Synthesis and properties of an EGF-like domain (residues 361-406) in the extreme N-terminal region of the mouse EGF precursor

• Published in: Growth factors (Chur, Switzerland) Vol. 17; no. 1; p. 37
• Main Authors: Diaugustine, R P, Henry, R, Sewall, C H, Suarez-Quian, C A, Walker, M P
• Format: Journal Article
• Language: English
• Published: England 1999
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Calcium Radioisotopes; Epidermal Growth Factor - chemical synthesis; Epidermal Growth Factor - metabolism; Epidermal Growth Factor - urine; Membrane Proteins - metabolism; Mice; Molecular Sequence Data; Peptide Fragments - chemical synthesis; Peptide Fragments - metabolism; Peptide Fragments - urine; Protein Precursors - chemical synthesis; Protein Precursors - metabolism; Protein Renaturation; Protein Structure, Tertiary; Rats; Receptor, Epidermal Growth Factor - metabolism
• ISSN: 0897-7194
• DOI: 10.3109/08977199909001061

Various proteins contain EGF-like domains that are not ligands for the EGF receptor. In the present study a cognate polypeptide for residues 361-406 of the mouse EGF precursor was synthesized by the solid-phase method. The product was renatured under oxidative conditions since it probably has an EGF-like array of three cystine disulfide bonds in its native state. HPLC analysis of the renaturation reaction revealed formation of a peak material with no apparent free-SH groups. Accordingly, the HPLC retention time of this product was readily increased by treatment (reduction of disulfides) with dithiothreitol. The renatured 46-mer (PEGF-1) did not displace 125I-EGF bound to rat liver membranes and 125I-PEGF-1 did not exhibit specific binding to membrane preparations from the mouse liver, mammary gland, or kidney, with or without Ca2+ in the binding medium. Although PEGF-1 contains a putative Ca2+ binding motif, specific binding of this cation by the polypeptide could not be demonstrated by electromobility shiff or incubation with 45Ca2+. Immunoassay of PEGF-1 and EGF in fractions obtained following gel filtration of mouse urine revealed multiple peaks of PEGF-1 immunoreactivity with the major peaks eluting at an Mr > 30 kDa. In contrast, virtually all the EGF immunoreactivity eluted at a volume similar to that of 125I-EGF. These data suggest that selective cleavage of the PEGF-1 domain from the precursor does not occur with the proclivity known for that of EGF. Instead, the PEGF-1 probably functions coordinately with other EGF-like domains while tethered to the precursor backbone. Finally, localization of PEGF-1 immunoreactivity occurred only in cell populations of the mouse previously demonstrated as sites for EGF/EGF precursor, which suggests that PEGF-1 is exclusively a domain of the EGF precursor.