Department of Chemistry, University of Kansas, Lawrence, Kansas, United States.
J Proteome Res. 2013 Mar 1;12(3):1223-34. doi: 10.1021/pr300870t. Epub 2013 Feb 20.
Glycosylation plays an essential role in regulating protein function by modulating biological, structural, and therapeutic properties. However, due to its inherent heterogeneity and diversity, the comprehensive analysis of protein glycosylation remains a challenge. As part of our continuing effort in the analysis of glycosylation profiles of recombinant HIV-1 envelope-based immunogens, we evaluated and compared the host-cell specific glycosylation pattern of recombinant HIV-1 surface glycoprotein, gp120, derived from clade C transmitted/founder virus 1086.C expressed in Chinese hamster ovary (CHO) and human embryonic kidney containing T antigen (293T) cell lines. We used an integrated glycopeptide-based mass mapping workflow that includes a partial deglycosylation step described in our previous study with the inclusion of a fragmentation technique, electron transfer dissociation (ETD), to complement collision-induced dissociation. The inclusion of ETD facilitated the analysis by providing additional validation for glycopeptide identification and expanding the identified glycopeptides to include coverage of O-linked glycosylation. The site-specific glycosylation analysis shows that the transmitted/founder 1086.C gp120 expressed in CHO and 293T displayed distinct similarities and differences. For N-linked glycosylation, two sites (N386 and N392) in the V4 region were populated with high mannose glycans in the CHO cell-derived 1086.C gp120, while these sites had a mixture of high mannose and processed glycans in the 293T cell-derived 1086.C gp120. Compositional analysis of O-linked glycans revealed that 293T cell-derived 1086.C gp120 consisted of core 1, 2, and 4 type O-linked glycans, while CHO cell-derived 1086.C exclusively consisted of core 1 type O-linked glycans. Overall, glycosylation site occupancy of the CHO and 293T cell-derived 1086.C gp120 showed a high degree of similarity except for one site at N88 in the C1 region. This site was partially occupied in 293T-gp120 but fully occupied in CHO-gp120. Site-specific glycopeptide analysis of transmitted/founder 1086.C gp120 expressed in CHO cells revealed the presence of phosphorylated glycans, while 293T cell-produced 1086.C gp120 glycans were not phosphorylated. While the influence of phosphorylated glycans on immunogenicity is unclear, distinguishing host-cell specific variations in glycosylation profiles provide insights into the similarity (or difference) in recombinant vaccine products. While these differences had minimal effect on envelope antigenicity, they may be important in considering immunogenicity and functional capacities of recombinant envelope proteins produced in different expression systems.
糖基化通过调节生物、结构和治疗特性在调节蛋白质功能方面发挥着重要作用。然而，由于其固有异质性和多样性，全面分析蛋白质糖基化仍然是一个挑战。作为我们分析重组 HIV-1 包膜基免疫原糖基化谱的持续努力的一部分，我们评估并比较了源自 C 型传播/创始病毒 1086.C 的重组 HIV-1 表面糖蛋白 gp120 的宿主细胞特异性糖基化模式，该蛋白在中华仓鼠卵巢 (CHO) 和含有 T 抗原的人胚肾 293T 细胞系中表达。我们使用了一种综合的基于糖肽的质量映射工作流程，其中包括我们之前研究中描述的部分去糖基化步骤，并纳入了碎裂技术电子转移解离 (ETD)，以补充碰撞诱导解离。ETD 的纳入通过为糖肽鉴定提供额外的验证，并扩展鉴定的糖肽以包括 O 连接糖基化的覆盖范围，从而促进了分析。位点特异性糖基化分析表明，在 CHO 和 293T 中表达的传播/创始 1086.C gp120 显示出明显的相似性和差异。对于 N 连接糖基化，V4 区域的两个位点 (N386 和 N392) 被 CHO 细胞衍生的 1086.C gp120 中的高甘露糖糖基化占据，而这些位点在 293T 细胞衍生的 1086.C gp120 中具有高甘露糖和加工糖基化的混合物。O 连接糖的组成分析表明，293T 细胞衍生的 1086.C gp120 由核心 1、2 和 4 型 O 连接糖组成，而 CHO 细胞衍生的 1086.C 仅由核心 1 型 O 连接糖组成。总体而言，除了 C1 区的 N88 位点外，CHO 和 293T 细胞衍生的 1086.C gp120 的糖基化位点占据程度非常相似。该位点在 293T-gp120 中部分占据，但在 CHO-gp120 中完全占据。在 CHO 细胞中表达的传播/创始 1086.C gp120 的位点特异性糖肽分析显示存在磷酸化聚糖，而 293T 细胞产生的 1086.C gp120 聚糖未磷酸化。虽然磷酸化聚糖对免疫原性的影响尚不清楚，但区分宿主细胞特异性糖基化谱的差异可以深入了解重组疫苗产品的相似性（或差异）。虽然这些差异对包膜抗原性的影响很小，但在考虑不同表达系统产生的重组包膜蛋白的免疫原性和功能能力时，它们可能很重要。
