• Aucun résultat trouvé

Structural evidences for a functional rôle of human tissue non-specific alkaline phosphatase in bone mineralization.

N/A
N/A
Protected

Academic year: 2021

Partager "Structural evidences for a functional rôle of human tissue non-specific alkaline phosphatase in bone mineralization."

Copied!
10
0
0

Texte intégral

(1)

HAL Id: hal-01211843

https://hal.archives-ouvertes.fr/hal-01211843

Submitted on 31 May 2020

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Copyright

Structural evidences for a functional rôle of human tissue non-specific alkaline phosphatase in bone mineralization.

E. Mornet, E. Stura, Anne-Sophie Lia-Baldini, T. Stigbrand, A. Menez, M.H.

Le Du

To cite this version:

E. Mornet, E. Stura, Anne-Sophie Lia-Baldini, T. Stigbrand, A. Menez, et al.. Structural evidences for

a functional rôle of human tissue non-specific alkaline phosphatase in bone mineralization.. Journal of

Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2001, 276, pp.31171-

31178. �hal-01211843�

(2)

Structural Evidence for a Functional Role of Human Tissue Nonspecific Alkaline Phosphatase in Bone Mineralization*

Received for publication, March 29, 2001, and in revised form, May 29, 2001 Published, JBC Papers in Press, June 6, 2001, DOI 10.1074/jbc.M102788200

Etienne Mornet‡, Enrico Stura§, Anne-Sophie Lia-Baldini‡, Torgny Stigbrand

, Andre´ Me´nez§, and Marie-He´le`ne Le Du§

From the ‡Centre d’Etudes de Biologie Pre´natale-SESEP, Universite´ de Versailles-Saint Quentin en Yvelines, 78000 Versailles, France, the §De´partement d’Inge´nierie et d’Etudes des Prote´ines (DIEP), CEA, C. E. Saclay, 91191 Gif-sur-Yvette Cedex, France, and the

Department of Immunology, U ¨ mea University, Umea, Sweden

The human tissue nonspecific alkaline phosphatase (TNAP) is found in liver, kidney, and bone. Mutations in the TNAP gene can lead to Hypophosphatasia, a rare inborn disease that is characterized by defective bone mineralization. TNAP is 74% homologous to human pla- cental alkaline phosphatase (PLAP) whose crystal struc- ture has been recently determined at atomic resolution (Le Du, M. H., Stigbrand, T., Taussig, M. J., Me´nez, A., and Stura, E. A. (2001)

J. Biol. Chem, 276, 9158 –9165). The

degree of homology allowed us to build a reliable TNAP model to investigate the relationship between muta- tions associated with hypophosphatasia and their prob- able consequences on the activity or the structure of the enzyme. The mutations are clustered within five crucial regions, namely the active site and its vicinity, the ac- tive site valley, the homodimer interface, the crown do- main, and the metal-binding site. The crown domain and the metal-binding domain are mammalian-specific and were observed for the first time in the PLAP structure.

The crown domain contains a collagen binding loop. A synchrotron radiation x-ray fluorescence study con- firms that the metal in the metal-binding site is a cal- cium ion. Several severe mutations in TNAP occur around this calcium site, suggesting that calcium may be of critical importance for the TNAP function. The presence of this extra metal-binding site gives new in- sights on the controversial role observed for calcium.

The alkaline phosphatases (EC 3.1.3.1) (APs)

1

form a large family of dimeric enzymes common to all organisms. They catalyze the hydrolysis of phosphomonoesters (1) with release of inorganic phosphate. Mammalian APs have low sequence identity with the Escherichia coli enzyme (25–30%), although the residues involved in the active site of the enzyme and the ligands coordinating the two zinc atoms and the magnesium ion are largely conserved. Therefore, the catalytic mechanism deduced from the structure of the E. coli AP is believed to be similar in eukaryotic APs (2). This mechanism involves the activation of the catalytic serine by a zinc atom, the formation of a covalent phosphoseryl intermediate, the hydrolysis of the phosphoseryl by a water molecule activated by a second zinc

atom, and the release of the phosphate product or its transfer to a phosphate acceptor (3).

In humans, three out of four AP isozymes are tissue-specific:

one is placental (PLAP), the second appears in germ cells (GCAP), and the third in the intestine (IAP). They are 90 –98%

homologous, and their genes are clustered on chromosome 2q37.1. The fourth, TNAP, 50% identical to the other three, is nonspecific and can be found in bone, liver, and kidney (4, 5, 6).

Its gene is located on chromosome 1p34 –36 (7), and mutations in the TNAP gene have been associated with hypophosphata- sia, a rare inherited disorder, characterized by defective bone mineralization. The disease is highly variable in its clinical expression, due to the strong allelic heterogeneity in the TNAP gene. Such expression ranges from stillbirth without mineral- ized bone to pathological fractures developing only late in adulthood (8). Depending on the age of onset, five clinical forms are currently recognized: perinatal, infantile, childhood, adult, and odontohypophosphatasia. To date, 89 different mutations associated with this disease have been characterized (9 –22).

