP (V406A), 5¢-TTC ACC GCC CAC TGC CTT GTA GCC AGG-3¢ and a sol-uble form of TNSALP (V406A), 5¢-GCA GCA AGG CTG CCT GCC TAG TGA TGG TGA TGG TGA TGG CTG GCA GGA GCA CA-3¢ TNSALP (V406L), TNSALP (V406I) and TNSALP (V406F) were created using the QuikChange II Site-Directed Mutagenesis kit with the following primers: V406L, 5¢-CCT GGC TAC AAG CTG GTG GGC GGT G-3¢ and 5¢-CAC CGC CCA CCA GCT TGT AGCCAG G-3¢; V406I, 5¢-CCT GGC TAC AAG ATA GTG GGC GGT GAA-3¢ and 5¢-TTC ACC GCC CAC TAT CTT GTA GCC AGG-3¢; and V406F, 5¢-CCT GGC TAC AAG TTC GTG GGC GGT G-3¢ and 5¢-CAC CGC CCA CGA ACT TGT AGC CAG G-3¢, respectively The DNA sequence of the mutation sites was verified by DNA sequence analyses The cDNA encoding TNSALP (V406A) was further subcloned into pTRE2 to establish stable cell lines Transfection and screening of stable cell lines were performed essentially according to the manufac-turer’s protocol CHO-K1 Tet-On cells, which successfully produced the mutant TNSALP in the presence of doxy-cycline, but not in its absence, were identified using immunofluorescence Establishment and characterization of CHO-K1 Tet-On cells expressing the wild-type TNSALP will be published elsewhere Established CHO-K1 Tet-On cells were cultured and passaged in the absence of doxycy-cline until they were used for experiments For immuno-blotting or immunofluorescence studies, the cells were cultured in the presence of 1 lgÆmL)1 of doxycycline for

24 h before use Alternatively, cells were cultured in 0.2–0.5 lgÆmL)1of doxycycline for 14 h before biosynthetic experiments For transient expression, COS-1 cells (1.0–1.3· 105

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

previously [14,15], and the transfected cells were incubated

for 24 h in a 5% CO2⁄ 95% air incubator before use For

purification of the soluble forms of enzymes, five 100-mm

dishes were transfected and cultured for 48 h COS-1 cells

were cultured in DMEM supplemented with 10% fetal

bovine serum [9]

Metabolic labeling and immunoprecipitation

For pulse-chase experiments, cells were pre-incubated for

0.5–1 h in methionine⁄ cysteine-free DMEM and labeled

with 50–100 lCi of [35S]methionine⁄ cysteine for 0.5 h in the

fresh methionine⁄ cysteine-free MEM After a pulse period,

cells were washed and chased in the DMEM as described

previously [15,29] After metabolic labeling, the medium

was removed and the cells were lysed in 0.5 mL of lysis

buffer [1% (w⁄ v) Triton X-100 ⁄ 0.5% (w ⁄ v) sodium

deoxy-cholate⁄ 0.05% (w ⁄ v) SDS in NaCl ⁄ Pi] For separation of

Triton X-100-soluble and Triton X-100-insoluble fractions,

labeled cells were lysed in 1% Triton X-100 in NaCl⁄ Pi

instead of in the lysis buffer and were centrifuged at

15 000 g for 10 min before lysing the insoluble fraction

fur-ther in the lysis buffer for immunoprecipitation A

prote-ase-inhibitor cocktail (antipain, aprotinin, chymostatin,

elastatinal, leupeptin, pepstatin A) was added to cell lysates

and media (10 lg of each protease inhibitor per mL) The

lysates were incubated for 20 min at 37C to extract

TNSALP The lysates and media were subjected to

immu-noisolation, as described previously [11,12] The immune

complexes⁄ Protein A beads were boiled in the absence or

presence of 1% (v⁄ v) 2-mercaptoethanol and were then

analyzed by SDS⁄ PAGE [9% (w ⁄ v) gels], followed by

fluo-rography [11]

Enzyme digestion

Endo H digestion and protease digestion using trypsin or

proteinase K were carried out as described previously

[11,12,29]

