Aberrant Mucin-1 (MUC1) glycosylation with the Thomsen-Friedenreich (TF) tumor-associated antigen (CD176) is a hallmark of epithelial carcinoma progression and poor patient prognosis. Recognition of TF by glycan-binding proteins, such as galectins, enables the pathological repercussions of this glycan presentation, yet the underlying binding specificities of different members of the galectin family is a matter of continual investigation. While Galectin-3 (Gal-3) recognition of TF has been well-documented at both the cellular and molecular level, Galectin-1 (Gal-1) recognition of TF has only truly been alluded to in cell-based platforms. Immunohistochemical analyses have purported Gal-1 binding to TF on MUC1 at the cell surface, however binding at the molecular level was inconclusive. We hypothesize that glycan scaffold (MUC1’s tandem repeat peptide sequence) and/or multivalency play a role in the binding recognition of TF antigen by Gal-1. In this study we have developed a method for large-scale expression of Gal-1 and its histidine-tagged analog for use in binding studies by isothermal titration calorimetry (ITC) and development of an analytical method based on AlphaScreen technology to screen for Gal-1 inhibitors. Surprisingly, neither glycan scaffold or multivalent presentation of TF antigen on the scaffold was able to entice Gal-1 recognition to the level of affinity expected for functional significance. Future evaluations of the Gal-1/TF binding interaction in order to draw connections between immunohistochemical data and analytical measurements are warranted.

异常粘蛋白 1 (MUC1) 与 Thomsen-Friedenreich (TF) 肿瘤相关抗原 (CD176) 的糖基化是上皮癌进展和患者预后不良的标志。聚糖结合蛋白（如半乳糖凝集素）对 TF 的识别使这种聚糖呈递的病理学影响成为可能，但半乳糖凝集素家族不同成员的潜在结合特异性是一个不断研究的问题。虽然 Galectin-3 (Gal-3) 对 TF 的识别已在细胞和分子水平上得到充分证明，但 Galectin-1 (Gal-1) 对 TF 的识别仅在基于细胞的平台中被真正提及。免疫组织化学分析表明，Gal-1 与细胞表面 MUC1 上的 TF 结合，但在分子水平上的结合尚无定论。我们假设聚糖支架（MUC1 的串联重复肽序列）和/或多价性在 Gal-1 对 TF 抗原的结合识别中起作用。在这项研究中，我们开发了一种大规模表达 Gal-1 及其组氨酸标签类似物的方法，用于等温滴定量热法 (ITC) 的结合研究，并开发了一种基于 AlphaScreen 技术的分析方法来筛选 Gal- 1 抑制剂。令人惊讶的是，聚糖支架或支架上 TF 抗原的多价呈递都不能将 Gal-1 识别到预期的功能重要性水平。有必要对 Gal-1/TF 结合相互作用进行未来评估，以便在免疫组织化学数据和分析测量之间建立联系。