Mechanism of glycoprotein sorting and transport in the cells and its application for drug design

用HA标记的VIPL或VIP36在293T细胞中表达FLAG标记的ERGIC-53，并对细胞裂解产物进行下拉实验。用ERGIC-53沉淀HA-VIPL和HA-VIP36表明这些蛋白与ERGIC-53相互作用。ERIGIC-53在钙离子存在下也能与MCFD2相互作用，但VIP36和VIPL都不能。表面等离子体共振(SPR)实验表明，ERGIC-53与MCFD2之间的相互作用对钙离子很敏感，尤其是在0.1~0.2 mM的浓度范围内。来自F5F8D患者的MCFD2突变体对ERGIC-53的Ka值降低了三到四个数量级。超速离心分析和可溶性VIP36的SPR实验表明，VIP36的糖结合活性受低聚物形成的调节，这也解释了VIP36对糖链亲和力的增强。进一步用YGFP和EGFP融合的VIP36进行Flet分析表明它们在细胞内相互作用。相反，ERGIC-53…的糖结合能力MCFD2的相互作用增强了更多。ERGIC-53与MCFD2的相互作用依赖于钙离子浓度。经VIP36下拉实验，确定与VIP36相互作用的新蛋白为伴侣蛋白Bip。这种相互作用具有不同于伴侣底物的特征。免疫电子显微镜分析表明，VIP36与Bip的结合发生在内质网。有趣的是，与Bip一起沉淀的VIP36具有抗Endo H的糖链，这表明逆行运输的VIP36可以与内质网中的Bip结合。PE标记的可溶性ERGIC-53、VIP36和VIPL四聚体与HeLaS3细胞的结合表明它们与高甘露糖型糖链有较好的结合。通过一组高甘露糖型糖衍生物的抑制分析，证明了它们的精确特异性。Vip36和vipl识别Manα2manα2man臂，其非还原末端的糖基化取消了它们的结合。ERGIC-53优先与内质网α-甘露糖苷酶I的产物M8B亚型结合，提示新合成的糖蛋白可能通过VIPL从钙粘蛋白转移到ERGIC-53。在HeLaS3细胞中，融合了CD8跨膜区和胞内区ζ的VIP36得到了表达。细胞表面展示系统使我们能够从随机突变的VIP36文库中挑选出具有不同糖结合特异性的突变VIP36的克隆。较少

FLAG-tagged ERGIC-53 was expressed with HA-tagged VIPL or VIP36 in 293T cells and pull down assay was performed from the cell lysates. HA-VIPL and HA-VIP36 were precipitated with ERGIC-53 indicating that these proteins interact with ERGIC-53. ERGIC-53 also interacted with MCFD2 in the presence of calcium, but neither VIP36 nor VIPL did. Surface plasmon resonance (SPR) experiment demonstrated that the interaction between ERGIC-53 and MCFD2 was susceptible to calcium ions, especially the concentration between 0.1 and 0.2 mM. MCFD2 mutants derived from F5F8D patients had three or four order of magnitude lower Ka values for ERGIC-53. Ultracentrifugation analysis and SPR experiment of soluble VIP36 indicating that sugar-binding activity of VIP36 was regulated by oligomer formation dependent on pH, which explains enhancement of avidity to sugar chains. Further FLET analysis using YGFP- and EGFP-fused VIP36 showed their interactions in the cells. In contrast, sugar-binding ability of ERGIC-53 … More was enhanced by MCFD2 interactions. The interaction of ERGIC-53 with MCFD2 depends on calcium concentration. By pull-down assay with VIP36, novel protein constitutively interacted with VIP36 was identified as a chaperone BiP. The interaction had different characteristics from those of chaperone substrates. Immunoelectron microscopic analysis showed that the binding of VIP36 to BiP was occurred especially in the endoplasmic reticulum (ER). Interestingly, VIP36 precipitated with BiP had sugar chains resistant to endo H suggesting that VIP36 transported retrogradely could bind to BiP in the ER. The binding of PE-labeled soluble ERGIC-53, VIP36, and VIPL tetramers to HeLaS3 cells showed their preferable binding to high mannose-type sugar chains. Inhibition analysis by a panel of high mannose-type sugar derivatives demonstrated the precise specificities of them. VIP36 and VIPL recognize Man α 2Man α 2Man arm and glucosylation of its non-reducing terminal abrogated their binding. ERGIC-53 preferencially bound to M8b isoform, which is a product of ER α-mannosidase I, indicating that newly synthesized glycoproteins may transferred from calnexin to ERGIC-53 via VIPL. VIP36 fused with CD8 transmembrane domain followed by intracellular domain of CD3ζ was expressed in HeLaS3 cells. The cell surface display system enabled us to pick up clones of mutated VIP36 having distinct sugar-binding specificities from random mutated VIP36 libraries. Less