Department of Anesthesia, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts.
Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts.
Am J Physiol Cell Physiol. 2021 Jan 1;320(1):C15-C29. doi: 10.1152/ajpcell.00430.2020. Epub 2020 Oct 14.
Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.
细胞外二磷酸核苷和三磷酸核苷从激活或损伤的细胞中释放出来，以触发血管和免疫 P2 嘌呤能受体，引发炎症和血管血栓形成。这些代谢产物被细胞外核苷三磷酸二磷酸水解酶-1（E-NTPDase1 或 CD39）清除。单磷酸核苷末端产物的进一步降解是通过表面外核苷酸酶（NMPase）或 CD73 完成的。这些细胞外酶促过程协同作用，促进具有免疫抑制和抗血栓形成作用的腺苷能反应。在氧化应激的情况下，这些维持内稳态的细胞外酶促机制丧失，从而加剧炎症过程。我们设计了由 CD39 和 CD73 的细胞外结构域（ECD）组成的双功能酶，这些 ECD 位于单个多肽中。还生成了人碱性磷酸酶-细胞外结构域（ALP-ECD）和人酸性磷酸酶-细胞外结构域（HAP-ECD）融合蛋白，并对其进行了表征，并与这些 CD39-ECD、CD73-ECD 和双功能融合蛋白进行了比较。通过应用比色功能测定法和高效液相色谱动力学测定法，我们证明了这些双功能细胞外酶表达高水平的 CD39 样 NTPDase 活性和 CD73 样 NMPase 活性。与 ALP-ECD 和 HAP-ECD 相比，嵌合 CD39-CD73-ECD 蛋白在将三磷酸核苷和二磷酸核苷转化为核苷方面表现出更高的活性。我们还注意到双功能融合蛋白与亲本融合蛋白之间存在 pH 敏感性差异以及细胞外酶特性差异。有趣的是，这些创新试剂在体外降低了血小板对外源性激动剂的激活。我们提出，这些嵌合融合蛋白可用作炎症性疾病的治疗剂，以清除促炎的 ATP，同时产生抗炎的腺苷。
