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

FLAG标记的ERGIC-53与HA标记的VIPL或VIP 36在293 T细胞中表达，并从细胞裂解物进行下拉测定。HA-VIPL和HA-VIP 36用ERGIC-53沉淀，表明这些蛋白质与ERGIC-53相互作用。ERGIC-53在钙存在下也与MCFD 2相互作用，但VIP 36和VIPL都不与MCFD 2相互作用。表面等离子体共振（SPR）实验表明ERGIC-53与MCFD 2之间的相互作用对钙离子敏感，特别是在0.1 ~ 0.2 mM浓度范围内。F5 F8 D患者来源的MCFD 2突变体对ERGIC-53的Ka值降低了3 ~ 4个数量级。可溶性VIP 36的超离心分析和SPR实验表明，VIP 36的糖结合活性受依赖于pH的寡聚体形成的调节，这解释了对糖链的亲合力的增强。使用YGFP-和EGFP-融合的VIP 36的进一步FLET分析显示它们在细胞中的相互作用。相反，ERGIC-53的糖结合能力 关于我们 通过MCFD 2相互作用增强。ERGIC-53与MCFD 2的相互作用取决于钙浓度。通过与VIP 36的pull-down分析，鉴定了与VIP 36组成型相互作用的新蛋白为伴侣BiP。这种相互作用具有与分子伴侣底物不同的特点。免疫电镜分析表明，VIP 36与BiP的结合主要发生在内质网。有趣的是，用BiP沉淀的VIP 36具有对endo H有抗性的糖链，这表明逆行转运的VIP 36可以在ER中结合BiP。PE标记的可溶性ERGIC-53、VIP 36和VIPL四聚体与HeLaS 3细胞的结合显示它们优选与高甘露糖型糖链结合。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。VIP 36和VIPL识别Man α 2 Man α 2 Man臂，其非还原末端的糖基化消除了它们的结合。ERGIC-53优先结合ER α-甘露糖苷酶I的产物M8 b亚型，表明新合成的糖蛋白可能通过VIPL从钙连接蛋白转移到ERGIC-53。在HeLaS 3细胞中表达了与CD 8跨膜结构域融合的VIP 36，随后是CD 3的胞内结构域。细胞表面展示系统使我们能够从随机突变的VIP 36文库中挑选具有不同糖结合特异性的突变的VIP 36的克隆。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