Abstract
Recombinant alkaline phosphatases are becoming promising protein therapeutics to prevent skeletal mineralization defects, inflammatory bowel diseases, and treat acute kidney injury. By substituting the flexible crown domain of human intestinal alkaline phosphatase (IAP) with that of the human placental isozyme (PLAP) we generated a chimeric enzyme (ChimAP) that retains the structural folding of IAP, but displays greatly increased stability, active site Zn2+ binding, increased transphosphorylation, a higher turnover number and narrower substrate specificity, with comparable selectivity for bacterial lipopolysaccharide (LPS), than the parent IAP isozyme. ChimAP shows promise as a protein therapeutic for indications such as inflammatory bowel diseases, gut dysbioses and acute kidney injury.
Highlights
Recombinant alkaline phosphatases (APs) (EC 3.1.3.1) show promise as therapeutic drugs for a variety of conditions ranging from soft-bone diseases such as hypophosphatasia (HPP) [1], inflammatory bowel diseases [2,3,4], gut dysbiosis [5,6] and acute kidney injury [7,8,9]
Production of FLAG-tagged enzymes To compare the kinetic properties of intestinal AP (IAP), placental AP (PLAP) and ChimAP
To test our hypothesis that introducing the PLAP crown domain in to IAP would affect enzyme stability and Figure 1. 3D modeling of the structure of ChimAP. (A) Schematic representation of the fusion of the first 359 amino acids of the mature human IAP structure to residues 360–430 of the mature human PLAP sequence followed by residues 431–484 of the human mature IAP sequence. (B) 3D representation of the ChimAP structure based on homology to human PLAP, visualized and analyzed using Chimera v1.7 and Swiss-PdbViewer. (C) Top view of the ChimAP structure, indicating the active site serine
Summary
Recombinant alkaline phosphatases (APs) (EC 3.1.3.1) show promise as therapeutic drugs for a variety of conditions ranging from soft-bone diseases such as hypophosphatasia (HPP) [1], inflammatory bowel diseases [2,3,4], gut dysbiosis [5,6] and acute kidney injury [7,8,9]. APs are encoded by a multigene family [10] that in humans comprises four loci: ALPL encodes an enzyme expressed primarily in bone, liver and kidney, referred to as tissue-nonspecific AP (TNAP). The other three loci are highly homologous and are expressed in a tissue-specific manner. These genes, ALPI, ALPP and ALPPL2, encode intestinal AP (IAP), placental AP (PLAP) and placental-like or germ cell AP (GCAP), respectively. In collaboration with Enobia Pharma ( Alexion Pharmaceuticals), our group developed the first therapeutic AP, a bone mineral-targeting recombinant TNAP (Asfotase alfa) that prevented seizures and all the skeletal and dental manifestations of HPP by normalizing PPi and PLP metabolism [1,13,14,15,16]. Clinical trials using asfotase alfa in patients with life-threatening HPP have shown remarkable results [17] (Clinicaltrials.gov Identifier NCT00744042) and are continuing with adolescents and adults with HPP (ClinicalTrials.gov Identifiers: NCT00952484 and NCT01163149)
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