Structural evidence for a functional role of human tissue nonspecific alkaline phosphatase in bone mineralization

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 placental alkaline phosphatase (PLAP) whose crystal structure has been recently determined at atomic resolution (Le Du, M. H., Stigbrand, T., Taussig, M. J., Ménez, 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 mutations associated with hypophosphatasia and their probable 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 active site valley, the homodimer interface, the crown domain, 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 confirms that the metal in the metal-binding site is a calcium 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 insights on the controversial role observed for calcium.     

An essential tryptophan residue in alkaline phosphatase from pearl oyster (<Emphasis Type="Italic">Pinctada fucata</Emphasis>)

Alkaline phosphatases are ubiquitous enzymes found in most species including the pearl oyster, Pinctada fucata, where it is presumably involved in nacreous biomineralization processes. In the present study, we have purified alkaline phosphatases from the pearl oyster and modified the tryptophan residues using N-bromosuccinimide (NBS). We show that the resulting inactivation of purified alkaline phosphatase by NBS is dependent on modification of only one of five tryptophan residues in the enzyme. Substrate protection experiments showed that the tryptophan residue was not located at the substrate-binding site but was involved in the catalytic activity. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 1, pp. 107–112.     

Characterization of Calcium Deposition and Shell Matrix Protein Secretion in Primary Mantle Tissue Culture from the Marine Pearl Oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis>

In this study, we established and characterized a long-term primary mantle tissue culture from the marine pearl oyster Pinctada fucata for in vitro investigation of nacre biomineralization. In this culture system, the viability of mantle tissue cells lasted up to 2 months. The tissue cells were demonstrated to express nacre matrix proteins by RT-PCR, and a soluble shell matrix protein, nacrein, was detected in the culture medium by Western blot analysis. On the other hand, 15 days after initiating culture, a large amount of calcium deposits with major elements, including calcium, carbon, and oxygen, were generated in the mantle explants and cell outgrowth area. The quantity and size of calcium deposits increased with the prolonged cultivation, and their location and nanogranular structure suggested their biogenic origin. These calcium deposits specifically appeared in mantle tissue cultures, but not in heart tissue cultures. Taken together, these results demonstrate that the mantle tissue culture functions similarly to mantle cells in vivo. This study provides a reliable approach for the further investigation on nacre biomineralization at the cellular level.     

Differential expression and activity of tissue-nonspecific alkaline phosphatase (TNAP) in rat odontogenic cells in vivo.

Among the four existing isoforms of alkaline phosphatase (AP), the present study is devoted to tissue-nonspecific alkaline phosphatase (TNAP) in mineralized dental tissues. Northern blot analysis and measurements of phosphohydrolase activity on microdissected epithelium and ectomesenchyme, in situ hybridization, and immunolabeling on incisors confirmed that the AP active in rodent teeth is TNAP. Whereas the developmental pattern of TNAP mRNA and protein and the previously described activity were similar in supra-ameloblastic and mesenchymal cells, they differed in enamel-secreting cells, the ameloblasts. As previously shown for other proteins involved in calcium and phosphate handling in ameloblasts, a biphasic pattern of steady-state TNAP mRNA levels was associated with additional variations in ameloblast TNAP protein levels during the cyclic modulation process. Although the association of TNAP upregulation and the initial phase of biomineralization appeared to be a basic feature of all mineralized tissues, ameloblasts (and to a lesser extent, odontoblasts) showed a second selectively prominent upregulation of TNAP mRNA/protein/activity during terminal growth of large enamel crystals only, i.e., the maturation stage. This differential expression/activity for TNAP in teeth vs bone may explain the striking dental phenotype vs bone reported in hypophosphatasia, a hereditary disorder related to TNAP mutation. (J Histochem Cytochem 47:1541-1552, 1999)     

Vaccinia topoisomerase mutants illuminate conformational changes during closure of the protein clamp and assembly of a functional active site.

