Structural Evidence of Functional Divergence in Human Alkaline Phosphatases

Artigo Acesso aberto Revisado por pares

Structural Evidence of Functional Divergence in Human Alkaline Phosphatases

2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês

10.1074/jbc.m207394200

ISSN

1083-351X

Autores

Marie‐Hélène Le Du, José Luís Millán,

Tópico(s)

Porphyrin Metabolism and Disorders

Resumo

The evolution of the alkaline phosphatase (AP) gene family has lead to the existence in humans of one tissue-nonspecific (TNAP) and three tissue-specific isozymes,i.e. intestinal (IAP), germ cell (GCAP), and placental AP (PLAP). To define the structural differences between these isozymes, we have built models of the TNAP, IAP, and GCAP molecules based on the 1.8-Å structure of PLAP (1Le Du M.H. Stigbrand T. Taussig M.J. Menez A. Stura E.A. J. Biol. Chem. 2001; 276: 9158-9165Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) and have performed a comparative structural analysis. We have examined the monomer-monomer interface as this area is crucial for protein stability and enzymatic activity. We found that the interface allows the formation of heterodimers among IAP, GCAP, and PLAP but not between TNAP with any of the three tissue-specific isozymes. Secondly, the active site cleft was mapped into three regions, i.e. the active site itself, the roof of the cleft, and the floor of the cleft. This analysis led to a structural fingerprint of the active site of each AP isozyme that suggests a diversification in substrate specificity for this isozyme family. The evolution of the alkaline phosphatase (AP) gene family has lead to the existence in humans of one tissue-nonspecific (TNAP) and three tissue-specific isozymes,i.e. intestinal (IAP), germ cell (GCAP), and placental AP (PLAP). To define the structural differences between these isozymes, we have built models of the TNAP, IAP, and GCAP molecules based on the 1.8-Å structure of PLAP (1Le Du M.H. Stigbrand T. Taussig M.J. Menez A. Stura E.A. J. Biol. Chem. 2001; 276: 9158-9165Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) and have performed a comparative structural analysis. We have examined the monomer-monomer interface as this area is crucial for protein stability and enzymatic activity. We found that the interface allows the formation of heterodimers among IAP, GCAP, and PLAP but not between TNAP with any of the three tissue-specific isozymes. Secondly, the active site cleft was mapped into three regions, i.e. the active site itself, the roof of the cleft, and the floor of the cleft. This analysis led to a structural fingerprint of the active site of each AP isozyme that suggests a diversification in substrate specificity for this isozyme family. Alkaline phosphatases (EC 3.1.3.1) (APs) 1The abbreviations used are: AP, alkaline phosphatase; GCAP, germ cell AP; IAP, intestinal AP; PLAP, placental AP; TNAP, tissue-nonspecific AP1The abbreviations used are: AP, alkaline phosphatase; GCAP, germ cell AP; IAP, intestinal AP; PLAP, placental AP; TNAP, tissue-nonspecific AP are dimeric enzymes present in most, if not all, organisms (2McComb R.B. Bowers G.N. Posen S. Alkaline Phosphatases. Plenum Press, New York1979: 986-988Google Scholar). They catalyze the hydrolysis of phosphomonoesters with release of inorganic phosphate (3Schwartz J.H. Lipmann F. Proc. Natl. Acad. Sci. U. S. A. 1961; 47: 1996-2005Crossref PubMed Scopus (118) Google Scholar). Mammalian APs have low sequence identity with theEscherichia coli enzyme (25–30%), but the residues involved in the active site of the enzyme and those coordinating the two zinc atoms and the magnesium ion are largely conserved, and the catalytic mechanism deduced from the structure of the E. coli AP was proposed to be similar in eukaryotic APs (4Kim E.E. Wyckoff H.W. J. Mol. Biol. 1991; 218: 449-464Crossref PubMed Scopus (928) Google Scholar). In humans, APs are encoded by four distinct loci. Three isozymes are tissue-specific, i.e. intestinal AP (IAP), placental AP (PLAP), and germ cell AP (GCAP). They are 