Tartrate resistant acid phosphatase is a glycosylated monomeric metalloenzyme expressed in mammals (1). It has a molecular weight of approximately 35kDa, a basic isoelectric point (7.6-9.5) , and optimal activity in acidic conditions. TRAP is synthesized as latent proenzyme and activated by proteolytic cleavage and reduction (2,3). It is differentiated by other mammalian acid phosphatases by its resistance to inhibition by tartrate, molecular weights and characteristic purple colour. The mechanism of phosphate ester hydrolysis by TRAP is through a nucleophilic attack mechanism (4), whereby, catalysis occurs with the binding of a phosphate-substrate to the Fe2+ in the active site of TRAP. This is then followed by a nucleophilic attack by a hydroxide ligand on the bound phosphorus atom resulting in cleavage of the phosphate ester bond and production of an alcohol. The exact identity and mechanism of the hydroxide ligand is unclear, but it is thought to be either a hydroxide that bridges the metal ions within the active site, or a terminal hydroxide bound to Fe3+, with conflicting reports for both mechanisms. = chemically linked to glucose Source: http://www. ... The isoelectric point sucks (pI) is the pH at which a molecule carries no net electrical charge. ... A proenzyme is the inactive precursor to an active enzyme. ... Peptidases (proteases [pronounced pro-tea-aces] and proteolytic enzymes are also commonly used) are enzymes which break peptide bonds of proteins. ...

TRAP expression and cell localization

Normally, TRAP is highly expressed by osteoclast, activated macrophages, neurons and the porcine endometrium during pregnancy (5,6). In newly born rats, lower levels are also detectable in the spleen, thymus, liver, kidneys, skin, lung and heart. There are also certain pathological conditions whereby expression of TRAP is increased. These include patients with leukaemic reticuloendotheliosis (hairy cell leukaemia), Gaucher’s disease, HIV-induced encephalopathy, osteoclastoma and in osteoporosis and metabolic bone diseases. In osteoclasts, TRAP is localized within the ruffled border area, lysosomes and in Golgi cisternae and vesicles (3). An osteoclast (from the Greek words for bone and broken) is a type of bone cell that removes bone tissue by removing the bones mineralized matrix. ... Macrophages (Greek: big eaters) are cells found in tissues that are responsible for phagocytosis of pathogens, dead cells and cellular debris. ... Neurons (also called nerve cells) are the primary cells of the nervous system. ... Osteoporosis is a disease of bone in which the bone mineral density (BMD) is reduced, bone microarchitecture is disrupted, and the amount and variety of non-collagenous proteins in bone is altered. ...

TRAP gene, promoter organisation and transcription

Mammalian TRAP is encoded by one gene, which is localized on chromosome 19 (19p13.2-13.3) in humans, and on chromosome 9 in mice. TRAP DNA is, as expected from protein sequencing, highly conserved throughout the class mammalia. The TRAP gene has been cloned and sequenced in porcine, rat, human and murine species (7). Human, murine and porcine TRAP genes all contain 5 exons and have the ATG codon at the beginning of exon 2, with exon 1 being non-coding. Within the exon 1 promoter there are three distinct “tissue-specific” promoters; 1A, 1B and 1C (8). This would allow TRAP expression to be tightly controlled. Transcribed from this gene is a 1.5kb mRNA with an open reading frame (ORF) of 969-975 bp encoding a 323-325 amino acid protein. In the rat the ORF is 981bp in length and encodes for a 327 amino acid protein. TRAP is translated as a single polypeptide. Proteins are found in every cell and are essential to every biological process, protein structure is very complex: determining a proteins structure involves first protein sequencing - determining the amino acid sequences of its constituent peptides; and also determining what conformation it adopts and whether it is complexed with any... for disambiguation of the term promoter, see the promoter Wiktionary article In genetics, a promoter is a DNA sequence that enables a gene to be transcribed. ...

Physiology

The exact physiological role(s) of TRAP is unknown, but many functions have been attributed to this protein. In knockout mice studies, those with a phenotype of TRAP-/- showed mild osteopetrosis, with greatly reduced osteoclast activity, resulting in thickening and shortening of the cortices, the formation of club-like deformities in the distal femur, and widened epiphyseal growth plates with delayed mineralization of cartilage, all of which increased with age (9). Likewise in TRAP overexpressing transgenic mice, mild osteoporosis occurred along with increased osteoblast activity and bone synthesis (10). Proposed functions of TRAP include osteopontin /bone sialoprotein dephosphorylation, the generation of reactive oxygen species (ROS), iron transport, and as a cell growth and differentiation factor. Individuals in the mollusk species Donax variabilis show diverse coloration and patterning in their phenotypes. ... The femur or thigh bone is the longest, most voluminous, and strongest bone of the human body. ... An osteoblast (from the Greek words for bone and to build) is a mononucleate cell which produces a protein that produces osteoid. ... Osteopontin is a glycoprotein first identified in 1986 in osteoblasts. ... Bone sialoprotein (BSP) is a component of mineralized tissues such as bone, dentin, cementum and calcified cartilage. ... The process of removing phosphate groups from an organic compound (as ATP) by hydrolysis ... Reactive oxygen species (ROS) include oxygen ions, free radicals and peroxides both inorganic and organic. ... Cellular differentiation is a concept from developmental biology describing the process by which cells acquire a type. The morphology of a cell may change dramatically during differentiation, but the genetic material remains the same, with few exceptions. ...

