Purification and characterization of a chemoattractant from electric shock-induced earthworm secretion, its receptor binding, and signal transduction through the vomeronasal system of garter snakes.

X C Jiang,J Inouchi,D Wang,M Halpern

doi:10.1016/s0021-9258(19)38950-1

Abstract

Following shocks with low voltage electric current, earthworms, Lumbricus terrestris, secrete a yellow mucus that has alarm properties for conspecifics and chemoattractive properties for garter snakes, Thamnophis sirtalis. A proteinaceous chemoattractant for garter snakes has been isolated and purified to homogeneity from such secretions by means of permeation chromatography and semipreparative nondenaturing polyacrylamide gel electrophoresis. The purified protein is highly attractive to garter snakes; it loses its activity after proteolytic digestion. It is a glycoprotein consisting of a single polypeptide chain with an NH2-terminal alanine. This chemoattractant has a minimum molecular mass of 15.4 kDa calculated from its amino acid and carbohydrate contents and an apparent molecular mass of about 20 kDa as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has a pI of about 4.0, and it binds wheat germ agglutinin but not concanavalin A. This chemoattractant shows a protein to carbohydrate ratio of 2.0 +/- 0.08 (n = 5) and a ratio of total sugar to amino sugar of 1.9 +/- 0.08 (n = 3). The sequence of its NH2-terminal 15 amino acid residues has been determined. Studies were also conducted on the chemosignal transduction through the vomeronasal sensory system of the garter snake. Dot blot analysis showed that the purified chemoattractant bound to snake vomeronasal sensory epithelial membrane fractions. It did not bind to membrane extracts of the nonsensory epithelium of the vomeronasal mushroom body. The chemoattractant also bound specifically to vomeronasal sensory epithelial membrane in a reversible and saturable fashion with Kd and Bmax values of about 0.3 microM and 0.4 nmol/mg of protein, respectively. In electrophysiological studies, the chemoattractant applied to the vomeronasal epithelium caused an increase in firing rate of individual neurons in the accessory olfactory bulb of garter snakes, the projection site for vomeronasal neurons. The present results are the first clear biochemical and electrophysiological evidence for a vomeronasal epithelium response to a purified nonvolatile odorant, and this makes the garter snake vomeronasal system ideal for studying the mechanisms of chemosignal transduction.

Highlights

It is a glycoprotein consisting of a single polypeptide chain with an NHzterminal alanine. This chemoattractant has a minimum molecular mass of 15.4 kDa calculated from its amino acid and carbohydrate contents and an apparent molecular mass of about 20 kDa as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis
The chemoattractant applied to the vomeronasal epithelium caused an increase in firing rate of individual neurons in the accessory olfactory bulb of garter snakes, the projection site for vomeronasal neurons
Species-specificNature of Chemoattractant(s) from Earthworms-Earthworm secretion produced by electrical stimulation consistently evoked chemoattractive responses in garter snakes

Summary

PROCEDURES

2 from Bio-Rad; 12’1 from Du Pant-New England Nuclear. All other reagents were the highest grade commercially available. Electric shock-induced earthworm secretion was prepared according to procedures described previously [14, 28] with modification as follows. During this time, the earthworms secreted viscous, cloudy, yellowish mucus-like fluid that drained and was collected into a beaker placed below the cone. (i) The electric shock-induced secretion (3 ml of 1 mg of protein/ml solution) was chromatographed on a Bio-Gel P-2 column (1.45 x 120 cm) preequilibrated with distilled water and eluted with the same solvent (this solvent was adopted because no difference in the separation had been found with saline or buffer and because we wished to recover simultaneously from the same run an earthworm alarm pheromone, a low molecular weight substance that would be lost upon dialysis [28]). The supernatants were used as membrane extracts in the dot blot procedure

Analytical Procedures

RESULTS

DISCUSSION