Type of Document Dissertation Author Negulescu, Ioan-Horia Author's Email Address email@example.com URN etd-01232012-115608 Title Defense Gland Protein from Coptotermes Formosanus Degree Doctor of Philosophy (Ph.D.) Department Biochemistry (Biological Sciences) Advisory Committee
Advisor Name Title Laine, Roger A. Committee Chair Henderson, Gregg R. Committee Member Lee, Yong-Hwan Committee Member Johnson, Charles E. Dean's Representative Keywords
- Defense gland protein
Date of Defense 2011-12-15 Availability unrestricted AbstractCoptotermes formosanus Shiraki is the most invasive termite species and has the largest economic impact of all the subterranean termites in the US. Although a lot of work has been done on the determination of chemical composition of frontal gland secretion from different families of termites, the protein component of those secretions was relatively overlooked.
This study started with the observation that proteins are present in the frontal gland secretion of Coptotermes formosanus Shiraki soldiers. The goal was to characterize one of these proteins by identifying its DNA sequence which would lead to protein sequence, function, structure and role it plays in the defense of Coptotermes formosanus Shiraki.
The protein cloned named TFP4 and analysis of its sequence revealed a molecular weight of 6853 Daltons. The sequence was compared with those of known proteins in NCBI's data base and similarities with other proteins analyzed. Two functions were assigned to TFP4, lysozyme and serine protease inhibitor.
The lysozyme function was assayed using a zymogram and a fluorometric assay. The zymogram showed clear zones where TFP4 had migrated indicating the β-(1,4) glucosidic linkages between N-acetyl-muramic acid and N-acetyl-D-glucosamine in the cell wall of Micrococcus lysodeikticus were hydrolysed. The fluorometric assay showed the fluorescent molecule which was embedded in the cell wall of Micrococcus lysodeikticus was released in solution upon addition of TFP4.
The serine protease assays tested the effect TFP 4 addition has on the reaction rates of trypsin, chymotrypsin and elastase with their respective substrates. The rate was unchanged in trypsinís assay but chymotrypsin and elastase were inhibited by TFP4.
In conclusion, we strongly suggest the existence of a protein of 6853 KDa with dual functions, lysozyme and serine protease inhibitor (chymotrypsin and elastase) in the defensive secretion of frontal gland of Coptotermes formosanus Shiraki soldiers. The inhibitor function is assigned to an exposed loop from Cys4 to Cys16 which functions as a substrate analog and employs a competitive inhibition mechanism for the inhibition of chymotrypsin while the lysozyme function is putatively assigned to a pocket between the β-hairpin structure containing Gln23 and the region at the C-terminus containing Glu45.
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