Hirabayashi Jun, Hayama Ko, Kaji Hiroyuki, Isobe Toshiaki, Kasai Ken-ichi
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan.
J Biochem. 2002 Jul;132(1):103-14. doi: 10.1093/oxfordjournals.jbchem.a003186.
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.
蛋白质糖基化是后基因组学（蛋白质组学）领域的核心问题。我们采用了一种系统的方法来分析线虫秀丽隐杆线虫中产生的可溶性糖蛋白。该方法旨在确定：（i）编码糖蛋白的基因，（ii）发生糖基化的位点，以及（iii）连接的聚糖结构类型。以秀丽隐杆线虫的可溶性提取物为起始材料，首先将其应用于伴刀豆球蛋白A（ConA）柱（对高甘露糖型N-聚糖具有特异性），然后将流过部分应用于半乳糖凝集素LEC-6（GaL6）柱（对复合型N-聚糖具有特异性）。吸附的糖蛋白用赖氨酰内肽酶消化，所得糖肽用相同的凝集素柱选择性回收。糖肽通过反相色谱分离，然后进行序列测定。结果，分别被ConA柱和GaL6柱捕获的44个和23个糖肽成功得到分析，并分别对应于32个和16个相应基因。对于这些糖肽，通过实验确认了49个N-糖基化位点，而21个位点仍为潜在位点。在已鉴定的基因中，约80%在其他物种中有明显的同源物，如典型的分泌蛋白所示。然而，为ConA和GaL6识别的糖肽指定的两组基因彼此之间只有1个重叠。糖捕获方法在模式生物秀丽隐杆线虫中的实际适用性证明，引导我们去探索更复杂的多细胞生物。
