Identification and Functional Characterization of Glycosylation of Recombinant Human Platelet-Derived Growth Factor-BB in Pichia pastoris.
Identification and Functional Characterization of Glycosylation of Recombinant Human Platelet-Derived Growth Factor-BB in Pichia pastoris.
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巴斯德毕赤酵母中重组人血小板衍生生长因子-BB 糖基化的鉴定和功能表征。
DOI：
10.1371/journal.pone.0145419
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Chen W
中科院分区：
文献类型：
--
作者：
Dai M;Yu C;Fang T;Fu L;Wang J;Zhang J;Ren J;Xu J;Zhang X;Chen W
巴斯德毕赤酵母是一种广泛应用的外源蛋白表达系统。然而，翻译后修饰，特别是糖基化，通常阻碍重组蛋白的药物应用，因为蛋白质结构和功能的意外改变。本研究的目的是鉴定重组人血小板源性生长因子-BB（rhPDGF-BB）的糖基化位点，并研究O-连接聚糖对PDGF-BB功能活性的影响。由巴斯德毕赤酵母分泌的PDGF-BB是非常异质的并且含有多种同种型。我们证明了PDGF-BB在分泌过程中是O-糖基化的，并使用糖基化染色和免疫印迹检测了推定的O-糖基化位点。通过定点突变和高分辨率LC/MS分析，我们首次鉴定了C-末端的两个苏氨酸残基为巴斯德毕赤酵母产生的rhPDGF-BB的主要O-糖基化位点。虽然O-糖基化导致异质性蛋白质表达，糖基化位点的去除并不影响rhPDGF-BB促有丝分裂活性。此外，未糖基化的PDGF-BBΔGly突变体表现出与野生型相当的免疫原性。此外，抗PDGF-BBΔGly的抗血清也识别糖基化的PDGF-BB，表明蛋白免疫原性不受糖基化的影响。这些发现阐明了糖基化对PDGF-BB结构和生物活性的影响，并可能有助于在巴斯德毕赤酵母中设计和生产用于药物应用的均匀表达的非糖基化或人型糖基化PDGF-BB。
Yeast Pichia pastoris is a widely used system for heterologous protein expression. However, post-translational modifications, especially glycosylation, usually impede pharmaceutical application of recombinant proteins because of unexpected alterations in protein structure and function. The aim of this study was to identify glycosylation sites on recombinant human platelet-derived growth factor-BB (rhPDGF-BB) secreted by P. pastoris, and investigate possible effects of O-linked glycans on PDGF-BB functional activity. PDGF-BB secreted by P. pastoris is very heterogeneous and contains multiple isoforms. We demonstrated that PDGF-BB was O-glycosylated during the secretion process and detected putative O-glycosylation sites using glycosylation staining and immunoblotting. By site-directed mutagenesis and high-resolution LC/MS analysis, we, for the first time, identified two threonine residues at the C-terminus as the major O-glycosylation sites on rhPDGF-BB produced in P. pastoris. Although O-glycosylation resulted in heterogeneous protein expression, the removal of glycosylation sites did not affect rhPDGF-BB mitogenic activity. In addition, the unglycosylated PDGF-BBΔGly mutant exhibited the immunogenicity comparable to that of the wild-type form. Furthermore, antiserum against PDGF-BBΔGly also recognized glycosylated PDGF-BB, indicating that protein immunogenicity was unaltered by glycosylation. These findings elucidate the effect of glycosylation on PDGF-BB structure and biological activity, and can potentially contribute to the design and production of homogeneously expressed unglycosylated or human-type glycosylated PDGF-BB in P. pastoris for pharmaceutical applications.