毕赤酵母UGGT1的功能验证、亚细胞定位及对外源糖蛋白TOX分泌表达的影响

UGGT1 is the key sensor of glycoprotein folding quality control system in the eukaryotic secretory pathway. Although the UGGT1 recognizes only unfolded glycoproteins and prevents them from entering the secretory pathway, the mechanism of recognition is not understood. According to the crystal structure of UGGT1, it can be speculate that the hydrophobic central cavity of UGGT1 molecular specifically recognizes the hydrophobic region of the unfolded glycoprotein, and the correctly folded glycoprotein has no exposed hydrophobic region and is not recognized. Therefore, the hydrophobicity of glycoprotein substrate is the key to the recognition mechanism of UGGT1, but there is no experimental verification. Studies have shown that glycosylation has an effect on the hydrophobicity of glycoproteins. In this study, a variety of tetanus toxin (TOX) mutants with different glycosylation levels were constructed, and their secretory levels in Pichia pastoris were significantly different. In order to clarify whether UGGT1 is the cause of the secretory difference between TOX mutants and verify the recognition mechanism of UGGT1. We’ll study the subcellular localization, activity analysis in vitro, and through gene knockout and overexpression, complementary expression and construction of mutant UGGT1 to determine its effect on the secretion of TOX as well as the verification of the mechanism of UGGT1 action. Pichia pastoris is an important industrial heterologous protein eukaryotic expression strain. Low secretory level of heterologous protein is the bottleneck of expanding its application range and reducing production cost. The study of UGGT1 gene cloning and functional identification will provide a theoretical basis for improving the secretion level of glycoproteins and some non-glycoproteins and breaking through the bottleneck of production technology.

UGGT1是真核细胞分泌途径中糖蛋白折叠质量监控系统的核心元件，它只识别错折叠糖蛋白，使其无法进入分泌途径，但识别机制尚不明确。由UGGT1的三维结构推测其分子中疏水腔能特异性识别未折叠糖蛋白疏水区，正确折叠糖蛋白无暴露的疏水区而不被识别，因此糖蛋白的疏水性是UGGT1识别机制的关键，但这一推测尚无实验验证。已有研究表明糖基化会影响糖蛋白的疏水性。本研究前期构建的不同糖基化的破伤风毒素（TOX）突变体在毕赤酵母中的分泌水平表现出明显的差异。本项目拟通过对毕赤酵母UGGT1亚细胞定位，体外活性测定，体内基因敲除、过表达、互补表达，定点突变来确定UGGT1对TOX分泌水平的影响，验证其作用机制。毕赤酵母作为重要的工业用外源蛋白真核表达菌株，外源蛋白分泌水平低是其扩大应用、降低成本的瓶颈。对其UGGT1的基因克隆和功能研究将有可能为提高糖蛋白及某些非糖蛋白的分泌水平，突破生产技术瓶颈提供理论基础

UGGT1是真核细胞，包括哺乳动物、昆虫细胞等分泌途径中糖蛋白折叠质量监控系统的核心元件，它只识别错折叠糖蛋白，使其无法进入分泌途径，但识别机制尚不明确。工业用外源蛋白表达宿主毕赤酵母基因组中也含有UGGT1同源基因，但其基因克隆、功能鉴定以及对外源糖蛋白分泌表达量的影响尚未被鉴定和表征。我们前期研究发现，具有不同N-糖基化位点的破伤风毒素（TOX）蛋白的突变体在毕赤酵母中的分泌表达水平表现出显著的差异。本项目通过对这些突变体分泌表达水平的鉴定和比较，以及在TOX重组酵母菌株基础上，敲除和过表达UGGT1，发现UGGT1对TOX突变体的分泌水平和宿主细胞生长无显著影响。为进一步确定这一结论是否适用于其它糖蛋白，本项目构建了一系列骆驼凝乳酶原（Chy）的N-糖基化突变体，并将野生型Chy及其突变体（mChys）分别导入到毕赤酵母中进行分泌表达，并将UGGT1及其同源基因UGGT2同时敲除，结果同样显示UGTT1/2对Chy和mChys的表达均无显著影响。由此可认为UGGT1/2不是影响外源糖蛋白在毕赤酵母中分泌表达水平的关键因素。为继续深入探究这一问题，本项目采用免疫共沉淀与质谱相结合的方法，鉴定了酵母宿主细胞中与Chy和其N-糖基化突变体Chy34的互作蛋白质组（Chy34的分泌表达量显著高于Chy），并进行比较分析，发现与Chy分泌表达水平密切相关的酵母胞内蛋白主要参与内质网定位的蛋白质量监控途径。该途径的关键蛋白BiP能够显著提高Chy的表达量，由此可以认为，BiP是影响外源糖蛋白Chy在毕赤酵母中分泌表达的关键因子之一，这一结论是否适用于其它糖蛋白以及是否还有其它关键影响因子，将在后续工作中进行深入研究。另外，本项目证明了N-糖基化不仅可以提高Chy在毕赤酵母中的表达量，而且不影响酶活，可以为降低Chy的工业化发酵生产成本提供新的研究思路。