Anderson Meagan E, Siahaan Teruna J
Department of Pharmaceutical Chemistry, The University of Kansas, Simons Research Laboratories, Lawrence, Kansas 66047, USA.
Pharm Res. 2003 Oct;20(10):1523-32. doi: 10.1023/a:1026188212126.
Peptides derived from the Domain 1 of the adhesion molecule ICAM-1(1-21) are being developed as targeting ligands for LFA-1 receptors expressed on activated T cells. This work aims to elucidate the binding and internalization of ICAM-1-derived cyclic peptides (cIBL, cIBC, and cIBR) to LFA-1.
Ninety-six-well plates coated with soluble LFA-1 (sLFA-1) were used to characterize the binding of FITC-labeled peptide. An anti-CD11a antibody to the I-domain of LFA-1 was used to inhibit the binding of these peptides, which was quantified using a fluorescence plate reader. An unrelated FITC-labeled cyclic peptide was used as a negative control, and PE-labeled anti-CD11a antibodies (PE-R3.2 and PE-R7.1) were used as positive controls. Peptide binding to cell surface LFA-1 was visualized using colocalization of FITC-cIBR peptide and PE-labeled anti-CD18 antibody (LFA-1 beta-subunit) on SKW-3 T cells by fluorescent microscopy. Inhibition of ICAM-1 binding to LFA-1 by peptides was evaluated using a Biacore assay. Binding and internalization of FITC-labeled peptides were evaluated by flow cytometry and confocal microscopy at 4 degrees C and 37 degrees C.
These FITC-labeled cyclic peptides bind to sLFA-1 and can be blocked by an anti-CD11a antibody to the I-domain, suggesting that their binding site is on the I-domain of LFA-1. The FITC-cIBR peptide was localized with an anti-CD18 antibody on the surface of T cells, indicating that the FITC-cIBR peptide binds to LFA-1 on the cell surface. Flow cytometry and confocal microscopy demonstrated that FITC-labeled peptides were internalized in a temperature-dependent manner. Biacore analysis demonstrated that these peptides did not inhibit sICAM-1 from binding to immobilized sLFA-1. However, the binding properties of the soluble forms of LFA-1 and ICAM-1 may not correlate to their interaction at the cell surface.
Cyclic ICAM-1-derived peptides (cIBL, cIBC, and cIBR) bind to the I-domain of LFA-1 and are internalized by LFA-1 receptors on the surface of T cells. Therefore, these peptides could be used to target and deliver drugs to the cytoplasmic domain of T cells.
源自黏附分子细胞间黏附分子-1（ICAM-1）第1结构域的肽（1-21）正被开发为活化T细胞上表达的淋巴细胞功能相关抗原-1（LFA-1）受体的靶向配体。这项工作旨在阐明源自ICAM-1的环肽（cIBL、cIBC和cIBR）与LFA-1的结合及内化过程。
使用包被有可溶性LFA-1（sLFA-1）的96孔板来表征异硫氰酸荧光素（FITC）标记肽的结合情况。使用针对LFA-1 I结构域的抗CD11a抗体来抑制这些肽的结合，通过荧光酶标仪对其进行定量。使用一种不相关的FITC标记环肽作为阴性对照，使用藻红蛋白（PE）标记的抗CD11a抗体（PE-R3.2和PE-R7.1）作为阳性对照。通过荧光显微镜观察FITC-cIBR肽与PE标记的抗CD18抗体（LFA-1β亚基）在SKW-3 T细胞上的共定位，以可视化肽与细胞表面LFA-1的结合。使用生物传感器分析评估肽对ICAM-1与LFA-1结合的抑制作用。在4℃和37℃下，通过流式细胞术和共聚焦显微镜评估FITC标记肽的结合和内化情况。
这些FITC标记的环肽与sLFA-1结合，并且可以被针对I结构域的抗CD11a抗体阻断，这表明它们的结合位点在LFA-1的I结构域上。FITC-cIBR肽与抗CD18抗体在T细胞表面共定位，表明FITC-cIBR肽与细胞表面的LFA-1结合。流式细胞术和共聚焦显微镜显示FITC标记的肽以温度依赖的方式被内化。生物传感器分析表明这些肽不会抑制可溶性细胞间黏附分子-1（sICAM-1）与固定化sLFA-1的结合。然而，LFA-1和ICAM-1可溶性形式的结合特性可能与其在细胞表面的相互作用不相关。
源自ICAM-1的环肽（cIBL、cIBC和cIBR）与LFA-1的I结构域结合，并被T细胞表面的LFA-1受体内化。因此，这些肽可用于将药物靶向递送至T细胞的胞质结构域。
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