Motomura Shou, Suzuki Tomohiko
Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi, 780-8520, Japan.
Protein J. 2016 Jun;35(3):212-7. doi: 10.1007/s10930-016-9663-0.
In this study, we confirmed N-terminal myristoylation of Tetrahymena pyriformis arginine kinase (AK1) by identifying a myristoylation signal sequence at the N-terminus. A sufficient amount of modified enzyme was synthesized using an insect cell-free protein synthesis system that contains all of the elements necessary for post-transcriptional modification by fatty acids. Subsequent peptide mass fingerprinting (PMF) analyses were performed after digestion with trypsin. The PMF data covered 39 % (143 residues) of internal peptides. The target N-myristoylated peptide had a theoretical mass of 832.4477 and was clearly observed with an experimental mass (m/z-H(+)) of 832.4747. The difference between the two masses was 0.0271, supporting the accuracy of identification and indicating that the synthesized T. pyriformis AK1 is myristoylated. The fixed specimens of T. pyriformis were reacted with an anti-AK1 peptide antibody followed by a secondary antibody with a fluorescent chromophore and were observed using immunofluorescence microscope. In agreement with previous western blotting analyses, microscopic observations suggested that AK1 is localized in the cilia. The present PMF and microscopic analyses indicate that T. pyriformis AK1 may be localized and anchored to ciliary membranes via N-terminal myristoyl groups.
在本研究中，我们通过鉴定梨形四膜虫精氨酸激酶（AK1）N端的肉豆蔻酰化信号序列，证实了其N端肉豆蔻酰化。使用包含脂肪酸转录后修饰所需所有元件的昆虫无细胞蛋白质合成系统合成了足量的修饰酶。用胰蛋白酶消化后进行后续的肽质量指纹图谱（PMF）分析。PMF数据覆盖了内部肽段的39%（143个残基）。目标N-肉豆蔻酰化肽的理论质量为832.4477，实验质量（m/z-H(+)）为832.4747时能清晰观察到，两者质量差为0.0271，支持了鉴定的准确性，表明合成的梨形四膜虫AK1被肉豆蔻酰化。将梨形四膜虫的固定标本与抗AK1肽抗体反应，然后与带有荧光发色团的二抗反应，并用免疫荧光显微镜观察。与之前的蛋白质印迹分析一致，显微镜观察表明AK1定位于纤毛中。目前的PMF和显微镜分析表明，梨形四膜虫AK1可能通过N端肉豆蔻酰基团定位于纤毛膜并锚定在其上。
