Dynamics of molecules involved in antigen presentation: effects of fixation
Dynamics of molecules involved in antigen presentation: effects of fixation
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DOI：
10.1016/s0161-5890(99)00091-7
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发表时间：
1999-08-01
影响因子：
3.6
通讯作者：
Roess, DA
中科院分区：
文献类型：
--
作者：
Barisas, BG;Wade, WF;Roess, DA
通过MHC II类分子的抗原呈递可以通过在测定之前多聚甲醛固定抗原呈递细胞来增强。这种处理可能会使膜蛋白聚集，从而稳定和加强参与细胞间合作的瞬时蛋白质-蛋白质相互作用。用荧光光漂白恢复法和时间分辨磷光各向异性法分别检测了不同浓度多聚甲醛固定的A20细胞上MHC Ⅱ类抗原I-A（d）的侧向和旋转动力学。探针是MKD 6 Fab片段的四甲基罗丹明和赤藓红缀合物。多聚甲醛浓度的增加导致I-A（d）在4 ℃时的极限各向异性从未处理细胞的0.042的值逐渐增加，表明大聚集体形成，同时保持29 μ s的旋转相关时间不变，这是未扰动分子的量度。另一方面，平移扩散常数从近似2 x 10（-10）cm（2）s（-1）降低，而部分回收率保持不变，约为40- 50%。综上所述，这些结果表明，固定将II类分子彼此交联或与其他膜蛋白交联成足够大的结构（> 500，000 kDa），以明显的尺寸依赖性速率进行扩散。固定效果II类旋转和横向扩散的半最大多聚甲醛浓度为0.2%，可能的关系II类的生物效应器功能和固定诱导的聚集体的物理尺寸进行了讨论。(C)1999爱思唯尔科技有限公司。保留所有权利。
Antigen presentation by MHC class II molecules can be enhanced by paraformaldehyde fixation of antigen-presenting cells prior to assay. This treatment might be expected to aggregate membrane proteins and thus stabilize and strengthen transient protein-protein interactions involved in intercellular cooperation. Lateral and rotational dynamics of the MHC class II antigen I-A(d) on A20 cells fixed with various concentrations of paraformaldehyde were examined by fluorescence photobleaching recovery and time-resolved phosphorescence anisotropy, respectively. Probes were tetramethylrhodamine and erythrosin conjugates of MKD6 Fab fragments. Increasing concentrations of paraformaldehyde led to a progressive increase in the limiting anisotropy of I-A(d) at 4 degrees C from the value of 0.042 for untreated cells, indicative of large aggregate formation, while leaving the rotational correlation time of 29 mu s unchanged, a measure of the unperturbed molecule. On the other hand, the translational diffusion constants decreased from similar to 2 x 10(-10) cm(2) s(-1), while me fractional recovery remained unchanged at about 40-50%. Taken together, these results suggest that fixation crosslinks class II molecules to each other or to other membrane proteins into structures large enough (> 500,000 kDa) to diffuse translationally with perceptibly size-dependent rates. The fixation effects on both class II rotation and lateral diffusion were half-maximal at paraformaldehyde concentrations of similar to 0.2%, Possible relations between the biological effector functions of class II and the physical sizes of fixation-induced aggregates are discussed. (C) 1999 Elsevier Science Ltd. All rights reserved.