90–98% homologous, and their genes are clustered on chromosome 2, bands q34-q37. The fourth AP isozyme is tissue-nonspecific (TNAP) and is expressed in a variety of tissues throughout development. TNAP is about 50% identical to the other three isozymes, and its gene is located on chromosome 1, bands p36.1-p34 (5Harris H. Clin. Chim. Acta. 1989; 186: 133-150Crossref Scopus (426) Google Scholar).Our current understanding of the functional properties of mammalian APs comes largely from studies using PLAP and TNAP as paradigms. Isozyme-specific properties, such as the characteristic uncompetitive inhibition properties of mammalian APs (6Hummer C. Millán J.L. Biochem. J. 1991; 274: 91-95Crossref PubMed Scopus (39) Google Scholar, 7Hoylaerts M.F. Millán J.L. Eur. J. Biochem. 1991; 202: 605-616Crossref PubMed Scopus (43) Google Scholar, 8Hoylaerts M.F. Manes T. Millán J.L. Biochem. J. 1992; 286: 23-30Crossref PubMed Scopus (74) Google Scholar), their variable heat stability (9Bossi M. Hoylaerts M.F. Millán J.L. J. Biol. Chem. 1993; 268: 25409-25416Abstract Full Text PDF PubMed Google Scholar), and even their allosteric properties (10Hoylaerts M.F. Manes T. Millán J.L. J. Biol. Chem. 1997; 272: 22781-22787Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), have been attributed to a top, flexible loop (or crown domain) unique to mammalian APs. This domain is also responsible for collagen binding in the case of TNAP (9Bossi M. Hoylaerts M.F. Millán J.L. J. Biol. Chem. 1993; 268: 25409-25416Abstract Full Text PDF PubMed Google Scholar, 11Wu L.N.Y. Genge B.R. Lloys G.C. Wuthier R.E. J. Biol. Chem. 1991; 266: 1195-1203Abstract Full Text PDF PubMed Google Scholar) but does not appear to mediate the reported binding of PLAP to IgG (12Makiya R. Stigbrand T. Eur. J. Biochem. 1992; 205: 341-345Crossref PubMed Scopus (61) Google Scholar, 13Makiya R. Stigbrand T. Biochem. Biophys. Res. Commun. 1992; 182: 624-630Crossref PubMed Scopus (44) Google Scholar).The recent elucidation of the 1.8-Å resolution structure of human PLAP (1Le Du M.H. Stigbrand T. Taussig M.J. Menez A. Stura E.A. J. Biol. Chem. 2001; 276: 9158-9165Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) has facilitated further studies on the structure and function of mammalian APs. An analysis of the structural-functional relationship of residues conserved between the E. coli AP and the PLAP structure revealed a conserved function for those residues that stabilize the active site zinc and magnesium metal ions, whereas the non-homologous disulphide bonds differ in their structural significance and non-conserved residues take part in determining the heat stability and uncompetitive inhibition properties of mammalian alkaline phosphatases (14Kozlenkov A. Manes T. Hoylaerts M.F. Millán J.L. J. Biol. Chem. 2002; 277: 22992-22999Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Deactivating mutations in the TNAP gene cause the inborn error of metabolism known as hypophosphatasia (15Henthorn P.S. Whyte MP Clin. Chem. 1992; 38: 2501-2505Crossref PubMed Scopus (86) Google Scholar), characterized by poorly mineralized cartilage and bones. The severity and expressivity of hypophosphatasia depends on the nature of the TNAP mutation (16Zurutuza L. Muller F. Gibrat J.F. Taillandier A. Simon-Bouy B. Serre J.L. Mornet E. Hum. Mol. Genet. 1999; 8: 1039-1046Crossref PubMed Scopus (160) Google Scholar). The mapping of hypophosphatasia mutations to specific three-dimensional locations on the TNAP molecule has provided clues as to the structural significance of these areas for enzyme structure and function (17Mornet E. Stura E. Lia-Baldini A.S. Stigbrand T. Menez A. Le Du M.H. J. Biol. Chem. 2001; 276: 31171-31178Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). It appears clear that the function of TNAP in bone tissue consists of hydrolizing inorganic pyrophosphate to maintain a proper concentration of this mineralization inhibitor to ensure proper bone mineralization (18Hessle L. Johnsson K.A. Anderson H.C. Narisawa S. Sali A. Goding J.W. Terkeltaub R. Millán J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9445-9449Crossref PubMed Scopus (666) Google Scholar). However, the physiological role of the three tissue-specific human APs remains to be clarified.It has been suggested that PLAP may be involved in the transfer of maternal IgG to the fetus (12Makiya R. Stigbrand T. Eur. J. Biochem. 