Site-specific N-glycosylation and oligosaccharide structures of recombinant HIV-1 gp120 derived from a baculovirus expression system.
Site-specific N-glycosylation and oligosaccharide structures of recombinant HIV-1 gp120 derived from a baculovirus expression system.
复制标题
源自杆状病毒表达系统的重组 HIV-1 gp120 的位点特异性 N-糖基化和寡糖结构。
DOI：
10.1021/bi00092a019
复制
发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Cummings,RD
中科院分区：
文献类型：
--
作者：
Yeh,JC;Seals,JR;Murphy,CI;vanHalbeek,H;Cummings,RD
1993年8月5日 * 摘要：我们报告了由杆状病毒表达系统产生的HIV-1 BH 8分离株的重组gp 120（rgp 120）的TV-连接寡糖的完整结构和TV-糖基化的位点特异性。糖肽来源于完整的rgp 120或溴化氰产生的rgp 120片段的胰蛋白酶解物，通过其与伴刀豆球蛋白A-琼脂糖凝胶的结合分离，并通过反相HPLC纯化。用PNGase F处理分离的糖肽，释放碳水化合物部分，同时将Asn转化为Asp，并通过氨基酸分析和/或肽测序进行鉴定。我们的研究结果表明，所有22个潜在的IV-糖基化位点的rgp 120序列被利用。我们在rgpl 20中没有检测到N-乙酰半乳糖胺，表明该糖蛋白缺乏典型的O-连接的寡糖。为了研究糖基化位点的寡糖结构，我们确定了每个位点的碳水化合物组成，并通过1H-NMR光谱和使用高pH阴离子交换色谱法的寡糖图谱表征了寡糖。甘露糖和N-乙酰葡糖胺是在完整的rgp 120中观察到的唯一糖，在单个糖肽中也是如此。所有来源于rgp 120的糖肽均含有高甘露糖型IV-连接的寡糖，范围从GlcNAc 2 Mans到GlcNAc 2 Mans。然而，不同的糖基化位点显示了不同程度的高甘露糖型寡糖的加工，如GlcNAc 2 Mang-9与GlcNAc2Man5_7的比例所表征的。这些结果表明，rgp 120在杆状病毒表达系统中的糖基化发生在所有可能的位点上，并且在寡糖结构上是位点特异性的。这两种糖蛋白通过前体蛋白gp 160的蛋白水解切割产生，并且通过非共价缔合保持缔合（Robey等人，1985年）。Gp 120在病毒感染的第一步中起重要作用，因为它充当T细胞受体CD 4的高亲和力配体（Dalgleish et al. 1984; Klatzman等人，1984年）。在与CD 4结合后，gp 41然后能够通过其IV末端疏水结构域介导病毒-细胞融合，随后病毒颗粒被释放到细胞中。Gp 120是一种高度糖基化的蛋白质，其约50%的分子量由碳水化合物贡献（Allan et
Revised Manuscript Received August 5, 1993* abstract: We report the complete structures of the TV-linked oligosaccharides and the site-specificity of the TV-glycosylation of recombinant gpl20 (rgpl20) of the HIV-1 BH8 isolate produced by a baculovirus expression system. Glycopeptides derived from the tryptic digests of intact rgp 120 or of cyanogen bromide-generated fragments of rgp 120 were isolated by their binding to concanavalin A-Sepharose and were purified by reversed-phase HPLC. The isolated glycopeptides were treated with PNGase F, releasing the carbohydrate moiety while converting Asn to Asp, and identified by amino acid analysis and/or peptide sequencing. Our results indicate that all 22 potential TV-glycosylation sites in the rgp 120 sequence are utilized. We did not detect TV-acetylgalactosamine in rgpl 20, indicating that the glycoprotein lacks typical O-linked oligosaccharides. To investigate the oligosaccharide structures at the sites of glycosylation, we determined the carbohydrate composition for each site and characterized the oligosaccharides by'H-NMR spectroscopy and by oligosaccharide mapping using high pH anion-exchange chromatography. Mannose and TV-acetylglucosamine were the only sugars observed in the intact rgp 120 and likewisein individual glycopeptides. All glycopeptides derived from rgp 120 contained high mannose-type TV-linked oligosaccharides, ranging from GlcNAc2Mans to GlcNAc2Mans. However, different glycosylation sites showedvaried degrees of processing of the high mannose-type oligosaccharides, as characterized by the ratio of GlcNAc2Mang-9 to GlcNAc2Man5_7. These results demonstrate that TV-glycosylation of rgpl 20 in the baculovirusexpression system occurs at all potential sites and is sitespecific in terms of oligosaccharide structures.Human immunodeficiency virus (HIV), 1 the causative agent of acquired immunodeficiency syndrome, contains glycopro-teins gpl20 and gp41 within its envelope. These two glycoproteins are produced by the proteolytic cleavage of a precursor protein, gpl60, and remain associated through noncovalent association (Robey et al., 1985). Gpl20 plays an important role in the first step of viral infection, because it serves as a high-affinity ligand for the T-cell receptor CD4 (Dalgleish et al., 1984; Klatzman et al., 1984). After binding to CD4, gp41 is then able to mediate virus-cell fusion via its TV-terminal hydrophobic domain, and subsequently the virus particle is released into the cells. Gpl20 is a heavily glycosylated protein with about 50% of its molecular weight contributed by carbohydrate (Allan et