Abstract
Hepatocyte growth factor (HGF) is critical for tissue homeostasis and repair in many organs including the lung, heart, kidney, liver, nervous system, and skin. HGF is a heterodimeric protein containing 20 disulfide bonds distributed among an amino-terminal hairpin, four kringle domains, and a serine protease-like domain. Due to its complex structure, recombinant production of HGF in prokaryotes requires denaturation and refolding, processes that are impractical for large-scale manufacture. Thus, pharmaceutical quantities of HGF are not available despite its potential applications. A fragment of the Listeria monocytogenes internalin B protein from amino acids 36-321 (InlB&sub3;₆₋&sub3;&sub2;&sub1;) was demonstrated to bind to and partially activate the HGF receptor Met. InlB&sub3;₆₋&sub3;&sub2;&sub1; has a stable β-sheet structure and is easily produced in its native conformation by Escherichia coli. We cloned InlB&sub3;₆₋&sub3;&sub2;&sub1; (1×InlB&sub3;₆₋&sub3;&sub2;&sub1;) and engineered a head-to-tail repeat of InlB&sub3;₆₋&sub3;&sub2;&sub1; with a linker peptide (2×InlB&sub3;₆₋&sub3;&sub2;&sub1;); 1×InlB&sub3;₆₋&sub3;&sub2;&sub1; and 2×InlB&sub3;₆₋&sub3;&sub2;&sub1; were purified from E. coli. Both 1× and 2×InlB&sub3;₆₋&sub3;&sub2;&sub1; activated the Met tyrosine kinase. We subsequently compared signal transduction of the two proteins in primary lung endothelial cells. 2×InlB&sub3;₆₋&sub3;&sub2;&sub1; activated ERK1/2, STAT3, and phosphatidylinositol 3-kinase/Akt pathways, whereas 1×InlB&sub3;₆₋&sub3;&sub2;&sub1; activated only STAT3 and ERK1/2. The 2×InlB&sub3;₆₋&sub3;&sub2;&sub1; promoted improved motility compared with 1×InlB&sub3;₆₋&sub3;&sub2;&sub1; and additionally stimulated proliferation equivalent to full-length HGF. Both the 1× and 2×InlB&sub3;₆₋&sub3;&sub2;&sub1; prevented apoptosis by the profibrotic peptide angiotensin II in cell culture and ex vivo lung slice cultures. The ease of large-scale production and capacity of 2×InlB&sub3;₆₋&sub3;&sub2;&sub1; to mimic HGF make it a potential candidate as a pharmaceutical agent for tissue repair.