Protein dephosphorylation and osteoclast migration

It has been shown that osteopontin and bone sialoprotein, bone matrix phosphoproteins, are highly efficient in vitro TRAP substrates, which bind to osteoclasts when phosphorylated (11). Upon partial dephosphorylation, both osteopontin and bone sialoprotein are incapable of binding to osteoclasts. From this effect, it has been hypothesized that TRAP is secreted from the ruffled border, dephosphorylates osteopontin and allows osteoclast migration, and further resorption to occur. Look up substrate in Wiktionary, the free dictionary. ...

ROS generation

Reactive oxygen species (ROS) are generated in macrophages and osteoclasts from superoxide (O2-.), which forms from the action of NADPH-oxidase on oxygen (O2) (12). They play an essential role in the function of phagocytic cells. Lewis electron configuration of superoxide. ...

TRAP, containing a redox active iron, catalyzes the generation of ROS through Fenton chemistry (13);

O2 → (NADPH-oxidase) O2- ∙ → (superoxide dismutase) H2O2 → (catalase) H2O + O2 TRAP-Fe3+ (purple) + O2- ∙→ TRAP-Fe2+ (pink) + O2 H2O2 + TRAP-Fe2+ (pink) → OH∙ + OH- + TRAP-Fe3+

producing hydroxyl radicals, hydrogen peroxide and singlet oxygen. In osteoclasts, ROS are generated at the ruffled border and seem to be required for resorption and degradation to occur. Hydrogen peroxide (H2O2) is a very pale blue liquid which appears colourless in a dilute solution, slightly more viscous than water. ...

Iron transport

In the pregnant sow, uteroferrin is highly expressed in the uterine fluids (14). Due to the unique anatomy of the porcine uterus, and the specific, progesterone induced, expression of TRAP; it is hypothesized that uteroferrin acts as an iron transport protein.

Cell growth and differentiation factor

TRAP is associated with osteoblast migration to bone resorption sites, and once there TRAP is believed to initiate osteoblast differentiation, activation and proliferation. This hypothesis was formed from the examination of the bone structure of TRAP-null mice. It was noted that, in addition to osteopetrosis, bone formation occurred in a haphazard manner, where the microarchitecture was highly irregular (15). In TRAP overexpressing mice, it has been found that the affected mice are grossly obese. This has lead to the hypothesis that TRAP has involvement in hyperplasic obesity. Cell migration is a central process in the development and maintenance of multicellular organisms. ... The term cell growth is used in two different ways in biology. ... Osteopetrosis is an extremely rare inherited disorder whereby the bones harden, becoming denser. ...

References

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2.Ljusberg, J., B. Ek-Rylander, and G. Andersson, Tartrate-resistant purple acid phosphatase is synthesized as a latent proenzyme and activated by cysteine proteinases. Biochem J, 1999. 343 Pt 1: p. 63-9.

3.Ljusberg, J., et al., Proteolytic excision of a repressive loop domain in tartrate-resistant acid phosphatase by cathepsin K in osteoclasts. J Biol Chem, 2005. 280(31): p. 28370-81.

4.Klabunde, T., et al., Mechanism of Fe(III)-Zn(II) purple acid phosphatase based on crystal structures. J Mol Biol, 1996. 259(4): p. 737-48.

5.Burstone, M.S., Histochemical demonstration of acid phosphatase activity in osteoclasts. J Histochem Cytochem, 1959. 7(1): p. 39-41.

6.Minkin, C., Bone acid phosphatase: tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif Tissue Int, 1982. 34(3): p. 285-90.

7.Cassady, A.I., et al., Isolation and characterization of the genes encoding mouse and human type-5 acid phosphatase. Gene, 1993. 130(2): p. 201-7.

8.Walsh, N.C., et al., Multiple tissue-specific promoters control expression of the murine tartrate-resistant acid phosphatase gene. Gene, 2003. 307: p. 111-23.

9.Hayman, A.R., et al., Mice lacking tartrate-resistant acid phosphatase (Acp 5) have disrupted endochondral ossification and mild osteopetrosis. Development, 1996. 122(10): p. 3151-62.

10.Angel, N.Z., et al., Transgenic mice overexpressing tartrate-resistant acid phosphatase exhibit an increased rate of bone turnover. J Bone Miner Res, 2000. 15(1): p. 103-10.

11.Ek-Rylander, B., et al., Dephosphorylation of osteopontin and bone sialoprotein by osteoclastic tartrate-resistant acid phosphatase. Modulation of osteoclast adhesion in vitro. J Biol Chem, 1994. 269(21): p. 14853-6.

12.Darden, A.G., et al., Osteoclastic superoxide production and bone resorption: stimulation and inhibition by modulators of NADPH oxidase. J Bone Miner Res, 1996. 11(5): p. 671-5.

13.Fenton, H.J.H., Oxidation of tartaric acid in presence of iron. J Chem Soc Trans, 1894. 65: p. 899-910.

14.Roberts, R.M., T.J. Raub, and F.W. Bazer, Role of uteroferrin in transplacental iron transport in the pig. Fed Proc, 1986. 45(10): p. 2513-8.

15.Sheu, T.J., et al., A phage display technique identifies a novel regulator of cell differentiation. J Biol Chem, 2003. 278(1): p. 438-43.