Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden.
Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden.
J Neurochem. 2021 May;157(3):816-833. doi: 10.1111/jnc.15252. Epub 2020 Dec 17.
Neurogranin (Ng) is a 78 amino acid neuronal protein and a biomarker candidate for Alzheimer's disease (AD). Ng has been suggested to bind to calmodulin and phosphatidic acid via its centrally located IQ domain. Ng is cleaved within this functionally important domain, yielding the majority of fragments identified in cerebrospinal fluid (CSF), suggesting that cleavage of Ng may be a mechanism to regulate its function. Up to now, Ng has been shown to be present in CSF as both C-terminal fragments as well as full-length protein. To obtain an overview of the different molecular forms of Ng present in CSF, we show by size exclusion chromatography (SEC), immunoblotting, immunoprecipitation, and MS that Ng is present in CSF as several molecular forms. Besides monomeric full-length Ng, also higher molecular weight forms of Ng, and C-terminal- and previously not identified N-terminal fragments were observed. We found by immunodepletion that C-terminal peptides contribute on average to ~50% of the total-Ng ELISA signal in CSF samples. There were no differences in the overall C-terminal fragment/total-Ng ratios between samples from AD and control groups. In addition, we found that monomeric Ng and its C-terminal fragments bind to heparin via a heparin-binding motif, which might be of relevance for their export mechanism from neurons. Taken together, this study highlights the presence of several molecular forms of Ng in CSF, comprising monomeric full-length Ng, and N- and C-terminal truncations of Ng, as well as larger forms of still unknown composition.
神经颗粒蛋白（Ng）是一种 78 个氨基酸的神经元蛋白，也是阿尔茨海默病（AD）的候选生物标志物。研究表明，Ng 通过其中央的 IQ 结构域与钙调蛋白和磷脂酸结合。Ng 在这个功能重要的结构域内被切割，产生了大多数在脑脊液（CSF）中鉴定到的片段，这表明 Ng 的切割可能是调节其功能的一种机制。到目前为止，已经证明 Ng 以 C 端片段和全长蛋白的形式存在于 CSF 中。为了全面了解 CSF 中存在的不同 Ng 分子形式，我们通过分子筛层析（SEC）、免疫印迹、免疫沉淀和 MS 显示 Ng 以几种分子形式存在于 CSF 中。除了单体全长 Ng 外，还观察到了更高分子量的 Ng 形式和以前未鉴定的 N 端片段。我们通过免疫耗竭发现 C 端肽平均贡献 CSF 样本中总 Ng ELISA 信号的~50%。AD 和对照组样本的总 C 端片段/总 Ng 比值没有差异。此外，我们发现单体 Ng 及其 C 端片段通过肝素结合基序与肝素结合，这可能与其从神经元中输出的机制有关。综上所述，本研究强调了 CSF 中存在几种 Ng 分子形式，包括单体全长 Ng 和 Ng 的 N 端和 C 端截断，以及组成仍未知的更大形式。
