Targeted metabolic labeling of yeast N-glycans with unnatural sugars
Targeted metabolic labeling of yeast N-glycans with unnatural sugars
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DOI：
10.1073/pnas.0911247107
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发表时间：
2010-03-02
影响因子：
11.1
通讯作者：
Bertozzi, Carolyn R.
中科院分区：
文献类型：
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作者：
Breidenbach, Mark A.;Gallagher, Jennifer E. G.;Bertozzi, Carolyn R.
用合成糖类似物对聚糖进行代谢标记已成为将非天然化学功能引入糖蛋白的有吸引力的手段。然而，聚糖生物合成的复杂性阻止了非天然部分在高水平掺入的糖蛋白内的确定的、可预测的位置处的安装。在这里，我们证明了保守的N-乙酰葡糖胺（GlcNAc）的N-聚糖在模式生物酿酒酵母的壳二糖核心内的残基可以特别针对非天然糖的代谢替代。我们将外源性GlcNAc补救途径引入酵母，使细胞代谢作为培养基补充剂提供的GlcNAc。然后，我们通过删除必需基因GNA 1使酵母产生供体核苷酸-糖尿苷二磷酸-GlcNAc（UDP-GlcNAc）的营养缺陷型。我们证明，gna 1三角洲菌株需要一个GlcNAc的补充和表达质粒含有外源成分的补救途径，GlcNAc转运蛋白NGT 1从白色念珠菌和GlcNAc激酶NAGK从智人，需要在这种情况下救援。此外，我们表明细胞成功地将合成的GlcNAc类似物N-叠氮基乙酰葡糖胺（GlcNAz）和N-（4-戊炔酰基）-葡糖胺（GlcNAl）掺入细胞表面聚糖和分泌的糖蛋白中。为了验证在N-聚糖核心位置处的非天然糖的掺入，通过质谱法分析来自纯化的分泌性糖蛋白Ygp 1的内切糖苷酶H（endoH）消化的肽。多个Ygp 1 N-糖基化位点轴承GlcNAc，同位素标记的GlcNAc，或GlcNAz进行了鉴定，这些修改是依赖于添加到培养基中的补充。该系统使得能够产生在其糖基化位点处针对特定化学修饰而官能化的糖蛋白。
Metabolic labeling of glycans with synthetic sugar analogs has emerged as an attractive means for introducing nonnatural chemical functionality into glycoproteins. However, the complexities of glycan biosynthesis prevent the installation of nonnatural moieties at defined, predictable locations within glycoproteins at high levels of incorporation. Here, we demonstrate that the conserved N-acetyglucosamine (GlcNAc) residues within chitobiose cores of N-glycans in the model organism Saccharomyces cerevisiae can be specifically targeted for metabolic replacement by unnatural sugars. We introduced an exogenous GlcNAc salvage pathway into yeast, allowing cells to metabolize GlcNAc provided as a supplement to the culture medium. We then rendered the yeast auxotrophic for production of the donor nucleotide-sugar uridine-diphosphate-GlcNAc (UDP-GlcNAc) by deletion of the essential gene GNA1. We demonstrate that gna1 Delta strains require a GlcNAc supplement and that expression plasmids containing both exogenous components of the salvage pathway, GlcNAc transporter NGT1 from Candida albicans and GlcNAc kinase NAGK from Homo sapiens, are required for rescue in this context. Further, we show that cells successfully incorporate synthetic GlcNAc analogs N-azidoacetyglucosamine (GlcNAz) and N-(4-pentynoyl)-glucosamine (GlcNAl) into cell-surface glycans and secreted glycoproteins. To verify incorporation of the nonnatural sugars at N-glycan core positions, endoglycosidase H (endoH)-digested peptides from a purified secretory glycoprotein, Ygp1, were analyzed by mass spectrometry. Multiple Ygp1 N-glycosylation sites bearing GlcNAc, isotopically labeled GlcNAc, or GlcNAz were identified; these modifications were dependent on the supplement added to the culture medium. This system enables the production of glycoproteins that are functionalized for specific chemical modifications at their glycosylation sites.