A tandem repeat of a fragment of Listeria monocytogenes internalin B protein induces cell survival and proliferation.
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. InlB₃₆₋₃₂₁ has 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 InlB₃₆₋₃₂₁ with a linker peptide (2×InlB₃₆₋₃₂₁); 1×InlB₃₆₋₃₂₁ and 2×InlB₃₆₋₃₂₁ were purified from E. coli. Both 1× and 2×InlB₃₆₋₃₂₁ activated the Met tyrosine kinase. We subsequently compared signal transduction of the two proteins in primary lung endothelial cells. 2×InlB₃₆₋₃₂₁ activated ERK1/2, STAT3, and phosphatidylinositol 3-kinase/Akt pathways, whereas 1×InlB₃₆₋₃₂₁ activated only STAT3 and ERK1/2. The 2×InlB₃₆₋₃₂₁ promoted improved motility compared with 1×InlB₃₆₋₃₂₁ and additionally stimulated proliferation equivalent to full-length HGF. Both the 1× and 2×InlB₃₆₋₃₂₁ 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×InlB₃₆₋₃₂₁ to mimic HGF make it a potential candidate as a pharmaceutical agent for tissue repair.
Mungunsukh
Ognoon
O
Dept. of Pharmacology, Uniformed Services University of the Health Sciences, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA. rday@usuhs.mil
Lee
Young H
YH
Marquez
Ana P
AP
Cecchi
Fabiola
F
Bottaro
Donald P
DP
Day
Regina M
RM
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D002465:Cell Movement; D049109:Cell Proliferation; D002470:Cell Survival; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D017228:Hepatocyte Growth Factor / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012091:Repetitive Sequences, Nucleic Acid; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
D001426:Bacterial Proteins; D008565:Membrane Proteins; D050796:STAT3 Transcription Factor; C095768:inlB protein, Listeria monocytogenes; D017228:Hepatocyte Growth Factor; D019859:Proto-Oncogene Proteins c-met; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases