recombinant tnap  (Addgene inc)

Journal: Communications Biology

Article Title: TNAP dephosphorylates phosphocholine and phosphoethanolamine and participates in triglyceride transport from the liver to the bloodstream

doi: 10.1038/s42003-025-08901-3

Figure Lengend Snippet: PC is sequentially hydrolyzed in the intestine by sPLA 2 -IB, PLB, and GDE5, or in the bloodstream by Lp-PLA 2 , lyso-PLA 1 , ENPP2, and/or ENPP6. TNAP could be the phosphatase dephosphorylating extracellular phosphocholine and phosphoethanolamine, allowing cellular choline and ethanolamine uptake. In hepatocytes, choline and ethanolamine generate PC and PEA via parallel metabolic pathways, and PEA can be methylated into PC by PEMT. PC is hydrolyzed into Lyso-PC and GPC in the endoplasmic reticulum by the sequential activity of PNPLA8 and PNPLA7, or by PLA 2 G15 in lysosomes. Choline serves in hepatocytes as a methyl donor via the production of SAM. BHMT betaine homocysteine methyltransferase, CDP-choline cytidine diphosphate choline, CTP cytidine triphosphate, DH dehydrogenase, ENPP ectonucleotide pyrophosphatase phosphodiesterase, GDE5 glycerophosphodiesterase 5, GPC glycerophosphocholine, Lp-PLA 2 lipoprotein-associated phospholipase A, PC phosphatidylcholine, PLB phospholipase B, PEA phosphatidylethanolamine, PEMT phosphatidylethanolamine N-methyltransferase, PLA 2 G15 phospholipase A, group XV, PNPLA patatin-like phospholipase domain containing, SAH S-Adenosyl-homocysteine, SAM S-Adenosyl-methionine, sPLA 2 -IB secreted phospholipase A IB.

Article Snippet: The sequence of the plasmid used to produce recombinant TNAP has been deposited at Addgene (ID 246682).

Techniques: Methylation, Activity Assay

Journal: Communications Biology

Article Title: TNAP dephosphorylates phosphocholine and phosphoethanolamine and participates in triglyceride transport from the liver to the bloodstream

doi: 10.1038/s42003-025-08901-3

Figure Lengend Snippet: A Michaelis-Menten representation, i.e., reaction rate (mM.h −1 ) of phosphocholine, phosphoethanolamine, and PP i hydrolysis by human TNAP, depending on the substrate concentration. Experimental data are presented, as well as the fit by a model with substrate inhibition, i.e., following the equation V 0 = V max [ S ]/([ S ]+ K m + [ S ] 2 / K i ), where V 0 is the initial reaction rate, V max the maximal reaction rate, and [ S ] the substrate concentration. All experiments were repeated at least three times. For the sake of clarity, individual data points are not presented in ( A ). However, source data are available in the . B Kinetic parameters ( K m , k cat , k cat / K m , and K i ) are given for phosphocholine, phosphoethanolamine, and PP i hydrolysis.

Article Snippet: The sequence of the plasmid used to produce recombinant TNAP has been deposited at Addgene (ID 246682).

Techniques: Concentration Assay, Inhibition

Journal: Communications Biology

Article Title: TNAP dephosphorylates phosphocholine and phosphoethanolamine and participates in triglyceride transport from the liver to the bloodstream

doi: 10.1038/s42003-025-08901-3

Figure Lengend Snippet: Substrate accommodation in TNAP’s active site. The best docking poses for phosphocholine and PP i obtained with the Apo enzyme are superimposed onto the TNAP structure containing P i : A Secondary structures and key residues that define the negatively charged zones (D109 and E452) and positively charged zones (R168 and R184). B The electrostatic surface is color-coded to depict the charge distribution, with red representing negatively charged regions (the groove) and blue representing positively charged regions (the pockets). C Two examples demonstrate the orientation of phosphoethanolamine within the active site: one positioned in the positively charged pocket and the other within the negatively charged groove. D Structure of the TNAPi MLS-0038949 (MLS) and global representation of MLS interaction with TNAP’s surface. E MLS forms P i –cation interactions between the para-di-methoxy-benzene group and Zn²⁺ (Zn2). This interaction is further stabilized by a hydrogen bond between His341 and the sulfonamide group of MLS. Additionally, cation– P i –stacking interactions are observed between Glu342, His338, and the quinolin-3-yl group. Finally, His338 contributes to the binding through a P i –stacking interaction with the quinolin-3-yl group of the MLS. F MLS positioning relative to the different substrates of TNAP. The structure in the presence of MLS was superimposed onto the structure containing P i , as well as the structure used for docking with phosphocholine and PP i . This comparison highlights the steric hindrance caused by the para-di-methoxy-benzene part of the inhibitor in relation to the phosphate group of the different substrates. G Superposition of the TNAP structure (in green) containing MLS with the AlphaFold model of IAP (in gray), illustrating the interaction specificity between TNAP and the MLS. The three residues interacting with MLS, which differ between TNAP and IAP, are indicated in the respective colors of the two structures.