1992; 205: 341-345Crossref PubMed Scopus (61) Google Scholar, 13Makiya R. Stigbrand T. Biochem. Biophys. Res. Commun. 1992; 182: 624-630Crossref PubMed Scopus (44) Google Scholar, 19Stefaner I. Stefanescu A. Hunziker W. Fuchs R. Biochem. J. 1997; 327: 585-592Crossref PubMed Scopus (24) Google Scholar), and evidence has accumulated indicating a role of PLAP in cell division in normal and transformed cells (20Stinson R.A. Chan J.R.A. Adv. Prot. Phosphatases. 1987; 4: 127-151Google Scholar, 21Telfer J.F. Green C.D. FEBS Lett. 1993; 329: 238-244Crossref PubMed Scopus (31) Google Scholar, 22She Q. Mukherjee J.J. Huang J. Crilly K.S. Kiss Z. FEBS Lett. 2000; 469: 163-167Crossref PubMed Scopus (32) Google Scholar, 23Dabare A.A. Nouri A.M. Cannell H. Moss T. Nigam A.K. Oliver R.T. Urol. Int. 2000; 63: 168-174Crossref Scopus (17) Google Scholar). Of considerable interest is the fact that human APs are abundantly expressed in tumor cells and that their serum levels are often used as tumor markers (24Millán J.L. Fishman W.H. Crit. Rev. Clin. Lab. Sci. 1995; 32: 1-39Crossref PubMed Scopus (107) Google Scholar). Plasma TNAP levels can indicate the presence of osteosarcomas (25Farley J.R. Hall S.L. Herring S. Tarbaux N.M. Matsuyama T. Wergedal J.E. Metabolism. 1991; 40: 664-671Abstract Full Text PDF PubMed Scopus (74) Google Scholar), Paget's disease (26Deftos L.J. Wolfert R.L. Hill C.S. Horm. Metab. Res. 1991; 23: 559-561Crossref PubMed Scopus (36) Google Scholar), and osteoblastic bone metastates (27Demers L.M. Costa L. Chinchilli V.M. Gaydos L. Curley E. Lipton A. Clin. Chem. 1995; 41: 1489-1494Crossref PubMed Scopus (135) Google Scholar). PLAP is a marker of cancer of the ovary, testis, lung, and the gastrointestinal tract (28Nathanson L. Fishman W.H. Cancer (Phila.). 1971; 27: 1388-1397Crossref PubMed Scopus (155) Google Scholar, 29Jacoby B. Bagshawe K.D. Clin. Chim. Acta. 1971; 35: 473-481Crossref PubMed Scopus (39) Google Scholar, 30Loose J.H. Damjanov I. Harris H. Am. J. Clin. Pathol. 1984; 82: 173-177Crossref PubMed Scopus (13) Google Scholar). GCAP is a particularly good marker of carcinoma (in situ) of the testis (31Wahren B. Homgren P.Å. Stigbrand T. Int. J. Cancer. 1979; 24: 749-753Crossref PubMed Scopus (73) Google Scholar, 32Jeppsson A. Wahren B. Brehmer-Andersson E. Silfverswärd C. Stigbrand T. Millán J.L. Int. J. Cancer. 1984; 34: 757-761Crossref PubMed Scopus (38) Google Scholar, 33Roelofs H. Manes T. Janszen T. Millán J.L. Oosterhuis W. Looijenga L. J. Pathol. 1999; 189: 236-244Crossref PubMed Scopus (34) Google Scholar), and IAP is a marker of hepatocellular carcinoma (34Higashino K. Otani R. Kudo S. Hashinotsume M. Hada T. Kang K.-Y. Ohkochi T. Takahashi Y. Yamamura Y. Ann. Intern. Med. 1975; 83: 74-78Crossref PubMed Scopus (23) Google Scholar).Although APs are homodimeric molecules, the re-expression in cancer cells of more than one AP isozyme often results in the formation and release into body fluids of heterodimeric enzymes. The Kasahara AP isoform was identified in a variety of human cancer cell lines (35Higashino K. Hashinotsume M. Kang K.-Y. Takahashi Y. Yamamura Y. Clin. Chim. Acta. 1972; 40: 67-81Crossref PubMed Scopus (105) Google Scholar, 36Higashino K. Otani R. Kudo S. Yamamura Y. Clin. Chem. 1977; 23: 1615-1623Crossref PubMed Scopus (43) Google Scholar) and cancer sera and was later found to consist of heterodimers of the IAP and PLAP (37Imanishi H. Hada T. Muratani K. Hirano K. Higashino K. Cancer Res. 1990; 50: 3408-3412PubMed Google Scholar). The human postnatal intestine also contains heterodimers of IAP and PLAP (38Behrens C.M. Enns C.A. Sussman H.H. Biochem. J. 1983; 211: 553-558Crossref PubMed Scopus (31) Google Scholar). Ovarian cancer cells often express both PLAP and GCAP (39Smans K.A. Ingvarsson M.B. Lindgren P. Canevari S. Walt H. Stigbrand T. Bäckström T. Millán J.L. Int. J. Cancer. 