TY - JOUR
T1 - Efficient Site-Specific Radiolabeling of a Modified C2A Domain of Synaptotagmin I with [Tc-99m(CO)(3)](+): A New Radiopharmaceutical for Imaging Cell Death
AU - Tavare, Richard
AU - T. M. de Rosales, Rafael
AU - Blower, Philip J.
AU - Mullen, Gregory E. D.
PY - 2009/11/18
Y1 - 2009/11/18
N2 - 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.
AB - 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.
U2 - 10.1021/bc900160j
DO - 10.1021/bc900160j
M3 - Article
VL - 20
SP - 2071
EP - 2081
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
IS - 11
ER -