3D structure of TNSALP

A 3D model based on the crystal structure of human

placental alkaline phosphatase (http://www.sesep.uvsq.fr./

Database.html) was downloaded and the program pymol

(http://pymol sourceforge.net/) was used to generate the

figure

Miscellaneous procedures

Immunofluorescence for alkaline phosphatase was

per-formed as described previously [12,15]

Sucrose-density-gra-dient centrifugation was performed as described previously

[14,25,29] Transfer of proteins and subsequent procedures

were as described previously [25,29] Proteins on mem-branes were detected using ECLwestern 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 37C

Acknowledgements

We are grateful to Dr Etienne Mornet for advice on the graphical presentation of 3D structure of TNSALP We thank Miyako Okamura for her technical assistance 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)

References

1 Harris H (1989) The human alkaline phosphatases: what we know and what we don’t know Clin Chim Acta 186, 133–150

2 Whyte MP (2001) Hypophosphatasia In The Metabolic and Molecular Basis of Inherited Disease (Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler KW & Vogelstein B, eds), 8th edn., vol 4 pp 5313–5329 McGraw-Hill, New York, NY

3 Mornet E (2000) Hypophosphatasia: the mutations in the tissue-nonspecific alkaline phosphatase gene Hum Mutat 15, 309–315

4 Millan JL (2006) Mammalian Alkaline Phosphatases: From Biology to Applications in Medicine and Biotech-nology WILEY-VCH Verlag GmbH & Co KGaA, Weinheim

5 Weiss MJ, Cole DE, Ray K, Whyte MP, Lafferty MA, Mulivor RA & Harris H (1988) A missense mutation in the human liver⁄ bone ⁄ kidney alkaline phosphatase gene causing a lethal form of hypophosphatasia Proc Natl Acad Sci USA 85, 7666–7669

6 Mumm S, Jones J, Finnegan P & Whyte MP (2001) Hypophosphatasia: molecular diagnosis of Rathbun’s original case J Bone Miner Res 16, 1724–1727

7 Zurutuza L, Muller F, Gibrat JF, Taillandier A, Simon-Bouy B, Serre JL & Mornet E (1999) Correla-tions of genotype and phenotype in hypophosphatasia Hum Mol Genet 8, 1039–1046

8 Waymire KG, Mahuren JD, Jaje JM, Guilarte TR, Coburn SP & MacGregor GR (1995) Mice lacking tissue non-specific alkaline phosphatase die from seizures due to defective metabolism of vitamin B-6 Nat Genet 11, 45–51

9 Narisawa S, Frohlander N & Millan JL (1997) Inactiva-tion of two mouse alkaline phosphatase genes and

establishment of a model of infantile hypophosphatasia.

Dev Dyn 208, 432–446

10 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

11 Shibata H, Fukushi M, Igarashi A, Misumi Y, Ikehara

Y, Ohashi Y & Oda K (1998) Defective intracellular

transport of tissue-nonspecific alkaline phosphatase

with an Ala162fi Thr mutation associated with

lethal hypophosphatasia J Biochem (Tokyo) 123,

968–977

12 Fukushi-Irie M, Ito M, Amaya Y, Amizuka N, Ozawa

H, Omura S, Ikehara Y & Oda K (2000) Possible

inter-ference between tissue-non-specific alkaline phosphatase

with an Arg54fi Cys substitution and a counterpart

with an Asp277fi Ala substitution found in a

com-pound heterozygote associated with severe

hypophos-phatasia Biochem J 348, 633–642

13 Fukushi M, Amizuka N, Hoshi K, Ozawa H, Kumagai

H, Omura S, Misumi Y, Ikehara Y & Oda K (1998)

