Department of Molecular Medicine, Mayo Clinic, College of Medicine, Rochester, MN 55905, USA.
Clin Chem. 2011 Jun;57(6):864-73. doi: 10.1373/clinchem.2010.157438. Epub 2011 Apr 11.
B-type natriuretic peptide (BNP), a key cardiac hormone in cardiorenal homeostasis, is produced as a 108 amino acid prohormone, proBNP1-108, which is converted to a biologically active peptide BNP1-32 and an inactive N-terminal (NT)-proBNP1-76. The widely accepted model is that the normal heart releases a proteolytically processed BNP1-32 and NT-proBNP, whereas the diseased heart secretes high amounts of unprocessed/glycosylated proBNP1-108 or inappropriately processed BNPs. In contrast, circulating proBNP1-108 has recently been identified in healthy individuals, indicating that the normal heart also secretes unprocessed proBNP1-108. However, the mechanism of proBNP1-108 secretion from the normal heart remains elusive. Our goal was to determine the molecular mechanisms underlying proBNP1-108 intracellular trafficking and secretion from the normal heart.
We expressed preproBNP in cardiomyocytes, and determined the subcellular localization and dominant intracellular and extracellular forms of BNP.
Intracellular immunoreactive BNPs were first accumulated in the Golgi apparatus, and then distributed throughout the cytoplasm as secretory vesicles. The predominant intracellular form of BNP was nonglycosylated proBNP1-108, rather than BNP1-32. Glycosylated proBNP1-108, but not nonglycosylated proBNP1-108, was detected as the major extracellular form in the culture supernatants of preproBNP-expressing cell lines and primary human cardiomyocytes. Ablation of O-glycosylation of proBNP1-108 at T71 residue, near the convertase recognition site, reduced the extracellular proBNP1-108 and increased extracellular BNP1-32.
Intracellular proBNP trafficking occurs through a conventional Golgi-endoplasmic reticulum pathway. Glycosylation of proBNP1-108 controls the stability and processing of extracellular proBNP1-108. Our data establish a new BNP secretion model in which the normal cardiac cells secrete glycosylated proBNP1-108.
B 型利钠肽（BNP）是心肾稳态中的一种关键心脏激素，作为 108 个氨基酸的前激素 proBNP1-108 产生，该前激素转化为生物活性肽 BNP1-32 和无活性的 N 端（NT）-proBNP1-76。广泛接受的模型是正常心脏释放经过蛋白水解加工的 BNP1-32 和 NT-proBNP，而患病心脏则分泌大量未经处理/糖基化的 proBNP1-108 或处理不当的 BNP。相反，循环中的 proBNP1-108 最近在健康个体中被鉴定出来，表明正常心脏也分泌未经处理的 proBNP1-108。然而，正常心脏中 proBNP1-108 分泌的机制仍不清楚。我们的目标是确定正常心脏中 proBNP1-108 细胞内运输和分泌的分子机制。
我们在心肌细胞中表达 preproBNP，并确定 BNP 的亚细胞定位和主要的细胞内和细胞外形式。
细胞内免疫反应性 BNPs 首先在高尔基体中积累，然后作为分泌小泡分布在细胞质中。BNP 的主要细胞内形式是未经糖基化的 proBNP1-108，而不是 BNP1-32。糖基化的 proBNP1-108，而不是未经糖基化的 proBNP1-108，被检测为表达 preproBNP 的细胞系和原代人心肌细胞培养上清液中的主要细胞外形式。在靠近转化酶识别位点的 T71 残基处切除 proBNP1-108 的 O-糖基化，减少了细胞外 proBNP1-108 并增加了细胞外 BNP1-32。
细胞内 proBNP 转运通过传统的高尔基体-内质网途径进行。proBNP1-108 的糖基化控制细胞外 proBNP1-108 的稳定性和加工。我们的数据建立了一个新的 BNP 分泌模型，其中正常心脏细胞分泌糖基化的 proBNP1-108。
