**2. TNAP: gene, structure of the protein, and its function as an enzyme**

There are four human alkaline phosphatase (ALP) isoenzymes (**Table 1**): TNAP, placental alkaline phosphatase (PLAP), intestinal alkaline phosphatase (IAP), and germ cell ALP [8, 9]. The latter three ALPs are tissue-specific and are expressed in the placenta, intestine, and germ cells (embryonic and cancer cells), respectively [9]. TNAP, also known as the liver/bone/kidney (LBK) alkaline phosphatase, is expressed ubiquitously; liver, bone, kidney, neuronal cells, and white blood cells in particular are tissues that show marked expression [10].

Human TNAP is encoded by the *ALPL* gene that is located on the short arm of chromosome 1 (1p36.1–34). The coding region of the gene is approximately 1.5 kb in length, and it is extended over more than 50 kb of genomic DNA [11]. The *ALPL* gene consists of 12 exons of which exons 2–12 are coding exons and there exist two alternative noncoding exons 1 (bone type and liver type) [12, 13]. The promoter region of the gene includes a TATA box, an Sp1 binding site, and a retinoic acid responsive element (RARE) [14, 15].


**Table 1.** Isoenzymes of human ALP.

Retinoic acid regulates the expression of TNAP via RARE [15], whereas another fat-soluble vitamin, active vitamin D (1,25-dihydroxycholecalciferol), regulates the expression of TNAP by modification of the stability of TNAP mRNA [16]. Furthermore, phosphates derived from ALP enzymatic activity are considered to regulate TNAP expression [17]. Epigenetic regulation by methylation of some of the promoter regions of the gene has been reported [18]. However, the precise regulatory mechanism of the *ALPL* gene regulation, especially its tissue-specific regulation, is not known. On the other hand, the genes encoding tissue-specific ALPs are located on the long arm of chromosome 2 and have a more compact gene structure [19–22].

of the surviving parents as a compound heterozygote of p.A114T and p.D294A [5]. Since then, a total of 335 mutations in the gene for TNAP (the *ALPL* gene) have been reported [6]. The symptoms of HPP vary and are classified into six HPP forms [1, 2]. The pathophysiology of HPP is basically due to a defect of bone mineralization. In severe forms, the patients show skeletal manifestations and respiratory failure derived from costal bone insufficiency, whereas in the mildest forms, they show only dental manifestations [1]. Recent development of enzyme replacement therapy (ERT) has opened up a new vista on the treatment of this

**2. TNAP: gene, structure of the protein, and its function as an enzyme**

There are four human alkaline phosphatase (ALP) isoenzymes (**Table 1**): TNAP, placental alkaline phosphatase (PLAP), intestinal alkaline phosphatase (IAP), and germ cell ALP [8, 9]. The latter three ALPs are tissue-specific and are expressed in the placenta, intestine, and germ cells (embryonic and cancer cells), respectively [9]. TNAP, also known as the liver/bone/kidney (LBK) alkaline phosphatase, is expressed ubiquitously; liver, bone, kidney, neuronal cells, and white blood cells in particular are tissues that show marked

Human TNAP is encoded by the *ALPL* gene that is located on the short arm of chromosome 1 (1p36.1–34). The coding region of the gene is approximately 1.5 kb in length, and it is extended over more than 50 kb of genomic DNA [11]. The *ALPL* gene consists of 12 exons of which exons 2–12 are coding exons and there exist two alternative noncoding exons 1 (bone type and liver type) [12, 13]. The promoter region of the gene includes a TATA box, an Sp1 binding site, and a retinoic acid responsive element (RARE) [14, 15].

**location**

Intestinal IAP *ALPI* 2q34–37 Intestine Degradation of LPS\*

Placental PLAP (PAP) *ALPP* 2q34–37 Placenta Degradation of

Germ cell (placental like) — *ALPP2* 2q34–37 Germ cells

**Sites of expression**

Cancer cells

*ALPL* 1p36.1–34 Ubiquitous Mineralization

**Function**

entrance of pyridoxal phosphate into the neuronal cells

lipid absorption

LPS\* (?)

**Common name Protein name Gene Chromosomal** 

TNAP (TNSALP)

previously untreatable disease [7].

102 Pathophysiology - Altered Physiological States

expression [10].

Tissue-nonspecific (liver/

Lipopolysaccharides.

**Table 1.** Isoenzymes of human ALP.

bone/kidney)

\*

The TNAP protein, which has a molecular weight of approximately 80 kDa, is linked to the outer cell membrane through a glycosylphosphatidylinositol (GPI) anchor [9]. The TNAP protein is initially synthesized as a 66 kDa peptide, and then *O*- and *N*-glycosides are attached in the endoplasmic reticulum. Eventually, TNAP is localized on the outer membrane of the cells via a GPI anchor [23]. This GPI anchor is added after hydrophobic amino acid residues at the C-terminus are eliminated. The GPI anchor consists of an ethanolamine phosphate, three residues of mannose, a glucosamine, and a phosphatidylinositol [9]. The precise amino acid residue in TNAP to which the GPI anchor is added has not been elucidated, whereas it is known to be an aspartate residue (D484) in PLAP [24, 25]. An active enzyme consists of a dimer and acts as an ectoenzyme. Approximately 58% of the amino acid residues in human TNAP sequences are conserved among mammalian ALPs [26]. On the other hand, approximately 90% of the amino acid residues are conserved among mammalian TNAPs, which allow prediction of missense mutations responsible for HPP [26]. Since the three dimensional structure of TNAP has not been solved, a simulation model based on human PLAP or mouse IAP is used to discuss TNAP structure [27–29]. The active site of the enzyme comprises a catalytic serine residue (S92 in the human PLAP), two Zn2+-binding sites, and an Mg2+-binding site. Ca2+ is also necessary as a cofactor. The crown domain is characteristic of mammalian ALPs and is considered to interact with extracellular proteins including collagen [30]. There are also isoforms of TNAP itself that depend on the tissue origin. Since these isoforms have different *O*-linked sugar chains, they show different patterns on the electrophoresis. [9, 31].

The systematic name of ALP is orthophosphoric-monoester phosphohydrolase [alkaline optimum] (EC 3.1.3.1) that hydrolyzes monophosphate esters, and the optimal pH is between 8 and 10 [9]. Inorganic pyrophosphate (PPi) and pyridoxal 5′-phosphate (PLP) are considered to be natural substrates of the enzyme [32]. PPi is an inhibitor of hydroxyapatite formation, which is essential for bone mineralization. PLP is an active vitamin B6 and is necessary in neuronal cells for the biosynthesis of γ-aminobutyric acid (GABA), which acts as an inhibitory neurotransmitter. PLP on the outside of neuronal cells must be dephosphorylated by TNAP at the cell membrane before it can enter the neuronal cells, and it is then be rephosphorylated within the neuronal cells [32, 33]. In laboratory testing, ALP enzymatic activity is usually estimated using *p*-nitrophenylphosphate as an artificial substrate [9].
