Simultaneous Retention of Thermostability and Specific Activity in Chimeric Human Alkaline Phosphatases

Abstract

Alkaline phosphatases (APs) are a family of dimeric metalloenzymes that has been utilized in many areas due to its ability to hydrolyze a variety of phosphomonoesters. While mammalian APs have higher specific activity than prokaryotic APs, they are generally less thermostable. To cultivate the possibility to confer mammalian APs with higher thermostability as well as high activity, we focused on human AP isozymes. Among the four isozymes of human APs, placental AP (PLAP) retains the highest thermostability, while intestinal AP (IAP) has the highest specific activity. Since the two APs display high homology, a series of chimeric enzymes were made in a secreted form to analyze their properties. Surprisingly, chimeric APs with IAP residues at the N-terminal and PLAP residues at the C-terminal regions showed higher specific activity than PLAP, while keeping thermostability as high as PLAP. Especially, one showed similar specific activity to IAP, while showing slower inactivation than PLAP after incubation at 75°C. Interestingly, the mutant also showed higher resistance to uncompetitive inhibitors Phe and Leu than their parent enzymes, possibly due to increased hydrophilicity of the active site entrance residues. The obtained chimera will be useful as a novel reporter in various assays including gene hybridization.

Protein-Grafted Polymers Prepared Through a Site-Specific Conjugation by Microbial Transglutaminase for an Immunosorbent Assay

“…It has been reported that the successive domains of protein G can increase the affinity to antibodies, and the addition of protein A in between protein G and the enzymatic domain can preserve the activities of each protein . As an alkaline phosphatase (AP) part, chimera alkaline phosphatase (IPP) composed of intestinal AP and placental AP was employed . The MTG-recognizable K-tag (MRH K GS) was introduced to the C-terminus of IPP-PG (Ktag-IPP-PG).…”

“…50 As an alkaline phosphatase (AP) part, chimera alkaline phosphatase (IPP) composed of intestinal AP and placental AP was employed. 51 The MTG-recognizable K-tag (MRHKGS) was introduced to the C-terminus of IPP-PG (Ktag-IPP-PG). After the confirmation of the produced protein showing the specific detection of biomolecules, the conjugation with the Z-QG polymer was examined.…”

Strategies for Making Multimeric and Polymeric Bifunctional Protein Conjugates and Their Applications as Bioanalytical Tools

“…Synthetic polymers containing the N-benzyloxycarbonyl-L-glutaminylglycine (Z-QG) motif have been used as scaffolds to conjugate numerous enzyme units via MTG-catalyzed conjugation reactions. A chimeric molecule comprising alkaline phosphatase (IPP), 47 pG (IPP-PG), and a K-tag (MRHKGS) was grafted onto the Z-QG polymer using MTG (Fig. 3A).…”

Pathophysiology of hypophosphatasia and the potential role of asfotase alfa

“… 87 , 88 Currently, the combination treatment is being attempted. In addition, a recent successful attempt of ERT in Akp2 −/− mice using a soluble chimeric alkaline phosphatase, which is a chimeric molecule of human intestinal alkaline phosphatase with the crown domain of human PLAP, 89 showed therapeutic benefit of the nonmineral-targeting recombinant enzyme. 90 Another method to reduce the injection burden is gene therapy, especially ex vivo gene therapy into bone marrow stem cells.…”