Ⅱ型核糖体失活蛋白(RIPs)是一类重要的蛋白毒素,由A链和B链通过链间二硫键连接而成。具有凝集素样活性的B链负责与真核细胞表面的含半乳糖受体结合,这对于通过内吞作用实现A链内化至关重要。A链具有N-糖苷酶活性,可从28S核糖体RNA(28SrRNA)中不可逆地纯化特定的腺嘌呤并终止蛋白质合成。A-B链的协同作用使核糖体失活,抑制蛋白质合成,并表现出高细胞毒性。蓖麻和Abrusprecatorius植物表达的蓖麻毒素和abrin,分别,是典型的Ⅱ型RIP。蓖麻毒素和abrin的毒性分别是385倍和2885倍,分别,更多的是神经毒剂VX。由于它们易于准备,广泛的可用性,以及作为生物恐怖主义代理人的潜在用途,Ⅱ型RIP近年来受到越来越多的关注。蓖麻毒素被列为《化学武器公约》(CWC)附表1A的违禁物质。近年来蓖麻毒素相关生物恐怖事件的发生,敏感,Ⅱ型RIPs的快速检测与识别技术。Ⅱ型RIPs的毒性机制和检测方法的研究取得了重大进展,主要涉及定性和定量分析方法,包括免疫测定,质谱分析方法,和基于脱嘌呤和细胞毒性的毒素活性检测方法。免疫测定通常涉及抗原和抗体的特异性识别,它基于称为适体的寡核苷酸分子识别元件。这些方法快速且高度灵敏,但是对于复杂样本中高度同源的蛋白质,他们提供假阳性结果。随着生物质谱检测技术的快速发展,电喷雾电离(ESI)和基质辅助激光解吸电离(MALDI)等技术被广泛用于蛋白质的鉴定。这些方法不仅提供了有关蛋白质分子量和结构的准确信息,但也证明了准确的量化。酶消化结合质谱是主要使用的检测方法。通过酶消化的肽的指纹分析可以实现蛋白质毒素的准确鉴定。为了分析复杂样品中的蛋白质毒素,使用多酶消化策略获得丰富的肽标记。肽标记的靶向质谱分析用于获得准确的定性和定量信息,有效地提高了Ⅱ型RIP毒素鉴定的准确性和灵敏度。虽然免疫分析和质谱检测方法可以准确鉴定Ⅱ型RIPs,他们无法确定毒素是否会保持效力。Ⅱ型RIPs活性分析中广泛使用的检测方法包括基于N-糖苷酶活性的脱嘌呤试验和细胞毒性试验。这两种方法都提供了简单的,快速,Ⅱ型RIP毒性的敏感性分析,并补充其他检测方法。由于Ⅱ型RIP毒素的重要性,禁止化学武器组织(OPCW)提出了明确的技术要求,以识别和分析相关样品。我们在这里回顾了结构特征,作用机制,以及Ⅱ型RIP检测方法的发展和应用;引用了近70项Ⅱ型RIP毒素及其检测方法的研究。除了禁化武组织明确鉴定生物毒素的技术要求外,探讨了Ⅱ型RIP检测技术的未来发展趋势。
Type Ⅱ ribosome-inactivating proteins (RIPs) are an important class of protein toxins that consist of A and B chains linked by an interchain disulfide bond. The B-chain with lectin-like activity is responsible for binding to the galactose-containing receptors on eukaryotic cell surfaces, which is essential for A-chain internalization by endocytosis. The A-chain has N-glycosidase activity that irreversibly depurinates a specific adenine from 28S ribosomal RNA (28S rRNA) and terminates protein synthesis. The synergistic effect of the A-B chain inactivates the ribosome, inhibits protein synthesis, and exhibits high cytotoxicity.
Ricin and abrin that are expressed by the plants Ricinus communis and Abrus precatorius, respectively, are typical type Ⅱ RIPs. The toxicity of
ricin and abrin are 385 times and 2885 times, respectively, more that of the nerve agent VX. Owing to their ease of preparation, wide availability, and potential use as a bioterrorism agent, type Ⅱ RIPs have garnered increasing attention in recent years.
Ricin is listed as a prohibited substance under schedule 1A of the Chemical Weapons Convention (CWC). The occurrence of
ricin-related bioterrorism incidents in recent years has promoted the development of accurate, sensitive, and rapid detection and identification technology for type Ⅱ RIPs. Significant progress has been made in the study of toxicity mechanisms and detection methods of type Ⅱ RIPs, which primarily involve qualitative and quantitative analysis methods including immunological assays, mass spectrometry analysis methods, and toxin activity detection methods based on depurination and cytotoxicity. Immunoassays generally involve the specific recognition of antigens and antibodies, which is based on oligonucleotide molecular recognition elements called aptamers. These methods are fast and highly sensitive, but for highly homologous proteins in complex samples, they provide false positive results. With the rapid development of biological mass spectrometry detection technology, techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are widely used in the identification of proteins. These methods not only provide accurate information on molecular weight and structure of proteins, but also demonstrate accurate quantification. Enzyme digestion combined with mass spectrometry is the predominantly used detection method. Accurate identification of protein toxins can be achieved by fingerprint analysis of enzymatically digested peptides. For analysis of protein toxins in complex samples, abundant peptide markers are obtained using a multi-enzyme digestion strategy. Targeted mass spectrometry analysis of peptide markers is used to obtain accurate qualitative and quantitative information, which effectively improves the accuracy and sensitivity of the identification of type Ⅱ RIP toxins. Although immunoassay and mass spectrometry detection methods can provide accurate identification of type Ⅱ RIPs, they cannot determine whether the toxins will retain potency. The widely used detection methods for activity analysis of type Ⅱ RIPs include depurination assay based on N-glycosidase activity and cytotoxicity assay. Both the methods provide simple, rapid, and sensitive analysis of type Ⅱ RIP toxicity, and complement other detection methods. Owing to the importance of type Ⅱ RIP toxins, the Organization for the Prohibition of Chemical Weapons (OPCW) has proposed clear technical requirements for the identification and analysis of relevant samples. We herein reviewed the structural characteristics, mechanism of action, and the development and application of type Ⅱ RIP detection methods; nearly 70 studies on type Ⅱ RIP toxins and their detection methods have been cited. In addition to the technical requirements of OPCW for the unambiguous identification of biotoxins, the trend of future development of type Ⅱ RIP-based detection technology has been explored.