Thermodynamic Switch in Binding of Adhesion/Growth Regulatory Human Galectin-3 to Tumor-Associated TF Antigen (CD176) and MUC1 Glycopeptides.
Thermodynamic Switch in Binding of Adhesion/Growth Regulatory Human Galectin-3 to Tumor-Associated TF Antigen (CD176) and MUC1 Glycopeptides.
复制标题
粘附/生长调节人半乳糖凝集素 3 与肿瘤相关 TF 抗原 (CD176) 和 MUC1 糖肽结合的热力学转换。
DOI：
10.1021/acs.biochem.5b00555
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发表时间：
2015
期刊：
影响因子：
2.9
通讯作者：
Cudic,Mare
中科院分区：
文献类型：
--
作者：
Rodriguez,MariaC;Yegorova,Svetlana;Pitteloud,Jean-Philippe;Chavaroche,AnaisE;André,Sabine;Ardá,Ana;Minond,Dimitriy;Jiménez-Barbero,Jesús;Gabius,Hans-Joachim;Cudic,Mare
在癌前和恶性上皮细胞中观察到粘蛋白型 O-糖基化发生向短链聚糖的转变。鉴于人半乳糖凝集素 3 可以与粘蛋白相互作用，并且与游离肿瘤相关 Thomsen-Friedenreich (TF) 抗原 (CD176) 相互作用也很弱，因此其与 MUC1 (糖) 肽相互作用的研究具有生物医学相关性。使用基于 Fmoc 的标准自动化固相肽化学合成携带通过 Thr 或 Ser 侧链连接的 TF 抗原的糖基化 MUC1 片段。通过等温滴定量热法测量，与游离 TF 二糖相比，galectin-3 与糖基化 MUC1 片段相互作用的解离常数 (Kd) 降低了 10 倍。没有观察到 MUC1 肽的非糖基化对照版本的结合。 MUC1 糖肽与 galectin-3 结合最显着的特征是从有利的焓转变为熵驱动的结合过程。对自由能 (ΔG) 的相对减少的焓贡献被熵项的相当大的增益所补偿。1H–15N 异核单量子相干光谱核磁共振数据揭示了规范位点的接触主要是通过 MUC1 糖肽的聚糖部分实现的。一种基于 AlphaScreen 技术的低亲和力聚糖-凝集素相互作用筛选新测定法也证实了结合亲和力的配体依赖性差异。另一个重要发现是，糖基化 MUC1 肽在基于细胞的检测中以浓度依赖性方式表现出活性，揭示了人半乳糖凝集素之间的选择性。因此，天然肽支架呈现的这种肿瘤相关碳水化合物配体增强了其亲和力，突出了人类凝集素与合成糖肽模型研究的重要性。
A shift to short-chain glycans is an observed change in mucin-type O-glycosylation in premalignant and malignant epithelia. Given the evidence that human galectin-3 can interact with mucins and also weakly with free tumor-associated Thomsen-Friedenreich (TF) antigen (CD176), the study of its interaction with MUC1 (glyco)peptides is of biomedical relevance. Glycosylated MUC1 fragments that carry the TF antigen attached through either Thr or Ser side chains were synthesized using standard Fmoc-based automated solid-phase peptide chemistry. The dissociation constants (Kd) for interaction of galectin-3 and the glycosylated MUC1 fragments measured by isothermal titration calorimetry decreased up to 10 times in comparison to that of the free TF disaccharide. No binding was observed for the nonglycosylated control version of the MUC1 peptide. The most notable feature of the binding of MUC1 glycopeptides to galectin-3 was a shift from a favorable enthalpy to an entropy-driven binding process. The comparatively diminished enthalpy contribution to the free energy (ΔG) was compensated by a considerable gain in the entropic term.1H–15N heteronuclear single-quantum coherence spectroscopy nuclear magnetic resonance data reveal contact at the canonical site mainly by the glycan moiety of the MUC1 glycopeptide. Ligand-dependent differences in binding affinities were also confirmed by a novel assay for screening of low-affinity glycan–lectin interactions based on AlphaScreen technology. Another key finding is that the glycosylated MUC1 peptides exhibited activity in a concentration-dependent manner in cell-based assays revealing selectivity among human galectins. Thus, the presentation of this tumor-associated carbohydrate ligand by the natural peptide scaffold enhances its affinity, highlighting the significance of model studies of human lectins with synthetic glycopeptides.