Rogers M, Serban D, Gyuris T, Scott M, Torchia T, Prusiner S B
Department of Neurology, University of California, San Francisco 94143.
J Immunol. 1991 Nov 15;147(10):3568-74.
The cellular prion protein (PrPc) is a host-encoded sialoglycoprotein bound to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor. A posttranslationally modified PrP isoform (PrPSc) is a component of the infectious particle causing scrapie and the other prion diseases. mAb have been raised against the protease-resistant core of Syrian hamster (SHa) PrPSc designated PrP 27-30. To map the epitopes within PrP reacting to these antibodies, we have expressed wild-type, chimeric mouse (Mo)/SHa and mutant MoPrP genes using recombinant vaccinia virus systems. The fidelity of the expression of recombinant PrPC was examined using vaccinia viruses expressing SHa-PrPC. It is full length, possesses Asn-linked carbohydrates and is attached to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor that is sensitive to cleavage by phosphatidylinositol-specific phospholipase C. We have tested 18 mAb for their ability to bind to chimeric prion proteins on immunoblots. Three distinct epitopes were identified that mapped to amino acid differences between SHa and MoPrP sequences. The first epitope, recognized by three of the antibodies tested, was defined by methionines at amino acids 108 and 111 in the mouse protein. The second epitope was dependent upon the presence of asparagines at positions 154 and 174 in MoPrP and was recognized by four of the antibodies tested. The third epitope mapped to a single amino acid substitution at residue 138 in MoPrP. mAb raised against SHaPrP 27-30 specific for this epitope are able to bind MoPrPC which has a single amino acid change (Ile to Met) at position 138. Eleven of the 18 antibodies tested mapped to this immunodominant epitope. It is located within a postulated amphipathic helix, a structure associated with immunodominant Ag. Inasmuch as PrPC, in its native form on the cell surface, is detected by the mAb 13A5 (a prototypic antibody of the immunodominant third epitope class), it is likely that this epitope is accessible in the native conformation of this protein.
细胞朊蛋白（PrPc）是一种宿主编码的唾液酸糖蛋白，通过糖基磷脂酰肌醇锚定在细胞膜外表面。一种翻译后修饰的PrP异构体（PrPSc）是导致羊瘙痒症和其他朊病毒疾病的感染性颗粒的组成成分。已经制备了针对叙利亚仓鼠（SHa）PrPSc的蛋白酶抗性核心（命名为PrP 27-30）的单克隆抗体（mAb）。为了绘制PrP中与这些抗体反应的表位图谱，我们使用重组痘苗病毒系统表达了野生型、嵌合小鼠（Mo）/SHa和突变型MoPrP基因。使用表达SHa-PrPC的痘苗病毒检测重组PrPC表达的保真度。它是全长的，具有天冬酰胺连接的碳水化合物，并通过对磷脂酰肌醇特异性磷脂酶C切割敏感的糖基磷脂酰肌醇锚定在细胞膜外表面。我们在免疫印迹上测试了18种mAb与嵌合朊蛋白结合的能力。鉴定出三个不同的表位，它们对应于SHa和MoPrP序列之间的氨基酸差异。第一个表位被测试的三种抗体识别，由小鼠蛋白中第108和111位氨基酸处的甲硫氨酸确定。第二个表位取决于MoPrP中第154和174位天冬酰胺的存在，被测试的四种抗体识别。第三个表位对应于MoPrP中第138位残基的单个氨基酸取代。针对该表位特异性的抗SHaPrP 27-30的mAb能够结合在第138位有单个氨基酸变化（异亮氨酸变为甲硫氨酸）的MoPrPC。测试的18种抗体中有11种对应于这个免疫显性表位。它位于一个假定的两亲性螺旋内，这是一种与免疫显性抗原相关的结构。由于在细胞表面天然形式的PrPC可被mAb 13A5（免疫显性第三表位类别的原型抗体）检测到，这个表位很可能在该蛋白的天然构象中是可及的。
