Demotz S, Danieli C
Basel Institute for Immunology, Switzerland.
Mol Immunol. 1993 Dec;30(18):1623-32. doi: 10.1016/0161-5890(93)90435-e.
To investigate how class II major histocompatibility complex (MHC) molecules are released from complexes with invariant chain (Ii), we studied a 25 kDa Ii fragment (p25) detected by Western blotting in affinity chromatographed DR preparations. The p25 species corresponds to the non-transmembrane, C-terminal Ii fragment 107-232. It was determined by gel filtration chromatography that the p25 fragment has a relative molecular mass (M(r)) of 46 kDa, indicating that this Ii fragment is present as dimers in B cell lysate. Two independent approaches were followed to demonstrate that generation of the p25 fragment takes place shortly before, or concomitantly to, loading of class II MHC molecules with antigen fragments. First, it was shown that a fraction of the p25 molecules is resistant to endoglycosidase H digestion, indicating that the p25 polypeptide can exit the endoplasmic reticulum (ER) and is transported at least to the cis-Golgi compartment. Second, treatment of class II MHC-positive B cells with leupeptin blocks the formation of p25, further indicating that this Ii fragment is generated in the endosomal compartment. The role of the p25 Ii species in the assembly of complexes between peptides and DR molecules was then investigated. While the p25 fragment was totally unable to prevent binding of a synthetic tetanus toxin peptide to DR molecules, the full-length Ii species (p33/35) effectively inhibited peptide binding, indicating that, by contrast with the p33/35 species, the p25 fragment does not occlude the peptide binding site of DR molecules. We concluded that the p25 fragment, which is produced by proteolytic cleavage at the N-terminal side of Methionine 107, has a decreased affinity for DR molecules as compared with the p33/35 species. Dissociation of the p25 fragment from DR molecules exposes the peptide binding site, which is thus made accessible for antigen fragments. This model of the complexes between DR and antigen fragments proposes that a stretch of Ii prevents peptide binding by occluding the peptide binding site without directly occupying it.
为了研究II类主要组织相容性复合体（MHC）分子是如何从与恒定链（Ii）的复合物中释放出来的，我们研究了在亲和层析的DR制剂中通过蛋白质免疫印迹法检测到的一个25 kDa的Ii片段（p25）。p25对应于非跨膜的C末端Ii片段107 - 232。通过凝胶过滤色谱法测定，p25片段的相对分子质量（M(r)）为46 kDa，表明该Ii片段在B细胞裂解物中以二聚体形式存在。我们采用了两种独立的方法来证明p25片段的产生发生在II类MHC分子加载抗原片段之前不久或同时。首先，结果表明一部分p25分子对内切糖苷酶H消化具有抗性，这表明p25多肽可以离开内质网（ER）并至少转运到顺式高尔基体区室。其次，用亮肽素处理II类MHC阳性B细胞会阻断p25的形成，进一步表明该Ii片段是在内体区室中产生的。然后研究了p25 Ii片段在肽与DR分子之间复合物组装中的作用。虽然p25片段完全无法阻止合成破伤风毒素肽与DR分子的结合，但全长Ii片段（p33/35）有效地抑制了肽的结合，这表明与p33/35片段相比，p25片段不会封闭DR分子的肽结合位点。我们得出结论，在甲硫氨酸107的N末端侧通过蛋白水解切割产生的p25片段与p33/35片段相比，对DR分子的亲和力降低。p25片段与DR分子的解离暴露了肽结合位点，从而使抗原片段能够接近该位点。DR与抗原片段之间复合物的这种模型表明，一段Ii通过封闭肽结合位点而不直接占据它来阻止肽的结合。
