Figure 2
From: Nanobodies targeting ABCC3 for immunotargeted applications in glioblastoma
Identification of nanobodies targeting ABCC3 following a peptide-based strategy for biopanning. (a) Proposed membrane topology of ABCC3 (UniProtKB reference: O15438). ABCC3 comprises two Membrane Spanning Domains (MSD-1 and MSD-2), each of them containing 6 transmembrane helices and followed by one Nucleotide-Binding Domain (NBD-1 and NBD-2), and an additional N-terminal domain containing 5 transmembrane helices (MSD-0). Extracellular N-glycosylation sites may regulate substrate specificity, stability, and localization of ABCC319. The location and amino acid sequence of the selected peptides in extracellular regions utilized to isolate specific nanobodies targeting ABCC3 from a phage display library is shown. (b) Amino acid sequence of the isolated nanobodies targeting ABCC3 depicting their Framework Regions (FR1-4, grey) and Complementarity Determining Regions (CDR1, pink; CDR2, green; and CDR3, given in alphabetic order, blue). Nanobodies NbA49, NbK2, NbK39, NbK229 and NbL115 present a VH-like VHH imprint in their framework 2 region (GLEW sequence). Nanobody NbL51 presents an extremely short CDR3 domain, which the longer CDR1 loop might compensate for, and a modified GLEW motif with a three-amino acid insertion. Some nanobodies targeting ABCC3 have an R118 residue instead of W118 as the first amino acid of the framework 4 region, which is exclusive of VHHs and not presented in humans, mouse nor camel VH. (c) Screening of the immunoreactivity of nanobodies targeting ABCC3 by flow cytometry. The immunoreactivity of 2 nanobodies targeting peptide ABCC3-A (NbA42, NbA213), 2 nanobodies targeting peptide ABCC3-K (NbK2, NbK39), and 1 nanobody targeting peptide ABCC3-L (NbL51) was confirmed. A549 cell line (A549 WT) and its ABCC3 knocked-out derivative cell line (A549ABCC3KO) were used as positive and negative controls. Secondary staining controls are also shown.
