A small subset of genes in the human genome encode peptides that have intrinsic therapeutic value. These genes, collectively known as the "theratome", most often encode ligands and are of particular interest to endocrinology. The genes encoding growth hormone, somatostatin, erythropoeitin, neuropeptides, placental and pituitary hormones are all examples of the theratome. Unfortunately, the sequences encoding many ligands are not always detectable within the sequences of their large precursors. As a case in point, twenty years after the identification of the common precursor for ACTH, β-endorphin and α and γ MSH, a new ligand for adrenocortical cells has been recently discovered in amino terminus of POMC (Lowry et al Cell. 2001, 105: 903-12). For these reasons, we developed a method to detect the presence of ligands in the genome and describe a novel placental gene product: POP1.
We fragmented genes into small sequences of 100 to 300 bp and expressed them as fusions to protein lll in filamentous bacteriophage. These particles, engineered so as to contain the gene for green fluorescent protein (GFP) under the cmv promoter, have the capacity to transduce cells if they can cross the plasma membrane. Accordingly, we reasoned that if ligands for cell surface receptors are displayed, the particles will transduce their target cells.
Fragmentation for the gene encoding the EGF precursor generates EGF-targeted particles which specifically transfect and transduce cells that express EGF receptors. When low numbers of particles (1: 106) are added to peptide libraries displayed on phage, the EGF targeted phage can be recovered in fewer than 3 rounds of biopanning and FACS analyses of GFP positive cells. When this technique was applied to a placental cDNA library, we identified a novel gene fragment called POP-1 that encodes a peptide with the ability to transfect and transduce prostate cancer cells (PC3) in culture. It also has the ability to transfect other epithelial cells but appears specific since it cannot transduce other cell type s (e.g. endothelial cells). Cloning of the POP1 gene establishes that the ligand is at the C-terminus of a large precursor. Immunohistochemical studies demonstrate that it is widely distributed in tissues including in cell bodies and axonal projections of selected neurons in the brain and in endocrine tissues like adrenal, testes, prostate and thyroid carcinoma. With an absence of classical sites to predict the cleavage of the POP1 ligand from its precursor (e.g. di-basic amino acids), the findings suggest that there may still remain unidentified ligands in cryptic sequences of genes with no known function.
08 - 11 Apr 2002
British Endocrine Societies