Characterization of Kunitz-type protease inhibitor purified from hemolymph of Galleria mellonella larvae

• Published in: Insect biochemistry and molecular biology 2010-12, Vol.40 (12), p.873-882
• Main Authors: Lee, Joon Ha, Kim, Chong Han, Shin, Yong Pyo, Park, Ho Jin, Park, Seungmi, Lee, Hwan Myung, Kim, Byung Sam, Lee, In Hee
• Format: Article
• Language: English
• Subjects: adipose tissue; amino acid sequences; Animals; biosynthesis; cDNA cloning; cell proliferation; complementary DNA; Epithelial Cells - enzymology; Epithelial Cells - immunology; Escherichia coli; Escherichia coli - immunology; Extracellular Signal-Regulated MAP Kinases - metabolism; fat body; Fat Body - enzymology; Fat Body - immunology; Galleria mellonella; Gene Expression; hemolymph; Hemolymph - chemistry; Hemolymph - metabolism; insect proteins; Insect Proteins - chemistry; Insect Proteins - genetics; Insect Proteins - metabolism; insects; integument; kinases; Kunitz-type protease inhibitor; Kunitz-type proteinase inhibitor; Larva - enzymology; Larva - immunology; larvae; midgut; mitogen-activated protein kinase; molecular sequence data; Moths - chemistry; Moths - cytology; Moths - metabolism; nucleotide sequences; phosphorylation; Physiological functions; proteolysis; Purification; sequence analysis; Serine Proteinase Inhibitors - chemistry; Serine Proteinase Inhibitors - genetics; Serine Proteinase Inhibitors - metabolism; signal transduction; transcriptome
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/j.ibmb.2010.08.007

We characterized a Kunitz-type protease inhibitor (Gm KTPI) obtained from the hemolymph of Galleria mellonella larvae immunized with Escherichia coli. The structural analysis of the cloned cDNA showed that it consists of 56 residues derived from the precursor of 75 amino acids. The peptide was constitutively produced in the fat bodies, but not in the midgut nor the integument of larvae. In our analysis of stage-dependent expression, its transcript was detected within the midgut, the fat bodies and the integument of the prepupae, which undergo tissue remodeling. The inhibition assays showed that Gm KTPI was capable of inhibiting only the trypsin-like activity of the larval midgut extracts. Furthermore, it was determined that Gm KTPI induced the activation of extracellular signal-regulated kinase (ERK) in the fat bodies and integument cells, and this kinase is known to perform a central role in cell proliferation signaling. Its effect on ERK activation was also verified in a control experiment using a human endothelial cell culture. Collectively, it was suggested that Gm KTPI might be responsible for the protection of other tissues against proteolytic attack by trypsin-like protease(s) from larval midgut during metamorphosis, and might play a role in the proliferation of cells in the fat body and integument. [Display omitted] ► Gm KTPI is expressed in the larval fat bodies and integument in response to E. coli. ► Gm KTPI inhibits the proteolytic activity of TLP(s) generated from the larval midgut. ► Gm KTPI induces ERK phosphorylation in the larval fat body and the integument.