Wang P, Raynoschek C, Svensson K, Ljunggren H G
Ludwig Institute for Cancer Research, Stockholm Branch, Box 240, S-171 77 Stockholm, Sweden.
J Biol Chem. 1996 Oct 4;271(40):24830-5. doi: 10.1074/jbc.271.40.24830.
Major histocompatibility complex (MHC) class I molecules are trimolecular complexes consisting of a heavy chain (HC), beta2-microglobulin (beta2m), and a short peptide. Assembly of MHC class I molecules is thought to take place early during biosynthesis. Deficiency in either beta2m or the transporter associated with antigen processing (TAP) results in accumulation of class I molecules in the endoplasmic reticulum (ER). In this study, we have assessed peptide binding to TAP and MHC class I in purified microsomes derived from wild-type, TAP1(-/-), beta2m-/-, and TAP1/beta2m-/- mice using a cross-linkable H-2Kb-binding peptide. This enabled us to study the influence of an intact TAP complex and beta2m on peptide binding to MHC class I and to analyze the stepwise interaction of peptide with TAP and MHC class I molecules. Peptide bound both immature and mature (terminally glycosylated) class I molecules in intact as well as permeabilized microsomes from wild-type mice. Efficient peptide binding to immature class I molecules was also detected in permeabilized microsomes from TAP1(-/-) mice. In contrast, no peptide binding to beta2m-free HC was detected in permeabilized microsomes from beta2m-/- and TAP1/beta2m-/- mice. However, the addition of exogenous beta2m allowed peptide binding to class I in permeabilized beta2m-/- and TAP1/beta2m-/- microsomes. These results demonstrate that a preformed class I HC middle dotbeta2m heterodimer is necessary for efficient peptide binding under physiological conditions. The observed peptide binding to class I in permeabilized TAP1(-/-) microsomes further suggests that TAP1 is not required for peptide binding to class I in the ER. Finally, kinetic studies allowed the demonstration of a stepwise binding of peptide to TAP, subsequent translocation across the ER membrane, a step that required ATP hydrolysis, and binding of peptide to preformed class I HC.beta2m heterodimers.
主要组织相容性复合体（MHC）I类分子是由重链（HC）、β2微球蛋白（β2m）和一个短肽组成的三分子复合物。MHC I类分子的组装被认为在生物合成早期发生。β2m或与抗原加工相关的转运体（TAP）的缺陷会导致I类分子在内质网（ER）中积累。在本研究中，我们使用一种可交联的H-2Kb结合肽，评估了从野生型、TAP1（-/-）、β2m-/-和TAP1/β2m-/-小鼠纯化的微粒体中肽与TAP和MHC I类分子的结合情况。这使我们能够研究完整的TAP复合物和β2m对肽与MHC I类分子结合的影响，并分析肽与TAP和MHC I类分子的逐步相互作用。肽能与野生型小鼠完整的以及通透化微粒体中的未成熟和成熟（终末糖基化）I类分子结合。在TAP1（-/-）小鼠的通透化微粒体中也检测到肽与未成熟I类分子的有效结合。相比之下，在β2m-/-和TAP1/β2m-/-小鼠的通透化微粒体中未检测到肽与无β2m的HC结合。然而，添加外源性β2m可使肽与β2m-/-和TAP1/β2m-/-通透化微粒体中的I类分子结合。这些结果表明，在生理条件下，预先形成的I类HC·β2m异二聚体对于有效的肽结合是必需的。在通透化的TAP1（-/-）微粒体中观察到的肽与I类分子的结合进一步表明，TAP1不是肽在内质网中与I类分子结合所必需的。最后，动力学研究表明肽与TAP逐步结合，随后跨内质网膜转运，这一步需要ATP水解，以及肽与预先形成的I类HC·β2m异二聚体结合。