1999; 83: 270-277Crossref PubMed Scopus (13) Google Scholar), and cell lines derived from these tumors have been shown to express PLAP/GCAP heterodimers (40Watanabe S. Watanabe T. Li W.B. Soong B.-W. Chou J.Y. J. Biol. Chem. 1989; 264: 12611-12619Abstract Full Text PDF PubMed Google Scholar, 41Hendrix P.G. Hoylaerts M.F. Nouwen E.J. De Broe M.E. Clin. Chem. 1990; 36: 1793-1799Crossref PubMed Scopus (25) Google Scholar). However, no heterodimers have ever been reported between any of the tissue-specific APs and TNAP. The fact that APs can form heterodimers is of structural significance since APs are non-cooperative allosteric enzymes in which the stability and the catalytic properties of each monomer are controlled by the conformation of the second subunit (10Hoylaerts M.F. Manes T. Millán J.L. J. Biol. Chem. 1997; 272: 22781-22787Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). This means that the properties of the heterodimeric enzymes do not correspond to the weighted average of each homodimeric counterpart. Understanding the behavior of AP heterodimers is also of biological significance since, in tissues such as the bovine intestine where up to seven IAP isozymes with differing kinetic properties are co-expressed (42Manes T. Hoylaerts M.F. Müller R. Lottspeich F. Hölke W. Millán J.L. J. Biol. Chem. 1998; 273: 23353-23360Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), the formation of heterodimers can give rise to significant functional complexity and novel substrate specificities.In this study, we have built three-dimensional models of GCAP, IAP, and TNAP based on the 1.8-Å PLAP structure. We have analyzed the homodimer interface and the active site cleft of each modeled isozyme structure. This analysis has allowed us to understand the restrictions observed in AP heterodimer formation, whereas also defining a fingerprint of the active site characteristic of each AP isozymeMATERIALS AND METHODSThe PLAP dimer was generated from the coordinates of the PLAP structure (Protein Data Bank accession code 1EW2 (1Le Du M.H. Stigbrand T. Taussig M.J. Menez A. Stura E.A. J. Biol. Chem. 2001; 276: 9158-9165Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar)) by using the symmetry operation corresponding to the C2221 space group. The GCAP, IAP, and TNAP sequences were aligned to the PLAP sequence using the program BLAST (43; www.ncbi.nlm.nih.gov/gorf/bl2.html). The PLAP and GCAP molecules display 98% identity with no insertion or deletion relative to PLAP. The IAP and PLAP molecules show 87% identity and 91% homology with no insertion or deletion relative to PLAP. The TNAP and PLAP molecules, however, display 57% identity and 74% homology, and TNAP has four insertions of one residue, one insertion of three residues, and one deletion of two residues relative to PLAP.The GCAP, IAP, and TNAP homodimeric models and the PLAP/GCAP, PLAP/IAP, and PLAP/TNAP heterodimeric models were constructed using the sequence alignment as found with BLAST, the coordinates of the PLAP dimer, and the program MODELLER (44Sanchez R. Sali A. Methods Mol. Biol. 2000; 143: 97-129PubMed Google Scholar). The quality of the model geometries was checked with PROCHECK (45Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Each model was superimposed to the structure of PLAP using the program ALIGN (46Satow Y. Cohen G.H. Padlan E.A. Davies D.R. J. Mol. Biol. 1986; 190: 593-604Crossref PubMed Scopus (532) Google Scholar).The protein surface, interface surface, and residue accessibility were calculated with the program AREAIMOL as implemented in the CCP4 package (47Collaborative Computational Project, Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19707) Google Scholar). The interactions between the two monomers were calculated with the program CONTACT as implemented in CCP4. The definition of a secondary structure of proteins given a set of three-dimensional