Correlation between genotype and phenotype are difficult to establish, because most patients are compound heterozygous for missense mutations, making difficult the determination of the respective roles of each mutation.

This difficulty arises mainly from the lack of data concerning the precise role that TNAP plays in bone mineralization. This may be partly solved by the use of site-directed mutagenesis of TNAP cDNA and cell transfection to assay residual activity of the mutant AP enzyme (16, 18, 20, 23–25). However, this only measures the ability of the enzyme to hydrolyze phosphomo- noesters. Transfection assays cannot distinguish structural mutations from functional ones, and mutations that exhibit residual activity in vitro may yield protein that in vivo is incompletely processed or incorrectly addressed to the cell membrane. To complement the study of TNAP missense mu- tations, a three-dimensional model was built from E. coli AP (ECAP) structure (18). However, ECAP and human TNAP are only 25% identical, and the resulting model allows the assign- ment of mutated residues mainly around the active site and at the homodimer interface. The model omits a large number of regions of deletions and insertions in TNAP relative to ECAP, in particular concerning the putative functional regions of TNAP. Such insertions are also present in human placental alkaline phosphatase (PLAP) which is 57% identical to TNAP in sequence and whose crystal structure has been recently solved to 1.8-Å resolution (26). PLAP contains fewer insertions and deletions and provides a better framework from which to interpret TNAP mutations. Compared with ECAP, PLAP has extra domains that have been acquired during evolution, prob- ably to mediate functional specialization of the enzyme. Among these domains, we point out the metal-binding domain with its

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked

“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

储To whom correspondence should be addressed. E-mail: mhledu@

cea.fr.

1The abbreviations used are: AP, alkaline phosphatase; PLAP, pla- cental alkaline phosphatase; ECAP,E. colialkaline phosphatase.

© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org

31171

at INRA Institut National de la Recherche Agronomique on June 18, 2018

http://www.jbc.org/

Downloaded from

(3)

bound calcium. The homology between PLAP and TNAP at this location suggests that such a site is also present in TNAP. The presence of calcium at this site was suggested on the basis of the metal coordination but remained to verify (26).

Here we report the analysis of the metal composition of a dehydrated solution of PLAP by x-ray fluorescence using a synchrotron source. This study confirms the presence of cal- cium in PLAP and, by homology of the sequence, also in TNAP.

We have built a three-dimensional model of TNAP on the basis of the PLAP structure, and we analyze the effect of the muta- tions that occur in TNAP and are associated with hypophos- phatasia. Such a model allows us to map the distribution of the amino acid substitutions within the molecule. The correlation between the degree of severity of the mutations and their location within the specific domains of TNAP suggests a prob- able role for the enzyme in bone mineralization. In particular, an additional mutagenesis study at the calcium binding site suggests that this atom plays a fundamental role in TNAP function.

MATERIALS AND METHODS

Mutations Studies—Fifty-four of the 73 missense mutations studied here have been reported previously (9 –22). The other mutations were found by our group and were not published or were directly submitted to the Human Gene Mutation Data base (Cardiff, United Kingdom).

Mutations associated with lethal or infantile hypophosphatasia were considered as severe, while mutations associated with less severe phe- notypes (childhood, adult, and odontohypophosphatasia) were classified as either severe or moderate depending on their residual enzymatic activity, measured by transfection experiments, and on the nature of the second mutation found in the observed genotype (Table I).

Sequence Alignment of TNAP and PLAP—The TNAP sequence was first aligned to that of PLAP using the program BLAST as implemented at the internet site www.ncbi.nlm.nih.gov/gorf/bl2.html (27). The two molecules display 57% identity and 74% homology. TNAP has four insertions of one residue, one insertion of three residues, and one deletion of two residues relative to PLAP (Fig. 1). The hydrophobicity profiles of the two molecules were calculated with the program HCA (28) to check the conservation of hydrophobic patches associated to the secondary structure elements.

Synchrotron Radiation X-ray Fluorescence with Human Placental Alkaline Phosphatase—PLAP was purified as described previously (26).

