Molecular Imaging Laboratory, MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA.
J Lipid Res. 2011 Sep;52(9):1660-71. doi: 10.1194/jlr.M017582. Epub 2011 Jul 11.
To devise successful imaging and therapeutic strategies, the identification of β-cell surface markers is one of the challenges in diabetes research that has to be resolved. We previously showed that IC2, a rat monoclonal IgM antibody, can be used for ex vivo determination of β-cell mass by imaging. Further progress toward the development of an antibody-based imaging agent was hampered by the lack of knowledge regarding the nature and composition of the IC2 antigen. Here, we show a series of systematic experiments involving classical lipid extraction and chromatography techniques combined with immunochemistry, which led to the identification of sphingomyelin as the target antigen assembled in the form of patches on the β-cell surface. Our findings were verified by modulating SM by enzymatic cleavage, downregulation, upregulation, and perturbation of membrane SM and observation of corresponding changes in IC2 binding. Cholesterol participates in stabilization of these patches, as its removal results in loss of IC2 binding. We believe that these findings have implications for identifying future ligands for the proposed antigen for imaging purposes as well as for potential therapy, as sphingomyelin has been shown to play a role in the apoptotic cascade in pancreatic β cells.
为了制定成功的成像和治疗策略，鉴定β细胞表面标志物是糖尿病研究中必须解决的挑战之一。我们之前表明，IC2 是一种大鼠单克隆 IgM 抗体，可用于通过成像来确定β细胞质量。由于缺乏关于 IC2 抗原的性质和组成的知识，基于抗体的成像剂的进一步开发受到阻碍。在这里，我们展示了一系列涉及经典脂质提取和色谱技术与免疫化学相结合的系统实验，这些实验导致鉴定出鞘磷脂作为靶抗原，以斑块的形式组装在β细胞表面上。我们的发现通过酶切、下调、上调和膜鞘磷脂的扰动来调节 SM，并观察到 IC2 结合的相应变化得到了验证。胆固醇参与这些斑块的稳定，因为其去除会导致 IC2 结合丧失。我们相信这些发现对于鉴定未来用于成像目的的拟议抗原的配体以及潜在的治疗具有重要意义，因为已经表明鞘磷脂在胰岛β细胞的凋亡级联中起作用。
