Tuesday, March 27, 2012

Understanding mineralization process: the role of TNSALP and Matrix Vescicles.

  

Alkaline phosphatases

Alkaline phosphatase (ALP) was discovered in 1923 by Robert Robinson in young rats and rabbits within ossifying bone and cartilage. However , never Robinson referred to this enzyme as “alkaline” phosphatase, term introduced only later. For most of the past eight decades physicians have recognized the important clinical insight that can come from measurement of ALP activity in serum. Detection and monitoring of hepatobiliary and skeletal disease are generally possible. In fact, since 1930 ALP detection and quantification in serum has been routine in hospital laboratories.

Nevertheless, the physiologiocal function of ALP, is largely unknown.

At the end of 1960s, electron microscopy helped to rejuvenate Robinson’s hypothesis, when the earliest site of hydroxyapatite crystal deposition in the developing skeleton was noted by E. Bonucci and HC Anderson to be within novel extracellular structures called matrix vescicles (1969). These vescicles were found to be rich in ALP activity and later they have been demonstrated to be replenished by many enzymes and constituents such as:

  1. Inorganic pyrophosphatase (PPi-ase)
  2. ATPase
  3. phospholipids
  4. polysaccarides
  5. glycolipids

During early phase (primary) of mineralization , hydroxyapatite crystals appear and grow within these structres. Soon after , the vescicles rupture and extravescicular (secondary) mineralization occurs as crystal propagation continues.

Actually the proposed biological roles of ALP in mammals are numerous including:

- hydrolysis of phosphate esters to supply the nonphosphate moiety

- transferase action for the synthesis of phosphate ester

- regulation of Pi metabolism

- maintenance of steady-state levels of phosphoryl metabolites

- action on phoshoprotein pshosphatases

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At plasmamembrane level it has been proposed that ALP can function not only such as an active P transporter but also for:

- calcium muvement regulator

- Na+/K+ exchange regulator

- Fat exchange

- Protein exchange

- Carbohydrate exchange

Interestingly sequence analysis of ALP demonstrated that this enzyme can be coupled with other proteins, for example adhering to collagen, and it has been suggested that this physical property of ALP should be considered when we examined the action for example of ALP on skeletal matrix such as phosphoprotein phosphatase.

It has to be outlined that ALP function mainly such as a cell surface enzyme, but at some stages during embryo formation ALP may acts also intracellularly.

The role of ALP in skeletal mineralization should be resumed considering ALP as “inhibitor” of pyrophosphate deposition (a potent inhibitor of hydroxyapatite formation):

  1. Locally increase P concentration
  2. Destruction of inhibitors of hydroxyapatite crystal growth
  3. Transport of P
  4. Calcium binding protein ( used by cells such as uptake enzyme for calcium)
  5. Ca++/Mg++ ATPase
  6. Tyrosine specific phosphoprotein phosphatase

Recently these different activities previously attribute only to Alkaline Phosphatase activity, have been studies in more details.

It is interesting to note that in normal adolescents 13 to 14 years old pyridoxal-5-phosphate concentrations have been reported to be approximately 40 nmol/L, also if this developmental stage is associated with high alkaline phosphatase activity. Low levels of Pyridoxal-5-phosphate are also observed in patients with hypophosphatemic rickets and the researchers attributed this data to increased activity of alkaline phosphatase enzymes. However most of alkaline phosphatase values are within the normal range for children. It has also been suggested the presence of so called “functional hypophosphatasia” in patients affected by renal osteodystrophy were normal alkaline phosphatase levels are coupled with high serum inorganic phosphate levels. In other words in these pathophysiological conditions no correlation exists between alkaline phosphatase activity and pyridoxal-5-phosphate concentrations. However in these conditions plasma levels of inorganic phosphate are also higher than normal suggesting that the main factor in decreased pyridoxal 5 phosphate concentration would be low phosphate concentration rather than high levels of alkaline phosphatase.