Intracellular retention and degradation of

tissue-nonspe-cific alkaline phosphatase with a Gly317fi Asp

substitu-tion associated with lethal hypophosphatasia Biochem

Biophys Res Commun 246, 613–618

14 Ito M, Amizuka N, Ozawa H & Oda K (2002)

Reten-tion at the cis-Golgi and delayed degradaReten-tion of

tissue-non-specific alkaline phosphatase with an

Asn153fi Asp substitution, a cause of perinatal

hypo-phosphatasia Biochem J 361, 473–480

15 Ishida Y, Komaru K, Ito M, Amaya Y, Kohno S &

Oda K (2003) Tissue-nonspecific alkaline phosphatase

with an Asp289fi Val mutation fails to reach the cell

surface and undergoes proteasome-mediated

degrada-tion J Biochem (Tokyo) 134, 63–70

16 Fleisch H, Straumann F, Schenk R, Bizaz S & Allgower

M (1966) Effect of condensed phosphates on

calcifica-tion of chick embryo femurs in tissue culture Am J

Physiol 211, 821–825

17 Russell RG, Bisaz S, Donath A, Morgan DB & Fleisch

H (1971) Inorganic pyrophosphate in plasma in normal

persons and in patients with hypophosphatasia,

osteo-genesis imperfect, and other disorders of bone J Clin

Invest 50, 961–969

18 Hessle L, Johnson KA, Anderson HC, Narisawa S, Sali

A, Goding JW, Terkeltaub R & Millan JL (2002)

Tissue-nonspecific alkaline phosphatase and plasma cell

membrane glycoprotein-1 are central antagonistic

regu-lators of bone mineralization Proc Natl Acad Sci USA

99, 9445–9449

19 Murshed M, Harmey D, Millan JL, MaKee MD &

Karsenty G (2005) Unique coexpression in osteoblasts

of broadly expressed genes accounts for the spatial

restriction of ECM mineralization to bone Genes Dev

19, 1093–1104

20 Taillandier A, Lia-Baldini AS, Mouchard M, Robin B, Muller F, Simon-Bouy B, Serre JL, Bera-Louville A, Bonduelle M, Eckhardt J et al (2001) Twelve novel mutations in the tissue-nonspecific alkaline phosphatase gene (ALPL) in patients with various forms of hypo-phosphatasia Hum Mutat 18, 83–84

21 Mornet E, Stura E, Lia-Baldin AS, Stigbrand T, Menez

A & Le Du MH (2001) Structural evidence for a func-tional role of human tissue nonspecific alkaline phos-phatase in bone mineralization J Biol Chem 276, 31171–31178

22 Bossi M, Hoylaerts MF & Millan JL (1993) Modifica-tions in a flexible surface modulate the isozyme-specific properties of mammalian alkaline phosphatase J Biol Chem 268, 25409–25416

23 Vittur FN, Stagni L, Moro L & der Bernard B (1984) Alkaline phosphatase binds to collagen; a hypothesis on the mechanism of extravascular mineralization in epihy-seal cartilage Experientia 40, 836–837

24 Wu LNY, Genge BR, Lloyd GC & Wuthier RE (1991) Collagen-binding proteins in collagenase-released matrix vesicles from cartilage Interaction between matrix vesi-cle proteins and different types of collagen J Biol Chem

266, 1195–1203

25 Komaru K, Ishida Y, Amaya Y, Goseki-Sone M, Orimo H & Oda K (2005) 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 secre-tion and proteasomal degradasecre-tion FEBS J 272, 1704–1717

26 Oda K, Amaya Y, Fukushi-Irie M, Kinameri Y, Ohsuye K, Kubota I, Fujimura S & Kobayashi J (1999)

A general method for rapid purification of soluble ver-sions of glycosylphosphatidylinositol-anchored proteins expressed in insect cells: an application for human tissue-nonspecific alkaline phosphatase J Biochem (Tokyo) 126, 694–699

27 Brun-Heath I, Lia-Baldini AS, Maillard S, Taillandier

A, Utsch B, Nunes ME, Serre JL & Mornet E (2007) Delayed transport of tissue-nonspecific alkaline phosphatase with missense mutations causing hypophosphatasia Eur J Med Genet 50, 367–378

28 Cai G, Michigami T, Yamamoto T, Yasui N, Stomura

K, Yamagata M, Shima M, Nakajima S, Mushiake S, Okada S et al (1998) Analysis of localization of mutated tissue-nonspecific alkaline phosphatase proteins associated with neonatal hypophosphatasia using green fluorescent protein chimeras J Clin Endocrinol Metab

83, 3936–3942

29 Nasu M, Ito M, Ishida Y, Numa N, Komaru K, Nomura S & Oda K (2006) Aberrant interchain disulfide bridge of tissue-nonspecific alkaline