Abstract

The extent of liver fibrosis predicts prognosis and is important for determining treatment strategies for chronic hepatitis. During the fibrosis progression, serum levels of Mac2 binding protein (M2BP) increase and the N-glycan structure changes to enable binding to Wisteria floribunda agglutinin (WFA) lectin. As a novel diagnostic marker, glycosylation isomer of M2BP (M2BPGi) has been developed. However, its glycan structures recognized by WFA are unclear. In this study, we analyzed site-specific N-glycan structures of serum M2BP using Glyco-RIDGE (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile) method. We evaluated five sample types: 1) M2BP immunoprecipitated from normal healthy sera (NHS-IP(+)), 2) M2BP immunoprecipitated from sera of patients with liver cirrhosis (stage 4; F4-IP(+)), 3) M2BP captured with WFA from serum of patients with liver cirrhosis (stage 4; F4-WFA(+)), 4) recombinant M2BP produced by HEK293 cells (rM2BP), and 5) WFA-captured rM2BP (rM2BP-WFA(+)). In NHS-IP(+) M2BP, bi-antennary N-glycan was the main structure, and LacNAc extended to its branches. In F4-IP(+) M2BP, many branched structures, including tri-antennary and tetra-antennary N-glycans, were found. F4-WFA(+) showed a remarkable increase in branched structures relative to the quantity before enrichment. In recombinant M2BP, both no sialylated-LacdiNAc and -branched LacNAc structures were emerged. The LacdiNAc structure was not found in serum M2BP. Glycosidase-assisted HISCL assays suggest that, reactivity with WFA of both serum and recombinant M2BP depends on unsialylated and branched LacNAc, and in part of recombinant, depends on LacdiNAc. On M2BPGi, the highly branched LacNAc, probably dense cluster of LacNAc, would be recognized by WFA.

中文翻译：

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肝纤维化患者血清中紫藤凝集素阳性Mac2结合蛋白的N-聚糖结构†

摘要

肝纤维化程度可预测预后，对于确定慢性肝炎的治疗策略很重要。在纤维化进展期间，Mac2 结合蛋白 (M2BP) 的血清水平增加，N-聚糖结构发生变化，从而能够与紫藤凝集素 (WFA) 凝集素结合。作为一种新型诊断标志物，M2BP 的糖基化异构体 (M2BPGi) 已被开发出来。然而，其被 WFA 识别的聚糖结构尚不清楚。在这项研究中，我们分析了特定地点的N-血清 M2BP 的聚糖结构使用 Glyco-RIDGE（基于聚糖异质性的洗脱曲线上的糖肽信号的关系识别）方法。我们评估了五种样本类型：1) 从正常健康血清 (NHS-IP(+)) 中免疫沉淀的 M2BP，2) 从肝硬化患者血清中免疫沉淀的 M2BP（第 4 期；F4-IP(+)），3) 捕获的 M2BP来自肝硬化患者血清的 WFA（第 4 期；F4-WFA(+)），4) HEK293 细胞产生的重组 M2BP (rM2BP)，和 5) WFA 捕获的 rM2BP (rM2BP-WFA(+))。在NHS-IP(+) M2BP中，双触角N-聚糖是主要结构，LacNAc延伸到其分支。在 F4-IP(+) M2BP 中，许多分支结构，包括三天线和四天线N-聚糖，被发现。F4-WFA(+) 显示分支结构相对于富集前的数量显着增加。在重组 M2BP 中，没有出现唾液酸化的 LacdiNAc 和分支的 LacNAc 结构。在血清 M2BP 中未发现 LacdiNAc 结构。糖苷酶辅助 HISCL 测定表明，血清和重组 M2BP 与 WFA 的反应性取决于非唾液酸化和分支的 LacNAc，部分重组取决于 LacdiNAc。在 M2BPGi 上，高度分支的 LacNAc，可能是密集的 LacNAc 簇，将被 WFA 识别。