The protein was concentrated to 10 mg/ml in 10 mMTris, 2 mMMgCl2, 0.02% NaN3, pH 7.0, using an Centricon Amicon 30. The experiment of synchrotron radiation x-ray fluorescence was performed at LURE on station D15. A 4-␮l droplet of protein was deposited on a 1-␮m mylar sheet and evaporated overnight. The elements (phosphorus, sulfur, chlorine, argon, potassium, calcium, titanium, vanadium, antimony, manganese, iron, nickel, copper, zinc) in the protein sample were as- sayed using highly sensitive synchrotron x-ray fluorescence. The sam- ple on the mylar sheet and the mylar alone were illuminated with a

10.10 KeV source, the sample was positioned at a 45° angle, and the detector positioned at 45° angle and 3.00 cm from the sample and 90°

from the source. The calcium/zinc concentration ratio was calculated after subtracting the mylar contribution. The area of a peak, corrected for the air absorption, for an element E, is given by the following formula,

SE

corrected

E

K,E)␻E(IK␣/(IK␣ ⫹IK␤))E (Eq. 1) with [E] being the concentration in element E,␻Ethe fluorescence yield, (IK␣/(IK␣

⫹I

K␤))Ethe intensity ratio of K␣ray,␴(K,E) K shell photo- electric cross-section␴(tot,E),

K,E)⫽␴

tot,E)(JK/(JK⫺1

兲兲

(Eq. 2) with␴(tot,E) being the total photoelectric cross-section andJKthe K edge jump ratio.

From the relation of proportionality (1), we cannot directly calculate the concentration in E. However, the ratio of the corrected areas of two peaks corresponding to elements E1 and E2 can be evaluated as the ratio of the concentrations of these elements (shown as follows).

E1]/[E2]⫽

SE1 corrected/SE2

corrected)(␴(K,E2)␻E2(IK␣/

(IK␣ ⫹IK␤))E2)/(␴(K,E1)␻E1(IK␣/(IK␣ ⫹IK␤))E1) (Eq. 3) We can therefore deduce the proportion present in the protein sample (Table II) (29, 30).

Modelization of TNAP—On the basis of the BLAST sequence align- ment and the coordinates of PLAP (entry code 1EW2; Ref. 26), the full atom model of TNAP was calculated with the program MODELLER (31). The program models three-dimensional protein structure by sat- isfying spatial restraints in the modeled molecule to accommodate the mutations made from the homologous structure (32). MODELLER pro- duces three-dimensional structure by minimization of the variable target function using the conjugate gradient method and molecular dynamics with simulated annealing. The program PROCHECK (33) was used to verify the model geometry. The resulting model corresponds to a crystal structure in the 1.5–2.0-Å resolution range, suggesting that none of the mutations between the two enzymes results in major struc- tural changes.

The range of action of the side chains of the mutated residues were visualized on a Silicon Graphic Octane station, with the program TURBO-FRODO (34).

Site-directed Mutagenesis and Transfection Study of Mutation E218G—The cDNA with mutation E218G was obtained directly from the normal cloned cDNA by using the QuikChange site-directed mu- tagenesis kit (Stratagene). Mutated and wild-type plasmids were transiently transfected in COS-1 cells. Transfections were performed with LipofectAMINE PLUS reagent (Life Technologies, Inc.) using the methodology recommended by the manufacturer. The plasmid pcDNA3.1/His/lacZ containing the␤-galactosidase gene was used as a positive control of transfection and expression. The cells were treated as described previously (23) and AP activities determined using a VP Abbott bichromatic autoanalyzer with commercially available kits.

TABLE I

Location and severity of the hypophosphatasia mutations

For mutations followed by “?” the severity remains unclear as these mutations were found in patients with moderate hypophosphatasia and compound heterozygotes for two missense mutations for which the respective effects were not determined by mutagenesis experiments. Few mutations appear more than once as they belong to overlapping domains.

Severe Moderate

Active site or active site vicinity:

G46V, T83M, A94T, A99T, G112R, N153D, H154Y, A159T, R167W, D277A, D277Y, G317D, A331T, A360V, D361V, H364R, R433C

M45L, H154R, R433H

Active site valley:

R433C, T117N R119H, E174K, E174G, R433H

Homodimer interface:

A23V, R54C, R54P, R54H, G58S, G103R, R374C, N400S, S428P, V442M, G456S, E459K, E459G, N461I, G474R

A16V?, V365I?, D389G, G439R?, I473F Crown domain:

R374C, N400S, V406A, G409C, S428P, R433C R433H, A382S, D389G, Y419H?

Calcium site or calcium site vicinity:

R206W, K207E, E218G, E274K, D277A, D277Y, E281K, D289V G203V?, L272F, M278V?

Others:

A34V, R135H, G145V, A162T, C184Y, Q190P, N194D, R229S, G232V, F310C, F310L, C472S

Q59R, A160T, S164L?, F310G?

Functional Role of Human Tissue Nonspecific Alkaline Phosphatase 31172

at INRA Institut National de la Recherche Agronomique on June 18, 2018

http://www.jbc.org/

Downloaded from

(4)

RESULTS AND DISCUSSION

Sequence Alignment and Synchrotron Radiation X-ray Flu- orescence—The sequence alignment of TNAP and PLAP shows 57% identity with four one residue insertions, one three resi- dues insertion, and one two residues deletion in TNAP (Fig. 1).