An emerging role in Pyrophosphate production has been recently attributed to Ectonucleotide Pyrophosphatase/phosphodiesterase 1 (NPP1), previously referred such as plasma cell membrane glycoprotein 1. This enzyme has been found in mineralizing tissues such as bones and teeth. Mutations in NPP1 cause the generalized arterial calcification of infancy due to inability of vascular cells to form pyrophosphate. Moreover, mutations in NPP1 have also been reported as a second cause of autosomal recessive Hypophosphatemic Rickets, the first being attributed to mutations in Dentin Matrix Protein 1 (DMP1). The role of NPP1 would be the hydrolyis from Adenosine Triphosphate (ATP) of Pyrosphosphate. NPP1 clearly has a role in PPi generation at the level of chondrocyte and osteoblast membranes, whereas at level of Matrix Vecicles NPP1 does not use ATP efficiently.

Another pathway for generation of pyrophosphate production is the secretion from cells by the transmembrane spanning cell surface protein Ankylosis Human homologue of the mouse progressive ankylosis protein (ANKH). Two autosomal dominant human diseases have to date been reported:

- Craniometaphyseal Dysplasia

- Chondrocalcinosis-2

Data conerning the possible presence of an autosomal recessive form linked to a mutation on exon 6 of the 12 exons constituting ANKH gene has to be clarified by further studies. Anyway ANKH protein seems to be important for mediating intracellular to extracellular channeling of pyrophosphate.

Interestingly another protein called Phosphatase PHOSPHO-1, first identified in chick as a member of the haloacid dehalogenase (HAD) superfamily of Magnesium dependent hydrolases, is expresed at levels 100-fold higher in mineralizing tissues compared to nonmineralizing ones. PHOSPHO-1 shows high phosphohydrolase activity toward Phosphoetanolamine (PEA) and Phosphocholine (PCho); it is active inside chondrocytes and osteoblast derived Matrix Vescicles. The role of PHOSPHO-1 is to maintain the concentration of inorganic Pyrophosphate (PP i) so that the ratio of inorganic phosphate to inorganic pyrophosphate would be permissive of a normal mineralization process. Inside Matrix Vescicles (MV) soluble phosphatase PHOSPHO-1 , with specificty for phosphoethanolamine and phosphocholine, increases the local intravescicular concentration of inorganic phosphate (P i) to change the Pi/PPi ratio in favor of precipitation of hydroxyapatite seed crystals.

In summary given the role of FGF23/Klotho pathway in inorganic phosphorus metabolism, as well’s of Vitamin D3 metabolites, more important role should be attributed to inorganic phosphate concentration that to enzymatic phosphatase activity for study derangements in bone mineralization.

ALP is found in nearly all plants and animals. In humans, four ALP isoenzymes are encoded by four separate genes. Three of these are expressed in a tissue-specific manner are they are called:

- placental

- intestinal

- germ-cell (placental-like)

- Tissue Non Specific

The fourth ALP isoenzyme is ubiquitous, but expecially abundant in hepatic, skeletal and renal tissues (liver/Bone/kiney ALP) and it is called tissue non specific ALP (TNSALP). Interestingly TNSALP is a family of “secondary” isoenzymes (isoforms), with the same polypeptide sequence, encoded by one gene (TNSALP) but different each other only by posttranslational modification involving a different glycosylation pattern (carbohydrate). TNSALP is located on chromosome 1p36.1-34 near the end of shot arm; the genes coding for placental, intestinal and germ-cells ALP are found near the tip of the long arm of chromosome 2q34-37. The TNSALP chromosome structure is represented by 12 exons, 11 of which are translated into a 507 aminoacid nascent enzyme. The promoter region of TNSALP is located within 610 nucleotides 5’ to the transcription start site and it contains TATA box and an Sp1 binding site acting as regulatory elements. It is believed that basal levels of TNSALP expression reflect inherent “housekeeping” promoter effects, whereas differential expression in various tissues should be mediated by a postranslational mechanism. Interestingly 5’ untranslated region differ between the bone and liver TSNALP isoforms. From phylogenetic point of view, the TNSALP should represent an ancestral gene, whereas the tissue-specific ALPs is likely originated from a series of gene duplications. Human ALP isoenzymes gene sequence indicates that the nascent polypeptide has a short signal sequence of 17 or 21 aminoacids residues and a hydrophobic domain at its c terminal site. The active site is coded by six exons and it is composed by 15 aminoacid residues with a nucleotidic sequence well conserved throught nature. ALPs is a metalloenzyme linking Zinc atom, the link of Zn++ atom stabilizes the tertiary structure. In summary the structure of these enzymes is formed to link a dinuclear metal cofactor structure so that a common cathalytic mechanism for enzymes involved in phosphotransfer reactions has been identified involving spin-coupled metal binding site formed by a scaffold structure at active metal linking site constituted by the same repeated tertiary spatial construct.

