枸杞多糖LBP1C-2靶向钾离子通道β亚基-2活性结构域的探索  被引量:1

Exploration of the Active Domain of Polysaccharide LBP1C-2 Targetingβ-Subunit-2 of Voltage-Gated Potassium Channel

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作  者:曾晖 杨纯历 靳灿 丁侃 ZENG Hui;YANG Chunli;JIN Can;DING Kan(Chinese Academy of Sciences Shanghai Institute of Materia Medica,Shanghai 201203,China;Zhongshan Institute for Drug Discovery/Shanghai Institute of Materia Medica,Zhongshan 528400,China;School of Science,China Pharmaceutical University,Nanjing 211198,China)

机构地区:[1]中国科学院上海药物研究所,上海201203 [2]中国科学院上海药物研究所中科中山药物创新研究院,中山528400 [3]中国药科大学理学院,南京211198

出  处:《世界科学技术-中医药现代化》2024年第5期1182-1191,共10页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基  金:国家自然科学基金委员会面上项目(31870801):蛋白聚糖Glypican-6在胰腺癌及其肿瘤组织纤维化发生中的功能机制研究,负责人:丁侃;上海市“超级博士后”(2022686):负责人:靳灿;广东省高水平创新研究院项目(2021B0909050003)

摘  要:目的本研究旨在通过对LBP1C-2靶向Kvβ.2构效关系的探索,阐明LBP1C-2靶向Kvβ.2的活性结构域,为开发具有潜在抗早老痴呆活性的创新药物提供科学依据。方法枸杞多糖LBP1C-2经部分酸水解得到不同的结构片段后,对其进行单糖组成和分子量分析。通过蛋白芯片挑选出潜在靶蛋白,再用表面等离子体共振(SPR)技术验证各结构片段的靶向性。蛋白质免疫印迹以及细胞免疫荧光测试验证其生物学功能。结果通过HuProtTM人类蛋白质组芯片高通量筛选到LBP1C-2的潜在靶蛋白Kvβ.2。SPR实验表明LBP1C-2与Kvβ.2蛋白有较强结合,其结合常数KD为1.9×10^(-7) M。而LBP1C-2的化学降解后各结构片段与靶蛋白Kvβ.2有不同强度结合。其中含鼠李糖和半乳糖醛酸摩尔比为1:1的片段LBP1C-2-1I(18.1kDa)与原多糖LBP1C-2对Kvβ.2结合强度相近为3.3×10^(-7) M。对LBP1C-2-1I结构解析表明其有1,2-Rha与1,4-GalA交替连接组成。而该片段对应的酸水解外液部分LBP1C-2-1O与Kvβ.2也有结合,但与其他片段与该蛋白解离相比,更容易解离。从BV2小胶质细胞中敲减KCNAB2基因(Kvβ.2)后,BV2细胞中Aβ的降解被抑制,表明蛋白Kvβ.2可能是早老痴呆发生发展中的一个功能蛋白。结论LBP1C-2靶向Kvβ.2的主要活性结构域为1,2-Rha与1,4-GalA交替连接组成的主链核心骨架结构。本研究为深化了解LBP1C-2潜在的抗早老痴呆活性提供了重要线索,为基于枸杞多糖抗早老痴呆靶向性药物的设计和开发提供了证据。Objective This study aims to elucidate the structure-activity domain of LBP1C-2 targeting Kvβ.2 through an exploration of the structure-activity relationship.This study may also provide the scientific basis for the development of drug candidate with anti-early-onset dementia activity.Methods After partial acid hydrolysis,various structural fragments were obtained and subjected to monosaccharide composition and molecular weight analysis.Potential target proteins were selected using a protein chip,followed by validation of the targeting specificity of each structural fragment using surface plasmon resonance(SPR)technology.Results Through high-throughput screening using the HuProtTM human protein array,potential target protein Kvβ.2 was identified for LBP1C-2.SPR experiments revealed a strong binding affinity between LBP1C-2 and Kvβ.2 protein,with a binding constant(KD)of 1.9×10^(-7) M.The various structural fragments of LBP1C-2 exhibited different binding strengths with the target protein Kvβ.2.Among them,the segment LBP1C-2-1I(18.1 k Da)with a molar ratio of rhamose to galecturonic acid of 1:1 showed a binding strength to Kvβ.2 similar to that of the polysaccharide LBP1C-2,with a KD of approximately 3.3×10^(-7) M.Structural analysis indicates that the structure of LBP1C-2-1I contains 1,2-linked Rha and 1,4-linked GalA which are alternatively linked.The acid-hydrolyzed extracellular portion corresponding to this segment,LBP1C-2-1O may also bind to Kvβ.2.However,compared to other segments,it demonstrated a higher tendency to dissociate from the protein.Knockdown of the KCNAB2 gene(Kvβ.2)in BV2 cells inhibited the uptake of Aβin BV2 cells,suggesting that protein Kvβ.2 may be a functional protein in the development of Alzheimer′s disease.Conclusion LBP1C-2-1I has been identified as the primary active domain through which LBP1C-2 targets Kvβ.2.This suggests that the active domain of LBP1C-2 predominantly resides on the main chain rather than the side chain.This study provides crucial insights for a deeper

关 键 词:枸杞多糖 Lycium barbarum Kvβ.2蛋白 活性结构域 靶向性研究 

分 类 号:R285.5[医药卫生—中药学]

 

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