The hydrophobicity profiles calculated with the program HCA

are similar between the two molecules, suggesting that the secondary structures are highly conserved. The extra domains observed in PLAP and not in ECAP are the N-terminal

-helix, the metal-binding domain, and the crown domain. They are respectively 35, 46, and 48% identical to the homologous re- gions of TNAP. In particular, in the metal-binding domain the metal ligands are strictly identical, in agreement with the presence of the same metal in both proteins.

By x-ray fluorescence, performed at LURE on station D15, the protein sample was found to contain chloride, zinc, sulfur, calcium, copper, and iron in order of peak height (Fig. 2). The chloride ion comes from the buffer solution, and the sulfur comes from the cysteines and methionines in the protein. In order of peak height, zinc is the second peak and calcium the fourth. The presence of iron and copper, in ionized form, are biological trace elements (Table II). Placenta, before purifica- tion is rich in mitochondria, which contains significant amounts of iron and copper ions, and the placenta is filled with fetal blood, containing trace amounts of iron and copper ions.

FIG. 1.Sequence alignment of PLAP and TNAP as used for the modelization of TNAP.Deletions are indicated with adot, basic residues are written inblue, acidic inred, and the catalytic serine is inboldface. In the intermediate line, identical residues are written, homologous residues are indicated by

, divergent residues by *, the␣-helices are highlighted inyellow,and the␤-strands ingreen. The metal-binding residues are indicated with anarrow.

TABLE II

Calculation of the concentration ratio of calcium versus iron, copper or zinc derived from relation (3). SE

correctedcorresponds to the averaged surface peak of calcium, copper, and zinc of the PLAP sample after

removing the mylar contribution and the air absorption.

E Calcium Iron Copper Zinc

SE

corrected 73,519 27,718 56,291 797,228

(tot,E) 89.9 165 209 226

JK 9.1 8.31 7.81 7.56

K␣/K␣

K␤) 0.887 0.882 0.881 0.844

E 0.163 0.340 0.440 0.474

[Calcium]/[E] 1 10.2 8.31 0.66

at INRA Institut National de la Recherche Agronomique on June 18, 2018

http://www.jbc.org/

Downloaded from

(5)

During the purification, heat denaturation of contaminating proteins could putatively cause the release of the bound metal ions. The coordination number observed in the metal-binding

site of PLAP is 7, clearly different from that usually observed for either copper or iron, which ranges from 4 to 6 (35). The presence of such metals is therefore unlikely. After subtraction

FIG. 2.a, emission x-ray fluorescence spectrum of human placental alkaline phosphatase on the mylar sheet (blue curves), and of the mylar alone (red curves).b, average differences curve of the emission x-ray fluorescence spectrum of human placental alkaline phosphatase on the mylar sheet minus the emission x-ray fluorescence spectrum of the mylar sheet. The peak corresponding to the K␣emission of the elements present in the protein are indicated with anarrow.

Functional Role of Human Tissue Nonspecific Alkaline Phosphatase 31174

at INRA Institut National de la Recherche Agronomique on June 18, 2018

http://www.jbc.org/

Downloaded from

(6)

of the signal on the mylar and correction of the surface peak for the air absorption, the ratio of calcium concentration over zinc concentration is 0.66 (Table II), which is in the proper range for one calcium atom and two zinc atom with the experimental errors. This ratio is in agreement with the presence of the two zinc atoms in the active site and one calcium atom in the metal-binding site. Since the coordination is also typical of a calcium atom, we conclude that the calcium is the natural ion that binds to the metal-binding domain, in agreement with the biochemical data (36). The presence of this calcium site is in agreement with the study of Leone et al. (37), which suggested that magnesium and calcium bind to different sites. A coordi- nation number of 7 is typical for calcium, suggesting that magnesium is also unlikely to bind at this site. On the contrary, the presence of calcium at this site does not exclude, at high concentration, calcium binding at the magnesium site as an octahedral coordination can be taken up by calcium. Therefore, whatever the role of the calcium site, high calcium concentra- tion may inhibit AP activity by replacing magnesium in the active site. The effects of calcium on AP activity should be reconsidered including in the analysis the presence of the cal- cium site (37–39). The precise biological role of calcium in TNAP remains to be addressed, in particular its binding con- stant. It is very interesting to observe that with the evolution and the specialization of the enzyme function, new features have been added: in E. coli, where there is no skeleton to mineralize, there is no calcium site in its AP.

Molecular Modeling—The model of TNAP shows that, al- though all the structural features are identical to those in

PLAP, the charge distributions at the protein surfaces are clearly different (Fig. 3). In particular, the active site valley located on both sides of the active site, which contains a large hydrophobic area in PLAP, is punctuated by more polar resi- dues in TNAP. Thus, the hydrophobic residues, Trp

168

, Tyr

169

, and Tyr

206

are conserved, but in TNAP, they are surrounded by ionic residues. Close to the active site, the acidic residue Glu

429

, which confers to PLAP the specificity toward the uncompetitive inhibitors

L

-Phe or

L

-Leu (40), is replaced by a basic one, Arg

433

, in TNAP. The hydrophobic pocket that is deeper in the active site of PLAP is not conserved in TNAP. Residues Phe

107

, Gln

108

, and Phe

118

are replaced in TNAP by Glu

108

, Gly

109

, and Arg

119

, respectively. However, the tyrosine, which enters in the active site of the other monomer (Tyr

367

in PLAP), is conserved in TNAP (Tyr

371

). This reinforces the idea that Tyr

371

may contribute to the allosteric properties shared by the two en- zymes (41). All residues that are essential for the hydrolytic activity of the bacterial and the other mammalian phospha- tases are preserved in TNAP, but those that confer substrate specificity are different.

