Liau B, Tan B, Teo G, Zhang P, Choo A, Rudd P M
Analytics Department, Bioprocessing Technology Institute, Agency for Science, Technology and Research, 20 Biopolis Way, Singapore, 138668, Republic of Singapore.
Sci Rep. 2017 Nov 3;7(1):14489. doi: 10.1038/s41598-017-15123-z.
Cancers display distinctive carbohydrate molecules (glycans) on their surface proteins and lipids. mAb A4, an in-house generated monoclonal IgM antibody, is capable of distinguishing malignant ovarian carcinoma cells from benign ovarian epithelia by binding specifically to cancer cell-associated glycans. However, the structural details of the glycan targets of mAb A4 have been elusive. Here we developed a novel approach of isolating and fractionating glycan molecules released from glycoproteins in cancer cell lysates using HILIC-UPLC, and used them as probes on a microarray for affinity-based identification of the binding targets, allowing full-size, difficult to synthesize, cancer-associated glycans to be directly studied. As a result of this "shotgun" glycomics approach, we corroborate the previously assigned specificity of mAb A4 by showing that mAb A4 binds primarily to large (>15 glucose units), sialylated N-glycans containing the H-type 1 antigen (Fuc-α1,2-Gal-β1,3-GlcNAc). Although mAb A4 was also capable of directly binding to type 1 N-acetyl-lactosamine, this epitope was mostly shielded by sialylation and thus relatively inaccessible to binding. Knowledge of the structure of mAb A4 antigen will facilitate its clinical development as well as its use as a diagnostic biomarker.
癌症在其表面蛋白和脂质上呈现出独特的碳水化合物分子（聚糖）。单克隆抗体A4是一种内部研发的单克隆IgM抗体，它能够通过特异性结合癌细胞相关聚糖，将恶性卵巢癌细胞与良性卵巢上皮细胞区分开来。然而，单克隆抗体A4的聚糖靶点的结构细节一直难以捉摸。在这里，我们开发了一种新方法，使用亲水相互作用液相色谱-超高效液相色谱（HILIC-UPLC）从癌细胞裂解物中的糖蛋白释放的聚糖分子进行分离和分级，并将它们用作微阵列上的探针，基于亲和力鉴定结合靶点，从而能够直接研究全长、难以合成的癌症相关聚糖。作为这种“鸟枪法”糖组学方法的结果，我们通过表明单克隆抗体A4主要结合含有H型1抗原（Fuc-α1,2-Gal-β1,3-GlcNAc）的大的（>15个葡萄糖单位）、唾液酸化的N-聚糖，证实了之前确定的单克隆抗体A4的特异性。尽管单克隆抗体A4也能够直接结合1型N-乙酰乳糖胺，但这个表位大多被唾液酸化屏蔽，因此相对难以结合。了解单克隆抗体A4抗原的结构将有助于其临床开发以及用作诊断生物标志物。