We present a mutational analysis of vaccinia topoisomerase that highlights the contributions of five residues in the catalytic domain (Phe-88 and Phe-101 in helix alpha1, Ser-204 in alpha5, and Lys-220 and Asn-228 in alpha6) to the DNA binding and transesterification steps. When augmented by structural information from exemplary type IB topoisomerases and tyrosine recombinases in different functional states, the results suggest how closure of the protein clamp around duplex DNA and assembly of a functional active site might be orchestrated by internal conformational changes in the catalytic domain. Lys-220 is a constituent of the active site, and a positive charge at this position is required for optimal DNA cleavage. Ser-204 and Asn-228 appear not to be directly involved in reaction chemistry at the scissile phosphodiester. We propose that (i) Asn-228 recruits the Tyr-274 nucleophile to the active site by forming a hydrogen bond to the main chain of the tyrosine-containing alpha8 helix and that (ii) contacts between Ser-204 and the DNA backbone upstream of the cleavage site trigger a separate conformational change required for active site assembly. Mutations of Phe-88 and Phe-101 affect DNA binding, most likely at the clamp closure step, which we posit to entail a distortion of helix alpha1.     

Structural studies of human alkaline phosphatase in complex with strontium: implication for its secondary effect in bones

Strontium is used in the treatment of osteoporosis as a ranelate compound, and in the treatment of painful scattered bone metastases as isotope. At very high doses and in certain conditions, it can lead to osteomalacia characterized by impairment of bone mineralization. The osteomalacia symptoms resemble those of hypophosphatasia, a rare inherited disorder associated with mutations in the gene encoding for tissue-nonspecific alkaline phosphatase (TNAP). Human alkaline phosphatases have four metal binding sites--two for zinc, one for magnesium, and one for calcium ion--that can be substituted by strontium. Here we present the crystal structure of strontium-substituted human placental alkaline phosphatase (PLAP), a related isozyme of TNAP, in which such replacement can have important physiological implications. The structure shows that strontium substitutes the calcium ion with concomitant modification of the metal coordination. The use of the flexible and polarizable force-field TCPEp (topological and classical polarization effects for proteins) predicts that calcium or strontium has similar interaction energies at the calcium-binding site of PLAP. Since calcium helps stabilize a large area that includes loops 210-228 and 250-297, its substitution by strontium could affect the stability of this region. Energy calculations suggest that only at high doses of strontium, comparable to those found for calcium, can strontium substitute for calcium. Since osteomalacia is observed after ingestion of high doses of strontium, alkaline phosphatase is likely to be one of the targets of strontium, and thus this enzyme might be involved in this disease.     

A Novel Matrix Protein p10 from the Nacre of Pearl Oyster (Pinctada fucata) and Its Effects on Both CaCO3 Crystal Formation and Mineralogenic Cells

A novel matrix protein, designated as p10 because of its apparent molecular mass of 10 kDa, was isolated from the nacreous layer of pearl oyster (Pinctada fucata) by reverse-phase high-performance liquid chromatography. In vitro crystallization experiments showed that p10 could accelerate the nucleation of calcium carbonate crystals and induce aragonite formation, suggesting that it might play a key role in nacre biomineralization. As nacre is known to contain osteogenic factors, two mineralogenic cell lines, MRC-5 fibroblasts and MC3T3-E1 preosteoblasts, were used to investigate the biological activity of p10. The results showed that p10 could increase alkaline phosphatase activity, an early marker of osteoblast differentiation, while the viability of MRC-5 and MC3T3-E1 remained unchanged after treatment of p10. Taken together, the findings led to identification of a novel matrix protein from the nacre of P. fucata that plays a role in both the mineral phase and in the differentiation of the cells involved in biomineralization.     

