Noro Erika, Matsuda Atsushi, Kyoutou Takuya, Sato Takashi, Tomioka Azusa, Nagai Misugi, Sogabe Maki, Tsuruno Chikayuki, Takahama Yoichi, Kuno Atsushi, Tanaka Yasuhito, Kaji Hiroyuki, Narimatsu Hisashi
Glycoscience and Glycotechnology Research Group, Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8568, Japan.
Life Innovation Business Headquarters, Yokogawa Electric Corporation, Musashino, Tokyo 180-8750, Japan.
Glycobiology. 2021 Nov 18;31(10):1268-1278. doi: 10.1093/glycob/cwab060.
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.
肝纤维化程度可预测预后，对确定慢性肝炎的治疗策略至关重要。在纤维化进展过程中，Mac2结合蛋白（M2BP）的血清水平升高，且N-聚糖结构发生变化，使其能够与紫藤凝集素（WFA）结合。作为一种新型诊断标志物，已开发出M2BP的糖基化异构体（M2BPGi）。然而，其被WFA识别的聚糖结构尚不清楚。在本研究中，我们使用Glyco-RIDGE（基于聚糖异质性的洗脱谱上糖肽信号的关系鉴定）方法分析了血清M2BP的位点特异性N-聚糖结构。我们评估了五种样本类型：（1）从正常健康血清中免疫沉淀的M2BP（NHS-IP(+)），（2）从肝硬化患者血清中免疫沉淀的M2BP（4期；F4-IP(+)），（3）用WFA从肝硬化患者血清中捕获的M2BP（4期；F4-WFA(+)），（4）由HEK293细胞产生的重组M2BP（rM2BP）和（5）WFA捕获的重组M2BP（rM2BP-WFA(+)）。在NHS-IP(+) M2BP中，双天线N-聚糖是主要结构，且LacNAc延伸至其分支。在F4-IP(+) M2BP中，发现了许多分支结构，包括三天线和四天线N-聚糖。F4-WFA(+)相对于富集前的量，分支结构显著增加。在重组M2BP中，未出现唾液酸化-LacdiNAc和分支LacNAc结构。在血清M2BP中未发现LacdiNAc结构。糖苷酶辅助的HISCL分析表明，血清和重组M2BP与WFA的反应性取决于未唾液酸化和分支的LacNAc，部分重组体还取决于LacdiNAc。对于M2BPGi，高度分支的LacNAc，可能是LacNAc的密集簇，将被WFA识别。
