Viguier M, Dornmair K, Clark B R, McConnell H M
Stauffer Laboratory of Physical Chemistry, Stanford University, CA 94305.
Proc Natl Acad Sci U S A. 1990 Sep;87(18):7170-4. doi: 10.1073/pnas.87.18.7170.
The binding of a chicken ovalbumin peptide (residues 323-339), Ova-(323-339), to I-Ad molecules was investigated in vitro and in vivo. By using antigenic peptides labeled either with a hapten or with fluorescein, complexes formed in vitro between I-Ad and antigenic peptides were detected by Western blot analysis with an antibody recognizing the hapten 7-nitrobenzo-2-oxa-1,3-diazole and by scanning gels for fluorescence emitted by fluoresceinated peptide. Both techniques reveal that Ova-(323-339) binds not only to I-Ad alpha/beta heterodimers and separated alpha and beta chains but also to complexes of higher molecular mass. Additional analysis shows that one of these additional complexes contains I-Ad heterodimers, antigenic peptides, and also invariant chain. To explore the physiological role of these complexes, cells were incubated with haptenated peptide and the I-Ad-peptide complexes formed in vivo were purified by affinity chromatography using hapten-specific antibody. The complexes formed migrate with a significantly higher apparent molecular mass than the alpha/beta heterodimers. A band at 180 kDa contained the alpha/beta heterodimer, the antigenic peptide, and the invariant chain. These results show that in vivo high molecular mass complexes formed by the I-Ad heterodimer and the invariant chain bind antigenic peptides.
对鸡卵清蛋白肽（第323 - 339位氨基酸残基）Ova-(323 - 339)与I-Ad分子的结合进行了体外和体内研究。通过使用用半抗原或荧光素标记的抗原肽，利用识别半抗原7 - 硝基苯并 - 2 - 恶唑 - 1,3 - 二氮杂茂的抗体进行蛋白质印迹分析，以及扫描凝胶以检测荧光素化肽发出的荧光，来检测体外I-Ad与抗原肽形成的复合物。这两种技术均表明，Ova-(323 - 339)不仅与I-Adα/β异二聚体以及分离的α链和β链结合，还与更高分子量的复合物结合。进一步分析表明，这些额外复合物之一包含I-Ad异二聚体、抗原肽以及恒定链。为了探究这些复合物的生理作用，将细胞与半抗原化肽一起孵育，然后使用半抗原特异性抗体通过亲和层析纯化体内形成的I-Ad - 肽复合物。形成的复合物迁移时的表观分子量明显高于α/β异二聚体。一条180 kDa的条带包含α/β异二聚体、抗原肽和恒定链。这些结果表明，在体内由I-Ad异二聚体和恒定链形成的高分子量复合物结合抗原肽。