Amorphous Calcium Carbonate Precipitation by Cellular Biomineralization in Mantle Cell Cultures of Pinctada fucata

The growth of molluscan shell crystals is generally thought to be initiated from the extrapallial fluid by matrix proteins, however, the cellular mechanisms of shell formation pathway remain unknown. Here, we first report amorphous calcium carbonate (ACC) precipitation by cellular biomineralization in primary mantle cell cultures of Pinctada fucata. Through real-time PCR and western blot analyses, we demonstrate that mantle cells retain the ability to synthesize and secrete ACCBP, Pif80 and nacrein in vitro. In addition, the cells also maintained high levels of alkaline phosphatase and carbonic anhydrase activity, enzymes responsible for shell formation. On the basis of polarized light microscopy and scanning electron microscopy, we observed intracellular crystals production by mantle cells in vitro. Fourier transform infrared spectroscopy and X-ray diffraction analyses revealed the crystals to be ACC, and de novo biomineralization was confirmed by following the incorporation of Sr into calcium carbonate. Our results demonstrate the ability of mantle cells to perform fundamental biomineralization processes via amorphous calcium carbonate, and these cells may be directly involved in pearl oyster shell formation.     

Cloning and characterization of an mRNA encoding a novel G protein α-subunit abundant in mantle and gill of pearl oyster Pinctada fucata

Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel G_sα (pfG_sα) from the pearl oyster was isolated. The G_sα cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus (Octopus vulgaris) G_sα. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfG_sα has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster G_sα mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfG_sα in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfG_sα that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the G_sα in MC3T3-E1 cells. These results indicated that G_sα may be involved in regulation of physiological function, particularly in biological biomineralization.     

Molecular cloning and characterization of perlucin from the freshwater pearl mussel, Hyriopsis cumingii

Perlucin is an important functional protein that regulates shell and pearl formation. In this study, we cloned the perlucin gene from the freshwater pearl mussel Hyriopsis cumingii, designated as Hcperlucin. The full-length cDNA transcribed from the Hcperlucin gene was 1460 bp long, encoding a putative signal peptide of 20 amino acids and a mature protein of 141 amino acids. The mature Hcperlucin peptide contained six conserved cysteine residues and a carbohydrate recognition domain, similar to other members of the C-type lectin families. In addition, a “QPS” and an invariant “WND” motif near the C-terminal region were also found, which are extremely important for polysaccharide recognition and calcium binding of lectins. The mRNA of Hcperlucin was constitutively expressed in all tested H. cumingii tissues, with the highest expression levels observed in the mantle, adductor, gill and hemocytes. In situ hybridization was used to detect the presence of Hcperlucin mRNA in the mantle, and the result showed that the mRNA was specifically expressed in the epithelial cells of the dorsal mantle pallial, an area known to express genes involved in the biosynthesis of the nacreous layer of the shell. The significant Hcperlucin mRNA expression was detected on day 14 post shell damage and implantation, suggesting that the Hcperlucin might be an important gene in shell nacreous layer and pearl formation. The change of perlucin expression in pearl sac also confirmed that the mantle transplantation results in a new expression pattern of perlucin genes in pearl sac cells that are required for pearl biomineralization. These findings could help better understanding the function of perlucin in the shell and pearl formation.     

Chemical modification studies on alkaline phosphatase from pearl oyster (Pinctada fucata): a substrate reaction course analysis and involvement of essential arginine and lysine residues at the active site

Alkaline phosphatases (ALP, EC 3.1.3.1) are ubiquitous enzymes found in most species. ALP from a pearl oyster, Pinctada fucata (PALP), is presumably involved in nacreous biomineralization processes. Here, chemical modification was used to investigate the involvement of basic residues in the catalytic activity of PALP. The Tsou's plot analysis indicated that the inactivation of PALP by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and phenylglyoxal (PG) is dependent upon modification of one essential lysine and one essential arginine residue, respectively. Substrate reaction course analysis showed that the TNBS and PG inactivation of PALP followed pseudo-first-order kinetics and the second-order inactivation constants for the enzyme with or without substrate binding were determined. It was found that binding substrate slowed the PG inactivation whereas had little effect on TNBS inactivation. Protection experiments showed that substrates and competitive inhibitors provided significant protection against PG inactivation, and the modified enzyme lost its ability to bind the specific affinity column. However, the TNBS-induced inactivation could not be prevented in presence of substrates or competitive inhibitors, and the modified enzyme retained the ability to bind the affinity column. In a conclusion, an arginine residue involved in substrate binding and a lysine residue involved in catalysis were present at the active site of PALP. This study will facilitate to illustrate the role ALP plays in pearl formation and the mechanism involved.     

