4.6 Article

The mAB 13A4 monoclonal antibody to the mouse PROM1 protein recognizes a structural epitope

Journal

PLOS ONE
Volume 17, Issue 10, Pages -

Publisher

PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0274958

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The study identified a structural epitope of PROM1 recognized by mAB 13A4 and demonstrated significant differences in affinity between mAB 13A4 and the PROM1 isoforms in photoreceptor cells. The results support the computationally predicted helical bundle structure of PROM1 and highlight the usefulness of mAB 13A4 in evaluating the impact of mutations on PROM1 structure.
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.

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