Catalytic Signature of a Heat-Stable, Chimeric Human Alkaline Phosphatase with Therapeutic Potential

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.

Evaluation of serum bone alkaline phosphatase activity in patients with liver disease: Comparison between electrophoresis and chemiluminescent enzyme immunoassay

“…Human ALP consists of a group of isozymes, placental, intestinal and three major tissue-nonspecific isoforms [3], which are encoded by 4 main gene loci: tissue-nonspecific, intestinal, placental, and germ-cell ALP [4][5][6]. Tissue-nonspecific boneand liver-type ALP are formed through posttranslational modifications and differences in carbohydrate composition, leading to different clinical phenotypes [4].…”

The Potential of Alkaline Phosphatase as a Treatment for Sepsis-Associated Acute Kidney Injury

“…This chimeric construct consists of the crown domain of placental AP, which is incorporated into the intestinal AP structure to improve enzyme stability, while maintaining its catalytic function [24]. Recently published in vitro data confirmed the dephosphorylating capacity of recAP towards LPS and ATP [24].…”

“…This chimeric construct consists of the crown domain of placental AP, which is incorporated into the intestinal AP structure to improve enzyme stability, while maintaining its catalytic function [24]. Recently published in vitro data confirmed the dephosphorylating capacity of recAP towards LPS and ATP [24]. Our preliminary in vitro results demonstrated that recAP can attenuate LPS-induced cytokine production in a human renal cell line [25], whereas recAP improves renal blood flow and vascular resistance and inhibits various parameters of renal inflammation and tissue damage during AKI induced by ischemia-reperfusion or by LPS injection in vivo [26].…”

Reconstituição da Anexina V em sistemas de lipossomos: associação com a fosfatase alcalina e correlação com estudos de biomineralização

“…No homem e nos primatas superiores, três isoformas distintas têm sido identificadas: a da placenta, a do intestino e a do fígado-osso-rim (Lowe et al, 1990;Le Du et al, 2001;Le Du e Millán, 2002;Kiffer-Moreira et al, 2014). As isoformas da placenta e do intestino são tecido-específicas e são expressas unicamente na placenta e no intestino, respectivamente, enquanto a isoforma do fígado-osso-rim é encontrada em quase todos os tecidos, sendo também denominada por esta razão de fosfatase alcalina não específica de tecido (TNAP).…”

“…A comparação de parâmetros cinéticos (V m , K 0,5 , n, k cat entre outros) é complexa e requer metodologias com rígidos controles e condições ótimas de pH, espécie tamponante, presença de diferentes íons, entre outros. Porem, os parâmetros cinéticos podem ser utilizados para comparar a atividade de enzimas (Hoylaerts et al, 1997;Leone et al, 2005Leone et al, , 2012Simão et al, 2013;Kiffer-Moreira et al, 2014 (Segel, 1975).…”