Mutagenesis of E477 or K505 in the B' domain of human topoisomerase II beta increases the requirement for magnesium ions during strand passage

A type II topoisomerase is essential for decatenating DNA replication products, and it accomplishes this task by passing one DNA duplex through a transient break in a second duplex. The B' domain of topoisomerase II contains three highly conserved motifs, EGDSA, PL(R/K)GK(I/L/M)LNVR, and IMTD(Q/A)DXD. We have investigated these motifs in topoisomerase II beta by mutagenesis, and report that they play a critical role in establishing the DNA cleavage-religation equilibrium. In addition, the mutations E477Q (EGDSA) and K505E (PLRGKILNVR) increase the optimal magnesium ion concentration for strand passage, without affecting the Mg(2+) dependence of ATP hydrolysis. It is likely that the binding affinity of the magnesium ion(s) specifically required for DNA cleavage has been reduced by these mutations. The crystal structure of yeast topo II indicates that residues E477 and K505 may help to position the three aspartate residues of the IMTD(Q/A)DXD motif for magnesium ion coordination, and we propose two possible locations for the magnesium ion binding site(s). These observations are consistent with a previous model in which the B' domain is positioned such that these acidic residues lie next to the active site tyrosine residue. A magnesium ion bound by these aspartate residues could therefore mediate the DNA cleavage-religation reaction.     

Crystal structure of a mini-intein reveals a conserved catalytic module involved in side chain cyclization of asparagine during protein splicing

We have determined the crystal structure of a 154-residue intein derived from the dnaB gene of Synechocystis sp. strain PCC6803 and refined it to a 2.0-A resolution. The x-ray structure suggests that this intein possesses two catalytic sites that appear to be separately responsible for splicing and cleavage of the N- and C-terminal scissile bonds. The conserved intein block F residues are the important components of a catalytic site for side chain cyclization of the last intein residue, Asn-154. The data suggest that the imidazole ring of His-143 is involved in the activation of the side chain Ndelta atom of Asn-154, leading to a nucleophilic attack on the carbonyl carbon of Asn-154. Substitution of His-143 with Ala or Gln resulted in the inhibition of C-terminal cleavage. His-153, Asp-136, and a water molecule appear to constitute an oxyanion binding site by contacting the carbonyl oxygen of Asn-154 to stabilize the transition state. The structure and mutagenesis data also support that the close contact between the hydroxyl groups of Thr-138 and Ser-155, whose side chain participates in an S --> O acyl shift, plays an important role in the nucleophile orientation. Our structural modeling suggests that this catalytic module is conserved in the C-terminal subdomains of inteins from diverse organisms.     

A Novel Matrix Protein Hic31 from the Prismatic Layer of Hyriopsis Cumingii Displays a Collagen-Like Structure

In this study, we clone and characterize a novel matrix protein, hic31, from the mantle of Hyriopsis cumingii. The amino acid composition of hic31 consists of a high proportion of Glycine residues (26.67%). Tissue expression detection by RT-PCR indicates that hic31 is expressed specifically at the mantle edge. In situ hybridization results reveals strong signals from the dorsal epithelial cells of the outer fold at the mantle edge, and weak signals from inner epithelial cells of the same fold, indicating that hic31 is a prismatic-layer matrix protein. Although BLASTP results identify no shared homology with other shell-matrix proteins or any other known proteins, the hic31 tertiary structure is similar to that of collagen I, alpha 1 and alpha 2. It has been well proved that collagen forms the basic organic frameworks in way of collagen fibrils and minerals present within or outside of these fibrils. Therefore, hic31 might be a framework-matrix protein involved in the prismatic-layer biomineralization. Besides, the gene expression of hic31 increase in the early stages of pearl sac development, indicating that hic31 may play important roles in biomineralization of the pearl prismatic layer.     

