Cellular and Molecular Life Sciences

, Volume 76, Issue 24, pp 5027–5039 | Cite as

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

  • Xitong Dang
  • Raul Coimbra
  • Liang Mao
  • Sonia Podvin
  • Xue Li
  • Hua Yu
  • Todd W. Costantini
  • Xiaorong Zeng
  • Dana Larocca
  • Brian P. Eliceiri
  • Andrew BairdEmail author
Original Article


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 establish that phage display can be used to identify cryptic domains within ORFs of the human secretome and identify a novel TLR4-targeted internalization domain in the amino terminus of ECRG4 that may contribute to its effects on cell migration, immune cell activation and tumor suppression.


Ligand Receptor Peptide targeting Secretome Phage display Cytokine precursor Tumor suppressor gene 



Research originally supported by the National Institutes of Health P20-GM078421 (AB/BPE), EY018479 (AB), HL73396 (BPE), the American Recovery Act (ARRA), and subsequently from GM121530 (TC), UC San Diego Department of Surgery Reinvestment Fund (RC) and completed with funding from grants 81-770-336 and 30-870-903 from the National Natural Science Foundation of China (XD). The authors declare no conflicts of interest and are indebted to Emelie Amburn’s expert laboratory support.


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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Department of SurgeryUniversity of California San DiegoSan DiegoUSA
  2. 2.The Key Laboratory of Medical Electrophysiology, Ministry of Education, Institute of Cardiovascular ResearchSouthwest Medical UniversityLuzhouChina
  3. 3.Mandala BiosciencesSan DiegoUSA
  4. 4.Department of SurgeryUniversity of California San DiegoSan DiegoUSA

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