Efficient Site-Specific Radiolabeling of a Modified C2A Domain of Synaptotagmin I with [99mTc(CO)3]+: A New Radiopharmaceutical for Imaging Cell Death
Efficient Site-Specific Radiolabeling of a Modified C2A Domain of Synaptotagmin I with [99mTc(CO)3]+: A New Radiopharmaceutical for Imaging Cell Death
复制标题
DOI：
10.1021/bc900160j
复制
发表时间：
2009-11-01
影响因子：
4.7
通讯作者：
Mullen, Gregory E. D.
中科院分区：
文献类型：
--
作者：
Tavare, Richard;De Rosales, Rafael Torres Martin;Mullen, Gregory E. D.
我们描述了一种新的Tc-99 m标记的生物缀合物的细胞死亡成像的基础上，C2 A，磷脂酰丝氨酸（PS）结合结构域的大鼠突触结合蛋白I的设计和合成。由于该蛋白质中的几个赖氨酸残基对于PS结合至关重要，我们设计了一种新的蛋白质C2 AcH，以包括C末端序列CKLAAALEHHHHHH，并掺入游离半胱氨酸（用于位点特异性共价修饰）和六组氨酸标签（对于用[Tc-99 m（CO）（3）（OH 2）（3）]（+）的位点特异性放射性标记），我们还设计了第二衍生物C2 Ac，其中C-末端序列仅包括C-末端半胱氨酸。这些蛋白质通过电喷雾质谱、SDS/PAGE和尺寸排阻色谱进行了表征，并用[Tc-99 m（CO）（3）（OH 2）（3）]（+）进行了放射性标记。位点特异性标记通过胰蛋白酶消化三羰基标记的C2 AcH来确认，并且仅含有His标签的肽含有[Re（CO）（3）]（+）。以钙依赖性方式测试标记的蛋白质与暴露的PS的红细胞（RBC）的结合。用[Tc-99 m（CO）（3）（OH 2）（3）]（+）在37 ℃标记100 μ g C2 AcH 30分钟，放射化学产率> 96%。然而，首先通过Cys残基与荧光素马来酰亚胺或碘乙酰胺缀合的C2 AcH在37 ℃孵育30分钟后分别仅得到50%和83%的放射化学产率。血清稳定性结果表明，>95%的放射性标记的C2 AcH在37 ℃下保持稳定至少18小时。位点特异性标记的C2 AcH表现出钙依赖性结合的红细胞上的PS，而非特异性修饰的衍生物，C2 AcH-B，其中赖氨酸已与苄氧基羰基氧基修饰，没有。我们的结论是（i）与单独的His-标签或Cys相比，Cys和His-标签的组合极大地提高了用[Tc-99 m（CO）（3）（OH 2）（3）]（+）标记的速率和效率，并且该序列值得作为放射性标记标签进一步评价;（ii）通过赖氨酸残基对C2 A的非位点特异性修饰损害靶结合亲和力，（iii）Tc-99 m-C2 AcH具有优异的放射性标记，稳定性和PS结合特性，并保证作为细胞死亡成像剂的体内评价。
We describe the design and synthesis of a new Tc-99m labeled bioconjugate for cell-death imaging, based on C2A, the phosphatidylserine (PS)-binding domain of rat synaptotagmin I. Since several lysine residues in this protein are critical for PS binding, we engineered a new protein, C2AcH, to include the C-terminal sequence CKLAAALEHHHHHH, incorporating a free cysteine (for site-specific covalent modification) and a hexahistidine tag (for site-specific radiolabeling with [Tc-99m(CO)(3)(OH2)(3)](+)), We also engineered a second derivative, C2Ac, in which the C-terminal sequence included only the C-terminal cysteine. These proteins were characterized by electrospray mass spectrometry, SDS/PAGE, and size exclusion chromatography and radiolabeled with [Tc-99m(CO)(3)(OH2)(3)](+), Conjugates of the proteins with the rhenium analogue [Re(CO)(3)(OH2)(3)](+) were also synthesized. Site-specific labeling was confirmed by performing a tryptic digest of rhenium tricarbonyl-labeled C2AcH, and only peptides containing the His-tag contained the [Re(CO)(3)](+). The labeled proteins were tested for binding to red blood cells (RBC) with exposed PS in a calcium dependent manner. Labeling 100 mu g of C2AcH with [Tc-99m(CO)(3)(OH2)(3)](+) at 37 degrees C for 30 min gave a radiochemical yield of >96%. However, C2AcH that had first been conjugated with fluorescein maleimide or iodoacetamide via the Cys residue gave only 50% and 83% radiochemical yield, respectively, after incubation for 30 min at 37 degrees C. Serum stability results indicated that >95% of radiolabeled C2AcH remained stable for at least 18 h at 37 degrees C. Site-specifically labeled C2AcH exhibited calcium-dependent binding to the PS on the RBC, whereas a nonspecifically modified derivative, C2AcH-B, in which lysines had been modified with benzyloxycarbonyloxy, did not. We conclude that (i) the combination of Cys and a His-tag greatly enhances the rate and efficiency of labeling with [Tc-99m(CO)(3)(OH2)(3)](+) compared to either the His-tag or the Cys alone, and this sequence deserves further evaluation as a radiolabeling tag; (ii) non-site-specific modification of C2A via lysine residues impairs target binding affinity, (iii) Tc-99m-C2AcH has excellent radiolabeling, stability and PS binding characteristics and warrants in vivo evaluation as a cell-death imaging agent.