Open reading frame mining identifies a TLR4 binding domain in the primary sequence of ECRG4.

Open reading frame mining identifies a TLR4 binding domain in the primary sequence of ECRG4. Cell Mol Life Sci. 2019 Jun 12;: Authors: Dang X, Coimbra R, Mao L, Podvin S, Li X, Yu H, Costantini TW, Zeng X, Larocca D, Eliceiri BP, Baird A Abstract The embedding of small peptide ligands within large inactive pre-pro-precursor proteins encoded by orphan open reading frames (ORFs) makes them difficult to identify and study. To address this problem, we generated oligonucleotide (< 100-400 base pair) combinatorial libraries from either the epidermal growth factor (EGF) ORF that encodes the > 1200 amino acid EGF precursor protein or the orphan ECRG4 ORF, that encodes a 148 amino acid Esophageal Cancer Related Gene 4 (ECRG4), a putative cytokine precursor protein of up to eight ligands. After phage display and 3-4 rounds of biopanning for phage internalization into prostate cancer epithelial cells, sequencing identified the 53-amino acid EGF ligand encoded by the 5' region of the EGF ORF and three distinct domains within the primary sequence of ECRG4: its membrane targeting hydrophobic signal peptide, an unanticipated amino terminus domain at ECRG437-63 and a C-terminus ECRG4133-148 domain. Using HEK-blue cells transfected with the innate immunity receptor complex, we show that both ECRG437-63 and ECRG4133-148 enter cells by interaction with the TLR4 immune complex but neither stimulate NFkB. Taken together, the results help establ...
Source: Cellular and Molecular Life Sciences : CMLS - Category: Cytology Authors: Tags: Cell Mol Life Sci Source Type: research