Β sheet – α helix - Β sheet - α helix - Β sheet

The 3 β strands of this structure form a parallel sheet that is capped by intervening α helices. Two metal ions are positioned at the apex of this fold forming a dinuclear metal center with 3.0 - 4.0 Ǻ between metal ions, with 4 of the metal ligands provided by residues in the loops between β sheets and α helices. ALP in E. Coli has been extensively studied and a Mg++ with a Zn-Zn dinuclear center reminiscent of the dinuclera metal site of seine/threonine phosphatase has been identified. In E.coli His 372 forms an hydrogen bond with Asp 327 , an aminoacid involved into didentate Zn stabilization) and it is thought to lower the pKa of the Zn atom involved to binding a water molecule. Cathalytic activity require multimeric configuration of identical subunits, each monmer having an active site and two Zn atoms. The role of Zn atoms is probably those of allowing the formation of a nucleophil reactant by hydroxyl group of serine residue located on cathalitic site, that attract the phosphoric group disrupting the esteric link. The mechanism of enzymatic reaction in ALP present in E. Coli has been elucidated for phosphate ester hydrolysis forming first an intermediate phosphoenzyme. In particular ALP of E. Coli cathalizes the transfert of phosphoryl group throught the formation of a transient link with a Serine residue located on active catalitic site. Later this phosphate group is released and the cathalitic site left free to react with anoter phosphoester group. If ALP in serum is present as a dimer with α/β topology with a 10 –stranded beta sheets in its center, ALP at membrane level is linked as a homotetramer. ALP is linked to plasmamembrane surface, through a polar head group of a phosphatidylinositol glycan and it can be released by a specific phospholipase. Intracellualr degradation of ALPs can involve proteasomal structures. Release from plasma membrane could involve phosphatidase C or D.

Clearance of circulating ALP, as for many plasma proteins is assumed to occur via uptake by the liver.

Whereas in children ALP plasma activity is mainly of bone origin and the remaining is of intestinal isotype; interestingly an old data report that blood type (0 and B are secretors) influences the level of placental isoenzyme of ALP in the blood after an ingestion of a fatty meal. In adult blood, ALP activity reflects equal amounts of hepatic and bone isotypes. Interestingly only recently on 2000 the crystal structure of placental isoform of ALP was isolated and studied on X ray crystallography.

TNSALP has a major role in two kind of reactions involved into mineralization process:

- Pyrophosphatase : hydrolizing pyrophosphate into two inorganic phosphate ions

- ATPase/ADPase: hydrolizing Adenosin triphosphate into Adenosin bisphosphate and one molecule of inorganic phosphate.

Accordingly TNSALP partecipates in the calcification process both by restricting the concentration of extracellular inorganic pyrophosphate PPi and by contributing to the inorganic phosphate (Pi) pool available for calcification.

ATP > ADP > AMP + 2 Pi

PPi > Pi

The working model in bone and cartilage supposed that bone mineralization is first initiated within the lumen of Matrix Vescicles (MVs). In a second time, hydroxyapatite crystals grow beyond the confines of the MVs and become exposed to the extracellular milieu, where they continue to propagate along collagen fibrils. Hydroxyapatite seed crystals are formed in the sheltered interior of MVs favored by the Pi-generating activity of PHOSPHO-1 fosfatase enzyme,as well’s by the transport function of Pyrophosphate (PPi) transporters, such as ANKH. The keys rate limiting step seems to be the ratio between Pi/PPi concentrations:

Pi/PPi > Mineralization

In other words an increased concentration of inorganic pyrophosphate (PPi) inhibits the crystalization process of hydroxyapatite, whereas increase increased concentration of inorganic phosphate ions (Pi) promote both crystalization and nucleation processes.