coordinates (DSSP) algorithm (48Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12108) Google Scholar) as implemented in TURBO was used to calculate secondary structures. Alkaline phosphatases (EC 3.1.3.1) (APs) 1The abbreviations used are: AP, alkaline phosphatase; GCAP, germ cell AP; IAP, intestinal AP; PLAP, placental AP; TNAP, tissue-nonspecific AP1The abbreviations used are: AP, alkaline phosphatase; GCAP, germ cell AP; IAP, intestinal AP; PLAP, placental AP; TNAP, tissue-nonspecific AP are dimeric enzymes present in most, if not all, organisms (2McComb R.B. Bowers G.N. Posen S. Alkaline Phosphatases. Plenum Press, New York1979: 986-988Google Scholar). They catalyze the hydrolysis of phosphomonoesters with release of inorganic phosphate (3Schwartz J.H. Lipmann F. Proc. Natl. Acad. Sci. U. S. A. 1961; 47: 1996-2005Crossref PubMed Scopus (118) Google Scholar). Mammalian APs have low sequence identity with theEscherichia coli enzyme (25–30%), but the residues involved in the active site of the enzyme and those coordinating the two zinc atoms and the magnesium ion are largely conserved, and the catalytic mechanism deduced from the structure of the E. coli AP was proposed to be similar in eukaryotic APs (4Kim E.E. Wyckoff H.W. J. Mol. Biol. 1991; 218: 449-464Crossref PubMed Scopus (928) Google Scholar). In humans, APs are encoded by four distinct loci. Three isozymes are tissue-specific, i.e. intestinal AP (IAP), placental AP (PLAP), and germ cell AP (GCAP). They are 90–98% homologous, and their genes are clustered on chromosome 2, bands q34-q37. The fourth AP isozyme is tissue-nonspecific (TNAP) and is expressed in a variety of tissues throughout development. TNAP is about 50% identical to the other three isozymes, and its gene is located on chromosome 1, bands p36.1-p34 (5Harris H. Clin. Chim. Acta. 1989; 186: 133-150Crossref Scopus (426) Google Scholar). Our current understanding of the functional properties of mammalian APs comes largely from studies using PLAP and TNAP as paradigms. Isozyme-specific properties, such as the characteristic uncompetitive inhibition properties of mammalian APs (6Hummer C. Millán J.L. Biochem. J. 1991; 274: 91-95Crossref PubMed Scopus (39) Google Scholar, 7Hoylaerts M.F. Millán J.L. Eur. J. Biochem. 1991; 202: 605-616Crossref PubMed Scopus (43) Google Scholar, 8Hoylaerts M.F. Manes T. Millán J.L. Biochem. J. 1992; 286: 23-30Crossref PubMed Scopus (74) Google Scholar), their variable heat stability (9Bossi M. Hoylaerts M.F. Millán J.L. J. Biol. Chem. 1993; 268: 25409-25416Abstract Full Text PDF PubMed Google Scholar), and even their allosteric properties (10Hoylaerts M.F. Manes T. Millán J.L. J. Biol. Chem. 1997; 272: 22781-22787Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), have been attributed to a top, flexible loop (or crown domain) unique to mammalian APs. This domain is also responsible for collagen binding in the case of TNAP (9Bossi M. Hoylaerts M.F. Millán J.L. J. Biol. Chem. 1993; 268: 25409-25416Abstract Full Text PDF PubMed Google Scholar, 11Wu L.N.Y. Genge B.R. Lloys G.C. Wuthier R.E. J. Biol. Chem. 1991; 266: 1195-1203Abstract Full Text PDF PubMed Google Scholar) but does not appear to mediate the reported binding of PLAP to IgG (12Makiya R. Stigbrand T. Eur. J. Biochem. 1992; 205: 341-345Crossref PubMed Scopus (61) Google Scholar, 13Makiya R. Stigbrand T. Biochem. Biophys. Res. Commun. 1992; 182: 624-630Crossref PubMed Scopus (44) Google Scholar). The recent elucidation of the 1.8-Å resolution structure of human PLAP (1Le Du M.H. Stigbrand T. Taussig M.J. Menez A. Stura E.A. J. Biol. Chem. 2001; 276: 9158-9165Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) has facilitated further studies on the structure and function of mammalian APs. An analysis of the structural-functional relationship of residues conserved between the E. coli AP and the PLAP structure revealed a conserved function for those residues that stabilize the active site zinc and magnesium metal ions, whereas the non-homologous disulphide bonds differ in their structural