A glycosylated form of the human cardiac hormone pro B-type natriuretic peptide is an intrinsically unstructured monomeric protein
A glycosylated form of the human cardiac hormone pro B-type natriuretic peptide is an intrinsically unstructured monomeric protein
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DOI：
10.1016/j.abb.2008.04.007
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发表时间：
2008-07-01
影响因子：
3.9
通讯作者：
Kao, Jeffrey L. -F.
中科院分区：
文献类型：
--
作者：
Crimmins, Dan L.;Kao, Jeffrey L. -F.
前B型利钠肽的N-末端片段（NT-proBNP）和proBNP被用作心肌功能障碍如心脏肥大和左心室心力衰竭的金标准临床标志物。这些肽的实际循环分子形式一直是深入研究的主题，特别是因为这些分析物是在临床测定中测量的。已报告了验证数据，但对分子种类的确切性质尚无明确的共识。因为这些临床测定是基于免疫测定的，所以检测特异性表位。因此可以想象，某些表位可能被掩蔽，因此不可用于抗体结合，因此确定这些分析物的循环分子形式的性质是重要的。这种情况通常是免疫测定中不可避免的阿喀琉斯之踵。重组O-连接的糖基化形式的proBNP已被证明模拟从心力衰竭患者提取的血浆的一些性质。特别是，重组和天然材料在SDS-PAGE上共迁移为弥漫性蛋白质免疫染色条带，并且每个条带在去糖基化后塌陷为表观均一条带。因此，糖基化-proBNP可能是一种这样的循环形式。在这里，我们提供了这种O-连接蛋白质的广泛理化表征，并将这些结果与其他描述的循环物质，非糖基化-proBNP和NT-proBNP进行比较。结果表明，糖基化对proBNP的二级和四级结构没有影响。事实上，在良性生理中性pH缓冲液中的中等浓度下，所有三种可能的循环种类基本上缺乏主要二级结构，即，非结构化蛋白质（IUP）。此外，所有三种蛋白质在溶液中均以单体形式存在。这些结果可能对NT-proBNP/BNP免疫测定的设计具有重要意义。(C)2008年爱思唯尔公司All rights reserved.
The N-terminal fragment of pro B-type natriuretic peptide (NT-proBNP) and proBNP are used as gold standard clinical markers of myocardial dysfunction such as cardiac hypertrophy and left ventricle heart failure. The actual circulating molecular forms of these peptides have been the subject of intense investigation particularly since these analytes are measured in clinical assays. Conflicting data has been reported and no firm consensus on the exact nature of the molecular species exists. Because these clinical assays are immunoassay-based, specific epitopes are detected. It is conceivable then that certain epitopes may be masked and therefore unavailable for antibody binding, thus the importance of determining the nature of the circulating molecular forms of these analytes. This situation is an unavoidable Achilles' heel of immunoassays in general.A recombinant O-linked glycosylated form of proBNP has been show to mimic some of the properties of extracted plasma from a heart failure patient. In particular the recombinant and native material co-migrated as diffuse Western-immunostained bands on SDS-PAGE and each band collapsed to an apparent homogeneous band following deglycosylation. Thus, glycosylated-proBNP may be one such circulating form. Here we provide extensive physiochemical characterization for this O-linked protein and compare these results to other described circulating species, non-glycosylated-proBNP and NT-proBNP. It will be shown that glycosylation has no influence on the secondary and quaternary structure of proBNP. In fact, at moderate concentration in benign physiological neutral pH buffer, all three likely circulating species are essentially devoid of major secondary structure, i.e., are intrinsically unstructured proteins (IUPs). Furthermore, all three proteins exist as monomers in solution. These results may have important implications in the design of NT-proBNP/BNP immunoassays. (C) 2008 Elsevier Inc. All rights reserved.