A tandem repeat of a fragment of Listeria monocytogenes internalin B protein induces cell survival and proliferation

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 (InlB36–321) was demonstrated to bind to and partially activate the HGF receptor Met. InlB36–321has a stable β-sheet structure and is easily produced in its native conformation by Escherichia coli . We cloned InlB36–321(1×InlB36–321) and engineered a head-to-tail repeat of InlB36–321with a linker peptide (2×InlB36–321); 1×InlB36–321and 2×InlB36–321were purified from E. coli . Both 1× and 2×InlB36–321activated the Met tyrosine kinase. We subsequently compared signal transduction of the two proteins in primary lung endothelial cells. 2×InlB36–321activated ERK1/2, STAT3, and phosphatidylinositol 3-kinase/Akt pathways, whereas 1×InlB36–321activated only STAT3 and ERK1/2. The 2×InlB36–321promoted improved motility compared with 1×InlB36–321and additionally stimulated proliferation equivalent to full-length HGF. Both the 1× and 2×InlB36–321prevented 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×InlB36–321to mimic HGF make it a potential candidate as a pharmaceutical agent for tissue repair.