Detection ofα_2u-globulin and its bound putative pheromones in the preputial gland of the Indian commensal rat (Rattus rattus) using mass spectrometry

Abstract

The role of pheromones and pheromone-binding proteins in the laboratory rat has been extensively investigated. However, we have previously reported that the preputial gland of the Indian commensal rat produces a variety of pheromonal molecules and preputial glands would seem to be the predominant source for pheromonal communication. The presence of pheromone-binding proteins has not yet been identified in the preputial gland of the Indian commensal rat; therefore, the experiments were designed to unravel the alpha(2u)-globulin (alpha2u) and its bound volatiles in the commensal rat. Total preputial glandular proteins were first fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently analyzed by mass spectrometry. Further, we purified alpha2u and screened for the presence of bound pheromonal molecules with the aid of gas chromatography/mass spectrometry (GC/MS). A novel alpha2u was identified with a high score and this protein has not been previously described as present in the preputial gland of Indian commensal rats. This novel alpha2u was then characterized by tandem mass spectrometry (MS/MS). Peptides with m/z values of 969, 1192, 1303 and 1876 were further fragmented with the aid of MS/MS and generated de novo sequences which provided additional evidence for the presence of alpha2u in the preputial gland. Finally, we identified the presence of farnesol 1 and 2 bound to alpha2u. The present investigation confirms the presence of alpha2u (18.54 kDa) in the preputial gland of the Indian commensal rat and identifies farnesol 1 and 2 as probably involved in chemo-communication by the Indian commensal rat.

Evaluating the binding efficiency of pheromone binding protein with its natural ligand using molecular docking and fluorescence analysis

“…Therefore, they would seem to carry information that leads to aggression when perceived by male mouse and to an attraction when perceived by females12. Farnesol has been identified as the major volatile compound in the preputial gland of albino rat1013 and detected the occurrence of α2u with its bound putative pheromones (i.e., Farnesol1 and 2) in the preputial gland of house rat14. The pheromonal compound farnesol identified in the preputial gland of a rat is found to attract the opposite sex very efficiently15.…”

Identification of Volatile and Protein Profiles in the Sting and Mandibular Glands of the Worker Honey Bee (Apis cerana indica)

“…In addition, the N-terminal sequence (LEDAPRVKTPGAIGYYKI) for the 60 kDa protein was determined in Apis mellifera L. and it was reported as which involved in the antennal functions characteristic of drons [21]. In vertebrates, the pheromone carrying protein, α 2u -globulin (18)(19)(20)(21) was reported in the preputial gland of both adult [45,46] and pup [47], and urine of house rat [48] while the intensity of this protein was higher in the preputial gland of adult than the prepubertal rat. Moreover, another protein, aphrodisin (17 kDa) and Bartholin gland was also reported to be a pheromone carrier and facilitated the male copulatory behaviour [49].…”

Structural elucidation of estrus urinary lipocalin protein (EULP) and evaluating binding affinity with pheromones using molecular docking and fluorescence study

“…Since functionally the protein would bind fatty acid or its derivatives, further analysis led us to infer that farnesol and IBMP would be the most appropriate ligands that would bind with the protein. Farnesol, in a form bound to α2u-globulin, has been identified in the perpetual gland of house rat13, and it was also shown to be an alarm pheromone for aphids38.…”

“…It has been reported that transport of these chemical messages occurs when they are bound with MUPs and generate broad-ranging effects in the congeners101112. The pheromone compounds farnesol 1 and 2 identified in the preputial gland of house rat, which secretes volatiles abundantly, are bound to α2u13. Female rodents and other mammals excrete some specific volatile compounds in urine during the estrus phase of reproductive cycle.…”

“…The rat MUPs have a lot of structural similarity to the members of lipocalin superfamily1321. The members of lipocalin family (MLF) proteins are highly diversified in terms of structure, sequence as well as function and play important roles in transport of pheromones and retinol, olfaction, synthesis of prostaglandins and regulation of immune response.…”