significance and non-conserved residues take part in determining the heat stability and uncompetitive inhibition properties of mammalian alkaline phosphatases (14Kozlenkov A. Manes T. Hoylaerts M.F. Millán J.L. J. Biol. Chem. 2002; 277: 22992-22999Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Deactivating mutations in the TNAP gene cause the inborn error of metabolism known as hypophosphatasia (15Henthorn P.S. Whyte MP Clin. Chem. 1992; 38: 2501-2505Crossref PubMed Scopus (86) Google Scholar), characterized by poorly mineralized cartilage and bones. The severity and expressivity of hypophosphatasia depends on the nature of the TNAP mutation (16Zurutuza L. Muller F. Gibrat J.F. Taillandier A. Simon-Bouy B. Serre J.L. Mornet E. Hum. Mol. Genet. 1999; 8: 1039-1046Crossref PubMed Scopus (160) Google Scholar). The mapping of hypophosphatasia mutations to specific three-dimensional locations on the TNAP molecule has provided clues as to the structural significance of these areas for enzyme structure and function (17Mornet E. Stura E. Lia-Baldini A.S. Stigbrand T. Menez A. Le Du M.H. J. Biol. Chem. 2001; 276: 31171-31178Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). It appears clear that the function of TNAP in bone tissue consists of hydrolizing inorganic pyrophosphate to maintain a proper concentration of this mineralization inhibitor to ensure proper bone mineralization (18Hessle L. Johnsson K.A. Anderson H.C. Narisawa S. Sali A. Goding J.W. Terkeltaub R. Millán J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9445-9449Crossref PubMed Scopus (666) Google Scholar). However, the physiological role of the three tissue-specific human APs remains to be clarified. It has been suggested that PLAP may be involved in the transfer of maternal IgG to the fetus (12Makiya R. Stigbrand T. Eur. J. Biochem. 1992; 205: 341-345Crossref PubMed Scopus (61) Google Scholar, 13Makiya R. Stigbrand T. Biochem. Biophys. Res. Commun. 1992; 182: 624-630Crossref PubMed Scopus (44) Google Scholar, 19Stefaner I. Stefanescu A. Hunziker W. Fuchs R. Biochem. J. 1997; 327: 585-592Crossref PubMed Scopus (24) Google Scholar), and evidence has accumulated indicating a role of PLAP in cell division in normal and transformed cells (20Stinson R.A. Chan J.R.A. Adv. Prot. Phosphatases. 1987; 4: 127-151Google Scholar, 21Telfer J.F. Green C.D. FEBS Lett. 1993; 329: 238-244Crossref PubMed Scopus (31) Google Scholar, 22She Q. Mukherjee J.J. Huang J. Crilly K.S. Kiss Z. FEBS Lett. 2000; 469: 163-167Crossref PubMed Scopus (32) Google Scholar, 23Dabare A.A. Nouri A.M. Cannell H. Moss T. Nigam A.K. Oliver R.T. Urol. Int. 2000; 63: 168-174Crossref Scopus (17) Google Scholar). Of considerable interest is the fact that human APs are abundantly expressed in tumor cells and that their serum levels are often used as tumor markers (24Millán J.L. Fishman W.H. Crit. Rev. Clin. Lab. Sci. 1995; 32: 1-39Crossref PubMed Scopus (107) Google Scholar). Plasma TNAP levels can indicate the presence of osteosarcomas (25Farley J.R. Hall S.L. Herring S. Tarbaux N.M. Matsuyama T. Wergedal J.E. Metabolism. 1991; 40: 664-671Abstract Full Text PDF PubMed Scopus (74) Google Scholar), Paget's disease (26Deftos L.J. Wolfert R.L. Hill C.S. Horm. Metab. Res. 1991; 23: 559-561Crossref PubMed Scopus (36) Google Scholar), and osteoblastic bone metastates (27Demers L.M. Costa L. Chinchilli V.M. Gaydos L. Curley E. Lipton A. Clin. Chem. 1995; 41: 1489-1494Crossref PubMed Scopus (135) Google Scholar). PLAP is a marker of cancer of the ovary, testis, lung, and the gastrointestinal tract (28Nathanson L. Fishman W.H. Cancer (Phila.). 1971; 27: 1388-1397Crossref PubMed Scopus (155) Google Scholar, 29Jacoby B. Bagshawe K.D. Clin. Chim. Acta. 1971; 35: 473-481Crossref PubMed Scopus (39) Google Scholar, 30Loose J.H. Damjanov I. Harris H. Am. J. Clin. Pathol. 1984; 82: 173-177Crossref PubMed Scopus (13) Google Scholar). GCAP is a particularly good marker of carcinoma (in situ) of the testis (31Wahren B. Homgren P.Å. Stigbrand T. Int. J. Cancer. 