The sequences alignment and the comparison of the hydro- phobicity profiles shows that the structural features that are present in PLAP, but not in the E. coli AP, also occur in TNAP.

These comprise the N-terminal

-helix involved in the dimer interface, the 76 residues of the calcium-binding-domain (res- idues 211–289), and the interfacial “crown-domain” formed by the insertion of a 60-residue segment (371– 431) from each monomer. Within the crown domain, a site for collagen attach- ment has been localized in loop 405– 435 (41). The TNAP model shows that this loop is highly accessible and located at the very tip of the crown domain. The functional relevance of these domains can be explain by an analysis of the mutations that lead to hypophosphatasia.

Functional Regions of TNAP—The location of the 73 mis- sense mutations associated with hypophosphatasia in the three-dimensional model allowed us to define five crucial re- gions in the TNAP molecule.

The first region of TNAP is the active site where 20 substi- tutions are located within a 15-Å sphere centered around the phosphate group. Except for M45L, H154R, and R433H, all other mutations found in this sphere are classified as severe (Table I). Mutation M45L is structurally conservative; the patient with this mutation was affected with infantile hy- pophosphatasia, and site-directed mutagenesis experiments suggested that this mutation corresponds to a moderate allele (18). H154R was associated with E174K in a patient with adult onset of the disease, and the substitution of a histidine by an arginine maintains the positive character of the residue, leav- ing some plausibility to the moderate nature of this mutation.

The same situation occurs for R433H that was associated with D389G in a patient with childhood hypophosphatasia, but with only odontologic symptoms. Apart from these more moderate mutations, most substitutions around the active site corre- spond to severe alleles as reported previously (18) (Fig. 4, upper panel).

The second region is located in the active site valley, which extends on both sides of the active site. Here, six mutations affecting four residues were observed: T117N, R119H, E174K, E174G, R433H, and R433C. The location of Arg

119

and Arg

433

in this region, both unique to TNAP, suggests that these two highly basic residues may be involved in regulating the ap- proach of the substrate and/or its stabilization (Fig. 4, upper panel). Amino acid substitutions seem to be better tolerated in this area, since at least four of the mutations, R119H, E174K, E174G, and R433H, have a moderate phenotype. Whatever role these polar residues have in the substrate approach and/or

FIG. 3. Surface representation of PLAP structure (a) and

TNAP model (b) with the acidic residues inredand the basic residues inblue.

at INRA Institut National de la Recherche Agronomique on June 18, 2018

http://www.jbc.org/

Downloaded from

(7)

stabilization, the same function could be performed by other polar residues, suggesting that polarity and not charge is es- sential for substrate steering.

The third important region is the homodimer interface.

Dimerization is particularly important for TNAP, not only for allosteric reasons (cannot exist without dimerization) but also because it appears that APs are active only in dimeric form (42). Since dimerization is such a fundamental aspect of APs, it is not surprising to find that of the 20 mutations in the ho- modimer interface, at least 15 of them are severe alleles. These residues may be directly involved in homodimer interactions or

play a role in maintaining the correct fold to allow these inter- actions to form. The PLAP-based model allowed us to locate some mutated homodimer interface residues, G456S, E459K, and G474R, that were not identified from the E. coli-based model (18). These residues, associated with a severe form of the disease, belong to the C-terminal part of the molecule that have no equivalent in the E. coli structure.

The fourth region is the loop 405– 435 within the crown domain, composed of a total of 65 residues. This region, which has been associated with the binding of collagen (40), corre- sponds to a long insertion loop with no counterpart in E. coli.

FIG. 4.Functional regions of TNAP.On theleft panelTNAP is shown in ribbon representation with the residues involved in hypophos- phatasia inred(active site and active site valley),purple(homodimer interface),blue(crown domain), oryellow(calcium binding site). On theright panel, the functional region are enlarged with the residues involved in hypophosphatasia inredif the mutation effect is severe oryellowif the mutation effect is moderate.

