STRUCTURAL CHARACTERIZATION OF HIGH AFFINITY GLYCOPEPTIDES OF GLYCAM 1

高亲和力糖肽的结构表征 Glycam-1，L-选择素的生理配体。 的结合 选择素（钙依赖性凝集素样受体） 内源性糖蛋白配体介导初始相互作用 白细胞和内皮细胞之间的关系。 L-选择素参与了 将白细胞运输至炎症部位和组织化 淋巴器官 一种糖基化细胞粘附分子（GlyCMA-1）， 来源于淋巴结的高内皮小静脉，是一种可溶性的， 选择素的生理配体。 GlyCAM-1似乎是一种 与淋巴细胞L-选择素相互作用以诱导 整合素LFA-1的高亲合力状态。 GlyCAM-1是一种粘蛋白样的 糖蛋白，具有6'端寡糖的异质阵列 和6个硫酸化唾液酸刘易斯X型末端。 多肽骨架 的133个氨基酸残基具有10簇连续的羟基-氨基 酸残基（二分体，三分体或一个三分体），这表明，一个密切 低聚糖的堆积集合形成了生物学上相关的 配体。 我们建议使用串联识别O-聚糖阵列 碰撞诱导解离-埃德曼质谱法 测序 我们的初步研究使用高效液相色谱-电喷雾 离子化质谱（LC-ESI/MS）显示胰蛋白酶 在两个糖基化区域之间切割GlyCAM-1。 我们将 使用L-选择素亲和性的GlyCAM-1的胰蛋白酶解 层析 每个区域糖肽的寡糖 将被b-和6-O-SO 3-GlcNAc释放。 高亲和力区域 糖肽将被一系列特异性内切蛋白酶消化 以产生含有不同组合的糖肽， Ser/Thr簇。 将使用LC-ESI/MS分析样品， 选择性离子监测（m/z204）定位糖基化 片段 这些较小的糖肽将通过 使用免疫球蛋白/L-选择素嵌合体的亲和层析。 低聚糖将通过b-消除从 L-选择素结合糖肽。 使用基质辅助激光器 碰撞诱导解离解吸电离 (CID)，我们将表征O-聚糖的分子结构， 重量，组成（根据Neu 5Ac，Hex，deoxyHex，NexNAc， Hex-SO 3和HexNAc-SO 3），序列，分支和残基位置 硫酸盐部分。 寡聚核苷酸将被分配给特定的 使用MALDI-CID的肽基因座。 光谱将通过串联 仪器 这些最后的分析将给出i）肽序列 从气相去糖基化肽和相关的 ii）完整寡糖链的质量;和 iii）所述寡糖链的肽位置， 糖基化肽片段的质量。

Structural Characterization of the High Affinity Glycopeptides of Glycam-1, a physiological ligand of L-selectin. The binding of the selectins (calcium-dependent lectin-like receptors) to their endogenous glycoprotein ligands mediates the initial interactions between leukocytes and endothelia. L-selectin is involved in the trafficking of leukocytes to inflammatory sites and to organized lymphoid organs. A glycosylated cell adhesion molecule (GlyCMA-1), derived from high endothelial venules of lymph nodes, is a soluble, physiological ligand for L-selectin. GlyCAM-1 appears to be a signaling molecule that interacts with lymphocyte L-selectin to induce a high avidity state of the integrin, LFA-1. GlyCAM-1 is a mucin-like glycoprotein with a heterogeneous array of oligosaccharides with 6' and 6 sulfated sialyl Lewis X typetermini. The polypeptide backbone of 133 amino acid residues has 10 clusters of contiguous hydroxy-amino acid residues (dyads, triads or a tretrad), suggesting that a closely packed ensemble of olgiosaccharides forms the biologically relevant ligand. We propose to identify the arrays of O-glycans using tandem mass spectrometry with collision-induced dissociation and Edman sequencing. Our preliminary studies using HPLC-electrospray ionization mass spectrometry (LC-ESI/MS) have shown that trypsin cleaves GlyCAM-1 between the two glycosylated regions. We will fractionate tryptic digests of GlyCAM-1 using L-selectin affinity chromatography. The oligosaccharides from each regional glycopeptide will be released by b-and 6- O-SO3-GlcNAc. The high affinity regional glycopeptides will be digested with an array of specific endoproteases to produce glycopeptides containing different combinations of the Ser/Thr clusters. The digests will be analyzed using LC-ESI/MS with selective ion monitoring (m/z204) to located the glycosylated fragments. These smaller glycopeptides will be fractionated by affinity chromatography using an immunoglobulin/L-selectin chimera. The oligosaccharides willbe released by b-elimination from the L-selectin binding glycopeptides. Using matrix assisted laser desorption ionization (MALDI) with collision induced dissociation (CID), we will characterize the O-glycans with respect to molecular weights, composition (in terms of Neu5Ac, Hex,deoxyHex, NexNAc, Hex-SO3 and HexNAc-SO3), sequence, branching and residue location of sulfate moieties. Oligosaccharides will be assigned to specific peptide loci using MALDI-CID. Spectra will be obtained with a tandem instrument. These last analyses will give i) the peptide sequence from the mass of the gas-phase de-glycosylated peptide and associated fragments; ii) the masses of the intact oligosaccharide chains; and iii) the peptide location of the oligosaccharide chains from the masses of the glycosylated peptide fragments.