Localization of α 2u-globulin in the acinar cells of preputial gland, and confirmation of its binding with farnesol, a putative pheromone, in field rat (Millardia meltada).

Ramachandran Rajamanickam,Archunan Govindaraju,Padmanabhan Parasuraman,Achiraman Shanmugam,Rajkumar Ramalingam,Ponmanickam Ponnirul,Kamalakkannan Soundararajan,Rajagopal Thangavel,Ramya Vaideki Ganesan,Sankarganesh Devaraj,Govindhaswamy Umapathy

doi:10.1371/journal.pone.0197287

Ramachandran Rajamanickam, Archunan Govindaraju + Show 9 more

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https://doi.org/10.1371/journal.pone.0197287

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Abstract

Pheromones, low molecular weight chemical entities that bind to pheromone carrier proteins, are chemical signals that play an important role in the communication system in animals. This has been rather fairly well-studied in the rodents. The preputial gland, a rich source of pheromones in many rodents, contains a low molecular mass protein (18–20 kDa) that acts as one such pheromone carrier. However, the presence of this protein in the notorious rodent pest Millardia meltada has not yet been proven. Therefore, we aimed at identifying this protein, and the pheromones that are bound to it, in this rodent so as to utilize the information in the control of this pest. Twenty volatile compounds were identified in the preputial gland using GC-MS. Total protein of the gland was fractioned by both one and two-dimensional electrophoresis when we identified a low molecular mass protein (19 kDa, pI-4.7). Adopting MALDI-TOF MS and LC-MS analyses, the protein was confirmed as α 2u-globulin. To identify the volatiles bound to this protein, we used column chromatography and GC-MS. We found that farnesol and 6-methyl-1-heptanol are the volatiles that would bind to the protein, which we propose to be putative pheromones. Immunohistochemical analysis confirmed localization of α 2u-globulin in the acinar cells of the preputial gland. Thus, we show that α 2u-globulin, a pheromone-carrier protein, is present in the preputial gland acinar cells of M. meltada and suggest farnesol and 6-methyl-1-heptanol to be the volatiles which would bind to it. The α 2u-globulin together with farnesol and 6-methyl-1-heptanol contribute to pheromonal communication of M. meltada.

Highlights

Pheromones play inevitable roles in mammalian reproduction and social behavior including sexual attraction [1], territorial marking [2], mother-young interactions [3], conspecifics identification [4] and aggression [5]
We identified volatiles bound with α 2u-globulin in the preputial gland [24] and urine [25] of house rat
The major difference between a specialised and an ordinary sebaceous gland is the presence of different cell types with continuous formation of small to large lipid droplets therein in the former [30, 8]

Summary

Introduction

Pheromones play inevitable roles in mammalian reproduction and social behavior including sexual attraction [1], territorial marking [2], mother-young interactions [3], conspecifics identification [4] and aggression [5]. They are mostly proteins with a low molecular mass [10, 11] These barrel-shaped (eight-stranded βbarrel) protein molecules belong to the superfamily of lipocalins and possess a hydrophobic cavity that serves as a “container” for the volatile ligands [12]. This cavity binds small hydrophobic molecules including a wide range of odorants [13]. The pheromone-protein complex slowly releases the odorants [17] and is crucial in protecting the pheromones from rapid evaporation, eventually extending the shelf-life of the scent mark [23]. These proteins resist high temperature and are not likely to be fast denatured when released into the environment

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