Expression, purification, and functional analysis of murine ectodomain fragments of CD8αα and CD8αβ dimers

被引:73
作者
Kern, P
Hussey, RE
Spoerl, R
Reinherz, EL
Chang, HC
机构
[1] Dana Farber Canc Inst, Immunol Lab, Boston, MA 02115 USA
[2] Harvard Univ, Sch Med, Dept Med, Boston, MA 02115 USA
关键词
D O I
10.1074/jbc.274.38.27237
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:27237 / 27243
页数:7
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