University of Louisville
Characterization of Plethodontid TIMP-like Protein (PTP) and Its Role in Tissue Remodeling During Pheromone Gland Development
Institution
University of Louisville
Faculty Advisor/ Mentor
Richard Feldhoff; Pamela W. Feldhoff
Abstract
The red-legged salamander, Plethodon shermani, is a terrestrial, lungless salamander indigenous to the mountains of western North Carolina, and is a valuable model for studying reproductive behavior and pheromone signaling. During a specialized courtship behavior, male salamanders deliver pheromones to a female using a submandibular gland known as the mental gland. The proteinaceous secretion from the mental gland is primarily composed of two pheromone proteins (~85% of the total protein), and a third protein (~10%) termed Plethodontid TIMP-like Protein (PTP). PTP is named based on its similarity to inhibitors of Matrix MetalloProteinases (MMPs), the Tissue Inhibitors of MetalloProteinases (TIMPs). MMPs digest structural proteins in the extracellular matrix, and MMP-TIMP interactions can regulate tissue remodeling. Because of its similarity to the TIMPs, PTP is hypothesized to have TIMP- like activity and possibly facilitate mental gland development. Consequently, regular secretion of PTP during tail straddling walk may serve as a unique chemical signal that the courtship season is ongoing, and its absence may alert surrounding cells and proteases that the tissue is no longer needed. Therefore, the general aims of this study were to characterize the biology and function of PTP. To better characterize the function of PTP, the activity of six different recombinant MMPs were assayed using a fluorescent substrate with different concentrations of PTP. MMP-1, MMP-8, and MMP-13 were the only MMPs to be partially inhibited with PTP. The results were confirmed for MMP-1 using a broader range of PTP concentrations. In order to observe the expression and localization of PTP at different stages of mental gland development, immunofluorescence confocal microscopy was used to visualize PTP and Collagen II, one substrate for MMP-1. Low PTP levels during an early stage of mental gland development may drive collagen proteolysis and permit mental gland expansion. Finally, the PTP gene was cloned into E. coli to allow expression of a recombinant PTP (rPTP). rPTP was fused to Maltose Binding Protein, which has previously been demonstrated to increase TIMP solubility in E. coli. Expression of rPTP will permit future NMR studies to determine the 3D structure of this novel TIMP-like protein.
Characterization of Plethodontid TIMP-like Protein (PTP) and Its Role in Tissue Remodeling During Pheromone Gland Development
The red-legged salamander, Plethodon shermani, is a terrestrial, lungless salamander indigenous to the mountains of western North Carolina, and is a valuable model for studying reproductive behavior and pheromone signaling. During a specialized courtship behavior, male salamanders deliver pheromones to a female using a submandibular gland known as the mental gland. The proteinaceous secretion from the mental gland is primarily composed of two pheromone proteins (~85% of the total protein), and a third protein (~10%) termed Plethodontid TIMP-like Protein (PTP). PTP is named based on its similarity to inhibitors of Matrix MetalloProteinases (MMPs), the Tissue Inhibitors of MetalloProteinases (TIMPs). MMPs digest structural proteins in the extracellular matrix, and MMP-TIMP interactions can regulate tissue remodeling. Because of its similarity to the TIMPs, PTP is hypothesized to have TIMP- like activity and possibly facilitate mental gland development. Consequently, regular secretion of PTP during tail straddling walk may serve as a unique chemical signal that the courtship season is ongoing, and its absence may alert surrounding cells and proteases that the tissue is no longer needed. Therefore, the general aims of this study were to characterize the biology and function of PTP. To better characterize the function of PTP, the activity of six different recombinant MMPs were assayed using a fluorescent substrate with different concentrations of PTP. MMP-1, MMP-8, and MMP-13 were the only MMPs to be partially inhibited with PTP. The results were confirmed for MMP-1 using a broader range of PTP concentrations. In order to observe the expression and localization of PTP at different stages of mental gland development, immunofluorescence confocal microscopy was used to visualize PTP and Collagen II, one substrate for MMP-1. Low PTP levels during an early stage of mental gland development may drive collagen proteolysis and permit mental gland expansion. Finally, the PTP gene was cloned into E. coli to allow expression of a recombinant PTP (rPTP). rPTP was fused to Maltose Binding Protein, which has previously been demonstrated to increase TIMP solubility in E. coli. Expression of rPTP will permit future NMR studies to determine the 3D structure of this novel TIMP-like protein.