Expression, purification, and functional analysis of murine ectodomain fragments of CD8αα and CD8αβ dimers
Expression, purification, and functional analysis of murine ectodomain fragments of CD8αα and CD8αβ dimers
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DOI：
10.1074/jbc.274.38.27237
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发表时间：
1999-09-17
影响因子：
4.8
通讯作者：
Chang, HC
中科院分区：
文献类型：
--
作者：
Kern, P;Hussey, RE;Chang, HC
使用亮氨酸拉链策略，在中国仓鼠卵巢细胞或糖基化变体Lec3.2.8.1细胞中表达可溶性小鼠CD 8 α α和CD 8 α β二聚体（对应于配对胞外域（CD 8（f））或其各自的Ig样结构域（CD 8）组分）作为分泌蛋白。如通过BIAcore测量的，CD 8 α α（f）对H-2 K（B）的亲和力显示类似于65 μ M K-d，类似于CD 8 α β（f）的亲和力。与该结果一致，CD 8 α α（f）以及CD 8 α β（f）以相当的剂量依赖性方式阻断N15 T细胞受体转基因溶细胞性T细胞的效应子功能。此外，Lec3.2.8.1产生的和中国仓鼠卵巢产生的CD 8同源二聚体和异源二聚体在抑制试验中均具有活性。这些结果表明，免疫球蛋白样结构域的CD 8分子本身是足以阻止必要的跨膜CD 8-pMHC之间的相互作用的溶细胞性T淋巴细胞和靶细胞。此外，考虑到pMHC的共受体亲和力的相似性，研究结果表明，CD 8 α β与CD 8 α α共受体在T细胞上的功能效率更高，这与它们的膜结合茎区和/或细胞内片段的差异有关。如最近对于sCD 8 α α所示，由该表达系统产生的去糖基化蛋白的产率、纯度和均一性足以用于结晶和原子分辨率的X射线衍射。
Soluble mouse CD8 alpha alpha and CD8 alpha beta dimers corresponding to the paired ectodomains (CD8(f)) or their respective component Ig-like domains (CD8) were expressed in Chinese hamster ovary cells or the glycosylation variant Lec3.2.8.1 cells as secreted proteins using a leucine zipper strategy. The affinity of CD8 alpha alpha(f) for H-2K(b) as measured by BIAcore revealed a similar to 65 mu M K-d, similar to that of CD8 alpha beta(f). Consistent with this result, CD8 alpha alpha(f) as well as CD8 alpha beta(f) blocked the effector function of N15 T cell receptor transgenic cytolytic T cells in a comparable, dose-dependent fashion. Furthermore, both Lec3.2.8.1-produced and Chinese hamster ovary-produced CD8 homodimers and heterodimers were active in the inhibition assay. These results suggest that the Ig-like domains of CD8 molecules are themselves sufficient to block the requisite transmembrane CD8-pMHC interaction between cytolytic T lymphocytes and target cells. Moreover, given the similarities in co-receptor affinities for pMHC, the findings suggest that the greater efficiency of CD8 alpha beta versus CD8 alpha alpha co-receptor function on T cells is linked to differences within their membrane-bound stalk regions and/or intracellular segments. As recently shown for sCD8 alpha alpha, the yield, purity and homogeneity of the deglycosylated protein resulting from this expression system is sufficient for crystallization and x-ray diffraction at atomic resolution.