Boesze-Battaglia Kathleen, Goldberg Andrew F X, Dispoto Janice, Katragadda Madan, Cesarone Gregory, Albert Arlene D
Department of Biochemistry, School of Dental Medicine, 4001 Spruce Street, University of Pennsylvania, Philadelphia 19104, USA.
Exp Eye Res. 2003 Oct;77(4):505-14. doi: 10.1016/s0014-4835(03)00151-9.
Photoreceptor rod cells contain a unique tetraspanin fusion protein known as peripherin/rds. This protein is important in membrane fusion events hypothesized to be essential to disk membrane morphogenesis and disk shedding. In vivo and in vitro fusogenic activity has been mapped to the C-terminal domain of peripherin/rds. Moreover, a fusion peptide domain localized to a 15 amino acid long region (residues 311-325) is essential for mediating lipid bilayer fusion of model membranes. To address the functional and structural properties required for peripherin/rds dependent membrane fusion, constructs of the entire C-terminal domain (residues 284-346) were generated and polypeptides expressed. A wild type-peripherin/rds C-terminal GST fusion construct that included the entire C-terminus (PERCTER) or a C-terminal truncation mutant (PERCTN) were engineered with a thrombin cleavage site. Protein expression was induced in E. coli with IPTG, expressed proteins cleaved from the GST with thrombin and purified to homogeneity on a Superdex 75 column. Purity was confirmed by SDS-PAGE and Western blot analysis. The purified wt C-terminal protein resolved as a monomer under reducing conditions on SDS-PAGE (15%) and was immunoreactive with anti peripherin/rds antibody 2B6 (gift from Dr R. Molday). The purified polypeptide promoted the requisite steps of fusion, membrane destabilization, lipid mixing and aqueous contents mixing. Conversely, the truncation mutant lacking a portion of the fusion domain was unable to promote these steps. A common feature of most membrane fusion proteins is a change in conformation upon membrane association. Structural changes in the C-terminal polypeptide were investigated using far UV CD. The far UV CD spectra of the purified C-terminal polypeptide indicated substantial alpha-helical content in the wt peptide in isotonic aqueous buffer. An increase in intensity of 208 and 222 nm CD bands upon addition of DPC vesicles indicated an increase in alpha-helical content of the polypeptide. These results demonstrate that a purified soluble form of the C-terminus of peripherin/rds can interact with biological phospholipids; moreover, this interaction promotes a conformational change that is most consistent with an increase in alpha-helical content.
光感受器视杆细胞含有一种独特的四跨膜蛋白融合蛋白，称为外周蛋白/视网膜变性慢（peripherin/rds）。该蛋白在膜融合事件中很重要，据推测这对盘膜形态发生和盘膜脱落至关重要。体内和体外的融合活性已定位到外周蛋白/视网膜变性慢的C末端结构域。此外，定位于一个15个氨基酸长区域（第311 - 325位残基）的融合肽结构域对于介导模型膜的脂质双层融合至关重要。为了研究外周蛋白/视网膜变性慢依赖性膜融合所需的功能和结构特性，构建了整个C末端结构域（第284 - 346位残基）的构建体并表达了多肽。一个包含整个C末端的野生型外周蛋白/视网膜变性慢C末端GST融合构建体（PERCTER）或一个C末端截短突变体（PERCTN）被设计带有凝血酶切割位点。用异丙基-β-D-硫代半乳糖苷（IPTG）在大肠杆菌中诱导蛋白表达，用凝血酶从GST上切割表达的蛋白，并在Superdex 75柱上纯化至均一。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹分析确认纯度。纯化的野生型C末端蛋白在还原条件下于SDS-PAGE（15%）上以单体形式解析，并且与抗外周蛋白/视网膜变性慢抗体2B6（由R. Molday博士馈赠）发生免疫反应。纯化的多肽促进了融合、膜去稳定化、脂质混合和水相内容物混合等必要步骤。相反，缺少部分融合结构域的截短突变体无法促进这些步骤。大多数膜融合蛋白的一个共同特征是膜结合后构象发生变化。使用远紫外圆二色性（far UV CD）研究了C末端多肽的结构变化。纯化的C末端多肽在等渗水性缓冲液中的远紫外CD光谱表明野生型肽中存在大量α-螺旋含量。加入二癸基磷脂酰胆碱（DPC）囊泡后，208和222 nm CD带的强度增加，表明多肽的α-螺旋含量增加。这些结果表明，纯化的外周蛋白/视网膜变性慢C末端的可溶性形式可以与生物磷脂相互作用；此外，这种相互作用促进了一种构象变化，这种变化与α-螺旋含量增加最为一致。