 

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Human diseases characterized by an abnormal decrease or increase in ALP blood levels are called respectively:

- Hypophosphatasias

- Aphosphatasia

- Hyperphosphatasias or Paget disease of bone

- Familial Expansile Osteolysis

- Expansile skeletal Hyperphosphatasias

- Early onset Paget’s disease of bone in Japan

- Hereditary Hyperphosphatasia (Juvenile Paget’s disease)

Hypophosphatasia

In 1948 a Canadian pediatrician John Campbell Rathbun coined the term hypophosphatasia reporting a boy who developed and died from severe rickets with epilepsy, whose ALP activity in serum, bone and other tissues was paradoxically subnormal.

Present in all races, however this condition is expecially frequent in inbred Mennonite families from Mannitoba, Canada, where about 1 every 25 individuals is a carrier and 1:2500 newborns manifests severe disease.

Six forms of hypophosphatasias have been individuated, the earlier is the presentation of symptoms and more severe is the skeletal disease and the biochemical manifestations:

  1. Perinatal : autosomal recessive
  2. Infantile : autosomal recessive
  3. Childhood : autosomal dominant or recessive
  4. adult: autosomal dominant or recessive
  5. odontohypophosphatasia: autosomal dominat or recessive
  6. pseudohypophosphatasia

Laboratory findings include elevated values of phosphoethanolamine, pyridoxalphosphate, inorganic pyrophosphate.

Hypophosphatasia is a rare heritable disordercaused by a loss-of-function mutation in the ALP gene encoding for the tissue non specific alkaline phosphatase (TNSALP). It is characterized by deficiency in serum and bone alkaline phosphatse and defective bone and tooth mineralization.

Nearly all babies with perinatal hypophosphatsia die in utero or shortly after birth.

Those with infantile form present before 6 months of age with rickets, failure to thrive, or vitamin B6-dependent seizures, and approximately 50% die for respiratory failure because of poor lung development or progressive hypomineralization of the rib cage.

Adult hypophosphtasia typicaly manifests during middle age as recurrent, slowly healing metatarsal fractures, followed by painful nonhealing proximal femur fractures or pseudofractures.

The bone symptoms are highly variable in their clinical expression, which ranges from stillbirth without mineralized bone to pathological fractures developing only late in adulthood.

Odontohypophosphatasia is characterized by premature exfoliation of primary teeth with roots intact and/or several dental caries, not associated with abnormalities of the skeletal system.

Severe forms of the disease such as perinatal and infantile forms are transmitted as an autosomal recessive trait, whereas both autosomal recessive and autosomal dominant transmission may be found in milder forms, especially odontohypophosphatasia.

The tissue nonspecific ALP (TNSALP) gene is localized on chromosome 1p36.1 and it consits of 12 exons distributed ober 50 Kbases. More than 160 mutations have been described to date in the TNSALP gene. In North American, Japanese, and European patients, indicating a very strong allelic heterogeneity in the disease. This variety of mutations results in highly variable clinical expression and a great number of compound heterozygous genotypes with missense mutations that account for 82% of mutations. The remaining mutations are:

- missense mutations (82%)

- microlesions (11%)

- splicing mutations (4%)

- nonsense mutations (3%)

- a nucleotide substitution on major transcription initiation site

- a denovo mutation on heterozygous carrier of a missense mutation

The affected individuals carry one or two loss-of-function mutations within the TNSALP gene alleles. This experiment of the nature, inherited as either an autosomal dominant or autosomal recessive trait, reveals a crucial role for TNSALP in skeletal mineralization. There is no established medical treatment for hypophosphatasia. Augmenting circulating alkaline phosphatase activity into or even above the normal range for several months using intravenously administered ALP from various tissues sources has had no convincing beneficial effects. Also transplantation therapy with cultured osteoblasts and bone fragments was quite unsuccessful and experiments suggested that we must lower PPi at mineralization sites. Accordingly TNSALP activity must be increased at mineralization sites more than at plasma level.

Recently a recombinant fusion protein including TNSALP ectodomain, the constant region of IgG1 Fc domain, and the terminal deca-aspartate motif has been admnistered in 11 patients with perinatal or infantile forms of hypophosphatasia. Treatment was associated with healing of skeletal manifestations of hypophosphatasia as well’s with improvement in respiratory and motor functions. Improvement is still being observed in patients receiving treatment for more than 3 years.