Article Snippet: The sequence of the plasmid used to produce recombinant TNAP has been deposited at Addgene (ID 246682).

Techniques: Binding Assay, Comparison

Journal: Regenerative Therapy

Article Title: Prenatal enzyme replacement therapy for Akp2 −/− mice with lethal hypophosphatasia

doi: 10.1016/j.reth.2021.06.002

Figure Lengend Snippet: Prenatal and postnatal ERT prolongs survival and improves growth and femur length in Akp2 −/− mice . (A) Survival rate of Akp2 +/+ mice ( n = 10), and untreated Akp2 −/− mice ( n = 10), prenatal ERT Akp2 −/− with TNAP mice ( n = 10), postnatal ERT Akp2 −/− with TNAP mice ( n = 10). (B) Growth curves of Akp2 +/+ mice ( n = 10), and untreated Akp2 −/− mice ( n = 10), prenatal ERT Akp2 −/− with TNAP mice ( n = 8), postnatal ERT Akp2 −/− with TNAP mice ( n = 8). (C) Body weight on day16, was measured in Akp2 +/+ mice ( n = 10) and untreated Akp2 −/− mice ( n = 10), prenatal ERT Akp2 −/− with TNAP mice ( n = 8), postnatal ERT Akp2 −/− with TNAP mice ( n = 8). Data represent the means ± SE. ∗∗ P < 0.01. (D) Serum ALP activity on day 19, was analyzed in Akp2 +/+ mice ( n = 10), and prenatal ERT Akp2 −/− with TNAP mice ( n = 5), postnatal ERT Akp2 −/− with TNAP mice ( n = 4), untreated Akp2 −/− mice ( n = 10). Data represent the means ± SE. ∗ P < 0.05, ∗∗ P < 0.01. (E) Femur Length on day 19, was measured in Akp2 +/+ mice ( n = 15), and untreated Akp2 −/− mice ( n = 9), prenatal ERT Akp2 −/− with TNAP mice ( n = 5), postnatal ERT Akp2 −/− with TNAP mice ( n = 9). Data represent the means ± SE. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Enzyme replacement therapy (ERT) with recombinant mineral-targeted TNAP (asfotase alfa: ENB-0040, Alexion Pharmaceuticals, Cheshire, CT) is effective in these HPP patients as it improves the shortened and bowed tubular bones, extends life expectancy, prevent seizures [ , , , , ] and can be administered right after birth [ ].

Techniques: Activity Assay

Journal: Regenerative Therapy

Article Title: Prenatal enzyme replacement therapy for Akp2 −/− mice with lethal hypophosphatasia

doi: 10.1016/j.reth.2021.06.002

Figure Lengend Snippet: Histological findings of the effect with prenatal and postnatal ERT to Akp2 −/− mice . Histological findings in Akp2 +/+ mice ( n = 15), and untreated Akp2 −/− mice ( n = 9), prenatal ERT Akp2 −/− with TNAP mice ( n = 5), postnatal ERT Akp2 −/− with TNAP mice ( n = 9). (A) Hematoxylin and Eosin stain (B) Alcian Blue stain (C) Enzyme histochemistry detecting alkaline phosphatase activity.

Article Snippet: Enzyme replacement therapy (ERT) with recombinant mineral-targeted TNAP (asfotase alfa: ENB-0040, Alexion Pharmaceuticals, Cheshire, CT) is effective in these HPP patients as it improves the shortened and bowed tubular bones, extends life expectancy, prevent seizures [ , , , , ] and can be administered right after birth [ ].

Techniques: H&E Stain, Staining, Activity Assay

Journal: Regenerative Therapy

Article Title: Prenatal enzyme replacement therapy for Akp2 −/− mice with lethal hypophosphatasia

doi: 10.1016/j.reth.2021.06.002

Figure Lengend Snippet: Comparison of prenatal and postnatal ERT effect by computed tomography analysis . Akp2 +/+ mice ( n = 15), untreated Akp2 −/− mice ( n = 9), prenatal ERT Akp2 −/− with TNAP mice ( n = 5), and postnatal ERT Akp2 −/− with TNAP mice ( n = 9) were analyzed. Data represent the means ± SE. ∗ P < 0.05, ∗∗ P < 0.01. ns: not significant. (A) Images of regions of interest (ROIs) in cancellous bone analysis. (B) Two-dimensional images of growth plate and cortical bone of femurs. Scale bars, 750 μm. (C) Cortical bone density. (D) Cancellous bone density. (E) Total bone density. (F) Cortical bone area ratio. (G) Cortical bone thickness. (H) Minimum Moment of inertia of area.

Article Snippet: Enzyme replacement therapy (ERT) with recombinant mineral-targeted TNAP (asfotase alfa: ENB-0040, Alexion Pharmaceuticals, Cheshire, CT) is effective in these HPP patients as it improves the shortened and bowed tubular bones, extends life expectancy, prevent seizures [ , , , , ] and can be administered right after birth [ ].

Techniques: Computed Tomography