Functional Role of Human Tissue Nonspecific Alkaline Phosphatase 31176

at INRA Institut National de la Recherche Agronomique on June 18, 2018

http://www.jbc.org/

Downloaded from

(8)

The 65 residues of the crown domain are well conserved (72%

homologous) with PLAP with the exception of loop 405– 435 where the two molecules share only 11 identical and 5 homol- ogous residues. However, the hydrophobicity profile shows that the hydrophobic and hydrophilic residues adopt similar distri- bution. Among the 10 mutations associated with hypophos- phatasia identified in this domain, V406A, G409C, Y419H, S428P, R433C, and R433H are located within this loop. The others, R374C, A382S, D389G, and N400S, in the portion of the crown domain involved in monomer-monomer interactions are disruptive of the interface, as is the case also for V406A. The substitution G409C rigidifies the loop backbone at the tip of the crown domain. Y419H belongs to an

-helix of the crown do- main, and S428P occurs within a

-strand. As mentioned pre- viously, Arg

433

is found at the entrance of the active site pocket with a role in the substrate positioning. Its mutation to His

433

is probably less severe than its mutation to Cys

433

, probably because it is more conservative. This is corroborated by the lethal effect associated to the homozygous genotype R433C and the moderate phenotype observed in patient R433H with child- hood hypophosphatasia but just odontologic symptoms. Fi- nally, we can observe that most of the mutations affecting this domain are severe, reinforcing the idea that collagen binding is crucial for the function of the enzyme.

The fifth region surrounds the calcium binding site (M4).

Eleven distinct mutations were found in this region. The cal- cium atom is coordinated by the carboxylates of Glu

218

, Glu

274

, and Asp

289

, by the carboxyl of Phe

273

, and by a water molecule.

Mutations of any of the three ligands Glu

218

, Glu

274

, or Asp

289

are related to a severe form of hypophosphatasia, probably because this abrogates calcium binding. E218G was found in a patient with the adult form of the disease and the genotype E218G/A382S (22). Thus, the second mutation could mask the effect of the mutation E218G, which could be either severe or moderate. To ascertain the role of the calcium atom at M4, we performed site-directed mutagenesis experiments at this posi- tion. We found that COS-1 cells transfected with E218G had no residual enzymatic activity, attesting to the severity of this mutation. The moderate phenotype of the patient was due to a compensatory effect of A382S. Mutation E274K was detected in a patient with childhood hypophosphatasia, carrying the gen- otype E274K/E174K (14). Previous site-directed mutagenesis experiments showed that this mutation allowed

8% of wild- type activity and was therefore classified as moderate, al- though close to the limit for severe alleles (18). Mutation D289V was found in a homozygous patient affected with lethal hypophosphatasia (17) and therefore had to be classified as severe. The other mutations in the vicinity of M4 are severe except for L272F that has 50% of wild-type activity in site- directed mutagenesis experiments (16) and G203V and M278V, which are relatively conservative. The severe character of the mutations surrounding the calcium site shows that this metal is fundamental to TNAP activity, suggesting a role of the en- zyme in bone mineralization. However, site-directed mutagen- esis experiments do not allow to distinguish structural muta- tions from functional ones. It remains, therefore, possible that some mutations may have a structural defect that results in protein misfolding and degradation, an effect that would not directly involve the metal-binding site function. However, the finding of a cluster of mutated residues in this particular region strongly suggests that at least some of these mutations are related to the function of the calcium binding site.

In addition to these five critical regions for hypophospha- tasia, severe mutations were also found in other particular regions. Cysteine mutations C184Y and C472S affect one or the other disulfide bridge, and mutations (R135H, G145V, Q190P,

N194D, F310C,L) are buried and important for secondary structure. Finally, mutation F310C results in the introduction of an extra cysteine that could affect the proper folding of the protein. The severity of these mutations is linked to their location within the core of the protein, likely to lead to struc- tural destabilization of the enzyme.

Conclusions—In this study, we have built a model of TNAP on the basis of the structure of PLAP. This model allowed us to position the substitutions responsible for hypophosphatasia.

The distribution of the substitutions highlights five regions comprising 78% of the mutations, most of them being severe alleles. These regions are namely the active site and its vicin- ity, the active site valley, the homodimer interface, the crown domain, and the calcium-binding domain, clearly indicating that these regions are crucial for the enzyme function and bone mineralization. Two of these regions, the crown domain and the calcium-binding domain, are absent in the E. coli structure and were ignored in our previous model. This new model highlights two distinct mammalian-specific regions, one involved in colla- gen binding and the other involved in a novel function, which probably involves calcium.

Acknowledgment—We are very grateful to P. Chevallier who helped us on station D15 at LURE to perform the experiment of synchrotron radiation x-ray fluorescence.