1979; 24: 749-753Crossref PubMed Scopus (73) Google Scholar, 32Jeppsson A. Wahren B. Brehmer-Andersson E. Silfverswärd C. Stigbrand T. Millán J.L. Int. J. Cancer. 1984; 34: 757-761Crossref PubMed Scopus (38) Google Scholar, 33Roelofs H. Manes T. Janszen T. Millán J.L. Oosterhuis W. Looijenga L. J. Pathol. 1999; 189: 236-244Crossref PubMed Scopus (34) Google Scholar), and IAP is a marker of hepatocellular carcinoma (34Higashino K. Otani R. Kudo S. Hashinotsume M. Hada T. Kang K.-Y. Ohkochi T. Takahashi Y. Yamamura Y. Ann. Intern. Med. 1975; 83: 74-78Crossref PubMed Scopus (23) Google Scholar). Although APs are homodimeric molecules, the re-expression in cancer cells of more than one AP isozyme often results in the formation and release into body fluids of heterodimeric enzymes. The Kasahara AP isoform was identified in a variety of human cancer cell lines (35Higashino K. Hashinotsume M. Kang K.-Y. Takahashi Y. Yamamura Y. Clin. Chim. Acta. 1972; 40: 67-81Crossref PubMed Scopus (105) Google Scholar, 36Higashino K. Otani R. Kudo S. Yamamura Y. Clin. Chem. 1977; 23: 1615-1623Crossref PubMed Scopus (43) Google Scholar) and cancer sera and was later found to consist of heterodimers of the IAP and PLAP (37Imanishi H. Hada T. Muratani K. Hirano K. Higashino K. Cancer Res. 1990; 50: 3408-3412PubMed Google Scholar). The human postnatal intestine also contains heterodimers of IAP and PLAP (38Behrens C.M. Enns C.A. Sussman H.H. Biochem. J. 1983; 211: 553-558Crossref PubMed Scopus (31) Google Scholar). Ovarian cancer cells often express both PLAP and GCAP (39Smans K.A. Ingvarsson M.B. Lindgren P. Canevari S. Walt H. Stigbrand T. Bäckström T. Millán J.L. Int. J. Cancer. 1999; 83: 270-277Crossref PubMed Scopus (13) Google Scholar), and cell lines derived from these tumors have been shown to express PLAP/GCAP heterodimers (40Watanabe S. Watanabe T. Li W.B. Soong B.-W. Chou J.Y. J. Biol. Chem. 1989; 264: 12611-12619Abstract Full Text PDF PubMed Google Scholar, 41Hendrix P.G. Hoylaerts M.F. Nouwen E.J. De Broe M.E. Clin. Chem. 1990; 36: 1793-1799Crossref PubMed Scopus (25) Google Scholar). However, no heterodimers have ever been reported between any of the tissue-specific APs and TNAP. The fact that APs can form heterodimers is of structural significance since APs are non-cooperative allosteric enzymes in which the stability and the catalytic properties of each monomer are controlled by the conformation of the second subunit (10Hoylaerts M.F. Manes T. Millán J.L. J. Biol. Chem. 1997; 272: 22781-22787Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). This means that the properties of the heterodimeric enzymes do not correspond to the weighted average of each homodimeric counterpart. Understanding the behavior of AP heterodimers is also of biological significance since, in tissues such as the bovine intestine where up to seven IAP isozymes with differing kinetic properties are co-expressed (42Manes T. Hoylaerts M.F. Müller R. Lottspeich F. Hölke W. Millán J.L. J. Biol. Chem. 1998; 273: 23353-23360Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), the formation of heterodimers can give rise to significant functional complexity and novel substrate specificities. In this study, we have built three-dimensional models of GCAP, IAP, and TNAP based on the 1.8-Å PLAP structure. We have analyzed the homodimer interface and the active site cleft of each modeled isozyme structure. This analysis has allowed us to understand the restrictions observed in AP heterodimer formation, whereas also defining a fingerprint of the active site characteristic of each AP isozyme MATERIALS AND METHODSThe PLAP dimer was generated from the coordinates of the PLAP structure (Protein Data Bank accession code 1EW2 (1Le Du M.H. Stigbrand T. Taussig M.J. Menez A. Stura E.A. J. Biol. Chem. 2001; 276: 9158-9165Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar)) by using the symmetry operation corresponding to the C2221 space group. The GCAP, IAP, and TNAP sequences were aligned to the PLAP sequence using the program BLAST (43; www.ncbi.nlm.nih.gov/gorf/bl2.html). The