Koopmann J O, Post M, Neefjes J J, Hämmerling G J, Momburg F
Department of Molecular Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Eur J Immunol. 1996 Aug;26(8):1720-8. doi: 10.1002/eji.1830260809.
The major histocompatibility complex (MHC)-encoded transporters associated with antigen processing (TAP) translocate peptides from the cytosol into the lumen of the endoplasmic reticulum (ER) where they associate with MHC class I molecules. The length of class I-binding peptides is usually 8-11 amino acids, but examples of significantly longer peptides have been described. The preferred lengths and upper and lower size limits for peptides translocated by TAP have not been determined in detail because in the currently used test systems, peptides are subject to proteolytic degradation. In the present study, three sets of individual peptides or partially randomized peptide libraries ranging between 6 and 40 residues were used that contained a radiolabeled tyrosine and a consensus sequence for ER-specific N-glycosylation at opposite ends, thus ensuring that only nondegraded peptides were monitored in the transport/glycosylation assay. For three different transporters, rat TAP1/2a, rat TAP1/2u and hTAP, the most efficient ATP-dependent transport was observed for peptides with 8-12 amino acids. Hexamers and longer peptides of up to 40 amino acids were also translocated, albeit less efficiently. For two of the three sets of peptides analyzed, rat TAP1/2a showed a less stringent length selection than rat TAP1/2u and human TAP. The superior transport of the decamer of the TNKT.. Y series was not due to faster degradation or less efficient glycosylation of shorter or longer length variants. A binding assay with TAP-containing microsomes revealed a high affinity for the radiolabeled decamer (KD = 580 nM), while other length variants were clearly inferior in their binding affinities. Thus, TAP binds and preferentially translocates peptides with a length suitable for binding to MHC class I molecules, but peptides that are considerably longer may also be substrates. About 10(5) peptide binding sites per cell equivalent of microsomes were determined, providing an estimate for the number of TAP complexes in the ER membrane.
主要组织相容性复合体（MHC）编码的与抗原加工相关的转运体（TAP）将肽段从细胞质转运至内质网（ER）腔，在那里它们与MHC I类分子结合。I类结合肽的长度通常为8 - 11个氨基酸，但也有明显更长肽段的例子。由于在当前使用的测试系统中肽段会发生蛋白水解降解，所以TAP转运的肽段的优选长度以及上下尺寸限制尚未详细确定。在本研究中，使用了三组分别包含6至40个残基的单个肽段或部分随机化的肽库，这些肽库在两端分别含有一个放射性标记的酪氨酸和一个内质网特异性N - 糖基化的共有序列，从而确保在转运/糖基化测定中仅监测未降解的肽段。对于三种不同的转运体，大鼠TAP1/2a、大鼠TAP1/2u和人TAP，观察到8 - 12个氨基酸的肽段具有最有效的ATP依赖性转运。六聚体以及长达40个氨基酸的更长肽段也能被转运，尽管效率较低。在分析的三组肽段中的两组中，大鼠TAP1/2a显示出比大鼠TAP1/2u和人TAP更宽松的长度选择。TNKT..Y系列十聚体的卓越转运并非由于较短或较长长度变体的更快降解或更低效糖基化。与含TAP的微粒体的结合测定显示对放射性标记的十聚体具有高亲和力（KD = 580 nM），而其他长度变体的结合亲和力明显较低。因此，TAP结合并优先转运长度适合与MHC I类分子结合的肽段，但长得多的肽段也可能是底物。每细胞当量的微粒体确定了约10^5个肽结合位点，为内质网膜中TAP复合物的数量提供了一个估计值。
