背景:序列的变化,三维结构,人血清白蛋白(HSA)的翻译后修饰(PTM)对其生理功能至关重要。本研究旨在分析和比较来自人血浆的HSA与重组基因来源在中国临床治疗中的PTM差异。
方法:六种不同的PTM,即乙酰化,琥珀酰化,巴豆酰化,磷酸化,β-羟基丁酰化,和乳酸化,通过蛋白质印迹分析使用泛特异性抗体进行鉴定。样品,包含来自六个不同制造商的人血浆来源的HSA(pHSA)和在酵母和水稻中表达的重组HSA(rHSA),对各种类型的PTM进行了检测。此外,进行4D无标记定量蛋白质组分析以鉴定pHSA和rHSA样品中的N-糖基化和前述PTM。该分析旨在辨别修饰位点和水平的差异。
结果:通过蛋白质印迹分析,AllsixpHSAandtworHSAsamplesdisplayedpositivebandsfor白蛋白(66.5kDa)acrossthesixPTM.30(32)琥珀酰化,41(50)丙二酸,15(23)磷酸化,36(30)β-羟基丁酰化,和27(34)在pHSA和rHSA样品的乳酸化修饰位点,没有检测到N-糖基化修饰位点。分析确定1个乙酰化(ALB_K160),2β-羟基丁酰化(ALB_K569,ALB_K426),和3个巴豆化(ALB_K264,ALB_K581,ALB_K560)特异性修饰位点,以及3个巴豆化(ALB_K560,ALB_K562,ALB_K75),1琥珀酰化(ALB_K490),和rHSA中的23个磷酸化特异性修饰位点。在pHSA(rHSA)中,2(6)乙酰化,10(12)琥珀酰化,0(9)巴豆化,1(9)磷酸化,6(0)β-羟基丁酰化,发现了0(7)个特定的乳酸化修饰位点。此外,在pHSA和rHSA之间的共享修饰位点中,pHSA在更多的位点表现出氨甲酰化(16:1)和β-羟基丁酰基化(12:2)的上调,和上调乙酰化(7:11),巴豆酰化(2:11),磷酸化(1:8),与rHSA相比,在更少的位点进行乳酸化(1:14)。
结论:在临床实践中,在中国使用的pHSA和rHSA通常显示乙酰化,琥珀酰化,巴豆酰化,磷酸化,β-羟基丁酰化,和乳酸化。值得注意的是,pHSA和rHSA之间在这些改变的位点特征和修饰水平上存在差异。进一步的实验调查是必要的,以深入研究这些差异在PTM的生物功能的影响,有效性,pHSA和rHSA的安全性。
BACKGROUND: The variations in sequence, three-dimensional structure, and post-translational modifications (PTMs) of human serum albumin (HSA) are crucial for its physiological functions. This study aims to analyze and compare the disparities in PTMs between HSA derived from human plasma and genetically
recombinant sources for clinical treatments in China.
METHODS: Six distinct PTMs, namely acetylation, succinylation, crotonylation, phosphorylation, beta-hydroxybutyrylation, and lactylation, were identified using pan-specific antibodies via Western blot analysis. The samples, comprising human plasma-derived HSA (pHSA) from six different manufacturers and
recombinant HSA (rHSA) expressed in yeast and Oryza sativa, underwent detection for various types of PTMs. Additionally, a 4D label-free quantitative proteomic analysis was performed to identify N-glycosylation and the aforementioned PTMs in both pHSA and rHSA samples. This analysis aimed to discern disparities in modification sites and levels.
RESULTS: Through Western blot analysis, all six pHSA and two rHSA samples displayed positive bands for albumin (66.5 kDa) across the six PTMs. Subsequent analysis using 4D label-free quantitative proteomics revealed 25 (29) acetylated, 30 (32) succinylated, 41 (50) malonylated, 15 (23) phosphorylated, 36 (30) beta-hydroxybutyrylated, and 27 (34) lactylated modification sites in pHSA and rHSA samples, with no N-glycosylation modification sites detected. The analysis identified 1 acetylation (ALB_K160), 2 beta-hydroxybutyrylation (ALB_K569, ALB_K426), and 3 crotonylation (ALB_K264, ALB_K581, ALB_K560) specific modification sites in pHSA, as well as 3 crotonylation (ALB_K560, ALB_K562, ALB_K75), 1 succinylation (ALB_K490), and 23 phosphorylation specific modification sites in rHSA. In pHSA (rHSA), 2 (6) acetylation, 10 (12) succinylation, 0 (9) crotonylation, 1 (9) phosphorylation, 6 (0) beta-hydroxybutyrylation, and 0 (7) lactylation specific modification sites were found. Moreover, in the shared modification sites between pHSA and rHSA, pHSA exhibited up-regulation of amberylation (16:1) and beta-hydroxybutyrylation (12:2) in more sites, and up-regulation of acetylation (7:11), crotonylation (2:11), phosphorylation (1:8), and lactylation (1:14) in fewer sites compared to rHSA.
CONCLUSIONS: In clinical practice, both pHSA and rHSA utilized in China commonly display acetylation, succinylation, crotonylation, phosphorylation, beta-hydroxybutyrylation, and lactylation. Notably, there exist distinctions in the site characteristics and modification levels of these alterations between pHSA and rHSA. Further experimental inquiries are imperative to delve into the implications of these disparities in PTMs on the biological functionality, effectiveness, and safety of pHSA and rHSA.