Zhang Jiang, Wang Guangli, He Wei-Wu, Losh Molly, Berry-Kravis Elizabeth, Funk William E
Department of Preventive Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
OriGene Technology, Inc., Rockville, MD, USA.
Proteomics Insights. 2019 Mar 3;10:1178641818825268. doi: 10.1177/1178641818825268. eCollection 2019.
Fragile X mental retardation protein is an mRNA-binding protein associated with phenotypic manifestations of fragile X syndrome, an X-linked disorder caused by mutation in the gene that is the most common inherited cause of intellectual disability. Despite the well-studied genetic mechanism of the disease, the proteoforms of fragile X mental retardation protein have not been thoroughly characterized. Here, we report the expression and mass spectrometric characterization of human fragile X mental retardation protein. cDNA clone was transfected into human HEK293 cells to express the full-length human fragile X mental retardation protein. Purified fragile X mental retardation protein was subjected to trypsin digestion and characterized by mass spectrometry. Results show 80.5% protein sequence coverage of fragile X mental retardation protein (Q06787, _HUMAN) including both the N- and C-terminal peptides, indicating successful expression of the full-length protein. Identified post-translational modifications include N-terminal acetylation, phosphorylation (Ser600), and methylation (Arg290, 471, and 474). In addition to the full-length fragile X mental retardation protein isoform (isoform 6), two endogenous fragile X mental retardation protein alternative splicing isoforms (isoforms 4 and 7), as well as fragile X mental retardation protein interacting proteins, were also identified in the co-purified samples, suggesting the interaction network of the human fragile X mental retardation protein. Quantification was performed at the peptide level, and this information provides important reference for the future development of a targeted assay for quantifying fragile X mental retardation protein in clinical samples. Collectively, this study provides the first comprehensive report of human fragile X mental retardation protein proteoforms and may help advance the mechanistic understanding of fragile X syndrome and related phenotypes associated with the mutation.
脆性X智力低下蛋白是一种与脆性X综合征表型表现相关的mRNA结合蛋白，脆性X综合征是一种X连锁疾病，由该基因的突变引起，是智力残疾最常见的遗传原因。尽管对该疾病的遗传机制已有充分研究，但脆性X智力低下蛋白的蛋白异构体尚未得到全面表征。在此，我们报告了人类脆性X智力低下蛋白的表达及质谱表征。将cDNA克隆转染到人HEK293细胞中以表达全长人类脆性X智力低下蛋白。对纯化的脆性X智力低下蛋白进行胰蛋白酶消化并通过质谱进行表征。结果显示脆性X智力低下蛋白（Q06787，_HUMAN）的蛋白质序列覆盖率为80.5%，包括N端和C端肽段，表明全长蛋白成功表达。鉴定出的翻译后修饰包括N端乙酰化、磷酸化（Ser600）和甲基化（Arg290、471和474）。除了全长脆性X智力低下蛋白异构体（异构体6）外，在共纯化样品中还鉴定出两种内源性脆性X智力低下蛋白可变剪接异构体（异构体4和7）以及脆性X智力低下蛋白相互作用蛋白，提示了人类脆性X智力低下蛋白的相互作用网络。在肽段水平进行了定量，该信息为未来开发用于定量临床样品中脆性X智力低下蛋白的靶向检测方法提供了重要参考。总体而言，本研究首次全面报道了人类脆性X智力低下蛋白的蛋白异构体，可能有助于推进对脆性X综合征及与该突变相关的相关表型的机制理解。
