Affinity capturing and gene assignment of soluble glycoproteins produced by the nematode Caenorhabditis elegans
Affinity capturing and gene assignment of soluble glycoproteins produced by the nematode Caenorhabditis elegans
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DOI：
10.1093/oxfordjournals.jbchem.a003186
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发表时间：
2002-07-01
影响因子：
2.7
通讯作者：
Kasai, K
中科院分区：
文献类型：
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作者：
Hirabayashi, J;Hayama, K;Kasai, K
蛋白质糖基化是后基因组(蛋白质组)科学的中心问题。我们采用了一种系统的方法来分析线虫产生的可溶性糖蛋白。该方法旨在指定(I)编码糖蛋白的基因，(Ii)发生糖基化的位置，以及(Iii)连接的糖链结构的类型。将线虫的可溶性提取物作为起始物质，首先应用于刀豆蛋白A(ConA)柱(专用于高甘露糖型N-聚糖)，然后将流过的部分应用于Galectin LEC-6(GaL6)柱(专用于复合型N-糖)。吸附的糖蛋白用赖氨酸内肽酶消化，得到的糖肽用相同的凝集素柱选择性地重新捕获。用反相色谱分离糖肽，然后进行序列测定。结果成功分析了ConA和GaL6柱捕获的44个和23个糖肽，并将其分别归属于32个和16个相应基因。对于这些糖肽，实验证实了49个N-糖基化位点，而21个位点仍然是潜在的位点。在已鉴定的基因中，约80%在其他物种中有明显的同源性，以典型的分泌蛋白为代表。然而，分配给ConA和GaL6识别的糖肽的两组基因只有1个相互重叠。糖链捕获方法对模式生物秀丽线虫的实际适用性的证明，指导我们探索更复杂的多细胞生物。
Protein glycosylation is a central issue for post-genomic (proteomic) sciences. We have taken a systematic approach for analyzing soluble glycoproteins produced in the nematode Caenorhabditis elegans. The approach aims at assigning (i) genes that encode glycoproteins, (ii) sites where glycosylation occurs, and (iii) types of attached glycan structures. A soluble extract of C. elegans, as a starting material, was applied first to a concanavalin A (ConA) column (specific for high-mannose type N-glycans), and then the flow-through fraction was applied to a galectin LEC-6 (GaL6) column (specific for complex-type N-glycans). The adsorbed glycoproteins were digested with lysylendopeptidase, and the resultant glycopeptides were selectively recaptured with the same lectin columns. The glycopeptides were separated by reversed-phase chromatography and then subjected to sequence determination. As a result, 44 and 23 glycopeptides captured by the ConA and GaL6 columns, respectively, were successfully analyzed and assigned to 32 and 16 corresponding genes, respectively. For these glycopeptides, 49 N-glycosylation sites were experimentally confirmed, whereas 21 sites remained as potential sites. Of the identified genes, about 80% had apparent homologues in other species, as represented by typical secreted proteins. However, the two sets of genes assigned for the ConA and GaL6-recognized glycopeptides showed only 1 overlap with each other. Proof of the practical applicability of the glyco-catch method to a model organism, C. elegans, directs us to explore more complex multicellular organisms.