REFERENCES

1. Schwartz, J. H., and Lipmann, F. (1961)Proc. Natl. Acad. Sci. U. S. A.47, 1996 –2005

2. Kim, E. E., and Wyckoff, H. W. (1991)J. Mol. Biol.218,449 – 464 3. Coleman, J. E. (1992)Annu. Rev. Biophys. Biomol. Struct.21,441– 483 4. Milla´n, J. L. (1988)Anticancer Res.8,995–1004

5. Milla´n, J. L. (1992)Clin. Chim. Acta209,123–129

6. Milla´n, J. L., and Fishman, W. H. (1995)Crit. Rev. Clin. Lab. Sci.32,1–39 7. Greenberg, C. R., Evans, J. A., McKendry-Smith, S., Redekopp, S., Haworth,

J. C., Mulivor, R., and Chordiket, B. N. (1990)Am. J. Hum. Genet.46, 286 –292

8. Whyte, M. P. (1994)Endocr. Rev.15,439 – 461

9. Weiss, M. J., Cole, D. E. C., Ray, K., Whyte, M. P., Lafferty, M. A., Mulivor, R. A., and Harris, H. (1988)Proc. Natl. Acad. Sci. U. S. A.85,7666 –7669 10. Henthorn, P. S., Raducha, M., Fedde, K. N., Lafferty, M. A., and Whyte, M. P.

(1992)Proc. Natl. Acad. Sci. U. S. A.89,9924 –9928

11. Orimo, H., Haysshi, Z., Watanabe, A., Hirayama, T., Hirayama, T., and Shimada, T. (1994)Hum. Mol. Genet.9,1683–1684

12. Ozono, K., Yamagata, M., Michigami, T., Nakajima, S., Sakai, N., Cai, G., Satomura, K., Yasui, N., Okada, S., and Nakayama, M. (1996)J. Clin.

Endocrinol. Metab.12,4458 – 4461

13. Orimo, H., Goseki-Sone, M., Sato, S., and Shimada, T. (1997)Genomics42, 364 –366

14. Mornet, E., Taillandier, A., Peyramaure, S., Kaper, F., Muller, F., Brenner, R., Bussie`re, P., Freisinger, P., Godard, J., Le Merrer, M., Oury, J. F., Plauchu, H., Puddu, R., Rival, J. M., Superti-Furga, A., Touraine, R. L., Serre, J. L., and Simon-Bouy, B. (1998)Eur. J. Hum. Genet.6,308 –314

15. Goseki-Sone, M., Orimo, H., Iimura, T., Takagi, Y., Watanabe, H., Taketa, K., Sato, S., Mayanagi, H., Shimada, T., and Oida, S. (1998)Hum. Mut.Suppl.

1, S263–S267

16. Sugimoto, N., Iwamoto, S., Hoshimo, Y., and Kajii, E. (1998)J. Hum. Genet.

43,160 –164

17. Taillandier, A., Zurutuza, L., Muller, F., Simon-Bouy, B., Serre, J. L., Bird, L., Brenner, R., Boute, O., Cousin, J., Gaillard, D., Heidemann, P. H., Steinmann, B., Wallot, and M., Mornet, E. (1999)Hum. Mut.13,171–172 18. Zurutuza, L., Muller, F., Gibrat, J. F., Taillandier, A., Simon-Bouy, B., Serre,

J. L., and Mornet, E. (1999)Hum. Mol. Genet.8,1039 –1046

19. Hu, J. C. C., Plaetke, R., Mornet, E., Zhang, C., Sun, X., Thomas, H. F., and Simmer, J. P. (2000)Eur. J. Oral Sci.108,189 –194

20. Taillandier, A., Cozien, E., Muller, F., Merrien, Y., Bonnin, E., Fribourg, C., Simon-Bouy, B., Serre, J. L., Bieth, E., Brenner, R., Cordier, M. P., De Bie, S., Fellmann, F., Freisinger, P., Golembowski, S., Hennekam, R. C. M., Josifova, D., Kerzin-Storrar, L., Leporrier, N., Zabot, M. T., and Mornet, E.

(2000)Hum. Mutat.15,293

21. Mochizuki, H., Saito, M., Michigami, T., Ohashi, H., Koda, N., Yamaguchi, S., and Ozono, K. (2000)Eur. J. Pediatr.159,375–379

22. Taillandier, A., Lia-Baldini, A. S., Mouchard, M., Robin, B., Muller, F., Simon-Bouy, B., Serre, J. L., Bera-Louville, A., Eckhardt, J., Gaillard, D., Myhre, A. G., Ko¨rtge-Jung, S., Larget-Piet, L., Libaers, I., Malou, E., Sillence, D., Temple, I. K., Viot, G., and Mornet, E. (2001)Hum. Mutat., in press

23. Fukushi, M., Amizuka, N., Hoshi, K., Ozawa, H., Kumagai, H., Omura, S., Misumi, Y., Ikehara, Y., and Oda, K. (1998)Biochem. Biophys. Res. Com- mun.246,613– 618

24. Shibata, H., Fukushi, M., Igarashi, A., Misumi, Y., Ikehara, Y., Ohashi, Y., and Oda, K. (1998)J. Biochem.(Tokyo)123,968 –977