Mammalian alkaline phosphatase catalysis requires active site structure stabilization via the N-terminal amino acid microenvironment

In mammalian alkaline phosphatase (AP) dimers, the N-terminus of one monomer embraces the other, stretching toward its active site. We have analyzed the role of the N-terminus and its microenvironment in determining the enzyme stability and catalysis using human placental (PLAP) and tissue-nonspecific AP (TNAP) as paradigms. Deletion of nine amino acid (aa) residues in PLAP reduced its AP activity and heat stability, while deletion of 25 aa resulted in an inactive enzyme. In turn, deletion of five and nine N-terminal aa in TNAP reduced and abolished AP activity, respectively. The N-terminal aa deletions in both isozymes affected the rate of substrate catalysis (k(cat)), with an only minor effect on the Michaelis constant (K(m)) explained by decelerated intramolecular transition rates in the active site. Arg370 in PLAP, and the corresponding Arg374 in TNAP, critically control the structure and function of the enzymes, but the Glu6-Arg370 bond predicted by the PLAP crystal structure appeared to be irrelevant with respect to PLAP stability or catalysis. Structural disruption was also noted in [R374A]TNAP, [Delta5]TNAP, [Delta9]TNAP, and [Delta25]TNAP using a panel of 19 anti-TNAP antibodies illustrating the structural role of the N-terminus. Our data reveal that the N-terminal alpha-helical folding is more crucial for the structural stability of the second monomer in TNAP than in PLAP. The correct folding of the N-terminus and of interacting loops in its immediate environment is essential for overall structural integrity and for execution of intramolecular transitions during enzyme catalysis. These findings provide a mechanistic interpretation for loss-of-function mutations of N-terminal TNAP residues in cases of hypophosphatasia.     

High-affinity and low-affinity calcium binding and stability of the multidomain extracellular 40-kDa basement membrane glycoprotein (BM-40/SPARC/osteonectin).

Reversible binding of calcium ions to a single high-affinity binding site in the 40-kDa basement membrane protein (BM-40) caused a 33% increase of alpha-helicity, an about 60% change in intrinsic fluorescence and a dramatic increase of the rate of cleavage by alpha-chymotrypsin. All these effects exhibited identical dependencies on calcium concentration from which a dissociation constant Kd = 0.6 microM was determined. Calcium release was accompanied by an increase of the frictional ratio in solution but not by denaturation which occurred at about equal guanidine.HCl concentration for both calcium-saturated and calcium-depleted protein (midpoint 1.5 M). The cleavage sites for alpha-chymotrypsin are located in or near to the EF-hand domain IV of calcium-depleted BM-40 (also known as SPARC, i.e. secreted protein acidic and rich in cysteine, and osteonectin). These and other data indicate that binding occurs in the EF-hand domain from which a large conformational change is transmitted. Low-affinity calcium-binding sites in the N-terminal glutamic-acid-rich domain I of BM-40 were identified by human leukocyte elastase which was found to cleave very specifically in the middle of this domain. From the increase of cleavage rate with increasing calcium concentration a Kd greater than or equal to 10 mM was estimated. It is suggested that variations of calcium levels in the extracellular space in this range may regulate functions of BM-40 such as collagen binding and that high-affinity binding is important for stabilization, folding and secretion during biosynthesis.     

Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-beta receptor system.

We describe the primary structure of rat betaglycan, a polymorphic membrane-anchored proteoglycan with high affinity for transforming growth factor-beta (TGF-beta). As deduced from its cDNA sequence, the 853 amino acid core protein of betaglycan has an extracellular domain with clustered sites for potential attachment of glycosaminoglycan chains. These chains are dispensable for TGF-beta binding to the core protein. The transmembrane region and the short cytoplasmic tail of betaglycan are very similar to these regions in human endoglin, an endothelial cell membrane glycoprotein involved in intercellular recognition. The ectodomain of betaglycan can be released as a soluble proteoglycan; a potential cleavage site near the transmembrane region is identical to the highly regulated cleavage site of the membrane-anchored transforming growth factor-alpha precursor. The unique features of betaglycan suggest important roles in cell interaction with TGF-beta.