Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, 1-7 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka, 560-0043, Japan; Project Research Center for Fundamental Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka, 560-0043, Japan.
Anal Biochem. 2020 Mar 15;593:113588. doi: 10.1016/j.ab.2020.113588. Epub 2020 Jan 22.
We previously identified fucosylated haptoglobin (Fuc-Hpt) as a clinical serum biomarker of pancreatic cancer and established the novel glycan monoclonal antibody (mAb) 10-7G. This antibody recognizes cancer-associated haptoglobin including Fuc-Hpt and the precursor of haptoglobin. Interestingly, Western blot analysis showed that the 10-7G mAb reacts with the haptoglobin α chain, which has no N-glycan potential sites; haptoglobin β chain has four N-glycan sites. In this study, we identified the epitope for the 10-7G mAb using haptoglobin deletion mutants, as well as inhibition ELISA with recombinant peptides. We illustrated molecular graphics to show a relationship between the epitope and the β chain. Furthermore, we hypothesized that the 10-7G mAb minimally recognizes normal haptoglobin, but aberrant glycosylation on the β chain causes conformational changes, enabling the 10-7G mAb to easily access the epitope within the α chain. Because 10-7G values, an enzyme-linked immunosorbent assay-immobilized 10-7G mAb, in patients with pancreatic cancer varied by haptoglobin phenotype, the amount of aberrant glycosylation needed to affect haptoglobin conformation probably depends on haptoglobin phenotype. Taken together, the 10-7G mAb recognized characteristic peptides on the haptoglobin α chain as a result of conformational changes and is a promising tool for diagnosing pancreatic cancer by haptoglobin phenotype.
我们之前发现岩藻糖化触珠蛋白（Fuc-Hpt）是胰腺癌的一种临床血清生物标志物，并建立了新型糖基单克隆抗体（mAb）10-7G。该抗体识别包括 Fuc-Hpt 在内的与癌症相关的触珠蛋白和触珠蛋白的前体。有趣的是，Western blot 分析表明，10-7G mAb 与触珠蛋白的α链反应，该链没有 N-糖基化潜在位点；触珠蛋白β链有四个 N-糖基化位点。在这项研究中，我们使用触珠蛋白缺失突变体以及用重组肽进行抑制 ELISA 来鉴定 10-7G mAb 的表位。我们用分子图形说明了表位与β链之间的关系。此外，我们假设 10-7G mAb 最小程度地识别正常触珠蛋白，但β链上的异常糖基化导致构象变化，使 10-7G mAb 能够轻松进入α链内的表位。因为 10-7G 值（酶联免疫吸附试验固定的 10-7G mAb）在胰腺癌患者中因触珠蛋白表型而异，所以影响触珠蛋白构象所需的异常糖基化量可能取决于触珠蛋白表型。总之，10-7G mAb 识别触珠蛋白α链上的特征肽是由于构象变化，是通过触珠蛋白表型诊断胰腺癌的有前途的工具。