25. Cai, G., Michigami, T., Yamamoto, T., Yasui, N., Satomura, K., Yamagata, M., Shima, M., Nakajima, S., Mushiake, S., Okada, S., and Ozono, K. (1998)

at INRA Institut National de la Recherche Agronomique on June 18, 2018

http://www.jbc.org/

Downloaded from

(9)

J. Clin. Endocrinol. Metab.83,3936 –3942

26. Le Du, M. H., Stigbrand, T., Taussig, M. J., Me´nez, A., and Stura, E. A. (2001) J. Biol. Chem.276,9158 –9165

27. Altschul, S. F., Madden, T. L., Scha¨ffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997)Nucleic Acids Res.25,3389 –3402

28. Callebaut, I., Labesse, G., Durand, P., Poupon, A., Canard, L., Chomilier, J., Henrissat, B., and Mornon, J. P. (1997)Cell. Mol. Life. Sci.53,621– 645 29. Bambynek, W., Crasemann, B., Fink, R. W., Freund, H. U., Mark, H., Swift,

C. D., Price, P. E., and Venugopola Rao, P. (1972)Rev. Modern Physics44, 4

30. Henke, B. L., Lee, P., Tanaka, T. J., Shimabukuro, R. L., and Fujikawa, B. K.

(1982)Atomic Data and Nuclear Data Tables27,1 31. Sanchez, R., and Sali, A. (2000)Methods Mol. Biol.143,97–129 32. Sali, A., and Blundell, T. L. (1993)J. Mol. Biol.234,779 – 815

33. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl Crystallogr.26,283–291

34. Roussel, A., and Cambillau, C. (1989) Silicon Graphics Geometry Partner Directory, pp. 77–78, Silicon Graphics, Mountain View, CA

35. Harding, M. M. (2001)Acta Crystallogr. Sect. D Biol. Crystallogr.57,401– 411 36. de Bernard, B., Bianco, P., Bonucci, E., Costantini, M., Lunazzi, G. C., Martinuzzi, P., Modricky, C., Moro, L., Panfili, E., Pollesello, P., Stagni, N., and Vittur, F. (1986)J. Cell Biol.103,1615–1623

37. Leone, F. A., Ciancaglini, P., and Pizauro, J. M. (1997)J. Inorg. Biochem.68, 123–127

38. Genge, B. R., Sauer, G. R., Wu, L. N. Y., McLean, F. M., and Wuthier, R. E.

(1988)J. Biol. Chem.263,18513–18519

39. Shinozaki, T., and Pritzker, K. P. H. (1996)J. Rheumatol.23,677– 683 40. Hoylaerts, M. F., Manes, T., and Milla´n, J. L. (1991)Biochem. J.274,91–95 41. Hoylaerts, M. F., Manes, T., and Milla´n, J. L. (1997)J. Biol. Chem.272,

22781–22787

42. Bossi, M., Hoylaerts, M. F., and Milla´n, J. L. (1993)J. Biol. Chem.268, 25409 –25416

Functional Role of Human Tissue Nonspecific Alkaline Phosphatase 31178

at INRA Institut National de la Recherche Agronomique on June 18, 2018

http://www.jbc.org/

Downloaded from

(10)

and Marie-Hélène Le Du

Etienne Mornet, Enrico Stura, Anne-Sophie Lia-Baldini, Torgny Stigbrand, André Ménez Phosphatase in Bone Mineralization

Structural Evidence for a Functional Role of Human Tissue Nonspecific Alkaline

doi: 10.1074/jbc.M102788200 originally published online June 6, 2001 2001, 276:31171-31178.

J. Biol. Chem.

10.1074/jbc.M102788200 Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted

When this article is cited

to choose from all of JBC's e-mail alerts Click here

http://www.jbc.org/content/276/33/31171.full.html#ref-list-1

This article cites 40 references, 9 of which can be accessed free at

at INRA Institut National de la Recherche Agronomique on June 18, 2018

http://www.jbc.org/

Downloaded from

Références

Documents relatifs

The results of this trial demonstrated that reducing the overall treatment time using accelerated PORT/CT by weekly concomitant boost (six fractions per week) combined with

[r]

These novel 3D quantitative results regarding the osteocyte lacunae and micro-cracks properties in osteonal and interstitial tissue highlight the major role

tonian parameters of table I.. MOSSBAUER STUDIES OF A SYNTHETIC ANALOG FOR ACTIVE SITE IN REDUCED RUBREDOXIN C6-161 in agreement with the observed practically

In the present work, we have measured for the first time the photoinduced flavin dynamics of BsTrmFO in both the WT and the C53A mutant, by ultrafast

We have also collected MAFS data on the catalyst with different chemical perturbations (reduction without CO /CO in the reducing gas and, in particular, reaction of the

These new outcrops, where an eastwards steeply dipping slip surface was exposed, together with the refined slope stability analysis, and newly acquired high resolution

Analysing the genomes of the mutator strains, we have been able to under- stand how the mutators manage to support the mutation rate increase.. First they reduce the number of