References

Robinson R. The possible significance of hexosephosphoric esters in ossification. Biochem 1923;17:286-293.

Anderson HC. Vescicles associated with calcification in the matrix of epiphyseal cartilage. J Cell Biol 1969;41:59-72.

deBernard B, Bianco P, Bonucci E et al. Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca++ -binding glycoprotein. J Cell Biol 1986;103:1615-23.

Coleman JE. Structure and mechanism of alkaline phosphatase. Ann Rev Biophys Biomol Struct 1992;21:441-83.

Coleman JE, Gettins P. Alkaline phosphatase p, solution structure, and mechanism . Adv Enzymol 1983;55:381-452.

Kim EE, Wyckoff HW. Reaction mechanism of alkaline phosphatase based on crystal structures: two metal ions catalysis. J Mol Biol 1991;218:449-64.

Xu X, Qin XQ, Kantrowitz ER. Probing the role of histidine-372 in zinc binding and the cathalitic mechanism of escherichia coli alkaline phsosphatase by site-specific mutagenesis. Biochemistry 1994;33:2279-84.

Whyte MP. Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization. Endocr Rev 1994;15:439-61.

Le Due HM, Stigbrand T, Taussig MJ et al. Crystal structure of alkaline phosphatase from human placenta at 1.8 Ǻ resolution. J Biological Chem 2000;275:9158-65.

Levy-Litan V, Hershkovitz E, Avizov L et al. Autosomal recessive hypophosphatemic rickets is associated with an inactivating mutation in the ENPP1 gene. Am J Hum Genet 2010;86:273-8.

Hessle L, Johnson KA, Anderson HC et al. Tissue non specific alkaline phosphatase and plasma cell membrane glycoprotein1 are central antagonistic regulators of bone mineralization. Proc Nat Acad Sci USA 2002;99:9445-9.

Lorenz-Depiereux B, Schnabel D, Tiosano D et al. Loss-of-function ENPP1 mutations cause both generalized arterial calcification of infancy and autosomal recessive hypophosphatemic rickets. Am J Hum Genet 2010;86:267-72.

Collins MT, Boehm M. It ANKH necessarily so. J Clin Endocrinol Metab 2011;96:72-4.

Ciancaglini P, Yadav MC, Simao AMS et al. Kinetic analysis of substrate utilization by native and TNAP, NPP or PHOSPHO1-Deficient matrix vescicles. J Bone Miner Res 2010;25:716-23.

Yadav MC, Simao AMS, Narisawa S et al. Loss of skeletal mineralization by the simultaneous ablation of PHOSPHO1 and Alkaline Phosphatase function: a unified model of the mechanisms of initiation of skeletal calcification. J Bone Miner Res 2011;26:286-97.

Whyte MP, Obrecht SE, Finnegan PM et al. Osteoprotegerin deficiency and Juvenile Paget’s disease. N Engl J Med 2002;347:175-84.

Whyte MP, Hughes AE. Expansile skeletal hyperphosphatasia is caused by a 15 base pair tandem duplication in TNFRSFIIA encoding RANK and is allelic to Familial Expansile Osteolysis. J Bone Min Res 2002;17:26-9.

Taillandier A, Sallinen SL, Brun-Heath P et al. Childhood hypophosphatasia due to a de novo missense mutation in the tissue nonspecific alkaline phosphatase gene. J Clin Endocrinol Metab 2005;90:2436-9.

Cahil RA, Wenkert D, Perlman SA et al. Infantile hypophosphatasia: transplantation therapy trial using bone fragments and cultured osteoblasts. J Clin Endocrinol Metab 2007;92:2923-30.

Schalin-Jantti C, Mornet E, Lamminen A et al. Parathyroid hormone treatment improves pain and fracture healing in adult hypophosphatasia. J Clin Endocrinol Metab 2010;95:5174-9.

Whyte MP, Greenberg CR, Salman NJ et al. Enzyme replacement therapy in life threatening hypophosphatasia. N Engl J Med 2012;366:904-13.

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