Display of the HIV envelope protein at the yeast cell surface for immunogen development.
Display of the HIV envelope protein at the yeast cell surface for immunogen development.
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DOI：
10.1371/journal.pone.0205756
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Dumont ME
中科院分区：
文献类型：
--
作者：
Mathew E;Zhu H;Connelly SM;Sullivan MA;Brewer MG;Piepenbrink MS;Kobie JJ;Dewhurst S;Dumont ME
作为开发具有增强的免疫原性的Env变体形式的一步，我们已经在酵母表面展示系统中以可以进行随机诱变的形式表达了糖蛋白，然后筛选与种系抗体结合增强的形式。为了优化酵母展示的Env蛋白的表达和免疫原性，我们测试了细胞壁锚定的不同方法、来自不同病毒株的gp 120和gp 140 Env的表达、引入旨在稳定Env的突变的效果以及改变Env的N-连接糖基化的程序的效果。我们发现，不同形式的HIV包膜糖蛋白可以有效地表达在酵母细胞表面和gp 140形式的Env有效地切割Kex 2 p，酵母弗林蛋白酶同源物。多种酵母展示的gp 120和gp 140蛋白能够结合针对V3可变环、CD 4结合位点和gp 41近膜区的抗体，包括已知其结合依赖于Env构象和N-连接聚糖的一些抗体。基于抗体识别和对糖苷酶的敏感性，酵母糖基化模式部分模拟哺乳动物细胞中的高甘露糖型N-糖基化。然而，酵母展示的Env不被一些对四级结构敏感的抗Env抗体识别，这表明展示的蛋白质以单体状态存在，或者对于这些抗体，某些区域中的酵母糖基化阻碍了识别或接近。与其他系统中的研究一致，重建的预测的抗Env抗体的未突变前体对酵母展示的包膜蛋白表现出很小的亲和力。
As a step toward the development of variant forms of Env with enhanced immunogenic properties, we have expressed the glycoprotein in the yeast surface display system in a form that can be subjected to random mutagenesis followed by screening for forms with enhanced binding to germline antibodies. To optimize the expression and immunogenicity of the yeast-displayed Env protein, we tested different approaches for cell wall anchoring, expression of gp120 and gp140 Env from different viral strains, the effects of introducing mutations designed to stabilize Env, and the effects of procedures for altering N-linked glycosylation of Env. We find that diverse forms of HIV envelope glycoprotein can be efficiently expressed at the yeast cell surface and that gp140 forms of Env are effectively cleaved by Kex2p, the yeast furin protease homolog. Multiple yeast-displayed gp120 and gp140 proteins are capable of binding to antibodies directed against the V3-variable loop, CD4 binding site, and gp41 membrane-proximal regions, including some antibodies whose binding is known to depend on Env conformation and N-linked glycan. Based on antibody recognition and sensitivity to glycosidases, yeast glycosylation patterns partially mimic high mannose-type N-glycosylation in mammalian cells. However, yeast-displayed Env is not recognized by some anti-Env antibodies sensitive to quaternary structure, suggesting either that the displayed protein exists in a monomeric state or that for these antibodies, yeast glycosylation in certain regions hinders recognition or access. Consistent with studies in other systems, reconstructed predicted unmutated precursors to anti-Env antibodies exhibit little affinity for the yeast-displayed envelope protein.
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