通过串联质谱法鉴定牛中、低分子量神经丝蛋白的内源性磷酸化位点

Identification of endogenous phosphorylation sites of bovine medium and low molecular weight neurofilament proteins by tandem mass spectrometry.

作者信息

Trimpin Sarah, Mixon April E, Stapels Martha D, Kim Moo-Young, Spencer Peter S, Deinzer Max L

机构信息

Department of Chemistry, Oregon State University, Corvallis, Oregon 97331, USA.

出版信息

Biochemistry. 2004 Feb 24;43(7):2091-105. doi: 10.1021/bi030196q.

DOI:10.1021/bi030196q

PMID:14967049

Abstract

Neurofilament proteins (NFP) are intermediate filaments found in the neuronal cytoskeleton. They are highly phosphorylated, a condition that is believed to be responsible for the assembly and stability of the filaments. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) shows molecular masses for bovine NFP subunits of 63, 105, and 125 kDa for NFL, NFM, and NFH. Mass spectrometric de novo sequencing was used to determine the N-terminal sequence of bovine NFM (115 amino acids), which was previously unknown. Molecular mass information shows that there is one-half equivalent phosphate group on NFL and 24 on NFM. For the first time, it is shown that bovine NFL has three phosphorylation sites (Ser(55), Ser(66), and Ser(472)) and NFM has 22 (Ser(512), Ser(546), Ser(554), Ser(560), Thr(627), Ser(629), Ser(634), Ser(639), Thr(646), Ser(649), Ser(654), Ser(664), Ser(669), Thr(676), Ser(679), Ser(684), Ser(694), Ser(726), Ser(750), Ser(756), Ser(770), and Ser(846)) and two tentative sites (Ser(659)/Thr(661) and Thr(840)). Ser(66) was previously not known to be phosphorylated in NFL of other species, while two sites (Ser(55) and Ser(472)) are consistent with the phosphorylations observed in other mammalian NFLs. The three sites, Ser(55), Ser(66), Ser(472), are heterogeneously phosphorylated. Phosphorylation in bovine NFM occurs mainly in the Lys-Ser-Pro (KSP) region, but the Val-Ser-Pro and Ser-Glu-Lys motifs are also phosphorylated. Most of the phosphorylation sites are in accordance with those previously identified in other mammalian NFMs. In bovine NFM, 16 out of the 22 sites are always phosphorylated (Ser(512), Thr(627), Ser(629), Ser(634), Ser(639), Thr(646), Ser(649), Ser(654), Ser(664), Ser(669), Thr(676), Ser(679), Ser(684), Ser(694), Ser(726), and Ser(750)), all of which are contained in the KSP region, and six are sometimes phosphorylated (Ser(546), Ser(554), Ser(560), Ser(756), Ser(770), and Ser(846)). The NFPs have other modifications, including deamidation, oxidation, and N-terminal acetylation. Pyroglutamic acid formation also occurs.

摘要

神经丝蛋白（NFP）是存在于神经元细胞骨架中的中间丝。它们高度磷酸化，这种状态被认为与丝的组装和稳定性有关。基质辅助激光解吸/电离（MALDI）飞行时间（TOF）质谱（MS）显示，牛NFP亚基NFL、NFM和NFH的分子量分别为63、105和125 kDa。采用质谱从头测序法确定了此前未知的牛NFM的N端序列（115个氨基酸）。分子量信息表明，NFL上有半个磷酸基团当量，NFM上有24个。首次表明，牛NFL有三个磷酸化位点（Ser(55)、Ser(66)和Ser(472)），NFM有22个（Ser(512)、Ser(546)、Ser(554)、Ser(560)、Thr(627)、Ser(629)、Ser(634)、Ser(639)、Thr(646)、Ser(649)、Ser(654)、Ser(664)、Ser(669)、Thr(676)、Ser(679)、Ser(684)、Ser(694)、Ser(726)、Ser(750)、Ser(756)、Ser(770)和Ser(846)）以及两个暂定位点（Ser(659)/Thr(661)和Thr(840)）。Ser(66)此前在其他物种的NFL中未知其被磷酸化，而两个位点（Ser(55)和Ser(472)）与在其他哺乳动物NFL中观察到的磷酸化情况一致。Ser(55)、Ser(66)、Ser(472)这三个位点存在异质性磷酸化。牛NFM中的磷酸化主要发生在Lys-Ser-Pro（KSP）区域，但Val-Ser-Pro和Ser-Glu-Lys基序也会被磷酸化。大多数磷酸化位点与此前在其他哺乳动物NFM中鉴定出的位点一致。在牛NFM中，22个位点中的16个总是被磷酸化（Ser(512)、Thr(627)、Ser(629)、Ser(634)、Ser(639)、Thr(646)、Ser(649)、Ser(654)、Ser(664)、Ser(669)、Thr(676)、Ser(679)、Ser(684)、Ser(694)、Ser(726)和Ser(750)），所有这些都包含在KSP区域，另外六个有时被磷酸化（Ser(546)、Ser(554)、Ser(560)、Ser(756)、Ser(770)和Ser(846)）。神经丝蛋白还有其他修饰，包括脱酰胺、氧化和N端乙酰化。焦谷氨酸的形成也会发生。