Abstract
The vomeronasal organ is a chemosensory organ present in most vertebrates and involved in chemical communication. In the last decade, the deciphering of the signal transduction process of this organ has progressed. However, less is known about the vomeronasal organ ligands and their structure-function relationships. Snakes possess a highly developed vomeronasal system that is used in various behaviors such as mating, predator detection, or prey selection, making this group a suitable model for study of the vomeronasal chemoreception. In this work, we used a proteomics approach to identify and characterize proteins from frog cutaneous mucus proteome involved in prey recognition by snakes of the genus Thamnophis. Herein we report the purification and characterization of two proteins isolated from the frog skin secretome that elicit the vomeronasal organ-mediated predatory behavior of Thamnophis marcianus. These proteins are members of the parvalbumin family, which are calcium-binding proteins generally associated to muscular and nervous tissues. This is the first report that demonstrates parvalbumins are not strictly restricted to intracellular compartments and can also be isolated from exocrine secretions. Purified parvalbumins from frog muscle and mucus revealed identical chemoattractive properties for T. marcianus. Snake bioassay revealed the Ca(2+)/Mg(2+) dependence of the bioactivity of parvalbumins. So parvalbumins appear to be new candidate ligands of the vomeronasal organ.
Highlights
The vomeronasal organ is a chemosensory organ present in most vertebrates and involved in chemical communication
Snakes possess a highly developed vomeronasal system that is used in various behaviors such as mating, predator detection, or prey selection, making this group a suitable model for study of the vomeronasal chemoreception
It is well established that Thamnophis predatory behavior involves detection by vomeronasal organ (VNO) of chemical cues on the prey surface [16, 26, 27]
Summary
Animals—This study was approved by the ethical review committee. Maintenance and care of animals were in compliance with guidelines set by the institutional animal care committee. Two-dimensional Polyacrylamide Gel Electrophoresis—After lyophilization, the mucus crude extract (about 0.1 g) was suspended in 500 l of denaturing buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, and 50 mM DTT) containing a complete set of protease inhibitors (Roche Applied Science) and centrifuged for 20 min at 20,000 ϫ g to discard the insoluble components. Western Blot Analysis—Proteins of the mucus crude extract (10 g) were separated by SDS-PAGE and blotted at 4 °C on a nitrocellulose membrane (Hybond ECL; Amersham Biosciences) using a semidry blotting device (Trans-Blot SD dry transfer cell, Bio-Rad) for 40 min at 500 mA and 25 V. Primary antibodies included a commercially available antibody raised against rat recombinant parvalbumin (1:1000; Swant, Bellinzona, Switzerland; this antibody equivalently recognizes ␣ and  parvalbumins as indicated by the manufacturer) and an anti-Ph14 polyclonal rabbit antibody (1:2000; Laboratoire d’Hormonologie, Marloie, Belgium) This antibody was produced from a SDS-PAGE protein band corresponding to mucus ␣ parvalbumin. The sections were counterstained with Luxol fast blue, dehydrated, and mounted with a permanent mounting medium
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