PLAP and GCAP molecules display 98% identity with no insertion or deletion relative to PLAP. The IAP and PLAP molecules show 87% identity and 91% homology with no insertion or deletion relative to PLAP. The TNAP and PLAP molecules, however, display 57% identity and 74% homology, and TNAP has four insertions of one residue, one insertion of three residues, and one deletion of two residues relative to PLAP.The GCAP, IAP, and TNAP homodimeric models and the PLAP/GCAP, PLAP/IAP, and PLAP/TNAP heterodimeric models were constructed using the sequence alignment as found with BLAST, the coordinates of the PLAP dimer, and the program MODELLER (44Sanchez R. Sali A. Methods Mol. Biol. 2000; 143: 97-129PubMed Google Scholar). The quality of the model geometries was checked with PROCHECK (45Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Each model was superimposed to the structure of PLAP using the program ALIGN (46Satow Y. Cohen G.H. Padlan E.A. Davies D.R. J. Mol. Biol. 1986; 190: 593-604Crossref PubMed Scopus (532) Google Scholar).The protein surface, interface surface, and residue accessibility were calculated with the program AREAIMOL as implemented in the CCP4 package (47Collaborative Computational Project, Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19707) Google Scholar). The interactions between the two monomers were calculated with the program CONTACT as implemented in CCP4. The definition of a secondary structure of proteins given a set of three-dimensional coordinates (DSSP) algorithm (48Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12108) Google Scholar) as implemented in TURBO was used to calculate secondary structures. The PLAP dimer was generated from the coordinates of the PLAP structure (Protein Data Bank accession code 1EW2 (1Le Du M.H. Stigbrand T. Taussig M.J. Menez A. Stura E.A. J. Biol. Chem. 2001; 276: 9158-9165Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar)) by using the symmetry operation corresponding to the C2221 space group. The GCAP, IAP, and TNAP sequences were aligned to the PLAP sequence using the program BLAST (43; www.ncbi.nlm.nih.gov/gorf/bl2.html). The PLAP and GCAP molecules display 98% identity with no insertion or deletion relative to PLAP. The IAP and PLAP molecules show 87% identity and 91% homology with no insertion or deletion relative to PLAP. The TNAP and PLAP molecules, however, display 57% identity and 74% homology, and TNAP has four insertions of one residue, one insertion of three residues, and one deletion of two residues relative to PLAP. The GCAP, IAP, and TNAP homodimeric models and the PLAP/GCAP, PLAP/IAP, and PLAP/TNAP heterodimeric models were constructed using the sequence alignment as found with BLAST, the coordinates of the PLAP dimer, and the program MODELLER (44Sanchez R. Sali A. Methods Mol. Biol. 2000; 143: 97-129PubMed Google Scholar). The quality of the model geometries was checked with PROCHECK (45Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Each model was superimposed to the structure of PLAP using the program ALIGN (46Satow Y. Cohen G.H. Padlan E.A. Davies D.R. J. Mol. Biol. 1986; 190: 593-604Crossref PubMed Scopus (532) Google Scholar). The protein surface, interface surface, and residue accessibility were calculated with the program AREAIMOL as implemented in the CCP4 package (47Collaborative Computational Project, Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19707) Google Scholar). The interactions between the two monomers were calculated with the program CONTACT as implemented in CCP4. The definition of a secondary structure of proteins given a set of three-dimensional coordinates (DSSP) algorithm (48Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12108) Google Scholar) as implemented in TURBO was used to calculate secondary structures. We are grateful to Prof. Marc F. Hoylaerts, Dr. Menetrey, and Marc Graille for careful reading of the manuscript.

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Structural Evidence of Functional Divergence in Human Alkaline Phosphatases