Li S, Paulsson K M, Sjögren H O, Wang P
Tumor Immunology, Lund University, Box 7031, S-22007 Lund, Sweden.
J Biol Chem. 1999 Mar 26;274(13):8649-54. doi: 10.1074/jbc.274.13.8649.
Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8 T cells. The peptides are generated in the cytosol, then translocated across the membrane of the endoplasmic reticulum by the transporter associated with antigen processing (TAP). TAP is a trimeric complex consisting of TAP1, TAP2, and tapasin (TAP-A) as indicated for human cells by reciprocal coprecipitation with anti-TAP1/2 and anti-tapasin antibodies, respectively. TAP1 and TAP2 are required for the peptide transport. Tapasin is involved in the association of class I with TAP and in the assembly of class I with peptide. The mechanisms of tapasin function are still unknown. Moreover, there has been no evidence for a murine tapasin analogue, which has led to the suggestion that murine MHC class I binds directly to TAP1/2. In this study, we have cloned the mouse analogue of tapasin. The predicted amino acid sequence showed 78% identity to human tapasin with identical consensus sequences of signal peptide, N-linked glycosylation site, transmembrane domain and double lysine motif. However, there was less homology (47%) found at the predicted cytosolic domain, and in addition, mouse tapasin is 14 amino acids longer than the human analogue at the C terminus. This part of the molecule may determine the species specificity for interaction with MHC class I or TAP1/2. Like human tapasin, mouse tapasin binds both to TAP1/2 and MHC class I. In TAP2-mutated RMA-S cells, both TAP1 and MHC class I were coprecipitated by anti-tapasin antiserum indicative of association of tapasin with TAP1 but not TAP2. With crosslinker-modified peptides and purified microsomes, anti-tapasin coprecipitated both peptide-bound MHC class I and TAP1/2. In contrast, anti-calreticulin only coprecipitated peptide-free MHC class I molecules. This difference in association with peptide-loaded class I suggests that tapasin functions later than calreticulin during MHC class I assembly, and controls peptide loading onto MHC class I molecules in the endoplasmic reticulum.
主要组织相容性复合体（MHC）I类分子将抗原肽呈递给CD8 T细胞。这些肽在胞质溶胶中产生，然后通过与抗原加工相关的转运体（TAP）跨内质网膜转运。TAP是一种三聚体复合物，由TAP1、TAP2和塔帕辛（TAP-A）组成，在人类细胞中分别通过与抗TAP1/2和抗塔帕辛抗体的相互共沉淀得以证实。TAP1和TAP2是肽转运所必需的。塔帕辛参与I类分子与TAP的结合以及I类分子与肽的组装。塔帕辛的功能机制仍然未知。此外，尚无小鼠塔帕辛类似物的证据，这导致有人提出小鼠MHC I类分子直接与TAP1/2结合。在本研究中，我们克隆了小鼠塔帕辛类似物。预测的氨基酸序列与人类塔帕辛有78%的同一性，信号肽、N-连接糖基化位点、跨膜结构域和双赖氨酸基序的共有序列相同。然而，在预测的胞质结构域发现的同源性较低（47%），此外，小鼠塔帕辛在C末端比人类类似物长14个氨基酸。分子的这一部分可能决定了与MHC I类分子或TAP1/2相互作用的物种特异性。与人类塔帕辛一样，小鼠塔帕辛既与TAP1/2结合，也与MHC I类分子结合。在TAP2突变的RMA-S细胞中，抗塔帕辛抗血清可共沉淀TAP1和MHC I类分子，表明塔帕辛与TAP1而非TAP2相关联。使用交联剂修饰的肽和纯化的微粒体，抗塔帕辛可共沉淀肽结合的MHC I类分子和TAP1/2。相比之下，抗钙网蛋白仅可共沉淀无肽的MHC I类分子。与负载肽的I类分子结合的这种差异表明，在MHC I类分子组装过程中，塔帕辛的作用晚于钙网蛋白，并在内质网中控制肽加载到MHC I类分子上。
