Structural requirements for in vivo detection of cell death with 99mTc-annexin V.
Structural requirements for in vivo detection of cell death with 99mTc-annexin V.
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发表时间：
2005-05
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通讯作者：
J. Tait;Christina Smith;F. Blankenberg
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作者：
J. Tait;Christina Smith;F. Blankenberg
未标记的（99 m）Tc-膜联蛋白V通过与暴露的磷脂酰丝氨酸的高亲和力结合用于体内细胞死亡成像。我们研究了膜结合亲和力、分子电荷和标记方法的变化如何影响其在正常小鼠中的生物分布和在凋亡组织中的摄取。方法在膜联蛋白V的N端增加一个内源性Tc螯合位点（Ala-Gly-Gly-Cys-Gly-His），得到膜联蛋白V-128。膜联蛋白V-128的膜结合亲和力，然后逐步减少1-4个突变的钙结合位点。此外，在其他残基中进行突变，改变分子电荷而不改变膜结合亲和力。所有突变蛋白均在相同的N-末端内源性螯合位点用（99 m）Tc标记。野生型膜联蛋白V在用肼基烟酰胺（HYNIC）衍生后也用（99 m）Tc标记。放射性标记的蛋白质在正常小鼠和经处理以诱导肝细胞凋亡的小鼠中测试生物分布。结果（99 m）Tc-annexin V-128与（99 m）Tc-HYNIC-annexin V在凋亡组织中的摄取量相同或更高，而在注射后60 min，肾脏摄取量降低88%。膜联蛋白V的血液清除率不受膜结合亲和力或分子电荷变化的影响。肾摄取不受结合亲和力变化的影响。与此形成鲜明对比的是，正常肝脏和脾脏中的摄取随着亲和力的降低而显著降低。在用放线菌酮处理以诱导细胞凋亡的动物中观察到相同的模式。电荷突变体的对照实验表明，亲和突变体的效应不是由于这些突变体中发生的分子电荷的伴随变化。结论（a）膜联蛋白V的所有四个结构域都是凋亡组织最佳摄取所必需的;仅具有1或2个活性结构域的分子不太可能适用于体内细胞死亡成像。(b)正常肝脏和脾脏的摄取是特异性的（依赖于磷脂酰丝氨酸结合亲和力），而肾脏的摄取是非特异性的。(c)（99 m）Tc-Annexin V-128与（99 m）Tc-HYNIC-annexin V一样检测细胞死亡，同时由于放射性的尿液排泄更快，肾放射性保留减少了88%。
UNLABELLED (99m)Tc-Annexin V is used to image cell death in vivo via high-affinity binding to exposed phosphatidylserine. We investigated how changes in membrane-binding affinity, molecular charge, and method of labeling affected its biodistribution in normal mice and its uptake in apoptotic tissues. METHODS An endogenous Tc chelation site (Ala-Gly-Gly-Cys-Gly-His) was added to the N-terminus of annexin V to create annexin V-128. The membrane-binding affinity of annexin V-128 was then progressively reduced by 1-4 mutations in calcium-binding sites. In addition, mutations were made in other residues that altered molecular charge without altering membrane-binding affinity. All mutant proteins were labeled with (99m)Tc at the same N-terminal endogenous chelation site. Wild-type annexin V was also labeled with (99m)Tc after derivatization with hydrazinonicotinamide (HYNIC). Radiolabeled proteins were tested for biodistribution in normal mice and in mice treated to induce apoptosis of the liver. RESULTS Comparison of (99m)Tc-annexin V-128 with (99m)Tc-HYNIC-annexin V showed that the protein labeled at the endogenous chelation site had the same or higher uptake in apoptotic tissues, while showing 88% lower renal uptake at 60 min after injection. The blood clearance of annexin V was unaffected by changes in either the membrane-binding affinity or the molecular charge. Kidney uptake was unaffected by changes in binding affinity. In marked contrast, uptake in normal liver and spleen decreased markedly as affinity decreased. The same pattern was observed in animals treated with cycloheximide to induce apoptosis. Control experiments with charge mutants showed that the effects seen with the affinity mutants were not due to the concomitant change in molecular charge that occurs in these mutants. CONCLUSION (a) All four domains of annexin V are required for optimal uptake in apoptotic tissues; molecules with only 1 or 2 active domains are unlikely to be suitable for imaging of cell death in vivo. (b) Uptake in normal liver and spleen is specific (dependent on phosphatidylserine-binding affinity), whereas renal uptake is nonspecific. (c) (99m)Tc-Annexin V-128 detects cell death as well as (99m)Tc-HYNIC-annexin V, while showing 88% less renal retention of radioactivity due to much more rapid urinary excretion of radioactivity.