Department of Biochemistry and Molecular Medicine, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia, United States of America.
Department of Ophthalmology and Visual Sciences, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia, United States of America.
PLoS One. 2022 Oct 10;17(10):e0274958. doi: 10.1371/journal.pone.0274958. eCollection 2022.
PROM1 (CD133, AC133) is a protein that is required for the maintenance of primary cilia. Mutation in the Prom1 gene in humans and animal models are associated with several forms of retinal degeneration. mAB 13A4 is the main reagent used to detect the mouse PROM1 protein. We endeavored to map the epitope of the rat monoclonal antibody mAB 13A4 to the mouse PROM1 protein. Deletion mutagenesis demonstrated that mAB 13A4 recognizes a structural epitope that is stabilized by two of the extracellular domains of PROM1. Furthermore, the affinity of mAB 13A4 to the major PROM1 isoform in photoreceptor cells is significantly reduced due to the inclusion of a photoreceptor-specific alternative exon in the third extracellular domain. Interestingly, a deletion in the photoreceptor specific isoform of six amino acids adjacent to the alternative exon restored the affinity of mAB 13A4 to PROM1. The results of the mutagenesis are consistent with the computationally predicted helical bundle structure of PROM1 and point to the utility of mAB 13A4 for evaluating the effect of mutations on the PROM1 structure. Our results show that the PROM1 isoform composition needs to be considered when interpreting tissue and developmental expression data produced by mAB 13A4.
PROM1（CD133、AC133）是一种维持初级纤毛所必需的蛋白。人类和动物模型中 Prom1 基因突变与几种形式的视网膜变性有关。mAB 13A4 是用于检测小鼠 PROM1 蛋白的主要试剂。我们努力确定大鼠单克隆抗体 mAB 13A4 针对小鼠 PROM1 蛋白的表位。缺失突变表明 mAB 13A4 识别一个结构表位，该表位由 PROM1 的两个细胞外结构域稳定。此外，由于第三个细胞外结构域中包含一个光感受器特异性的替代外显子，mAB 13A4 与光感受器细胞中主要 PROM1 同工型的亲和力显著降低。有趣的是，在与替代外显子相邻的光感受器特异性同工型中缺失六个氨基酸恢复了 mAB 13A4 对 PROM1 的亲和力。突变的结果与 PROM1 的计算预测的螺旋束结构一致，并指出 mAB 13A4 可用于评估突变对 PROM1 结构的影响。我们的结果表明，在解释由 mAB 13A4 产生的组织和发育表达数据时，需要考虑 PROM1 同工型的组成。
